annals of microscopy vol 16, april 2017 · 2017-06-06 · annals of microscopy vol 16, april 2017 3...

ANNALS OF MICROSCOPY Vol 16, April 2017

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SCOPE OF THE JOURNALThe “Annals of Microscopy” provides an international forum for researchers in biological, physical

and materials sciences to present and discuss new research on microscopy.

Fields of interest include: all forms of microscopy. Image acquisition and improvement techniques, along with computer-aided microscopy and analysis are included.

The journal publishes short communications, technical or scientifi c articles and reviews. Regular articles feature reports of new instrumentation, new theoretical methods and their applications to microstructural analysis in a broad range of fi elds in biological, physical and material sciences.

The journal also publishes selected news and commentaries of interest to members of microscopy societies and others working in the fi eld of microscopy and microanalysis.


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EDITORIAL BOARDEditor-in-chief Samuel Sam-Wah Tay

Associate Editors(Life Sciences) Boon-Huat Bay S. Thameem Dheen Goplakrishnakone P Eng-Ang Ling Mah-Lee Ng Yee-Kong Ng Sek-Mun Wong

(Physical Sciences) Thomas Osipowicz Frank Watt

Editorial Assistant Tuck-Yong Yick

International Advisory Board

Sadakazu Aiso, Japan Geoffrey Burnstock, UK Hiang Lian Hing, Malaysia Kuo-Shyan Lu, Taiwan John F Morris, UK Tetsuji Nagata, Japan Harumichi Seguchi, Japan Terence Heaton Williams, USA

ISSN: 0219-2209


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ContentsScope of the Journal ...............................................................................................................................................1

Editorial Board .........................................................................................................................................................2

REM Sleep Deprivation is Associated with Morphological and Functional Changes of Vascular Endothelium

Tengku Farah Adilah Tengku Adnan, Anis Amin, Che Badariah Ab Aziz, Wan Amir Nizam Wan Ahmad & Liza Noordin ...................................................................................................................................................................4

Preliminary Evaluation on the Effect of Methanolic Extract from Syzygium Polyanthum on Improvement of Hypertensive-Renal Damage among Spontaneous Hypertensive Rat Models

Nurul Syahida Ramli, Nor ´Atiqah Muhammad, Sabreena Safuan, Liza Noordin & Wan Amir Nizam Wan Ahmad ....................................................................................................................................... 15

Identifi cation of Particulate Iron Contamination from Na-Feldspar Production Using SEM/EDX Pornpot Nuthong ................................................................................................................................................. 23

Morphological Changes in the Liver of Rats (Rattus norvegicus) treated with different Doses of Doxorubicin

Ilham A. Al-Saleem, Hamad J. Jumaa, Imad M. Al-Ani, Hanaa Kh. Ismael ..............................................29

Association between Helicobacter Pylori Chronic Gastritis and Atherosclerosis of Carotid and Coronary Arteries in Autopsy Sample of Iraqi Population

Wafaa K. Ibraheem, Salim R.Hamoudi, Imad M.Al-Ani, Mohammed I.A. Mustafa .............................................37

Role of Microscopic Imaging and its Analytical Techniques in the Optimization of Geochemical Signature Natural Products for Infectious Disease Screening, Drug Discovery and Medicinal Effi cacy Studies.

Farid Che Ghazali, Norizzati Mohd Noor, Azreen Mohammad .............................................................. 46

Instructions To Contributors ............................................................................................................................. 76

Membership Application Form ........................................................................................................................ 78


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REM Sleep Deprivation is Associated with Morphological and Functional Changes of Vascular Endothelium

Tengku Farah Adilah Tengku Adnan1, Anis Amin2, Che Badariah Ab Aziz1, Wan Amir Nizam Wan Ahmad2 & Liza Noordin1*

1Department of Physiology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. 2Biomedicine Programme, School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.*Corresponding author: Tel: 609-7676175, Fax: 609-7653370, email: [email protected]

ABSTRACT

Rapid eye movement (REM) sleep deprivation increased free radicals that can cause endothelial dysfunction. The present study aimed to investigate the effect of 72-hour REM sleep deprivation on the serum levels of oxidative stress markers and to assess the morphological and functional changes in vascular endothelium. Twenty four (24) male Sprague–Dawley rats were divided equally into three groups: free-moving control rats (FMC), 72-h REM sleep-deprived rats (REMsd) and tank control rats (TC). The rats were deprived from REM sleep using inverted fl owerpot technique. Levels of oxidative stress markers; superoxide dismutase (SOD), malondialdehyde (MDA), glutathione reductase (GRx) were measured using ELISA kits. The descending thoracic aorta was examined under the scanning electron microscope (SEM). The descending thoracic aortic ring was exposed to acethylcoline (ACh) in the functional myographic study. There were no signifi cant differences in the levels of oxidative stress markers between the groups. The endothelium of REMsd group demonstrated a rough surface with numerous fi brin networks. The arterial ring from REMsd rats show endothelium-dependent vasomotion, suggesting an early stage of endothelial dysfunction. This study demonstrates that REM sleep deprivation is associated with endothelial damage and dysfunction. It provides a preliminary understanding of the association of REM sleep deprivation and endothelial dysfunction.

INTRODUCTION

Cardiovascular disease (CVD) is the leading cause of mortality worldwide (Sabanayagam & Shankar, 2010). The mortality rate of cardiovascular disease was higher in people who had sleep deprivation (Kario et al., 2010). Sleep deprivation, which includes insuffi cient duration, irregular timing of sleep, poor sleep quality and sleep/circadian disorders, is highly prevalent in modern society (Laposky et al., 2016). Although the physiologic mechanisms where sleep deprivation adversely affects the human life are poorly understood, a considerable body of clinical evidence has revealed a relationship between sleep deprivation and obesity (Gangwisch et al., 2005), hypertension (Tochikubo et al., 1996), atherosclerosis (Miller, 2011) and diabetes (Gottlieb et al., 2005). In addition, insuffi cient sleep leads to perception impairment, diffi culties in concentration, visual disturbances, slower reactions and poor memorizing (Orzeł-gryglewska, 2010). Thus, sleep deprivation results not only in potentially increased CVD, but also infl uence one’s emotional well being and mental health (Marks & Landaira, 2015.

Sleep has an antioxidative role as it removes free radicals that accumulate during wakefulness (Reimund, 1994). Numerous studies have been done on humans and animals to determine the physiological signifi cance of rapid eye movement (REM) sleep. REM sleep deprivation is widely known as a stressor that affect physical and biochemical changes in human that result in health consequences. Sleep deprivation in rats caused a signifi cant reduction in antioxidant enzymes including superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) in the hippocampus (Alzoubi et al., 2012). Signifi cant changes on oxidative stress markers have also been found in the hypothalamus and thalamus (D’Almeidaet al., 1998) and brainstem (Ramanathan et al., 2002) in sleep deprived rats. However, others have found that sleep deprivation did not affect


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oxidant production, antioxidant enzyme activity, lipid peroxidation or protein oxidation in the brain of a rat model (Gopalakrishnan et al., 2004).

Vascular endothelial dysfunction is one of the early changes in the pathophysiology of cardiovascular disease (Lum & Roebuck, 2001), which can serve as an independent predictor of future cardiovascular events. Oxidative stress has been attributed as one of the causes of endothelial dysfunction (Endemann & Schiffrin, 2004). Although sleep deprivation has been associated with cardiovascular diseases, the pathogenesis of cardiovascular disease in people with insuffi cient sleep is still poorly understood. To date, there is not a single study that has evaluated the association between oxidative stress that is produced during REM sleep deprivation with vascular endothelial dysfunction. The effects of oxidative stress induced by REM sleep deprivation could be an important contributor to cardiovascular disease. The aims of the study were to evaluate the effects of REM sleep deprivation on the serum levels of oxidative stress markers and to assess the morphological and functional changes in vascular endothelium following REM sleep deprivation.

MATERIAL AND METHODS Animals

The present study was approved by Animals Ethics Committee, Universiti Sains Malaysia. Twenty four, 8-10 week old Sprague-Dawley (SD) male rats, weighing between 200-250 g each were purchased from the Laboratory Animal Research Unit (ARASC), Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia. The rats were placed in polypropylene solid-fl oor cages and were maintained in a room with 12:12-h light dark cycle and controlled environment (temperature; 25 ºC, 60-70% humidity). Food and water were provided ad libitum. All rats were divided into three main groups (n= 8 for each group) consisting of free moving controls (FMC), REM sleep deprivation for 72 h (REMsd) and tank controls (TC) for 72 h.

Adaptation period:For REMsd and TC groups, rats were isolated and adapted individually in a dry tank model

(without water) for 72 hours before being exposed to the REM sleep deprivation model. The adaptation period was to expose and adapt the rats to the glass tank environment. Meanwhile, the FMC rats were adapted in their normal dry cage for 72 hours.

REM sleep deprivation modelThe REM sleep deprivation model was carried out by modifi cation of the inverted fl ower-

pot technique (Siran et al., 2014). It was shown to selectively deprive 90-99% of REM sleep in rats (May et al., 2005) and widely used for investigating the effects of REM sleep deprivation on psychological, physiological, biochemical and behavioral parameters. Two small platforms of 6.5-cm diameter each were placed in the middle of a glass tank, immersed in water. The height of the platform is 1-cm above a pool of water and only one rat was placed in a tank at one time. The glass tank measuring 30-cm in height, 30-cm in width and 60-cm in length and the top was covered by a wire mesh. This model is based on the principle that during REM sleep, animals lose their muscle tone that caused the rats to fall off the platform into the water, and wake up. Thus, this will prevent the development of the REM sleep component of the sleep cycle. Food and water were provided ad libitum by placing pellets and water bottles on a grid located on top of the chamber.

Control groupsRats were maintained in normal dry cages with one rat to a cage (Siran et al., 2014). For

TC group, this is similar model to REM sleep deprivation except the diameter of the platform was larger, 14 cm (Siran et al., 2014). A wide platform allowed the rats to sleep without falling into the water, thus rats experienced both NREM and REM sleep. Food and water were provided similarly as for the REM sleep deprivation model.


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Measurement of oxidative stress markers in serum At the end of the experiment, rats were subjected to terminal anaesthesia using sodium

pentobarbital (100 mg/kg; intraperitoneally). Blood was collected by cardiac puncture into plain tubes. The activity of superoxide dismutase (SOD) and glutathione reductase (GRx) were measured using EnzyChromTM Superoxide Dismutase Assay kit and EnzyChromTM Glutathione Reductase Kit (BioAssays, USA) respectively. Level of malondialdehyde (MDA) was measured using Malondialdehyde Assay Kit (North West Life Sciences, USA).

Histological analysis of endotheliumA 1-cm segment of the thoracic descending aorta was isolated (Izumi et al., 1998). This

aortic ring was cut into vertical section. Each sample was put directly into the McDowell-Trump Fixative at 4 ºC for 24 hours. Following fi xation, the sample was washed with Phosphate Buffer Saline (PBS) and fi xed in 1% osmium tetroxide. It was washed again with distilled water for two times, 10 minutes each. Subsequently, the sample was dehydrated with ascending grades of acetone (35% to 100% acetone), immersed into Hexamethyldisilazane (HDMS)/ethanol (1:1) for 15 minutes and 100% HDM for three times, 15 minutes each. The HDMS was then decanted from the specimen vial and the endothelium was left to air-dry at room temperature overnight. Finally, it was coated with gold and viewed under Quanta, FEG 450 Scanning Electron Microscopy (SEM) at 20kV. Normal thoracic descending aorta endothelium is characterised by a smooth surface, a regular arrangement of the endothelial cell that corresponds with the direction of the blood fl ow and without dilatation of intercellular clefts (Izumi et al., 2005).

Functional study of endotheliumSample preparation

The thoracic descending aorta was carefully isolated, cleaned from the adherent fat and connective tissues and cut into 2–4 mm-long ring. The aortic rings were immersed in Krebs’ solution. In all instances, extreme care was taken to avoid stretching and contact with the luminal surface to avoid damage to the endothelium during isolation. The aorta was either used the same day or kept overnight at 4º C in Krebs solution (viability as demonstrated by McIntyre et al., 1998). Krebs’ solution was prepared fresh on the day of the experiment. Four ring segments were prepared from each rat.

Subsequently, the aortic ring was suspended horizontally between parallel hooks in tissue chambers containing 10 ml of Kreb’s Henseleit solution (The constitution was NaCl 118.4; KCl 4.7; CaCl 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCO4 25.0; glucose 11.1 mM). The tissue-bath solution was continuously aerated with 95% O2/5%CO2 (carbogen) at 37 ⁰C. The aortic ring was allowed to equilibrate at a resting tension of 1.5 g for half an hour. Changes in isometric force were recorded by using of LabChart program (version 7.0) and a PowerLab data acquisition system (ADInstruments Ltd, Oxfordshire, UK). The preparations were allowed to equilibrate for half an hour, and then exposed twice to KCl at 40 mM for viability assessment. Only rings that gave contractile responses of more than 50% of maximum contraction by KCl (40 mM) were considered. Any aortic rings not contracted by more than 0.5 g (representing 50% of 40 mM normal KCl response) were excluded from the study.

Myographic experimentFour preparations can be used at one time. Phenylephrine was used as the precontractile agent.

Rings were pre-contracted with 1 μm of phenylephrine. At the plateau of tension development to phenylephrine, concentration-dependent relaxation responses to cumulative addition of acetylcholine (1, 10, 100 nM) was used to evaluate endothelium-dependent vasodilation (Furchgott and Zawadzki, 1980). The arterial rings were challenged with this acetylcholine twice interspersed by washing, equilibration and re-exposure to the phenylephrine.


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Statistical analysisData were analysed using one way analysis of variance (ANOVA) with Bonferroni’s post

hoc test. The level of signifi cance was set at p<0.05 and the results were expressed as mean ± SEM. Data analysis and graphs were plotted using GraphPad Prism version 6.00.

RESULTS

REM sleep deprivation has no effect on the serum level of oxidative stress markersIn order to determine the effects of REM sleep deprivation on oxidative stress markers, serum

levels of SOD, MDA and GRx were measured by ELISA. There was no signifi cant difference in the serum levels of SOD, MDA and GRx between groups as shown in Figure 1, 2 and 3 respectively.

Figure 1. The effect of 72-h REM sleep deprivation on serum levels of SOD in FMC (free-moving control) rats, 72-h REMsd (REM sleep-deprived rats) and TC (tank control) rats. Each value represents means ± S.E.M derived from eight animals.

Figure 2. The effect of 72-h REM sleep deprivation on serum levels of MDA in FMC (free-moving control) rats, 72-h REMsd (REM sleep-deprived) rats and TC (tank control) rats. Each value represents means ± S.E.M derived from eight animals.

Figure 3 The effect of 72-h REM sleep deprivation on serum levels of GPx in FMC (free-moving control) rats, 72-h REMsd (REM sleep-deprived) rats; TC (tank control) rats. Each value represents means ± S.E.M derived from eight animals.


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REM sleep deprivation induces endothelial damage

In order to determine the effects of REM sleep deprivation on the vascular endothelium, the endothelium of thoracic descending aorta was examined under the Scanning Electron Microscopy (SEM). The endothelial surface of both control groups appeared normal as shown in Figure 4a (FMC rats) and Figure 4b (TC rats). The surface was smooth with a regular arrangement of the endothelial cells and without dilatation of intercellular clefts. In contrast, the endothelial surface of REMsd rats was rough with irregular arrangement of endothelial cells and dilatations of intercellular clefts (Figure 5a). Interestingly, there were also numerous fi brin networks with rich interlacing and trapped red blood cells in the blood vessels of REMsd rats (Figure 5b).

Figure 4. Scanning electron micrographs of the endothelial surface of the thoracic descending aorta endothelium (transverse section) of (a) free-moving control rats and (b) tank control rats (magnifi cation, X2000). The endothelium is smooth with regular arrangement of the endothelial cells and no dilatation of intercellular clefts seen.


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REM sleep deprivation induces endothelial dysfunctionThe function of thoracic descending aorta endothelium between groups was determined

by using myographic test. Endothelium from all the groups relaxed to 10 nM of acetylcholine (ACh); Figure 6, 7 and 8 for FMC rats, REMsd rats and TC rats respectively. In addition, the pre-contracted aortic rings from all groups showed relaxation until 75% when exposed to 100 nM of acetylcholine. However, the tracing from 72-h REMsd group showed vasomotion as seen in Figure 7.

Figure 5. Scanning electron photomicrographs of the endothelial surface of the thoracic descending aorta endothelium (transverse section) of REM sleep deprived rats (magnifi cation, X2000). a) There is an irregular arrangement of endothelial cells and dilatations of intercellular clefts. b) Dense fi brin networks with rich interlacing and trapped red blood cells are clearly observed.


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Figure 6. Tracing from free-moving control rat shows the aortic ring was slightly relaxed when exposed to 10 nM acetylcholine and approximately 75% relaxation was observed when exposed to 100 nM of acetylcholine.

Figure 7. Tracing from free-moving control rat shows the aortic ring was slightly relaxed when exposed to 10 nM acetylcholine and approximately 75% relaxation was observed when exposed to 100 nM of acetylcholine. Vasomotion (as outlined by the oval shape) is shown in the tracing.


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DISCUSSION

Vascular endothelial dysfunction is widely accepted as the early changes in cardiovascular diseases. The present study looks at the role of oxidative stress that leads to vascular endothelial dysfunction in REM sleep deprived rats. In this study, three oxidative stress markers; SOD, MDA and GR were measured in the serum to identify possible factors that induced endothelial dysfunction. The morphological and functional changes of the endothelium of the thoracic aorta were also examined to confi rm the presence of endothelial dysfunction. Early sleep deprivation studies suggest that sleep deprivation is associated with oxidative stress. Thus, this study was carried out to determine whether REM sleep deprivation induced endothelial dysfunction via oxidative stress.

The prevalence of sleep disorder is increasing in modern societies, thus adequate sleep is crucial for preventing cardiovascular disease. Various cardiovascular risk factors have been reported to be associated with endothelial dysfunction such as smoking, aging, hypercholesterolaemia, hyperglycaemia and hypertension (Hadi et al., 2005). Accumulating evidence indicates that oxidative stress plays a major role in the initiation and progression of cardiovascular disease such as hypertension, coronary artery disease, chronic heart failure, and peripheral artery disease (Endemann & Schiffrin, 2004). Reactive oxygen species (ROS) play a physiological role in controlling endothelial function, vascular tone and vascular integrity. Several studies have demonstrated that enhanced ROS contributes to the pathogenesis of endothelial dysfunction (Annuk et al., 2003; Hadi et al., 2005). ROS are involved in the mechanisms of hypertrophy, proliferation, apoptosis, constriction, migration, fi brosis and angiogenesis, which contribute to endothelial dysfunction and arterial remodeling in cardiovascular diseases (Montezano & Touyz,

Figure 8. Tracing from free-moving control rat shows the aortic ring was slightly relaxed when exposed to 10 nM acetylcholine and approximately 75% relaxation was observed when exposed to 100 nM of acetylcholine.


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2011). With evidence implicating the development of oxidative stress in the hippocampus (Alzoubi

et al., 2012) and liver (Chang et al., 2008) in REM sleep deprived rats, it is essential to identify the roles of oxidative stress markers in REM sleep deprivation. However, in this study, there was no signifi cant differences in the levels of oxidative stress markers between the groups. In contrast to this study, Alzoubi et al., (2012) have demonstrated a signifi cant reduction in the levels of antioxidant enzymes including SOD, catalase and glutathione peroxidase in the hippocampus of sleep deprived rats. However, the duration of sleep deprivation in their study was for 96 hours. A degradation of SOD enzymes after prolonged activation during wakefulness may contribute to the decrease in the levels of SOD (Ramanathan et al., 2002). Other studies have demonstrated a signifi cant increase of MDA level in hepatocytes after 5 days of sleep deprivation (Chang et al., 2008). Thus, further studies are needed to identify other oxidative stress markers that might induce endothelial dysfunction. Measurement of oxidative stress markers in the blood vessel remains to be elucidated.

Interestingly, this study demonstrated REM sleep deprivation induced endothelial damage that was characterised by a dilatation of intercellular clefts, an irregular arrangement of endothelial cells and a rough surface. Besides, there were numerous fi brin (with trapped red blood cells) observed which indicated that coagulation process was taking place. The occurrence of endothelial damage was proven by the presence of fi brin as its formation usually occurs when there is a damage to the endothelium. Following injury, platelet tissue factor that is present in the subendothelial tissue is activated and initiates the blood coagulation cascades. Subsequently, platelet thrombus is produced together with fi brin clot formation (Furie & Furie, 2005). Damage to the endothelium promotes alteration in endothelium-mediated vasodilation, increased vascular reactivity, platelet aggregation, thrombus formation, increased permeability, leucocyte adhesion and monocyte migration (Minodora Andor, 2005). Based on the structural changes of the endothelium in the present study, it is confi rmed that REM sleep deprivation induced endothelial damage. Measurement of coagulation factors such as prostacyclin (PGI2), Thromboxane A2 (TxA2) and von Willerbrand factor (vWF) will be considered in future studies.

Vasomotion in vivo is rhythmic oscillations in vascular tone leading to fl ow motion. Vasomotion requires coordination of the activity of the vascular smooth muscle cells in the vessel wall. It is caused by local changes in smooth muscle; however its physiological role and its underlying mechanism remain elusive. In order to assess the functions of the vessel, a myographic study was done. The pre-contracted aortic rings from all groups showed relaxation until 75% when exposed to acetylcholine. However, the presence of vasomotion was shown in the tracing from 72-h REMsd rats. The presence of vasomotion in vitro indicates that the mechanism initiating vasomotion is inherent to the vascular wall (Peng et al., 2001). Vasomotion is suggestive of early stage of endothelial dysfunction and has been proposed as a predictive marker for atherosclerosis and hypercholesterolemia (Anderson et al., 1996). Further study is needed to determine the mechanism of vasomotion in REM sleep deprived rats.

CONCLUSION

From the results obtained in this study, we suggest that REM sleep deprivation is associated with endothelial damage and dysfunction. Fibrin formation might produce thrombus formation in the presence of prolonged or repeated exposure to cardiovascular risk factors. The damage has produced early endothelial dysfunction in REM sleep deprived rats that was demonstrated by the presence of vasomotion. Although the oxidative stress markers in the serum were not signifi cant, the occurence of oxidative stress cannot be excluded at this stage as there are many other oxidative stress markers which were not measured. Besides oxidative stress markers, other factors such as infl ammatory markers, coagulation factors and adhesion molecules


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may be considered for future experiments. This study suggests that sleep disturbance is a potential risk factor for the development of cardiovascular diseases. Poor sleep is considered a modifi able risk factor, thus, it is necessary to have adequate sleep to reduce the cardiovascular risk factors. Adequate sleep is also a key part of a healthy lifestyle. This study provides a preliminary understanding of the association of the REM sleep deprivation and endothelial dysfunction.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Dr. Sabreena Safuan from School of Health Sciences, Universiti Sains Malaysia for her expert advise on histology fi ndings. This work was funded by a grant from the Universiti Sains Malaysia (USM Short Term Grant: 304/PPSP/61312095).

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Preliminary Evaluation on the Effect of Methanolic Extract from Syzygium polyanthum on Improvement of Hypertensive-Renal Damage among Spontaneous Hypertensive Rat Models

Nurul Syahida Ramli1*, Nor ´Atiqah Muhammad1, Sabreena Safuan1, Liza Noordin2 & Wan Amir Nizam Wan Ahmad1

1Biomedicine Programme, School of Health Sciences,2Department of Physiology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan.*No. phone: +060145211351 Fax: +06097677515 Email address: [email protected]

ABSTRACT

Evidence has shown that there is a strong relationship between hypertension and kidney-disorder. Syzygium polyanthum, a local plant that has been claimed traditionally as anti-hypertensive and believed to-be an effective remedy for kidney problems as well. This study aimed to investigate the oral effect of 4-weeks administration of methanolic extract from S. polyanthum (MESP) leaves towards renal structural improvement in hypertensive-renal damage among spontaneous-hypertensive (SHR) and normotensive-rats (WKY). The study utilized 15-male; 10 SHR and 5 WKY rats. Methanolic-extract was successively prepared via ultrasound-assisted method. Both Group 1 (n=5) and Group 2 (n=5) received distilled water and served as negative control group of WKY and SHR respectively. The treatment group of SHR; Group 3 (n=5) received 2000 mg/kg MESP. At the end of study, all rats were euthanized and kidney tissues were isolated for histopathological studies using light microscopy (H&E-stain) and scanning electron microscopy. The study revealed that there was improvement of renal structure of the MESP-treated group; similar like normal structure WKY. The Bowman’s capsule is well capsulated and the Bowman’s space is well-defi ned. Meanwhile, the podocyte had better morphology. In contrast to untreated-SHR, the appearance of kidney structure is slightly attenuated due-to distortion and engorgement of the glomerulus. In conclusion, the result suggests that oral-administration of S. polyanthum has a renoprotective effect in improving renal morphology in hypertensive-renal damage of SHR rats.

Keywords: Hypertension, Syzygium polyanthum, Kidney disorder, Hypertensive rats, Histopathology

INTRODUCTION

Hypertension is one of the chronic diseases that can kill or reduce life expectancy and/or increased health problems. If left untreated, it will result in serious heart failure (Gaddam et al., 2009), stroke (White, 2009), renal disease and retinal complications (Cohuet and Struijker-Boudier, 2006). According to the National Health and Morbidity Survey (NHMS) 2015, hypertension prevalence in Malaysia was a general increasing trend with age from 0.7% in 18-19 year age populations, reaching a peak of 50.4% among 70 to 74 years age populations. Kearney and co-authors (2005) projected that, by year 2025, the number of adults with hypertension is estimated to increase up to 1.56 billion.

In the literature, there are many risk factors that infl uence the increment of the blood pressure (Ritchey, 2016). Of the great interest, the studies have proposed that damage of kidney as the major causative factor. Several studies have also proven that is a strong relationship between hypertension and kidney disorder (Barri, 2008; Zamo et al., 2010). Moreover, it is well-documented that chronic kidney disease (CKD) is a life-threatening disease frequently associated with hypertension, which eventually progress into renal fi brosis and renal failure (Kaze et al., 2015; Parnham et al., 2015).

Recent studies warrant specifi cally a fresh look at the development of renal damage in spontaneously hypertensive rats (SHR) (Hultstro¨m, 2012). This occurrence is probably due to SHR kidneys required a greater arterial pressure to excrete a given amount of salt and water than kidneys of Wistar Kyoto (WKY) control rats (Arendshorst and Beierwaltes, 1979). In addition to


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this, Norman et al., (1978) observed that relationship between SHR to those of WKY in term of arterial pressure-urinary output is also differed. They suggested a genetically determined renal structural alteration might be considered for future investigation since fewer studies have been carried out on hypertensive-kidney damage.

Previous studies done by our group have shown that Syzygium polyanthum, a local plant that has been claimed traditionally as anti-hypertensive; help in reducing the blood pressure increment; both in vitro and in vivo study (Ismail, 2015). However, the exact mechanisms on how it reduces the blood pressure still remain to be elucidated. Even reliable blood pressure measurements had been made in identifying the level of blood pressure, however its structural effect either improving the renal function and morphology of hypertensive rats or not are still unknown.

Thus, we suspected that if there is a linkage between hypertension and renal morphology, the structure of treated-kidney will improve along with decline of blood pressure. Therefore, the present study aimed to investigate the oral effect of 28 days administration of methanolic extract from S. polyanthum (MESP) on renal structural of hypertensive rats (SHR). In order to compare the results with control normotensive rats (WKY), the studies had utilised light microscopy and scanning electron microscopy.

MATERIALS AND METHODS

Plant Material Syzygium polyanthum leaves were collected at District of Bachok Kelantan, Malaysia. The

leaves were authenticated by Dr. Richard Chung at Forest Research Institute Malaysia (FRIM). The voucher specimen of S. polyanthum was deposited into FRIM herbarium (Sample number: PID-171011-10) at School of Biology, USM Penang.

Plant Extraction One kilogram (1 kg) of fresh S. polyanthum leaves was initially weighed, washed, air-

dried and oven-dried. The dried leaves were then grinded into powder form. Subsequently, the specimens were extracted via ultrasound-assisted (Sonicor Instrument Corporation, Copiague, N. Y.) using two solvents; n-hexane (Merck) and ethyl acetate (Merck) accordingly for 30 minutes each at room temperature (25 ± 1 °C). After that, the specimens were repeatedly sonicated three times using methanol (Merck) until clear extract observed. The fi nal fi ltered of methanolic extract was then concentrated in vacuo and stored in amber vial at -20 °C until further used. Methanolic extracts was chosen as it proved to show higher extractability and enhance higher antioxidant compounds as compare to other solvents (Annegowda et al., 2012).

AnimalsThe procedure and protocol described below had been approved by Animal Ethic Committee

of Universiti Sains Malaysia (Ref USM/ Animal Ethics Approval/2015 (95) (635)). This study utilised 10 male Spontaneous Hypertensive rats (SHR) and 5 male Wistar Kyoto rats (WKY). All rats were three months old weighing between 200g-250g. The animals were supplied and provided by Animal Research and Service Centre (ARASC), Health Campus, Universiti Sains Malaysia, Kelantan. Prior to the experiments, the animals were kept in standard rat cages and allowed to acclimatize for 7 days in a standard environmental condition (25 °C with 60–70% humidity) on a 12 hour light-dark cycle, and food ad libitum.

All rats were then equally divided into three different groups of fi ve rats each. Both Group 1 (n=5) and Group 2 (n=5) received distilled water and represented as negative control group of WKY and SHR respectively. The treatment group; Group 3 (n=5) received 2000 mg/kg methanolic extract of S. polyanthum (MESP). The selection of MESP dosage was based on a preliminary study by a previous group member (Ismail, 2015). All treatments were given daily via oral-gavage up to


17

four weeks with single administration per day. At the end of the treatment, all rats were euthanized via intraperitoneal injection with 100

mg/kg sodium pentobarbitone (Alfasan Woerden-Holland). After confi rming the death of rats, a portion of kidney was dissected out in order to further analyze the histopathological study using light microscope and scanning electron microscope.

Histopathology Examination

Hematoxylin and Eosin staining After dissection, small portions of kidney from the control and experimental animals were

fi xed immediately in 10% neutral buffered formalin until the structure of specimen looked stiff and hard. After that, the specimens were preceded with tissue processing, embedded in paraffi n wax and serially sectioned at 4 μm thickness using a microtome (Leica®; Germany). Finally, the stained specimen slides were then examined under light microscope for histopathological changes of the kidney structure.

Scanning Electron Microscopy The protocol for scanning electron microscopy was prepared by the Scanning Electron

Microscope Laboratory, PPSK, USM. Once kidney tissues were obtained from the euthanized rat, the tissues were immediately fi xated using McDowel-Trump Fixative at 4 °C for 24 hours. The fi xed tissues were then cut, washed, post-fi xed and dehydrated. After that, the tissues were allowed to be air dried using a critical point dryer, coated with gold and lastly viewed under Quanta FEG 450 Scanning Electron Microscope (SEM) by using XTm Product Version 4.1.7.2095 viewer software.

RESULTS

Histological Examination of the Kidney under Light Microscope (H&E stain) Figure 1 shows the histological assessment of treated and untreated kidney sections of

spontaneous hypertensive rats and Wistar Kyoto rats after 4 weeks administration under light microscopy (40x magnifi cation). The morphology of normotensive WKY kidney (Figure 1a) demonstrates normal condition of glomerulus where the Bowman’s capsule is well capsulated and the Bowman’s space is well defi ned. The glomerulus is well placed within the capsule with typical number of mesangial cells. However, in untreated SHR (Figure 1b), the appearance of the kidney structure is slightly attenuated due to distortion and engorgement of the glomerulus with diminishing number of mesangial cells. In addition, the Bowman’s space is also reduced and shows thickened capsular membrane. In contrast to treated SHR group (Figure 1c), the results

Figure 1. Histology slides showing the effects of different treatments on kidney of SHR and WKY under light microscope (H&E stain). (a) normal kidney WKY; (b) Untreated SHR kidney; (c) MESP-treated kidney SHR, with magnifi cation 40x.


18

reveal that there is a signifi cant improvement of kidney observed in comparison to untreated SHR in (Figure 1b); where the glomerulus size of treated MESP is much smaller and the membrane is slightly thinner. In addition, its morphology is nearly the same as that seen in the kidney WKY (Figure 1a) with well defi ned Bowman’s space.

Histological Examination of the Kidney under Scanning Electron Microscope (SEM)Figure 2 demonstrates the images of kidney section of the rats under 2500x and 10000x

magnifi cation via scanning electron microscope. At 2500x magnifi cation, 3D image of glomerulus was observed. In normal kidney of WKY (Figure 2a) (i), the glomerulus is illustrated as a tight ball of capillaries surrounded by podocytes (pedicals). The glomerulus is placed within the capsule where it is well surrounded by glomerular membrane. The podocytes exhibit as normal foot processes; which arrange in parallel, longer in size and show numerous footlike radiating surrounds the capillary loop. Furthermore, no distortions or fusions have been identifi ed when viewed at higher magnifi cation of 10000x (Figure 2a) (ii).

However, in untreated SHR (Figure 2b) (i), the appearance of kidney surface is distorted and

Figure 2. Scanning electron microscope images showing the effects of different treatments on kidney. (a) (i-ii) normal kidney WKY; (b) (i-ii) Untreated kidney SHR; (c) (i-ii) MESP-treated kidney SHR. (i) glomerulus; (ii) podocytes (pedicals), under magnifi cation 2500x and 10000x respectively.

a) Normal Kidney WKY

b) Untreated Kidney SHR

c) MESP-treated Kidney SHR


19

the glomerulus is largely shrink. There is also a slight space between the glomerulus as compared to normal kidney WKY due to loose of structure. Whereas, the podocytes present as enlarge in size, blunted, irregular in thickness, not in parallel arrangement and the pedicels indicate shorter and tortouos appearance (Figure 2b) (ii).

With 4-weeks treatment of 2000 mg/kg MESP on SHR (Figure 2c) (i), there are marked improvements of the glomerulus appearance seen; depicted by showing normal-like structure of the kidney WKY. The shape of glomelurus is well organised and the glomerulus is unshrunk. Meanwhile, the pedicels appearance is slightly improved (Figure 2c) (ii) compared with (Figure 2b), although it is not well preserved in normal WKY (Figure 2a).

DISCUSSION

Hypertension is defi ned as having elevation of blood pressure by means of increase systolic blood pressure (SBP) greater than 140 mmHg and/or having diastolic blood pressure (DBP) exceeding 90 mmHg in adults (Jones and Hall, 2004). It is well documented that hypertension might affect the major target organs (heart, arteries, kidneys) (Mensah et al., 2002) both structurally and functionally through pressure-independent mechanisms (Matavelli et al., 2007; Cavanagh et al., 2010). Among the target organs, much evidence has shown a close relationship between hypertension and kidney function (Barri, 2008; Zamo et al., 2010).

This study utilised spontaneous hypertensive rats (SHR) since the rats have been considered as model for human essential hypertension (Okamoto and Aoki, 1963; Doggrell and Brown, 1998). Other than that, there is also natural progression of kidney damage occur in SHR which mimic human pathophysiology (Zhou and Frohlich, 2007). Hence, selection of SHR is the most suitable models to look into the relationship between hypertension and kidney damage (Pinto et al., 1998; Chandran et al., 2014).

Results obtained previously by Ismail (2015) showed that SBP of untreated SHR started to show hypertensive levels at age of 8 weeks onwards (Dickhout and Lee, 1998; Lee et al., 2010). With similar study design as in previous study by Ismail (2015), our current histology studies has confi rmed that there is development of renal abnormalities observed at age of 3 months; both under light microscopy (HE stain) and scanning electron microscopy (SEM). The kidney of SHR is characterised by showing marked changes to the glomerulus size and structure as well as alteration to podocyte appearance when compared to their corresponding group WKY. These structural fi ndings are also consistent and in agreement to previous study reported by Garcia-Pinto et al., (2011) on the effect of low physical exercise among SHR models.

Collectively, these alterations happened probably due to increase intraglomerular pressure with possible reason for how hypertension affects the glomerular structure (Garcia-Pinto et al., 2011). Likewise, the integrity of podocytes also showed to be affected among hypertensive people (Wang et al., 2009). Palmer & Fenves (2010) have suggested that in damaged kidneys, the myogenic refl ex is commonly blunted. Whereas the pressure-natriuresis mechanism becomes dysfunction; requiring high pressure of renal perfusion to excrete out sodium and water (Kost et al., 1998). As a result, it will impair the renal autoregulation and loss the ability to prevent the transmission of systemic blood pressure changes into the glomerular circulation (Palmer and Fenves, 2010).

Unexpectedly, with 4-weeks treatment of 2000 mg/kg methanolic extract of S. polyanthum (MESP) on SHR, our current fi ndings revealed that there is marked improvement of the renal histology seen; depicted by showing similar-like normal structure as in the kidney of WKY. The shape and structure of the glomerulus is said to be more preserved as compared to untreated SHR. Meanwhile, the podocyte shows much better appearance.

The exact mechanism by which MESP improves the renal structure in hypertensive rats is still unknown. The possible reasons might be due to S. polyanthum leaves that showed mild antioxidant property (Har & Ismail, 2012). Oral gavage of this extracts (in vivo) by our previous group (Ismail,


20

2015), when acutely, or repeatedly administered for 3 weeks has caused signifi cant reduction in SBP. Thus, we suggest that the abundance of phenolic rich contents in crude MESP extract (Ismail, 2015) might act as an anti-hypertensive contributor (Ranilla et al., 2010) which simultaneously provide benefi t as renal protection as well. This is because we believe that there is a relationship between hypertension and renal morphology as the structure of treated-kidney has improved along with a decline of the blood pressure. This expectation is similar to agreement reported by Goth (1991) and Pinto et al., (1998) on other antihypertensive medications in improving renal damage. However, the contribution of these potential phenolic compounds towards hypotensive and renal effects by S. polyanthum leaves extracts remains not conclusive and requires further testing.

CONCLUSION

In conclusion, the result suggests that oral administration of S. polyanthum have a renoprotective effect in improving renal morphology in hypertensive rats.

ACKNOWLEDGEMENT

This study was supported by Short Term Grant (304/PPSK/61312059) from Universiti Sains Malaysia.

REFERENCES

Annegowda HV, Bhat R, Min-Tze L, Karim AA, Mansor SM. (2012). Infl uence of sonication treatments and extraction solvents on the phenolics and antioxidants in star fruits. Journal of Food Science and Technology, 49(4): 510–514.

Arendshorst WJ, Beicrwaltcs WH. (1979). Renal tubular reabsorption in spontaneously hypertensive rats. Am J Physiol, 273: F38.

Barri YM. (2008). Hypertension and kidney disease: a deadly connection. Curr Hypertens Rep., 10: 39-45.

Cavanagh EM, Ferder LF, Ferder MD, Stella IY, Toblli JE, Inserra F. (2010). Vascular structure and oxidative stress in salt-loaded spontaneously hypertensive rats: effects of losartan and atenolol. Am. J. Hypertens. 23(12): 1318-1325.

Chandran G, Sirajudeen KNS, Nik Syamimi Nik Yusoff, Swamy M, Samarendra Mutum S. (2014). Effect of the antihypertensive drug enalapril on oxidative stress markers and antioxidant enzymes in kidney of spontaneously hypertensive Rat. Evidence-Based Complementary and Alternative Medicine, 2014, 1-10.

Cohuet G, Struijker-Boudier H. (2006). Mechanisms of target organ damage caused by hypertension: therapeutic potential. Pharmacology & Therapeutics, (111): 81-98.

Dickhout JG, Lee RM. (1998). Blood pressure and heart rate development in young spontaneously hypertensive rats. The American Journal of Physiology—Heart and Circulatory Physiology, 274(3): H794–H800.

Doggrell SA, Brown L. (1998). Rat models of hypertension, cardiac hypertrophy and failure. Cardiovasc Res., 39: 89–105.

Foley RN, Collins AJ. (2013). The USRDS: what you need to know about what it can and can’t tell us about ESRD. Clin. J. Am. Soc. Nephrol., 8: 845–851

Gaddam KK, Verma A, Thompson M, Amin R, Ventura H. (2009). Hypertension and cardiac


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failure in its various forms. The Medical Clinics of North America, 93(3): 665–80.Garcia-Pinto AB, de Matos VS, Rocha V, Moraes-Teixeira J, Carvalho JJ. (2011). Basic research:

low-intensity physical activity benefi cially alters the ultrastructural renal morphology of spontaneously hypertensive rats. Clinics, 66(5): 855-863.

Goth L. (1991). A simple method for determination of serum catalase activity and revision of reference range. Clinica Chimica Acta, 196(2-3): 143–151.

Grolllman A. (1972). The spontaneously hypertensive rat: An experimental analogue of essential hypertension in the human being, in Spontaneous Hypertension, edited by OKAMOTO K, Tokyo, Igaku Sham, p. 238

Har LW, Ismail IS. (2012). Antioxidant activity, total phenolics and total fl avonoids of Syzygium polyanthum (Wight) Walp leaves. International Journal of Medicinal and Aromatic Plants ISSN 2249-4340, 2(2): 219-228.

Hultstro¨m M. (2012). Review: Development of structural kidney damage in spontaneously hypertensive rats. Journal of Hypertension, 30: 1087–1091.

Ismail A, Mohamed M, Sulaiman SA, Wan Ahmad WAN. (2013). Autonomic nervous system mediates the hypotensive effects of aqueous and residual methanolic extracts of Syzygium polyanthum (Wight) Walp. var. polyanthum leaves in anaesthetized rats. Evidence-Based Complementary and Alternative Medicine, 1-17.

Ismail A. (2015). Pharmacological evaluation on the effect of Syzygium polyanthum (Wight) Walp. Leaves on rat’s blood pressure and related parameters. PhD Thesis, Universiti Sains Malaysia, Malaysia.

Jones DW, Hall JE. (2004). Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure and evidence from new hypertension trials. Hypertension, 43: 1–3.

Kaze FF, Meto DT, Halle MP, Ngogang J, Kengne AP. (2015). Prevalence and determinants of chronic kidney disease in rural and urban Cameroonians: a cross-sectional study. BMC Nephrol., 16: 117.

Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J. (2005). Global burden of hypertension: analysis of worldwide data. Lancet, 365(9455):217-23.

Kost Jr CK, Li P, Willians S, Jackson EK. (1998). Renal vascular responses to angiotensin II in conscious spontaneously hypertensive and normotensive rats. J Cardiovasc Pharmacol., 31: 854-61.

Lee SK, Arunkumar S, Sirajudeen KNS, Singh HJ. (2010). Glutathione system in young spontaneously hypertensive rats. Journal of Physiology and Biochemistry, 66(4): 321– 327.

Matavelli LC, Zhou X, Varagic J, Susic D, Frohlich ED. (2007). High-salt diet produces severe renal dysfunction independently of changes in arterial pressure in spontaneously hypertensive rats (SHR). Am. J. Physiol. Heart. Circ. Physiol., 292(H): 814–819.

Mensah GA, Croft JB, Giles WH. (2002). The heart, kidney, and brain as target organs in hypertension. Cardiol Clin., 20(2): 225-47.

National Health and Morbidity Survey (NHMS). (2015). Volume II: Non-Communicable Diseases, Risk Factors & Other Health Problems. Ministry Of Health Malaysia. Available from URL http://www.iku.gov.my/images/IKU/Document/REPORT/nhmsreport2015vol2.pdf

Norman RA, Enobakhare JA, De Clue JW, Douglas BH, Guyton AC. (1978). Arterial pressure-urinary output relationship in hypertensive rats. Am J Physiol., 243: R98.

Okamoto K, Aoki K. (1963). Development of a strain of spontaneously hypertensive rats. Jap Circ J, 27: 282-293.

Palmer BF, Fenves AZ. (2010). Optimizing blood pressure control in patients with chronic kidney disease. Proc (Bayl Univ Med Cent). 23: 239-45.


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Parnham S, Gleadle JM, Bangalore S, Grover S, Perry R, Woodman RJ, De Pasquale CG, Selvanayagam JB. (2015). Impaired myocardial oxygenation response to stress in patients with chronic kidney disease. J. Am. Heart Assoc., 4: e002249.

Pinto YM, Paul M, Ganten D. (1998). Lessons from rat models of hypertension: from Goldblatt to genetic engineering. Cardiovascular Research, 39(1): 77–88.

Ranillaa LG, Kwonb Y, Emmanouil Apostolidis E, Shetty K. (2010). Phenolic compounds, antioxidant activity and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension of commonly used medicinal plants, herbs and spices in Latin America. Bioresource Technology, 101(12): 4676-4689.

Ritchey M, Yuan K, Gillespie C, Zhang G, Ostchega Y. (2016). Development and validation of a hypertension prevalence estimator tool for use in clinical settings. The Journal of Clinical Hypertension.

Wang G, Lai FM, Kwan BC, Lai KB, Chow KM, Li PK, Stezo CC. (2009). Podocyte loss in human hypertensive nephrosclerosis. Am J Hypertens., 22: 300–306.

White WB. (2009). Defi ning the problem of treating the patient with hypertension and arthritis pain. The American Journal of Medicine, 122(5 Suppl): S3–9.

Zamo FS, Lacchini S, Mostarda C, Chiavegatto S, Silva IC, Oliveira EM, Irigoyen MC. (2010). Hemodynamic, morphometric and autonomic patterns in hypertensive rats - Renin-Angiotensin system modulation. Clinics (Sao Paulo), 65: 85-92.

Zhou X, Frohlich ED. (2007). Analogy of cardiac and renal complications in essential hypertension and aged SHR or L-NAME/ SHR. Medicinal Chemistry, 3(1): 61–65.


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Identifi cation of Particulate Iron Contamination from Na-Feldspar Production Using SEM/EDX

Pornpot Nuthong*Scientifi c Equipment Center, Prince of Songkla University, Songkhla, Thailand*Corresponding author: Email: [email protected]

ABSTRACT

Na-feldspar products from the crushing plant can be contaminated with Fe-particles, caused by inappropriate process used. Na-feldspar particles and contaminants, characterized by SEM-EDX, were irregular in structure with the diameter of 2-150 μm and 3-80 μm, respectively. Based on EDX result, the concentration and distribution of Fe, Cr and Mn elements could be discriminated between Na-feldspar and contaminant particles. The wear and tear of jaw crusher used in crushing process wares therefore a major cause of Fe-contamination in Na-feldspar.

Key words: Na-feldspar, SEM-EDX, Contamination, Jaw crusher

INTRODUCTION

Thailand is one of the important countries in Asia for feldspar production. The current production of Thailand is around 800,000 metric tons per year (Heyes et al., 2012) Feldspar minerals have been used as important industrial raw material. They are widely used in ceramic, porcelain and glass industries. Sixty percent of the world feldspar have been used for ceramic industry. A purity of feldspar plays an important role in the ultimate color and quality of the ceramic products. White color is a requirement for the production of good quality colored ceramic products (Roy et al., 2010). Therefore, the enhancement of feldspars quality by removing an impurity from the minerals is required. The impurity minerals found in feldspar ores are TiO2 and Fe2O3. If amounts of these minerals are higher than the critical content (0.50%), the quality of ceramic products decreases, mainly associated with the color changes (Hacifazlioglu et al., 2012).

Flotation is the conventional method that is still used for removal of the impurity in feldspar. The separation procedure of feldspar ore by fl otation starts from grinding the rocks. Then, crushed rocks are de-slimed and sieved for the selected particle size and particles with the size below 20 microns are rejected. Fine grains of rocks are treated with acid medium for impurity removal in the fl otation process, such as Mica, Fe minerals. The feldspar is subsequently dewatered with a rotary dryer. Then Fe2O3 in feldspar is removed by a magnetic separator (Gaudin, 1957).

The effi ciency of feldspar processing depends on the confi guration of the process, equipment design and the chemical and physical properties of milled aqueous mineral suspensions (Jamett et al., 2012). The combination of fl otation and magnetic separation process can remove Fe2O3 in feldspar as high as 90% (Hacifazlioglu et al., 2012). Although the particle size screening with sieving machine is the suitable method for enrichment of feldspars in terms of cost and simplicity, but a few impurity of Fe particle are contaminated during the process, especially, crushing process.

In general, the metal component from mechanical parts of grinding machine could be contaminated together with feldspar mineral before the enrichment. Therefore, crusher, jaws crushers, cone crusher and vibrator screen can affect the impurity of Na-feldspar. The aim of this study was to assess the impurity of feldspar using SEM/EDX.


24


Sample collection and preparationSodium feldspar ores (NaAlSi3O8) were supplied from a crushing plant in the south of

Thailand. The ores were crushed by the primary and secondary crushers, followed by grinding using the jaw crushers and cone crushers. Then, the ground ores were sieved with vibrator screen. The fractions with the size less than 841 micron were used. The prepared samples were dried in the hot air oven at 60°C for 24 h. The dried feldspar ores were dispersed on the Tefl on stub with a double sided conductive adhesive tape. Samples were coated with carbon by arc discharge method (Vacuum Evaporator, JEE-400, JEOL, Japan).

Scanning Electron Microscopy (SEM)The scanning electron microscope (Quanta400, FEI, Netherland) was used. The morphological

information was operated by tungsten hot electron emission at the accelerating voltage of 20 kV in high vacuum mode. The sample was scanned in the secondary electrons (SE) and backscatter electrons (BE) with the magnifi cation of 100x and 500x.

Energy Dispersive X-ray Spectrometry (EDX)The chemical analysis of Na-feldspar was recorded by energy dispersive x-ray spectrometer

(X-Max80, Oxford, England) at Scientifi c Equipment Center, Prince of Songkla University. The working conditions included an accelerating voltage of 20 kV with high vacuum mode, probe current varying between 500 pA and 1 nA, working distance of 10 mm, X-ray acquisition time of 95s, X-ray exit angle of 35 and SDD sensors 80 mm2 away from the samples to be analyzed. X-ray detection limit was 0.1%. The X-Max EDS system with 123 eV resolutions was capable of collecting spectrum from points and elemental mapping. The particle (spot marked as Fe) was analyzed by EDX to ascertain the presence of metal (Fe). The distribution of Fe element in the sample was obtained with elemental mapping analysis mode. Elemental mapping images were captured after a hundred frame of mapping and recorded with the magnifi cation of 500x.

RESULTS

Na-feldspar ores collected from crushed rocks after vibrating screen process were observed by SEM and elemental compositions were analyzed by EDX. From SE micrograph, the Na-feldspar particles had irregular morphology (Fig.1A). Na-feldspar structure consisted of several ores aggregated or complexed into varying sizes and shapes (Fig.1A). The diameter of particles ranged from 2 to 150 μm. The BE image of particles were composed of high atomic number (Z) as illustrated in Fig.1B. Z-particles with the proportional diameter ranging from 3 to 80 μm were observed.

The chemical compositions of Na-feldspar ore are given in Table 1. Oxygen (O), sodium (Na), aluminum (Al), silicon (Si) and iron (Fe) constituted as the major components. EDX analysis revealed the presence of various peak intensities of O, Na, Mg, Al, Si, K, Ca and Fe in Na-feldspar (Fig.3). The elements of O, Na, Al, Si, Cr, Mn and Fe were obtained and mapped using AZtec program. X-ray mapping images of elemental composition and the distribution of elements are presented in Fig. 4.

DISCUSSION

Fe was the main component used to identify Fe-particle contamination in Na-feldspar ore. High Z particles were observed with BE image, indicating that these particles were rich in metal (Fig.1B, 2). The EDX results showed the dominant metalliferous particles, in which Fe was the


25

major component (>90%) along with Cr (>3%). Na, Al and Si were found at a trace amount (Table 1, Fig.2). In addition, particles rich in Fe, Cr and Mn associated with contaminant were localized in high Z particles of this region (Fig.2). These results corresponded to Fe-mapping analysis in combination with SE micrograph of particles (Fig.3). Therefore, these metal particles are Fe-rich. The major source attributed to these metals was the metallic machine, especially jaw crusher used for crushing process.

O, Na, Al, and Si were observed in the same area, indicating that the particles were Na-feldspar (NaAlSi3O8). Earlier studies on chemical characterization of feldspar ore showed that the main impurity minerals found in feldspar ores was Fe oxides from garnet, hematite, hornblende, tourmaline, biotite and muscovite (Hacifazlioglu et al., 2012). From our observation, some Fe minerals were aggregated together with O, Na, Al, and Si, indicating that Fe was of natural origins. Nevertheless, some Fe particles were not localized within these elements. Those contaminants were from the environment during crushing or screening processes.

To remove the impurities, the feldspars are generally subjected to benefi ciation by magnetic separation, fl oatation or specifi cally with a bio-benefi ciation. However, the section with a coarse size screening by vibrator screen cannot be benefi ciated alone and should be combined with other iron removal techniques to increase the effi ciency of feldspars processing. Hassan et al., (2012) reported that the size fraction of 1.2-0.6 mm cannot be separated by magnetic separator because the presence of Fe2O3 and mica are compacted with the light minerals. Furthermore, the grinding

Figure 1. SEM micrographs of Na-feldspar ores. A: Secondary Electron (SE) image, B: Backscatter Electron (BE) image. Bar = 500 μm

Table 1. Chemical analysis of Na-feldspar ores


26

Figure 2. Backscatter Electron (BE) micrograph and EDX spectrum of contaminant particles (Fe-rich).

Figure 3 SEM micrograph with Fe-mapping location of Na-feldspar (Fe-rich particles) and corresponding EDX spectrum of element composition.


27

fi neness is one of the most important factors affecting the iron removal from feldspar ore (Yanjie et al., 2013). Therefore, the effectiveness of size screening process could be improved with re-milling a coarse-grain and reducing mesh size of machine before transferring though the feldspar treatment process.

In conclusion, the SEM-EDX technique is a valuable tool for the characterization of feldspar contaminant. Additionally, the identifi cation of the morphology and chemical composition of contamination particles provided the valuable information for the determination of their origin. The abundance of Fe and Cr elements represented by elemental mapping analysis in combination with point scan analysis could be used to identify and distinguish the contaminated particles in feldspar ore.

ACKNOWLEDGEMENTS

This work is supported by Scientifi c Equipment Center, Prince of Songkla University, Hat Yai, Thailand.

Figure 4. EDX mapping of Na-feldspar.


28

REFERENCES

Gaudin A.M (1957) Flotation. 2nd edn. New York, McGraw Hill Co., Inc., p. 491.Hacifazlioglu H, Kursun I, Terzi M (2012) Benefi ciation of low-grade feldspar ore using cyclojet

fl otation cell, conventional cell and magnetic separator. Physicochem. Probl. Miner. Process., 48(2):381-392.

Hassan M.H, Marzouki A, Jeffery C, Khater G.A (2012) Evaluation of Saudi pegmatite and its use in porcelain industry. Proc. Applic.of Ceramics, 6(3):133-140.

Heyes G.W, Allan G.C, Bruckard W.J, Sparrow G.J (2012) Review of fl otation of feldspar. Trans. Inst. Min. Metall. C, 121(2):72-78.

Jamett N.E, Vielma J.P, Cisternas L.S (2012) Design of fl otation circuits including uncertainty and water effi ciency. In: Bogle IDL, Fairweather M (eds). Proceeding of the 22nd European Symposium on Computer Aided Process Engineering, 17-20 June 2012, London.

Roy A, Singh S.K, Banerjee P.C, Dana K, Das S.K (2010) Bio-benefi ciation of kaolin and feldspar and its effect on fi red characteristics of triaxial porcelain. Bull. Mater. Sci., 33(3):333-338.

Yanjie L, Huiqing P, Mingzhen H (2013) Removing iron by magnetic separation from a potash feldspar ore. J. Wuhan University of Technology-Mater. Sci. Ed. 28(2):362-366.


29

Morphological Changes in the Liver of Rats (Rattus norvegicus) treated with different Doses of Doxorubicin

Ilham A. Al-Saleem1, Hamad J. Jumaa1, Imad M. Al-Ani2*, Hanaa Kh. Ismael3

1 Department of Biology, College of Science, University of Mosul, Mosul, Iraq.2 Department of basic Medical Science, Kulliyyah of Medicine, International Islamic University Malaysia, Kuantan, Pahang, Malaysia.3Department of pathology and poultry diseases, College of Veterinary Medicine, University of Mosul, Mosul, Iraq.*Correspondence author: E-mail : [email protected], Contact Number: 0060179776014

ABSTRACT

The aim of the present study was to investigate the hepatoxicity of doxorubicin in albino rats (Rattus norvegicus) using different doses and variable durations. Fifty fi ve male rats were divided into two groups, the fi rst group include 35 rats treated intraperitoneal twice a week (every 84 hr) for 3 weeks, 30 rats were treated with doxorubicin at dose (1, 2, 3, 4, 5, 6) mg/kg, and 5 rats served as control and treated with normal saline. The second group includes 20 rats treated twice a week (every 84hr) for 6 weeks, 15 rats were treated with doxorubicin at the dose (1, 2, 3) mg/kg, while 5 rats served as control. The animals were sacrifi ced at 48 hours postoperative last treatment day; specimens of liver were fi xed in 10% neutral buffered formalin for 72 hours, and processed for light microscopy. The liver parenchyma of DOX treated rats for 3 weeks revealed degenerative changes in most of the hepatic lobules; there was vacuolar degeneration of hepatocytes, haemorrhage in the portal area, dilation of the central vein associated with mononuclear infi ltration, haemorrhage and centrilobular coagulative necrosis of the of the hepatocytes. Administration of DOX for 6 weeks induced severe degenerative changes showing distorted liver architecture in most of the hepatic lobules. These pathological changes of the liver tissue were more severe with the higher doses and the duration of the treatment.

Keywords: doxorubicin, hepatotoxicity, liver, rats.

INTRODUCTION

Doxorubicin (DXR) is an anthracycline glycoside antibiotic isolated from cultures of Streptomyces peucetius (Tam, 2013). It has been widely used in the treatment of various cancers, such as acute leukemia neuroblastoma, carcinoma of breast, ovary, bladder, lymphomas, Hodgkin’s disease, soft tissue and primary bone sarcomas (Thippeswamy et al., 2011; Rashid et al., 2013). In spite of its high antitumor effi cacy, use of doxorubicin in chemotherapy has been restricted mostly due to its diverse complications that can remarkably affect quality of life, such as, development of irreversible cardiotoxicity, renal, pulmonary, testicular, hematological toxicities and cerebrovascular accidents (Bray et al., 2010; Mohan et al., 2011).

Histopathological studies of the liver showed disruption of the histological architecture, there was massive degeneration of hepatic cords, including dissolution of hepatic cords, focal infl ammation and necrotic tissues and increased apoptosis, vacuolations of hepatocytes with round to oval and elliptical shaped nuclei and sinusoidal dilatation, hemorrhage and congested portal vein and mononuclear cells infi ltration in rats treated with intraperitoneally with 10 mg/kg doxorubicin (Chaudhary et al., 2016), rats given six intraperitoneal injections (each containing 2.5 mg/kg) over a period of two weeks(Ahmed, 3013), rats treated intraperitoneally with 20 mg/kg doxorubicin (Yagmurca et al., 2007) and mice treated with intraperitoneal single of doze 25 mg/kg doxorubicin (Rasha and Abdella, 2010). The present study was conducted to investigate the effect of different doses of DOX in different duration on the histology of the liver.


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AnimalsFifty fi ve healthy adult male albino rats (Rattus norvegicus) weighing (200 - 225g) each, were

kept in well ventilated room at controlled temperature (24±2◦C). They had free access to laboratory chow and water, and were exposed to a 12 hour/day light program. All experimental animals were treated in compliance with the animal care standards outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals of Mosul University.

ChemicalsDoxorubicin (Ebewe pharma, Ges.m.b.H.Nfg.KG, A-4866 Unterach, Austria) which is being

used in hospitals of the Neneva province. It was purchased from local pharmacy in the form of 50 mg/25ml vials.

Experimental designThe animals were randomly divided into two major groups (A and B) according to the number

of injections (table 1). Group A consists of 35 rats and further divided into 7 subgroups according to the administered dose, the experimental animals were intraperitoneally administered 6 doses of doxorubicin twice a week for 3 weeks. Group B consists of 20 rats and further divided into 4 subgroups according to the administered dose, the experimental animals were intraperitoneally administered 12 doses of doxorubicin twice a week for 6 weeks (table 1).

The animals were sacrifi ced with light ether anesthesia at 48 hours postoperative last treatment day. Specimens of liver were rapidly removed, fi xed in 10% neutral buffered formalin for 72 hours, and processed for light microscopy. Paraffi n sections of 5-6μm thick were stained with Haematoxylin and Eosin (H & E).

Table 1: Distribution of animals by experimental groups.

Group A Treatmenttwice a week for 3 weeks Group B Treatment

twice a week for 12 weeksA1

(control) 1.5 ml/ kg normal saline B1 (control) 1.5 ml/ kg normal saline

A2 1mg/kg doxorubicin B2 1mg/kg doxorubicinA3 2mg/kg doxorubicin B3 2mg/kg doxorubicinA4 3mg/kg doxorubicin B4 3mg/kg doxorubicinA5 4mg/kg doxorubicinA6 5mg/kg doxorubicinA7 6mg/kg doxorubicin


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RESULTS

Microscopic examination of liver sections of control group rats exhibited a normal hepatic architecture of regular hepatocytes, each with well-defi ned cytoplasm, and a prominent nucleus. The polygonal hepatocytes arranged in cords extending radially from the central vein with blood sinusoids in between them toward the peripheral portal area (Figure 1-A).

The liver parenchyma of experimental rats treated with DOX for 3 weeks revealed degenerative

Figure 1. Haematoxylin and eosin stained sections of liver. A; Normal control group rats exhibited a normal hepatic architecture of regular hepatocytes. B, C, D; Group A2, received 1mg/kg DOX, showed the vacuolar degeneration of hepatocytes, haemorrhage in the portal area, dilation of the central vein associated with mononuclear infi ltration. E; Group A3 rats treated with 2mg/kg DOX, showed the necrosis and nuclear pyknosis of the hepatocytes associated with mononuclear infi ltration. F; A4 receiving 3 mg/kg DOX showing dilation of the central vein, mononuclear infi ltration; severe hepatocytic necrosis. (A, B, D, E, F x 350; C x 142). (COAN; coagulative necrosis, DIL; dilation of central vein, FIB; fi brosis, H; haemorrhage, INF; infl ammatory cells, V; vacuolations of hepatocytes).


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changes in most of the hepatic lobules. Group A2, received 1mg/kg doxorubicin showed marked vacuolar degeneration of hepatocytes, haemorrhage in the portal area, dilation of the central vein associated with mononuclear infi ltration, haemorrhage and centrilobular coagulative necrosis of the of the hepatocytes (Figure 1-B,C,D). The lesions increased in group A3 rats treated with 2mgkg doxorubicin; there was necrosis and nuclear pyknosis of the hepatocytes associated with mononuclear infi ltration (Figure 1-E). In contrast, group A4 receiving doxorubicin at doses of 3 mg/kg showed massive hepatotoxicity, particularly characterized by dilation of the central vein, mononuclear infi ltration; severe hepatocytic necrosis with pyknosis, karyorrhexis or disappearance of the nuclei. Acute vacuolar degeneration of the hepatocytes and per-central vein mononuclear infi ltration were also observed in this group (Figure 1-F). Doses of doxorubicin (4 and 5 mg/

Figure 2. Haematoxylin and eosin stained sections of liver. A,B,C,D; group 5, received 4mg/kg DOX, notice the dilatation and vacuolar degeneration in the sinusoids, centrilobular mononuclear infi ltration haemorrhage and thickness of the capsule and subcapsular infl ammation. E; Group 6, received 5mg/kg DOX, notice the coagulation necrosis in the portal area with thickening of the walls of some interlobular attires. (A, B, C x 350; D, E x 142). (COAN; coagulative necrosis, CON; congestion of portal area vein, DIL; dilation of sinusoids, FIB; fi brosis, INF; infl ammatory cells, THR; thrombus formation ,N; necrosis of hepatocytes).


33

kg) of groups A5 and A6 respectively greatly disrupted the histological architecture; there was dilatation and vacuolar degeneration in the sinusoids, heavy centrilobular mononuclear infi ltration haemorrhage and coagulation necrosis in addition to thickness of the capsule and subcapsular infl ammation (Figure 2-A, B, C, D) and coagulation necrosis in the portal area with thickening of the walls of some interlobular attires (Figure 2-F). On the other hand, administration of DOX for 6 weeks induced severs degenerative changes showing distorted liver architecture in most of the hepatic lobules. Doses of doxorubicin (5 and 6 mg/ kg) of groups A6 and A7 respectively also induced morphological changes in the liver; the three lobes of the liver coalesce forming on lobe only (Figure 3).

Liver of group B2 treated with 1mg/kg doxorubicin revealed severe haemorrhage, vacuolization, necrosis and degeneration of the hepatocytes, congestion of the blood sinusoids in addition to of infl ammatory cells (Figure 4-A). Rats of group B3 treated with 2mg/kg doxorubicin also showed severe histopathological changes in their liver; there was fatty degeneration and mononuclear infi ltration (Figure 4-B). Treatment with 3mg/kg doxorubicin in group B4 prompted more severe hepatic changes; there was dilatation and congestion of the sinusoids, increased necrotic hepatocytes, thickening of liver capsule associated with mononuclear infi ltration(Figure 4-C, D), and thickening of some veins wall in the portal area (Figure 4-E).

Figure 3. Liver of rats treated with 5 and 6 mg/ kg doxorubicin showing the coalescence of three lobes forming one lobe only.


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DISCUSSION

Doxorubicin is a powerful anticancer drug that is used in treating both hematological and solid tumors. However, severe cardiomyopathy and heart failure have been detected in cancer patients (Ibrahim et al., 2010) and experimental animals (Bartoli et al., 2011; Hozayen et al., 2014) treated with Dox. The liver may play an important role in heart injury by forming Dox metabolites that are more cardiotoxic than Dox (Danz et al., 2009; Panda et al., 2009; Hozayen et al., 2014). NADPH-dependent cellular reductases convert Dox to semiquinone free radicals which

Figure 4. Haematoxylin and eosin stained sections of liver. A; group B2, received 1mg/kg DOX for 6weeks, notice the degeneration of the hepatocytes and congestion of the blood sinusoids. B; group B3 treated with 2mg/kg DOX showed fatty degeneration and mononuclear infi ltration. C, D & E; group B4 treated with 3mg/kg DOX showing dilatation and congestion of the sinusoids, batches of necrotic hepatocytes, thickening of liver capsule associated with mononuclear infi ltration. (A, B, x 350; C, D, E, x 142). (FATT; Fatty changes, FIB; fi brosis, FON; necrotic spots, INF; infl ammatory cells, N; necrosis of hepatocytes, THI, thinking of the capsule “fi g 3D” and the vein in “fi g 3E”).


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can produce reactive oxygen species (ROS) including superoxide, hydroxyl radicals and hydrogen peroxide. These free radicals are critical to Dox medicated cytotoxicity (Domitrović et al., 2009). Such ROS can damage liver membranes and activate phospholipases via lipid peroxide which can elevate intracellular Ca2+ and can also lead to ALT release and liver cell death (Guicciardi et al., 2013).

The present study revealed that treatment with DOX at doses of 1 mg/kg twice a week for 3 weeks caused disruption of normal architecture of the liver, including dissolution of hepatic cords, dilatation of the sinusoidal spaces and central veins and haemorrhage in the portal area. The severities of these changes were increased with the elevation of the doses and with duration of treatments; there was necrosis and nuclear pyknosis of the hepatocytes, vacuolar degeneration of the hepatocytes and congestion of the blood sinusoids in addition to infl ammatory cells, fatty degeneration and coagulation necrosis. These observations coincide with previous studies that showed pronounced histological abnormalities in the rats treated with doxorubicin at a dose of 1mg/kg body weight on alternate days for 20 days (El-Sayyad et al., 2009), destruction of the hepatic cords, cytoplasmic vacuolization, cellular degeneration, pyknotic and atrophic nuclei, and accumulation of lipid droplets in the hepatocytes, with dilated intercellular spaces in rats treated with 2.5 mg/kg DOX in six intraperitoneal injections over a period of two weeks (Ahmed, 3013), and progressive destruction of liver architecture such as degeneration of hepatic cords, vacuolated and necrotic hepatocytes in rats given a single dose of doxorubicin of 10 mg/kg and sacrifi ced after 7 days and 14 days (Chaudhary et al., 2016).

Previous studies have shown that the hepatotoxicity of DOX was also associated with changes in biochemical properties of the liver; there was a signifi cant increased levels of liver enzymes such as alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT) activities alongside an increase in serum total bilirubin, α-fetoprotein (AFP) and sialic acid levels. DOX also caused a signifi cant decrease in the levels of antioxidant defense enzymes superoxide dismutase (SOD) and catalase (CAT) (Mohan et al., 2010; Rašković et al., 2011; Hozayen et al., 2014 ). The effect of different doses of DOX on liver enzymes is in progress.

CONCLUSION

It is concluded the hepatotoxicity of DOX is proportional to the administered dose and duration of the treatment.

REFERENCES

Ahmed MA (3013). The protective effect of ginger (Zingiber Offi cinale) against adriamycin-induced hepatotoxicity in rats: Histological study. Life Science Journal; 10(1): 1412-1422.

Bartoli CR, Brittian KR, Giridharan GA, Koenig SC, Hamid T, Prabhu SD (2011). Bovine Model of Doxorubicin-Induced Cardiomyopathy. Journal of Biomedicine and Biotechnology. doi:10.1155/2011/758736

Bray J, Sludden J, Griffi n M J, Cole M, Verrill M, Jamieson D (2010). Infl uence of pharmacogenetics on response and toxicity in breast cancer patients treated with doxorubicin and cyclophosphamide. British Journal Cancer, 102: 1003–1009.

Chaudhary D, Khatiwada S, Sah SK, Tamang MK, Bhattacharya S, Jha CB (2016). Effect of Doxorubicin on histomorphology of liver of wistar albino rats. Journal of Pharmacy and Pharmacology; 4: 186-190.

Danz ED, Skramsted J, Henry N, Bennett JA and Keller RS (2009). Resveratrol prevents Doxorubicin cardiotoxicity through mitochondrial stabilization and the Sirt1 pathway. Free


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Radic. Biol. Med; 46: 1589-1597.Domitrović R, Jakovac H, Tomac J and Sain I (2009). Liver fi brosis in mice induced by carbon

tetrachloride and its reversion by luteolin. Toxicology and Applied Pharmacolog; 241: 311-321.

EI-Sayyad HI, Ismail MF, Shalaby FM, Abou-EI-Magd RF, Gaur RL, Fernando A (2009). Histopathological effects of cisplatin, doxorubicin and 5-fl urouracil (5-FU) on the liver of male albino rats. Int. J. Biol. Sci; 5 (5): 466-473.

Guicciardi ME, Malhi H, Mott JL, Gores GJ (2013). Apoptosis and necrosis in the liver. Compr Physiol; 3(2): . doi:10.1002/cphy.c120020.

Hozayen WG, Abou Seif HS, Amin S (2014). Protective effects of ruitn and / or hesperidin against doxorubicin-induced hepatotoxicity. International Journal of Clinical Nutrition; 2: 11-17.

Ibrahim SS, Barakat MA, Helmy HT (2010). Modulating effect of cardilol on doxorubicin-induced cardiomyopathy and hepatic damage. Journal of American Science; 6 (12): 20-32.

Mohan M, Kamble S, Gadhi P, Kasture S (2010). Protective effect of Solanum torvum on Doxorubicin- induced hepatotoxicity in rats. Food Chem Toxicol; 48(1):436-40.

Panda S, Kar A (2009). Periplogenin-3-O-D-glucopyranosyl-(1-N6)-D-glucopyaranosyl-(1–N4)-D-cymaropyranoside, isolated from Aegle marmelos protects doxorubicin induced cardiovascular problems and hepatotoxicity in rats. Cardiovascular therapeutics; 27; 108-116.

Rasha AR, Abdella EM (2010). Modulatory effects of rosemary leaves aqueous extract on doxorubicin-induced histological lesions, apoptosis and oxidative stress in mice. Iranian Journal of Cancer Prevention; 1: 1-22.

Rashid S, Ali N, Nafees S, Ahmad ST, Arjumand W, Hasan SK, Sultana S (2013). Alleviation of doxorubicin-induced nephrotoxicity and hepatotoxicity by chrysin in Wistar rats. Toxicol Mech Methods; 23(5): 337–345.

Rašković A, Stilinović N, Kolarović J, Vasović V, Vukmirović S, Mikov M (2011). The protective effects of silymarin against doxorubicin-induced cardiotoxicity and hepatotoxicity in rats. Molecules; 16: 8601-8613.

Tam K (2013). The roles of doxorubicin in hepatocellular carcinoma. ADMET & DMPK; 1(3): 29-44.

Thippeswamy AH, Shirodkar A, Koti BC, (2011). Protective role of Phyllantus niruri extract in doxorubicin-induced myocardial toxicity in rats. Indian J Pharmacol; 43:31–35.

Yagmurca M, Bas O, Mollaoglu H, Sahin O, Nacar A, Karaman O, Songur A (2007). Protective effects of erdosteine on doxorubicin-induced hepatotoxicity in rats. Arch Med Res; 38(4):380-385.


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Association between Helicobacter Pylori Chronic Gastritis and Atherosclerosis of Carotid and Coronary Arteries in Autopsy Sample of Iraqi Population

Wafaa K. Ibraheem1, Salim R.Hamoudi2, Imad M.Al-Ani3*, Mohammed I.A. Mustafa3

1 Department of Pathology, college of Medicine, University of Baghdad, Baghdad, Iraq.2 Department of Pathology, college of Medicine, University of Baghdad, Baghdad, Iraq.3 Department of Basic Medical Science, Kulliyyah of Medicine, International Islamic University Malaysia, Kuantan 25710, Pahang, Malaysia.* Correspondence: Imad M.Al-Ani, Tel. +60179776014. E-mail: [email protected]

ABSTRACT

Introduction: Chronic infection with Helicobacter pylori (H. pylori) had been implicated in the pathogenesis of atherosclerosis. The Aim of the study was to assess the relationship between H. pylori chronic gastritis, and the severity of atherosclerosis of the carotid and coronary arteries in a sample of Iraqi population. Methods: Autopsy tissue samples were taken from eighty four sequentially selected cadavers collected at the Institute of Forensic Medicine, Baghdad, Iraq. Samples were taken from the gastric antrum to study histopathological changes and detect the presence of H. pylori, and the common carotid and the coronary arteries to observe for the presence of atherosclerosis. The tissues were fi xed in 10% formal saline and processed for light microscopy; the wall thickness of the atherosclerotic vessels (intima and media) was measured using specifi c morphometric scale. Monoclonal mouse antihuman antibody CD34 was used for the detection of the adhesion molecule on the endothelial cells of the atheromatous vessels. Results: There was a signifi cant relationship between H. pylori induced chronic gastritis and carotid artery atherosclerosis (p<0.05), but a statistically non-signifi cant relationship (p>0.05) between coronary artery atherosclerosis and H. pylori chronic gastritis. CD34 adhesion molecule CD34 was signifi cantly expressed on endothelial cells in atherosclerotic lesions of both carotid and coronary arteries as compared to non-atherosclerotic vessels (p<0.05). Conclusion: There was a signifi cant crude association between presence of H. pylori with chronic gastritis and carotid artery atherosclerosis. On the other hand the association with coronary artery atherosclerosis was not signifi cant.

Keywords: Atherosclerosis, Carotid artery, Coronary artery, Gastritis, H. pylori.

INTRODUCTION

Arteriosclerosis refers to the thickening and loss of elasticity of arterial walls. Conversely, atherosclerotic lesions are characterized by focal accumulation of lipoproteins, monocyte-derived macrophages and lymphocytes on the inner lining of the arterial blood vessels (Sriniva et al., 2008). The immunomodulatory cytokines produced by lymphocytes promote the accumulation of cholesterol from the lipoproteins by intimal-associated macrophages; over time, this process leads to the formation of large unstable lesions that are prone to rupture, thrombus formation, occlusion of the affected vessels and the death of tissue distal to the blockage (Whitman, 2004). Many conventional risk factors including hyperlipidemia, hypertension, diabetes, tobacco use, gender, age and family history of premature vascular disease are relevant for approximately half of the patients with clinically apparent atherosclerosis (Nieminen et al., 1993). Other factors which are associated with the induction of arteriosclerosis include leukocyte activation, accumulation of infl ammatory cells, within the vascular wall and chronic infections such as with Helicobacter pylori (H. pylori) which can consequently be linked to ischemic damage of the relevant tissue (Lindsberg and Grau, 2003).

H. pylori, a Gram–negative spiral pathogenic bacterium that colonizes the gastrointestinal tract (Warren et al., 1983). Now a days H. pylori is recognized as the most common cause of chronic gastritis and peptic and duodenal ulcer disease (Khalifa et al., 2010). The mechanisms involved in pathogenesis of gastritis and peptic ulceration induced by H. pylori are not well elucidated. Adhesion molecules, interleukins and other infl ammatory mediators, participate in the whole process (Charalabopoulos et al., 2003). Increased serum concentrations of interleukin-6 (IL6) and


38

tumor necrotic factor (TNF α) in particular show a linear correlation with some cardiovascular risk factor (Stöllberger and Finsterer, 2002).8 These mediators and others promote release of acute phase proteins, such as fi brinogen, C-reactive protein (Ridker et al., 2001).

Previous studies have explored the association between infection with H pylori and risk of coronary heart disease and cerebrovascular arteriosclerosis (Singh et al., 2002). It has been suggested that the immune system is involved in the process that leads to progression of atherosclerotic plaques. In particular autoimmune mechanisms have been shown to be involved in directing and sustaining the infl ammatory state within the atheroma (Stöllberger and Finsterer, 2002), as the infection is known to trigger a vigorous immune response, resulting in high titers of H. pylori-specifi c antibodies (Bergma and D’Elios, 2010).

The prevalence of H. pylori infection varies widely by geographic area, age, race, and socioeconomic status (Brown, 2000). In industrialized countries, as many as 50% of adults are infected with the pathogen, while in the developing and many Middle Eastern countries, the prevalence rates of about 70 to 90% have been reported (Novis et al., 1998). This is likely due to the easier transmission of the infection in crowded living conditions associated with poor socioeconomic status and is most likely transmitted by ingesting contaminated food and water, and through oral–oral and faecal–oral routes (Salih, 2009). Iraq is one of the developing Middle Eastern countries. Decades of war, sanctions since 1980, and more recent post-invasion sectorial confl icts and impasses, had infl icted a major blow on Iraq’s health system that lead to the decline of public health and health care. As a result, the quality of life of the average Iraqi citizen has been adversely affected (Al Hilfi et al., 2013), resulting in a relatively high prevalence of H. pylori infection as compared to other countries (Baqir et al., 2002; Mohsun et al., 2011). The present study has been conducted to assess the association between H. pylori chronic gastritis with atherosclerosis of the carotid and coronary arteries in postmortem tissue samples of Iraqi population.

SUBJECTS AND METHODS

SubjectsEighty four male cadavers aging between 18-68 years, undergoing post-mortem examination

at the Institute of Forensic Medicine, Ministry of Health, Baghdad, were randomly selected during the period from February to May 2008. The cadavers were grouped and compared based on the following two criteria. The fi rst was to compare those with and those without histopathological features of H. pylori chronic gastritis, for morphometric evidence of atherosclerotic involvement of the carotid and/or coronary arteries. The second was to compare those having morphometric features of atherosclerosis of the carotid and/or coronary arteries, with those infected or not infected with H. pylori, for the presence or absence of immunohistopathological evidence of CD34 expression on endothelial cell.

Tissue preparationTissues from the antrum of the stomach, common carotid arteries and anterior descending

branch of the coronary artery were fi xed in 10% formal saline for 72 hrs, and processed for light microscopy. Paraffi n sections of 5μm thickness were stained by Hematoxylin and Eosin (H&E), Verhoeff ‘s hematoxylin, Giemsa, van Gieson’s and PAS stains.

Morphometric AnalysisCross section from the carotid and coronary arteries were used for morphometric analysis.

Area measurements were obtained from different layers of carotid arteries; external elastic lamina, internal elastic laminae, and the vessel lumina, were indicated for measurements of tunica intima and media and luminal measurements by ocular micrometer.


39

Statistical analysisStatistical analysis was done using SPSS system version 15. Descriptive statistics: Mean,

Standard deviation (SD), percentage histogram and Pie charts were used. The statistical signifi cance of the difference in mean between two groups was assessed by using Student t-test for quantitative measure and chi - square test for qualitative measure. A value of p < 0.05 was considered as statistically signifi cant at 95% confi dence interval.

RESULTS

Positive association was demonstrated between presence of H. pylori chronic gastritis and carotid artery atherosclerosis ((Table 1). H. pylori chronic gastritis was found in 44 cadavers (52.3%), while the other 40 (47.6%) showed no evidence of gastritis or infection. Out of those 44 H. pylori +ve cases 26 (59.09%) were associated with atherosclerotic changes in the carotid arteries while eighteen (40.9%) had no evident atherosclerotic lesions in both of these arteries. Also it was observed that 41/84 (48.8%) cadavers had coronary artery atherosclerosis, 22 (53%) of which were associated with H. pylori chronic gastritis (Table 2). The association of H. pylori chronic gastritis and coronary atherosclerotic disease was found to be non-signifi cant (p>0.05).The mouse antihuman CD-34 class ΙΙ monoclonal antibody was used for the detection of CD34 adhesion molecule expression CD34 on the endothelium of coronary and carotid arteries. Seventeen out of twenty-six (65.3%) cadavers with carotid atherosclerotic lesions and H. pylori gastritis were CD34 CD34 +ve; while only 3/14 (21.4%) cadavers with atherosclerotic but H. pylori -ve were CD34 +ve. Alternatively, 13/22(54.09%) of cadavers with coronary atherosclerosis and H. pylori +ve gastritis were CD34 +ve (Table 3). The wall thickness of the blood vessels “IMT”, was higher in the atherosclerotic artery associated with gastric H. pylori infection than that without gastric H. pylori infection; statistically this difference was not signifi cant with a p-value of (0.391) for the carotid arteries and (0.24) for the coronary arteries (Table 4).

Histological sections of the antral part of the stomach stained with H&E and Giemsa stains revealed slender, curved, microorganisms resembling H. pylori on the epithelial surface and in the subepithelial clefts (Figure 1.A).The atherosclerotic carotid and coronary arteries showed alterations in the tunica intima and tunica media. Sections from these arteries stained with van

Table 1. Relationship between H. pylori chronic gastritis & carotid atherosclerosis

CadaversWith carotid artery

atherosclerosisWithout carotid artery

atherosclerosis Total No.

*Hp +ve chronic gastritis 26 (59.09%) 18 (40.9%) 44 Hp-ve, no chronic

gastritis14 (35%) 26 (65%) 40

Total No. 40 (47.6%) 44 (52.3%) 84

Odds ratio - - 2.68 P-value - - 0.046673**

95% Confi dence interval(CI)

- - 1.04-2.42

*Hp- H.pylori. ** Signifi cant.


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Table (2). Relationship between H. pylori chronic gastritis and coronary artery atherosclerosis.

Cadavers With coronary artery atherosclerosis

Without coronary artery atherosclerosis Total No.

*Hp+ve chronic gastritis 22(53.65%) 22(51.16%) 44 (52.38%)Hp-ve chronic gastritis 19(46.34%) 21(48.83%) 40 (47.61%)Total N 41 43 84Odds ratio - - 0.98P-value - - 0.82*95% confi dence interval(CI)

- - 0.36- 2.69

*Not signifi cant.

Table (3). CD34 expression in atheromatous plaques of both carotid & coronary arteries in H. pylori gastritis positive and negative cadavers.

H. pylori Carotidatherosclerosis

CD34 ٭+ve CD34 -ve Odds

ratio P-value 95%confi dence interval (CI)

+ve +ve 17 9 6.93 0.020* 1.08-8.64-ve +ve 3 11 - 0.002 --ve -ve 0 10 - - -Total N0. 20 30 - - -

H. pylori CoronaryAtherosclerosis

CD34 +ve CD34 -ve -

+ve +ve 13 11 6.3 0.023* 1.45-27.46-ve +ve 3 16 - 0.010 --ve -ve - 10 - - -Total N0. 16 37

* Signifi cant.

Table (4). The IMT of atherosclerotic coronary and carotid arteries in H. pylori gastritis +ve group and H. pylori gastritis -ve group.

No. Mean Std. Deviation Std. Error Mean

IMT carotid arteryH. pylori +ve 26 1.813 0.354 0.086

H. pylori-ve 14 1.702 0.355 0.095Total No. 40

IMT Coronary arteryH. pylori +ve 22 1.940 0.511 0.142

H. pylori –ve 19 1.711 0.329 0.099Total No. 41

P value for carotid IMT=0.391 &P value for coronary IMT=0.24


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Gieson, PAS and Verhoffe;s stains demonstrated the atherosclerotic vascular wall. There were elongated and rounded plaques fi rmly attached to the arterial wall, (Figure 1.B), the intima was thickened and clearly distinguished from the media. The intimal thickening was protruding into the lumen but did not involve the whole inner circumference of the vessels (Figure 1.B). There was disruption of the internal elastic layer and vacuolations of cells in the tunica media (Figure 1.B) associated with infl ammatory reaction and infi ltration by s macrophages (Figure 2.A). There were extensive areas of denudation (Figure 2.B) and focal desquamation of the endothelial cells lining the tunica intima (Figure 3.A & B). Immunohistochemical staining for CD34 was positive in the atherosclerotic carotid artery (Figure 2.B); while such staining reaction was absent in the atherosclerotic coronary arteries (Figure 3.B).

Figure 1. (A) Section of antral part of the stomach showing spiral shape H. pylori bacteria (arrow), Geimsa stain, X1000. (B) Cross section of carotid artery showing elastic fi ber in tunica intima, (T.I) tunica media (T.M) and internal elastic membrane (arrow), TA; tunica adventitia,Verhoeff stain X200.

Figure 2. Cross section of carotid artery (A) showing infl ammatory cells, like macrophage (arrows) in tunica intima of atherosclerotic plaque. PAS stain, X1000; (B) showing brownishdiscoloration of endothelial cells (arrows), indicating positive staining for retromembranous ICAM– 1. Immunostaining with CD34, X400.


42

DISCUSSION

The present study searched for the association of H. pylori chronic gastritis with atherosclerotic lesions of carotid and coronary arteries in 84 male cadavers of various age groups. There are many published epidemiological studies on the association between H. pylori antibody titer and ischemic vascular disease, some showing positive relationship regarding the role of H. pylori in the pathogenesis of carotid and coronary artery disease in many countries and H. pylori appear to be implicated in the occurrence of atherosclerosis (Ridker et al., 2001; Torres and Gaensly, 2002; Khalil, 2004; Saijo et al., 2005; Akbas et al., 2010; Longo-Mbenza et al., 2012).

Our study is the fi rst preliminary investigation in Iraq exploring the association between H. pylori and the development of atheromatous plaques in the carotid and the coronary arteries. We demonstrated the presence of H. pylori gastric infection in 44 out of 84 (52.3%) subjects. Previous studies in Iraq, using the serodiagnostic ELISA test showed positive serological evidence of H. pylori infection in (73.91%) of patients attending the Gastrointestinal Tract Center in Baghdad Medical City (Mohsun et al., 2011 ), and 70% of patients attending Al-Anbar General Hospital (Baqir et al., 2002). These differences between our results and other studies is due to the nature and number of study subjects and the diagnostic methods used where it is an indirect sensitive test indicating previous infection in live patients in case of serology while the other is a direct test for current infection (Premjeet et al., 2011). The high percentage of H. pylori infections indicated that H. pylori gastritis is common in Iraq and might be attributed to non-hygienic practices among large sector of the population that facilitate the transmission of the organism which is postulated to be via water or food contaminated with animal or human feces.

Coronary artery atherosclerosis was observed in 41/84 (48.8%) cadavers; 22/41 (53.6%) of the cadavers had associated H. pylori gastritis, while the rest 19/41 (46.4%) tested negative, the difference however is statistically non-signifi cant. On the other hand, signifi cant crude association has been demonstrated between carotid artery atherosclerosis and chronic H. pylori gastritis; 26/40 (65%) subjects with carotid artery atherosclerosis had H. pylori gastritis, while only 35% did not show evidence of H. pylori gastritis. From epidemiological viewpoint, the role of H. pylori in the pathogenesis of coronary or carotid arteries disease is still controversial as several investigators failed to confi rm an association (Rumyantsev et al., 2014). Molecular studies looking for the presence of H. pylori specifi c base sequences in peripheral blood and/or in atherosclerotic plaques

Figure 3. Cross section of coronary artery (A) showing atherosclerotic changes, thick tunica intima (T.I), compressed tunica media (T.M), TA; tunica adventitia, Van giesons stain X100; (B) showing no evidence of retromembranous ICAM-1 in the endothelial cell of atherosclerotic vessels wall, Immunostaining by CD34, X400.


43

also failed to show an association; H. pylori was not detected by the polymerase chain reaction (PCR) in atherosclerotic plaques from carotid endarterectomy samples of 10 male patients (Malnick et al., 1999). Danesh et al., (1999) fi ndings did not support the hypothesis that high prevalence of the H. pylori is responsible for carotid artery disease; they studied by PCR the presence of H. pylori genomic sequences in buffy coat of blood samples and in diseased arterial segments and found only 1 of the 39 atheromatous specimens tested was positive. On the other hand, morphological and immunohistochemical studies have demonstrated H. pylori in 20/38 (52%) of atherosclerotic plaques obtained from carotid arteries (Ameriso et al., 2001). The presence of H. pylori DNA in atherosclerotic plaque samples of coronary arteries by PCR was found in 17/46 (37%) of patients who had acute coronary syndrome or transient ischemic attack (Farsak et al., 2000). Iriz et al., (2008) investigated by PCR the association between H. pylori-related atherosclerosis and the development of atheromatous plaques in the aorta, and the coronary arteries; H. pylori DNA was detected in 11 (26.19%) of the aortic biopsies of 42 patients who underwent coronary artery bypass grafting. H. pylori seropositivity and/or H. pylori seropositivity with elevated C-reactive protein levels were signifi cantly associated with an increase of arterial stiffness in 95% of 3412 Japanese males (Saijo et al., 2005).

The maximum values of the IMT of the carotid and coronary arteries of the present study were more increased in H. pylori infected subjects than the non-infected, but the difference between the two groups was not signifi cant. Previous studies have shown that the maximum values of the internal and common carotid artery IMT of patients with dyspepsia were signifi cantly increased in H. pylori infection as compared with non-infected patients (Akbas et al., 2010; Ibrahim et al., 2014). Mete et al., (2013) measured carotid IMT using a grey scale high resolution color Doppler ultrasound of 134 patients with upper gastrointestinal diseases and reported that IMTs were signifi cantly higher in 103 H. pylori positive group versus H. pylori negative group. These fi ndings support ours, however there are differences due to the nature of the subjects studied; they used patients attending their clinics while, we used cadavers.

In the present study, the CD34 adhesion molecule was signifi cantly more frequently expressed in carotid artery plaques in H. pylori positive subjects as compared to those who are H. pylori negative. (p=0.020, odds ratio=6.9, CI=1.08–8.64). Similarly signifi cant positive association was observed for coronary artery plaques (Table 3) (Odds ratio 6.30, 95% CI=1.47-27.46 p value=0.023). CD34 adhesion molecule is a member of a family of single-pass transmembrane sialomucin proteins that show expression on early hematopoietic and vascular-associated tissue. It has been utilized as a marker for angiogenesis in H. pylori gastritis and gastric cancer as it is considered as an endothelial growth factor in association with vascular endothelial growth factor (VEGF). It is postulated that increased VEGF expression and neo-angiogenesis may contribute to H. pylori-related gastric carcinogenesis (Ananiev et al., 2014).

The unstable present situation in Iraq faces us with many obstacles to proceed with more scientifi cally solid research. The present study has several limitations. First, it involved a limited number of cadavers, most of them have no detailed medical records, and thus we do not have enough information of the many confounding factors that may have infl uenced the development of atherosclerosis in the study subjects. Second, we did not determine the presence of H. pylori specifi c genomic sequences or specifi c proteins in the vascular wall. Thirdly, we couldn’t measure the seropositivity for H. pylori infection in the cadavers. Fourth, no female cadavers were available. Thus, the analyses were adjusted for the mere association of H. pylori with atherosclerosis in male subjects. Further studies on Iraqi patients with dyspepsia and related gastrointestinal diseases are needed to properly investigate the association of infection by H. pylori and perhaps other organisms with cardiovascular diseases using more sensitive and specifi c genomic and proteomic techniques; also to explore and exclude the bias originating from the many confounding risk factors.

CONCLUSIONS


44

The present study showed a signifi cant relationship between chronic H. pylori gastritis and carotid arteries atherosclerosis. CD34 together with other markers such as VEGF and proinfl ammatory cytokines may be of value in elucidating the role of infl ammatory reaction induced by infection with H. pylori or other agents in the pathogenesis of atherosclerotic lesions.

ACKNOWLEDGMENT

The authors are grateful to the staff of the Institute of Forensic Medicine, Ministry of Health, Baghdad, Iraq for allowing us access to the studied cadavers, and to their technical assistance during the period of the study.

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Ibrahim HI, Mohammed MO, Dhahir HA, et al., (2014). Impact of H. pylori Infection on serum lipid profi le and atherosclerosis of carotid artery. Internat. J. Clinical. Med., 5: 933-939.

Iriz E, Cirak MY, Engin ED, et al., (2008). Detection of Helicobacter pylori DNA in Aortic and Left Internal Mammary Artery Biopsies. Tex. Heart Inst. J., 35, 130-135.

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Role of Microscopic Imaging and its Analytical Techniques in the Optimization of Geochemical Signature Natural Products for Infectious Disease Screening, Drug Discovery and Medicinal Effi cacy Studies.

Farid Che Ghazali, Norizzati Mohd Noor, Azreen MohammadSchool of Health Sciences (Biomedicine), Universiti Sains Malaysia, Health Campus.16150 Kubang Kerian, Kelantan DN, Malaysia

ABSTRACT

Natural products based medicinal plants contained a variety of health attributes biocompounds that are benefi cial to mankind health. While diseases that developed and are contributed from the global human lifestyle and infl uenced by the environmental factors cause health dilemmas to the human body system. Per se, out sourcing for tangible taxonomized medical plants is still a universal agenda and in tandem to this natural product of geochemical signatures as purported via folk medicine is highly sourced. As such, a complication of literatures is available pertaining to diverse discussions on advantages and benefi ts obtained such as antimicrobial, antifungal, antioxidant, have been the millennium manuscripts. However there are still lacunae’s. This article reviews the putative issues to the said matter especially in where microscopy techniques and methodologies were orchestrated with these research activities.

Keywords: Geochemical natural product, high-resolution imaging, Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM).

INTRODUCTION

Traditional medicine has long established itself globally as an integral parts of world communities’ wellbeing. For centuries, traditional folk medicine uses herbal and local geochemical signature natural products in a large exploitable scale because of the fact that the pharmacologically active compounds in them do provide protection against microbial and insect attacks (Kuruvilla, 2002; Savita, 2013). Plant-based systems continue to play an essential role in healthcare, and their use by different ethnics and cultures has been extensively documented (Moerman, 1986) and (Johnson, 1999). The World Health Organization (WHO) estimated in 1985 that approximately 65% of the population of the world predominately believed and relied upon plant-derived traditional medicines for their primary health care and wellness. As such, these plant products also play an important, though more indirect role in the health care systems of the remaining population who mainly reside in developed countries (Farnsworth et al., 1985). A survey of plant-derived pure compounds used as drugs in countries hosting WHO-Traditional Medicine Centre indicated that, of 122 compounds identifi ed, 80% were used for the same or related ethno-medical purposes and were derived from only 94 plant species (Farnsworth et al., 1985). Vis a vis relevant examples includes khellin, from Ammi visnaga (L) Lamk., which led to the development of chromolyn (in the form of sodium chromoglycate) as a bronchodilator; galegine, from Galega offi cinalis L., which was the model for the synthesis of metformin and other bisguanidine-type antidiabetic drugs (Fabricant & Farnsworth, 2001); and papaverine from Papaver somniferum which formed the basis for verapamil used in the treatment of hypertension (Fabricant & Farnsworth, 2001). The latter plant is better known as being the source of painkillers such as morphine and codeine (Buss et al., 1995), but probably the best example of ethno- medicine’s role in guiding drug discovery and effi cacies development is that of the antimalarial drugs, particularly quinine and artemisinin (Cragg & Newman, 2013). Much of these works have been revealed under the microscope.

Microorganisms and plants do have an extraordinary capability to produce tangible structural diverse and unique natural product properties. For centuries, these compounds (also called secondary metabolites) have been used as colorants, perfumes, fl avours, antibiotics and other drugs. With the


47

rise of the molecular-based genomic era, it is now much clearer that these nature’s potential to produce natural products is much larger than previously estimated and purported. As such, many biosynthetic pathways which were not known then have been dynamically extrapolated under modern laboratory conditions. The plant based medical therapeutics implemented by the primitive man has generated a great awareness amongst the present scientists to validate the folk medicine claims by focusing on the plant based crude drug studies (Pradhan et al., 2012). Per se, wider society acceptance of herbal medicines in relation to modern drugs that also possess side effects have been a magnifi cent infl uence for continuous research of alternative medicine. However, in order to develop a plant-based drug, it is very important to specifi cally determine the anatomical and taxonomical properties of the plant of interest. Microscopy techniques had been widely used in the study of medicinal plants considering the microscopic features of the specifi c plant species is a fundamental knowledge for the standardization of plants used (Sá et al., 2015). As such, it also allows the discovery of characters that are not described by the macroscopic study by imaging the cellular and subcellular events incurred (Ahmad and Khan, 2012).

Geochemical signature natural products thus represent a signifi cant reservoir of much wanted unexplored chemical diversity for various optimization stages of drug discoveries and effi cacies. These products are derived from a variety of sources such as marine organisms, fungi, bacteria and plants that have been rhetorically purported and scientifi cally proven to have incorporated a large group of tangible bioactive substances (Katz & Baltz, 2016; Lam, 2007). The term environs relate not only to the complex extract of these products but also the isolated bioactive compounds derived from the said extract. Historically, what was derived from these said natural sources, which tend to be mostly plants, now involve also minerals, animals sources and microorganisms such as fungi. This has led to the development in the fi eld of pharmacy identifi ed as pharmacognosy (Yun et al., 2012). The application of these geochemical natural products as identifi ed traditional medicine or remedies especially in the form of lotion and oil has been proven over history and documented in the pharmacopoeia together with their usage and preparation methodologies (Cragg & Newman, 2013; Sunil & Pandey, 2014). In tandem to this, Egyptian medicine from about 2900 BC record the documentation of over 700 plant-based drugs, known as the “Ebers Papyrus”. Vis a Vis, the Greeks and Romans also contributed signifi cantly in the development of herbal drugs usage in the ancient Western world. The collection, storage, and use of these medicinal herbs have been recorded by a Greek physician, Dioscorides (100 B.C.) during his travels with Roman armies throughout the then “unknown world”.

The dominant source of tangible knowledge of local geochemical natural product usage and rule from accessible medicinal plants actually comes from man himself while searching for available foods for the treatment of diseases by trial and error experimentation for hundreds of years through palatability trials or untimely deaths (Dias et al., 2012). World Health Organization claimed that a medicinal plant is any plant containing substances in one or more of its organs that can be used for therapeutic purposes (Doughari, 2012). This type of plants will have parts that include leaves, roots, rhizomes, stems, barks, fl owers, fruits, grains or seeds that have been employed in the control and treatment for disease condition, therefore contains various chemical compounds that seems medicinally active. These non-nutrient plant chemical compounds or bioactive components are then referred as phytochemicals (‘phyto-‘ from Greek - phyto meaning ‘plant’) or phytoconstituents and are responsible for protecting the plant against microbial infections or infestations by pest organisms (Amin & Thakur, 2014; Doughari et al., 2009). As such globally, the scientifi c communities have been deeply focused to document the therapeutic worth of these natural products so as to validate the purported claims of medicinal values as rhetorically purported by the folk medicine practitioners.

Per se, infectious diseases account for a substantial proportion of deaths worldwide. Infectious diseases possess unique characteristics that separate them far apart from the other diseases (Fauci and Morens, 2012). The most signifi cant clinical features are their unpredictability and worsening potential to post an explosive health global impact. As such, the infectious diseases are acute and


48

ambiguous in nature where their onset especially in healthy person is sudden and distinctive. Infectious diseases based on their nature can be prevented by implementing personal protection, general public health awareness measures or incorporating immunological approaches such as vaccination. As such, effective and effi cient control measures have curtailed the local transmission of human infectious diseases such as measles, malaria and polio, and saved and improved billions of lives globally (Klepac et al., 2013). Since the discovery of penicillin by a Nobel laureate Scottish scientist, Alexander Fleming, hundreds of antibiotics have been developed till today which have been very helpful in the treatment of infectious diseases considered dreadful for centuries (Nigam et al., 2014).

However, the current ease of global travel and increased global interdependence has been said to have defeated the extraordinary advances in development of counter measures (diagnostics, therapeutics, and vaccines), adding layers of complexity to the infectious diseases that affect not only the global health issue but the economic stability of various societies (Fauci and Morens, 2013). Infectious pathogens have high capacities of replication and mutation that provides them with evolutionary advantages that are infl uenced by the environmental factors, antimicrobial drugs and human immune response (Fauci and Morens, 2012; Beceiro et al., 2013). Continuing progress in the clinical treatment of many said infections is threatened by the growing resistance of those pathogens to now highly exploited antimicrobial drugs. For example, in the European Union (EU) it is estimated that more than 25,000 people have died annually of various sepsis tragedies caused by resistant bacteria (Lim et al., 2016). The epidemiology of resistance to pathogens is however considered very complex and the problem is further compounded by recent lack of success in developing novel antibiotic classes worldwide. As such, more scientifi c innovations and transformation are indeed needed in the identifi cation of tangible lead compounds of geochemical natural product origin that would be of benefi t from therapeutically relevant bioassays activities and screening, thus facilitating the isolation of bioactive molecules from these said multi-constituent extracts.

Reactive oxygen species and reactive nitrogen species are generated during metabolism and other activities in a biological system, such as reactions catalysed by the electron carriers in the mitochondria, irradiation by ultraviolet light, X-rays, and gamma rays, reactions catalysed by metals, and infl ammation process, etc. They have sparked great interest in biomedical research because an overproduction of these reactive species in geochemical natural product biological systems beyond antioxidant defence systems, result in oxidative stress and cause oxidative damage to biomolecules (Cárdenas-Rodríguez et al., 2013; Valko et al., 2006). This imbalance between free radicals and the antioxidants has been proposed as one of the mechanisms that correlate with many clinically chronic diseases, including cancer (Sosa et al., 2013), coronary heart diseases (Juni et al., 2013), diabetes, neurodegenerative diseases, and aging (Gutowski & Kowalczyk, 2013).

ANTIOXIDANTS SCREENING AND PROFILING AS REVEALED UNDER THE MICROSCOPE.

There has been much interest in research on phytochemical of medicinal plants in recent years. In addition, it is generally accepted that antioxidants from natural products have effi cacy and safety advantages when compared to synthetic products (Karre et al., 2013). The presence of plant-derived bioactive chemicals, such as phenolics and fl avonoids are correlated with multiple biological and antioxidant effects. As such the presence of various different categories of phenolics will resulted in differences steps in radical scavenging activities (Bernhoft, 2010; C. Rice-Evans et al., 1997). Therefore, the tangible relationship between the various chemical constituents and antioxidant activity of geochemical natural preparations are very much in need to be properly determined. This is because the structural features of the said fl avonoids and phenolic compounds that are reportedly responsible for various antioxidant activity, via the measurements of its total


49

phenolic and fl avonoid contents in these geochemical signatures extracts can thus be optimized.Currently, about 25% of the active components was identifi ed from plants that are used as

prescribed medicines (Sahoo et al., 2013). Literature has proven that a lot of diseases evolved due to the reaction of the reactive oxygen species in the body system (Pham-Huy et al., 2008; Wiseman & Halliwell, 1996). Thus, the most practical way to conquer the problem is by increasing the antioxidant activity in the body which could possibly be achieved by consuming a lot of vegetables, fruits or edible plants (Adefegha & Oboh, 2011). Shukla et al., (2015), declared that plants-derived phenolic compounds and fl avonoids are radical scavengers and act as good antioxidants agents. However, clinical trial and screening of the natural products are mandatory to demonstrate the effectiveness of a bioactive compound to verify the traditional claim (Sasidharan et al., 2011). Hence the assessment of such properties remains an interesting and useful task, particularly for fi nding new sources for natural antioxidants (Lee et al., 2003).

The assessments of the antioxidant activity are categorized in two methods which are in vivo and in vitro methods. For in vivo assessment, various method can be used to screen for antioxidant activity (AOA) included 2,2’-diphenyl-1-picrylhydrazyl radical (DPPH) method, the static head space gas chromatographic (HS-GC) method, and the b-carotene bleaching test (BCBT) (Koleva et al., 2002), hydrogen peroxide scavenging (H2O2) assay, ABTS radical cation decolorization assay, peroxynitrite radical scavenging activity, total radical-trapping antioxidants parameter (TRAP) method, ferric reducing-antioxidants power (FRAP) assay, superoxide radical scavenging activity (SOD) assay, hydroxyl radical scavenging activity, reducing power method (RP), phosphomolybdenum, oxygen radical absorbance capacity (ORAC) method, ferric thiocyanate (FTC) method, thiobarbituric acid (TBA) method, xanthine oxidase method, DMPD (N,N-dimethyl-p-phenylene diamine dihydrochloride) method, Cupric ion reducing antioxidant capacity (CUPRAC) method and metal chelating activity (Alam et al., 2013; Pisoschi & Negulescu, 2012).

Rhetorically and still in practice today, the method for determination and quantifi cation of volatile components is via chromatography studies. The samples are sealed in chromatography vial containing either gas phase or the vapour portion. The rule of thumb is applied as when the biological samples are being heated (for a specifi c period of time), the said solvents will volatize and the compounds within will undergo structural separation that differ in weight, polarity and volatility (Sriseadka et al., 2006; Koleva et al., 2003; Huie, 2002; Koleva et al., 2002). Thus this method will disclose and highlight the different bioactive compounds present in each natural product sample (Rahul, 2014).

Over the past decade, DPPH (2, 2-diphenyl-1-picryl-hydrazyl-hydrate) method had gained high popularity due to its rapidity and analytical sensitivity. It is a representative method employing model radical in the evaluation of radical scavengers by ascertaining their inhibitory effect against free radicals and their discoloration. DPPH produces violet solution in ethanol, however the violet coloration will be reduced if there is presence of antioxidant molecule (such as a hydrogen-donating antioxidant ) which turns the solution form violet to yellow, which is measured spectrophotometrically (Chusri et al., 2015). DPPH is a free radical substance and stable at room temperature hence this method is an easy and rapid design to evaluate antioxidants present in a plant extract or substance (Mensor et al., 2001). The DPPH method is widely used to determine antiradical/antioxidant activity of purifi ed phenolic compounds as well as natural plant extracts (Das & Srivastav, 2014; El-Baky et al., 2008; Bendini et al., 2006; Awika et al., 2003). For example, the antioxidant capacity of Algerian propolis were evaluated spectrophotometrically via DPPH method. The free radical scavenging of propolis was measured and the percentage of inhibition capacity was calculated. The antioxidant capacity of the propolis extract was expressed as IC50 which defi nes as the concentration of extract that inhibits the formation of DPPH radical by 50% (Rebiai et al., 2011).

Whereas in the b-carotene bleaching tests (BCBT) method, assay is based on the loss of the yellow colour of β-carotene due to its reaction with radicals which are formed by linoleic acid oxidation in an emulsion. The rate of β-carotene bleaching is slowed down in the presence


50

of antioxidants (Ueno et al., 2014; Lai & Lim, 2011; Kulisic et al., 2004). H2O2 is a non-radical reactive oxygen species (ROS) which can infl uence several cellular processes, including DNA damage (Mukhopadhyay et al., 2016). The ability of natural product in plant extract to scavenge the hydrogen peroxide that enters the body can be estimate by the H2O2 scavenging method. In vitro method is performed by mixing the hydrogen peroxide solution with phosphate buffer then addition with plants extract. The absorbance being measured at 230nm using a spectrophotometer. The percentage of hydrogen peroxide scavenging is calculated as follows:

% scavenged (H2O2) = (Ai –At) / Ai X 100

where Ai is the absorbance of control and At is the absorbance of test (Mukhopadhyay et al., 2016; Alam et al., 2013; Keser et al., 2012).

Per se ABTS radical cation decolorization assay being determined by using 2,2’- azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) free radical to measures the ability of antioxidants to scavenge the ABTS generated in aqueous phase when compared with Trolox reagents standard (Shalaby & Shanab, 2013). The color change form blue to green as the antioxidants were added to the ABTS reagents is measured using a spectrophotometer at 750nm (Alam et al., 2013). However, several lacunae was noted pertaining to this method that include poor reproducibility, lack of specifi city, inaccuracy, high cost and others. As a matter of discussion, most the available method used in the determination of antioxidants activities from the various compounds analysed, employed the chemical reaction principle of the reagent used that occur when the antioxidant compounds were added to the said reagents. The said activities will be measured using spectrophotometer absorbance analysis reaction at specifi c wavelength.

Modern use of the method has improved rapidly and recently, the MTT method is the most popular method used in numerous studies for assaying of antioxidants capacity which is based on the reduction of MTT by antioxidants compounds. This method aid in most studies as it is simple, rapid, inexpensive, sensitive and accurate method. Its employs the cell culture work which the results are reproducible and a little material needed in for detection and quantifi cation of the antioxidants activity (Muraina et al., 2009). There are as many methods developed for assessing antioxidant activity in vitro, as there are vice versa for the in vivo method. Basically in vivo method used to evaluate the ability of the antioxidants to reduce the radical which can be measured using both serum sample as well as tissue homogenate. The method included lipid peroxidation (LPO), reduced glutathione (GSH), glutathione peroxidase, gluthathione reductase, glutathione-s-transferase (GST), superoxide dismutase (SOD), catalase (CAT), total protein, lactate dehydrogenase, ornithine decarboxylase, glucose 6 phosphate (G6P) and many more (Rahul, 2014; Lee et al., 2003).

Commonly, a method developed for antioxidants assay uses assessment via spectrophotometry to measure the capacity of an antioxidant in the reduction of an oxidant, which changes colour when reduced (Apak et al., 2013). Another available method by electrochemical method based on cylicvoltammetry techniques. This technique involve the reaction of electrochemical cell with glassy carbon electrode, as working electrode and mercury/mercury (I) chloride as reference electrode in small volumetric capacity. The potential reaction occurred is measured under inverse scanning mode starting from-200 to +800 mV with a scanning rate of 100 mV sec. The reaction of antioxidant capacity is denoted with the area below the anodic curve plotted from the voltammogram (Rebiai et al., 2011).

The observation of antioxidant activity of the natural product cannot be see under the naked view; however, the activity can be determine by the interaction of the product or substance with living cell especially cancerous cell. This method allows the observation whether the multiplication or growth of cancer cell can be inhibited with the presence of natural product in plants extract. The free radicals and antioxidants imbalance has been tendered as one of the mechanism that dovetails with many chronic diseases including cancer (Rice-Evans et al., 1996). Ferlay et al., (2015),


51

stated that in average of 8.2 million people has died from cancer and an estimated around 13% of all deaths worldwide and ranked among fi ve leading causes of death in both males and females (Fadeyi et al., 2013; Sreelatha et al., 2011). Cancer is characterized by the uncontrolled growth of abnormal cells (Society, 2016) and disruptions of cellular balance or dysregulation of controlling mechanism (Sreelatha et al., 2011).The disruption of mechanism infl uenced the reactive oxygen species (ROS) and cellular oxidants stress production (Schumacker, 2006) which the existence of these reaction can be balance by the biochemical antioxidants (Waris & Ahsan, 2006). Ergo the screening for antioxidants and free radical scavenging activities of natural products is crucial owing to free radical production has enmesh in a number of pathological conditions such as infl ammatory diseases, atherosclerosis, cerebral ischemia, AIDS and cancer (Diallo et al., 2001).

Recently, there are plenty of methods being applied to prove and justify the effectiveness of the antioxidants form many plants extracts against cancerous cells as revealed under microscope. As side of it, enormous diversity of compounds that can be isolated and elucidated from plants screened. Confocal laser scanning microscopy (CLSM) is one of microscopic technique used to study the localization of phenolic compounds in plants tissues. The principle of CLSM is the confocal optical system has a pinhole that conjugated the focal position of the objective lens to detect light at focused position. The light emitted form point light source is radiated by the objective lens therefore the light converges to one point on the sample. The plants tissues are stained using Naturstoffreagenz (NA) and the fl uorescence of NA-stained phenolic compounds are observed. NA is a reagent that can visualized fl avonoid compounds in plant tissue (Ferchaud, Qi, & Chin, 2016), as it appeared as a green fl uorescence when viewed under CLSM (Hutzler et al., 1998).

Research form Abdelwahab et al., (2009), which study the effect Goniothalamus umbrosus hexane plant extract on MCF (human breast adenocarcinoma) cell line revealed that the morphological assessment of apoptotic reaction of bioactive compound properties against human cancer cells viewed under inverted microscope denoted by the presence of shrunken cells with surface blebbing, nuclear condensation and fragmentation. Another mode of observation is via monitoring apoptotic reaction cancer cells that has been stained with acridine orange (AO) and propium iodide (PI) when viewed under a fl uorescence microscope. The staining reagents will stained nucleic acid or DNA in the cell and appear green fl uorescence for AO while orange fl uorescence for PI staining (Abdelwahab et al., 2009; Lee et al., 2003).

As revealed under the transmission electron microscope (TEM) and scanning electron microscope (SEM), cancerous cells treated with plants extract consists of diversity of natural products especially presence of antioxidants compounds seem to produced structural alterations of chromatin aggregation, mitochondrial denaturation and apoptotic body formation as well as cytoplasmic compartments, swelling and disappearance of mitochondrial cristae (Stewart, Boonsiri, Puthong, & Rojpibulstit, 2013). The morphological showed by Figure 4.

Although natural products have greatly contributed to the therapeutic treatment of numerous human diseases, their impact as anticancer drugs has been profound. Natural products or their semi-synthetic analogues constitute approximately 74% of all new chemical entities marketed as anti-cancer drugs between 1981 and 2006 (Newman and Cragg, 2007). Current examples of natural product-derived anticancer agents include: paclitaxel (breast/ovarian/lung), etoposide (testicular/lung), idarubicin (leukemia), topotecan (ovarian/lung) and irinotecan (colon) (Newman and Cragg, 2005).Unfortunately, although numerous drugs are available for the treatment of certain cancers, there is still a need to develop new cancer therapeutic agents due to the resistance of many tumour types to current therapies.

Microscopic imaging and its analytical technique role.

Microscopy has played an important role in the analysis of microbial cells since Antonie van Leeuwenhoek described microorganisms using a compound microscope in the late 1600s.


52

Microscopy that falls under the nanotechnology category can be employed as a tool in the search for new antimicrobials, for example, by using the atomic force microscope using its cantilevers to detect low concentrations of bacteria and screen their response to the said antibiotics. Since then enormous improvements have been done in microscopy techniques such as phase contrast principle, invention of a variety of fl uorescence imaging microscopes, the confocal microscope, as well as relatively new super-resolution microscopy methods like electron microscope, the atomic force microscope (Haagensen et al., 2011 ), and still the advancement is going on. Imaging the cellular and sub-cellular events has been an important technique required to answer many cell biological questions. In olden times, authentications of the herbal plants depended on the experienced collector’s knowledge with the plants organoleptic characteristics, such as colour, texture and odor. This technique of evaluation by experienced collectors became less reliable, as the sources of medicinal plants broadened and adulteration became more complicated.

Development of reliable, easy, cheap, quality and convenient methods for authentication is important for the identifi cation and standardization of medicinal plants, predominantly, toxic plants. Since the advancement in the microscopy technique could help to achieve the goal of standardizations of the herbal products, authentication methods of medicinal plants have been enhanced by the macroscopic and microscopic identifi cation methods. The light microscopic method (LMM) of identifi cation of medicinal plants has been applied for many years and was published by Chinese Pharmacopoeia, as well as in other reference books such as: Chinese Materia Medica, New compendium of Chinese Materia Medica, British Herbal Pharmacopoeia, American Herbal Pharmacopoeia, Japanese Pharmacopoeia, Korea Herbal Pharmacopoeia and Indian Ayurvedic Pharmacopoeia (Juni et al., 2013). Light microscopic method offers several advantages over conventional authentications of the medicinal plants including the effectiveness, simplicity, and low cost, and has been widely adopted as an offi cial method in many international herbal pharmacopoeias as mention earlier. However, there are some limitations of this method such as usage of outdated instruments and equipment, microscopic characters were simply described by words or coupled with pictures drawn by hands, and none of them provide detailed illustrations or photos of both transverse sections and powder characteristics. On the other hand, the advancement in the microscopy technique and the development of digital imaging techniques, much detailed and accurate information about microscopic features can be obtained.

Optical microscopy technique and authentication

Various available techniques that are currently applied in plant morphology studies and the related and anatomical features were rhetorically developed more than a century ago (Yeung et al., 2015). The advancement of present scientifi c knowledge and understanding of these geochemical wild plants inert cellular and histological organization have been a stepping stone for the ongoing development of better preparatory techniques to harness taxonomy bloom used in optimization of the plants as commercial-able bioactive compound extracts at sustainable level and with application of green approaches techniques.

Light microscopy has been documented and used for the structural characterization via cross-sectional inspection of the natural plant. Per se, this plant via cross- sectional inspection includes characterization of the root, stem, petiole and leaf blade that was chemically fi xed prior to the light microscope observation. This is important to study signifi cant anatomical parts in comparison to other studies as well as to distinguish the specifi c plants from different species of the same family (Kahraman et al., 2009; Celep et al., 2014). Despite advances in the knowledge of the biological and pharmacological potential of essential oils, few studies have been described and related the morphological characteristics of each individual cell or organ involved in the production of the said plant bioactive substances that can be the source of therapeutic benefi t and the specifi c substances that are present in certain natural plant parts that can be identifi ed by using a light microscope histochemistry staining (Sá et al., 2015). Below is a montage of photomicrograph image of cross-


53

sectional inspection of the plant Dysphania ambrosioides under the light microscopy technology.Santiago et al., (2016) revealed that terpene compound in the essential oil of Chenopodium

ambrosioides L. by the glandular trichomes as observed under the light microscope. The chemical analysis of C. ambrosioides essential oil showed that monoterpene hydrocarbon and oxygenated monoterpenes is the two most abundant compounds. In order to locate the production and storage site of this compound, histochemistry staining under light microscope was done by using Sudan IV stain and Nadi reagent which specifi cally stained the lipid substance. As observed under the microscope, the glandular trichome shows positive reaction of blue colour after treated with Nadi reagent and red colour stained with Sudan IV indicating the presence of terpenes. These fi ndings suggested that the cellular secretory glandular trichomes are responsible for storing substances present in the essential oil of C. ambrosioides. In modern taxonomy, one of the most important taxonomic tools for describing a natural product geochemical plant or plant part is the powder microscopy instead of examining the whole fi xed plant sample. Bijeshmon and George (2014) had focused on the screening of Tabernaemontana divaricata fl owers to detect the antibacterial effect and also to detect the powder microscopic characteristics of the fl ower for the taxonomic evaluation of the fl owers. In the said powder microscopic study, it was revealed that the said fl ower has light fragrant spirally thickened xylem vessels, prismatic crystals of calcium oxalate, trichomes, small starch grains and pollen grains that suggest it as probable source of signifi cant antibacterial property of T. divaricata.

In the fi eld of microbiology, under the effort to optimized and characterized the antimicrobial properties of natural product plants, light microscopy technique have also been exploited. In such studies, the characterization of fungal structure as well as biofi lm formation under stress condition imparted by the natural products with signifi cant antimicrobial activities was observed. Literatures have documented the microscopic techniques to unfold the mechanism of action of natural products and have given much needed insight of ultra-structural alterations occurring in the hyphae or fungal cell in the presence of such kinds of antimicrobial agents (Sharma & Tripathi, 2008; Khan & Ahmad, 2011; Chaieb et al., 2011). Ghahfarokhi et al., (2004) elucidated the toxic effects of onion extracts on the hyphal morphology of Trichophyton rubrum and T. mentagrophytes. The observation under light microscope revealed damaging effects within the fungal hyphae, and there was budding of the hyphal tip, thinner hyphal wall, fl attened and empty hyphal tips.

Similar observations were reported by Sharma and Tripathi (2008) for oils of Citrus sinensis against A. fumigatus. Oils of Origanum syriacum, Thymbra spicata, Lavandula stoechas, Rosmarinus offi cinalis, Foeniculum vulgare, and Laurus nobilis were assessed for their antifungal activity against phytopathogens namely Phytophthora infestans and Botrytis cinerea by Soylu et al., research (Soylu et al., 2006; Soylu et al., 2010). They exploited light microscope observation on pathogen hyphae, exposed to both volatile and contact phase of oil. Their study revealed considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage. Recently, Khan and Ahmad (2011) have reported the antifungal activity of oils of Cinnamomum verum and Syzygium aromaticum and their major active compounds cinnamaldehyde and eugenol. This study has highlighted the time and concentration dependent toxic effects of these oils on to the growth and morphology of drug resistant strains of Tricophyton rubrum and Aspergillus fumigatus, where the light microscopic observation revealed the presence of hyphal necrosis, blistering and autolysis of its cytoplasm. A signifi cant inhibitory effect on biofi lm formation by Staphylococcus aureus and Staphylococcus epidermidis was noted after thymoquinone of Nigella sativa supplementation by Chaieb et al., (2011). This effect on biofi lm formation was confi rmed by microscopic analysis of strains grown on the surface of glass slides covers that reveals the biofi lm inhibition in a dose-dependent manner.

Soukup and Tylova (2014) stated that there are numerous preparatory techniques for various geochemical plant samples to be subjected for light microscopy analysis that permit the access to the plant inner structure and it’s characteristic. For this, it is not only the availability of the right microscopical equipment and its specifi cations, but the said nature self optical character,


54

complexity, size of the object as well as the purpose of preparation have also been a crucial factor that must be taken into consideration when choosing the correct method of preparation. The rule of thumb is that regardless of any microscopy techniques, all of them required the sample to be a true representative, thus specimen preparation is the crucial step and a basic concern in each microscopy analysis (Mitra, 2003). Several sample preparation are suggested according to the size of the sample which are whole-mount technique and sectioning technique.

Whole mount technique is used to gain information regarding the inner arrangement of a minute specimen where the deepness is within few hundred micrometers (Soukup and Tylova, 2014). Yeung et al., (2015) stated that this technique does not require any special equipment as the specimen will be simply examine by using stereomicroscope that enable the image to be captured and recorded. However, in order to permit access into the inner structure, the optical density must be lowered and the tissue transparency needs to be improved. Thus the specimen needs to be cleared by using several chemical reagents to remove the pigments, inclusions and cellular contents (O’ Brian and Mccully, 1981; Gardner, 1975). The plant specimen can be cleared by using sodium chloride, hydrogen peroxide, strong alkali or acids, phenol, lactic acid, chloral hydrate and their combinations are commonly used. An alternative method to increase the tissue transparency is by treating the specimen with a high refractive index compound such as sodium iodide solution (Bougourd, Marrison, Haseloff, 2000; Dubrovsky et al., 2009). The solution is prepared by dissolving sodium thiosulphate in 65 % (aq. v/v) glycerol. Then, add the sodium iodide and 2 % (v/v) of dimethyl sulfoxide to fi nal solution. The fi nal solution should be clear and colorless with refractive index close to 1.5.

A voluminous specimen might need to be processed by various sectioning techniques in order to gain the information of inner structure. Normally, the sample will be cut to an adequate size to allow rapid access of fi xative, considering the size of structure of interest by using a sharp razor blade to minimize damage in the vicinity of cut edge. The process need to be carried with care to avoid signifi cant changes in the natural appearance (Konstankiewicz & Zdunek 2005). Fixation and embedding into supporting matrix are common initial steps involved in most of sectioning methods that determine the fi nal microscopic sections. According to Bancroft and Gamble (2008), the tissue section must maintain the microscopic relationship among cells, cellular components as well as extracellular material with little disruption on the tissue organization in order to view the tissue microanatomy. This principle has led to the development of various fi xatives over the last century.

The most commonly used fi xative is formaldehyde and a mixture solution of formalin, acetic acid and alcohol (FAA). After the sample has been cut into adequate size, it will then fi x in 4% formaldehyde in 50 mM phosphate buffer pH 7.2. The fi xative can be prepared by dissolving 8% (w/v) of paraformaldehyde (PFA) in distilled water and warm the solution up to 60oC in a fume hood. When PFA was dissolved, add equal volume of 100 mM phosphate buffer of proper pH (Soukup & Tylová, 2014). The mixture of 50% FAA (formalin-acetic acid-alcohol) can be obtained by mixing 50% (v/v) of ethanol, 5% (v/v) of acetic acid, 5% (v/v) of formalin and 40% (v/v) distilled water. Each fi xative has their own advantage where formaldehyde stabilizes the tissue arrangement via the formation of Methylene Bridge as it reacts with the tissue component. Meanwhile, formalin–acetic acid–alcohol (FAA) is the most common botanical fi xative for the paraffi n-embedded method. The fi xative penetrates tissue quickly, and the ethanol will produce shrinkage effect on the tissue but counterbalance by the acetic acid which has swelling effect (Yeung et al., 2015).

The specimens were then sequentially rehydrated at room temperature in 70%, 85%, 95% and 100% ethanol (30 min each step), vitrifi ed with a decreasing gradient from 100% ethanol and increasing gradient to 100% xylene. After that, the specimens were infi ltrated and embedded in a supporting matrix such as paraffi n and glycol- methacrylate (GMA). Gamborg and Phillips (1995) stated that paraffi n embedding is the fi rst method developed (Johansen 1940) which allow rapid examination of wall pattern during tissue development and usage of rather large specimen


55

(more than 1cm3) is possible. The tissue sections were obtained by using microtome which were subsequently mounted on to microscopic slides. Sections were then rehydrated and allowed to dry. Mounting medium was added then covering the section with a cover slip (Tao et al., 2009). Yet, the extent of damage due to the processing method making it less reliable for study of cellular detail. Thus, second method has been developed (Feder and O’Brien 1968) to overcome this problem. According to Chiarini-Garcia and Melo (2011), GMA resin preserves the cellular and subcellular details by reducing damage during processing. The polymerized plastic need not to be removed before staining as it is non-crystalline, homogenous and transparent, thus reducing the tissue damage. The specimens were dehydrated through a graded ethanol series (70%, 95%, 100%, 2 h each) and infi ltrated in a mixture of glycol- methacrylate resin and alcohol 95% at room temperature, and then embedded in pure activated and polymerized resin. The blocks were sectioned in a rotary semiautomatic microtome to obtain 2–8 mm thick sections (Barbosa et al., 2009).

Posteriorly, cross-sections were stained with safranin and astra blue, while paradermal sections were stained by using 1% methylene blue before subjected to analysis by light microscopy (Kahraman, et al., 2009). The sections can also be stained with toulidine blue, methylene blue, cotton clue or safranin/fast green according to component of interest and mounted between glass slides and cover slips (Barbosa et al., 2009).

Powder microscopy is done by using wet mount when the constituents of the herbal medicine are unknown; hence it is important to know the microscopic features of the powdered plant material in order to know which one(s) is present for further identifi cation (Diaz & Mendez-Vilas, 2010). The preparation involve pre-treating and mounting. During pre-treating, the cleaned sample is pulverized and the powder should pass through a No. 60 sieve where to obtain fi ne granules. The powdered material is then mounted onto the slides followed by addition of 1-3 drops of testing agents and stir to distribute it evenly. Place the cover slip over the sample and remove any excess liquid by blotting around its edges gently with a fi lter paper (Kagithoju et al., 2013).

Electron microscopy

Modern electron microscopy has been utilized for examination of biological changes at the subcellular or nano-resolution where both scanning and transmission electron microscope are predominant for observing the cell structure and its enclosure of singular organelles (Zajmi et al., 2015). Per se, electron microscope produces a higher in-depth resolution as compared to that is possible when using a light microscope. The images are three-dimensional and thus allowing investigation and extrapolation of the samples surface topography and play a vital role in authentication of entire botanicals either in fragmentized or in powder form (Serrano et al., 2010). Rhetorically, microscopic evaluation and chemical profi ling of the herbal materials especially for quality control audit and standardization tends to involve microscopical techniques such as comparative microscopic inspection of powdered herbal drug (Choudhary and Sekhon, 2011; Nikam et al., 2012). In tandem to this, the dynamic technological advances in microscope technology have increased accuracy and characterization capabilities as a mean and critical step in incorporating taxonomy bloom in herbal crude material detection and identifi cation as factors for optimization. Thus the use of modern light and electron microscopic techniques is essential in the advancement of this research area where any toxic effects (natural or synthetic compound) can be analysed especially when drug effi cacy development studies are involved (Ahmad and Khan, 2012) .

Both the scanning electron microscope (SEM) and the transmission electron microscope (TEM) are useful in such evaluations where SEM provides a tri- dimensional view of the cell surface. By contrast, the TEM produces a bi-dimensional image of the interior of the cell by detecting differences in density between the various parts of the sample (Fontanetti et al., 2010; Chatterjee et al., 2013). Hence, SEM and TEM have a potential advantage to encompass a number


56

of bacteriostatic or bactericidal properties of a substance, including plant extract.Per se plant samples can be prepared by the following procedures such as the samples is cut

in a small section the will be soak in mixed solution of glutaraldehyde and Sorencen’s buffered phosphate in an hour. After that the samples will be soaked in a prepared mixed solution of osmium and distilled water for about 14 hours at low temperature. The samples then soak in a series of alcohol (ethanol) concentration for 20 minutes respectively. Critical point dryer is the followed critical step and the specimen will be mount in stubs and coat with gold before being viewed under SEM (Sandalio et al., 2001; Veeramohan & Haron, 2014). With the samples preparation, examples imaged that can be captured under SEM include the presence of air pocket in rough structure of leaves specimen, and the hairy structures of the leaves such as Lady Mantle, also known as Alchemilla vulgaris L. (Otten & Herminghaus, 2004).

The correlation between SEM visual and TEM signature analysis, allowed us to compare the structure of the plasma membrane, the roughness of the outer surface as well as the irregularity of the subcellular structure. The electron microscopy allows the identifi cation of the visual changes of the cell architecture in correlation to the presence or absence of the targeted molecules at certain dose and thus highlighted the potential effects of the different antibacterial agent.

Study by Dholvitayakhun et al., (2015) revealed the antibacterial effect of Annona squamosa extract mechanism of action on several common Gram positive and Gram negative bacteria, while a study carried by Yossa et al., (2012) claimed the antibacterial effect of food chemical such as cinnamaldehyde and sporan on the food borne pathogens, E. coli and Salmonella sp. The SEM images showed the surface deformation of the treated E. coli where they have wrinkled and shrunken including morphological rod alterations. Similarly, treated Salmonella cells collapsed and appeared empty of content. TEM image of treated E. coli evidenced the deformation and disruption of the outer membrane with granulation on surface of some cells. On the other hand, TEM of Salmonella cells revealed severe damages including disruption of the plasma membrane, cell wall lyses and empty content without visible changes in the outer membrane.

The independent morphological parameters of the cell and its organelles described both the susceptibility and sensitivity status of the bacteria to the target compound. Furthermore, electron microscopy allowed the identifi cation of the visual changes of the cell architecture in correlation to the presence or absence of the targeted molecules at certain doses and thus highlighted the potential effects of the different antibacterial agents.

Scanning electron microscopy technique and authentication

The scanning electron microscope (SEM) offers a relatively simple method of studying the surface morphology of micro-organisms at high magnifi cation with a resolution approaching 15 to 20 nm under optimal conditions. One of the potentials of the instrument which remains largely unexplored is in the study of the morphological consequences of exposure of bacteria to antimicrobial agents.

A summary of various methodologies for chemical fi xation and sample preparation for microscopy investigation are as follows: Basically the fi rst step of sample processing to view sample under scanning electron microscope (SEM) is sample fi xative which aims to preserve and stabilize the structure of the cells, tissues or organism in keeping them structurally possible to its living state. Fixation method in preparation of live cells and tissue purposely done to stop the metabolism, to fi x structures of organelles and molecules in their current position and to make material accessible and stable during further processing (Wisse et al., 2010). In this step, the samples are placed in the chemical fi xative formulation such as 4% glutaraldehyde, Karnovsky‘s fi xative, McDowell-Trump fi xative and 1% osmium tetroxide. The purpose of this fi xative is to provide an optimal environmental condition in terms of pH, temperature and osmolarity and routinely used fi xative by most laboratories is glutaraldehyde and osmium tetroxide, a lipid cross-linker. For a better penetration into the cells, McDowell-Trump fi xative is recommended as it allows larger


57

sample to be processed. The volume of fi xative used in about 15-20 greater than the volume of the tissue as it allow full penetration into the tissue or cells, poorly penetration of fi xative into the tissue will result in shrinking and structural damage. The pH of buffers for the ultrastructure preservation is generally rectify between pH 7.2 and 7.4 while the concentrations of the buffers are rectify between 0.5 and 1.0 molar solution. Phosphate and cacodylate buffers are commonly used buffer in the sample processing. Routinely fi xation procedure carried out at room temperature and the duration of fi xation depends on size and density of the sample to be fi xed. Mainly the duration for fi xation require is 1 hour per 1mm of the sample thus larger samples required in about 6-24 hours for processing (Mogana and Patchamuthu, 2016).

The universal protocol for SEM sample preparation include fi xation with suitable fi xative solution which prepare in 0.1M phosphate buffer with pH 7.2 at 4 ºC for 2-24 hours. Then the procedure proceeds with washing with buffer (same buffer as fi xative preparation) three times and 10 minutes estimate for each washing steps. 1% Osmium tetroxide used as post fi xative at room temperature for 1-2 hours. After post-fi xation another washing procedure is require, however this steps require washing in water two times and 10 minutes for each times. The next step is dehydration procedure in the increasing concentration of ethanol ranging from 35%, 50%, 75%, 95% (2 times), absolute ethanol (2 times) and lastly in acetone. For each concentration of ethanol duration takes in about 15 minutes. Moving to the next step which is drying procedure and commonly used method is critical point drying (CPD), the samples is transfer into the CPD specimen holder which contain enough acetone to cover the samples. Last but not least the dried samples are then mounted on to a SEM sample stub with double-sided sticky tape and sputter the sample with gold before view under SEM (Mogana and Patchamuthu, 2016).

There are two method of drying for sample applied for decades which are critical point drying (CPD), freeze-drying and air-drying, all these method of drying infl uence the morphology of the sample eventually. Drying technique apply for the purpose to remove liquids form the specimen and avoid surface tension effect by blocked a liquid or gas interface to develop. Air drying procedure is possible however some biological tissue will collapse, fl atten or shrink. Thus recently to avoid the complication and to avoid tissue collapse, freeze drying technique and CPD being introduced. CPD techniques urge to provide fast and reliable result in most of the cases. Basically after the sample being dehydrated it will be transferred into the cooled vessels containing dehydrant usually ethanol which undergo pressurized with transitional fl uid such as liquid carbon dioxide and neon. Specifi c temperature and pressure applied to the sealed vessels until the pressure inside the vessels started to rise in response to the heat. This is the point where it reaches its critical point. Whereas freeze drying method, the frozen samples being freezed in a specifi c chamber at -80ºC and evacuated into the 10-1 Pascal (Pa) range or higher (Bozzola & Russell, 1999; Pathan, Bond, & Gaskin, 2010).

Predominantly, there are times when laboratory sample processing critical steps are similar for SEM and for TEM imaging. This rule of thumb processing protocol is applicable when the types of samples are usually of hydrated samples, frozen hydrated samples or dehydrated dried samples. This rule of thumb should be noted as resolution can be pseudo-interpreted and visualized. (Table 2 below, indicates this dilemma as when different type of samples are being processed from domain tissue or organism). There are pros and cons of each types of sample processing which hydrated samples usually benefi ts to provide excellent low magnifi cation images of plants surfaces and allows the fl exibility to alter temperature and vapour pressure in the specimen chamber. Besides that, it is widely used to study trichomes and glandular morphology and function of plant surfaces (Pathan et al., 2008).

Per se for frozen hydrated samples, it is quick and suited to view samples with higher water content and useful for preservation of life-like morphology of cells and tissues. For frozen hydrated samples, the samples fi rst undergo general protocol fi xation and then proceed by freezing the samples because ultrarapid freezing is thought to provide superior ultrastructure preservation which no combination of chemical fi xative could achieve. Ultra-rapid freezing usually done at a


58

rate of 104 to 105 ºC per second to avoid the formation of damaging ice crystals within the cell. The common cryo method used include plunge freezing (plunging the specimen in the liquid cryogen), slam freezing (directly slamming the specimen onto the polished surface of copper or silver block that has been chilled with liquid nitrogen), propane freezing (the specimen clamped with two metal plates that direct jets of liquid propane chilled with liquid nitrogen against both sides), and high pressure freezing, similar to propane het freezing except it used higher atmospheres pressure to enhance freezing (Bozzola & Russell, 1999; Bullen et al., 2014; Thompson et al., 2016). High pressure freezing allows possibility to work with larger biological structures, such as tissue from biopsies as well as with cells in suspension (Hayles et al., 2010). Advantages for dynamic biological processes such as fungal spore discharge or fungal infection on leaf plus being able to avoid deleterious changes associated with preparative steps that remove water and produce extraction/shrinkage of tissue (Erlandsen et al., 2001). Disadvantages for frozen hydrated samples include frozen specimens becoming very beam sensitive and beam damage may cause cracking of the specimen surface and required for specialized cryo-chamber which is very costly. The method is unsuitable for detailed visual examinations (Pathan et al., 2008). Simple and fast but do not allow viewing of the whole sample in a cross section due to damage structure caused by liquid nitrogen. In addition to that, drying without fi xation caused deformation of the tissue through evaporation of the water which has a high surface tension (Spricigo et al., 2015). Cryofi xed bacteria samples can be better visualized with the SEM examined under very low temperature (usually below -120 ºC). A technique carried out with the said specimen placed in high vacuum chamber. Drying or freezing of the specimen at very low temperature ensured there are no releases of any volatile substances from the specimen. (Kalab et al., 2008).

Transmission electron microscopy technique and authentication

Mielańczyk et al., (2015) stated that the transmission electron microscope (TEM) invented by Ernst Ruska and Max Knoll in early 1930’s is one of the approaches that able to provide a detailed structure up to the nanoscale level. In combination with chemical staining and immunolabelling, TEM contributes to the knowledge of ultrastructure and the topochemical details for cell wall study. A better and further understanding of cell and tissue functions can be obtained through the ultrastructural information provided by the TEM.

TEM has a higher resolution but is more diffi cult to implement, the image analysis is less straightforward and whereas bulk information is obtained, samples have to be thinned to electron transparency (less than about 500 nm thick). The specimen thickness is crucial for the highly absorbable electrons to penetrate the solid and form an image (Mitra, 2003). Yet, it is a very complicated problem in preparing such a thin solid specimen with minimal artifacts, thus sample preparation is a crucial step in this technique. As TEM is unable to view a large sample, hence it is important to direct the fi eld to the target of interested.

In TEM, tissue ware prepared follow several steps starting with primary fi xation, washing, secondary fi xation, dehydration, and infi ltration with transitional solvents, infi ltration with resin, embedding and curing. The fi rst always begin with hydrated tissue which eventually become water free at the end of the process and will be preserve in a static state within a plastic resin matrix. The hydrated tissue being faxed with an ideal fi xative such as osmium tetroxide, potassium permanganate, and standard glutaraldehyde-osmium tetroxide which is important to maintain the tissue against disruption during embedding, sectioning and during exposure to electron beam.

The general protocol for tissue preparation includes primary fi xation with 2-4% glutaraldehyde in buffer for 1-2 hours. Then washing procedure takes place in which the samples are being washed with buffer (3 changes) at 4ºC in duration of 1-12 hour or sometimes it takes overnight procedure. Secondary fi xation normally with osmium tetroxide (1-2%, usually buffered) for 1-2 hour is followed with dehydration procedure with increase percentage of ethanol started with 30%, 50%, 70% 95% (2 changesfor 5 minutes each) and absolute ethanol (2 changes for 20 minutes each).


59

Table 1: Preparatory critical methods for optical microscopy analysis.

Method Description Advantage & Disadvantage Procedures References

Whole mount

Preserve the structur-al relationships be-tween individual cells and extracellular components of min-ute sample (deepness within few hundred micrometres).

Advantage: Does not need special equip-ment, simply examine underthe stereomicro-scope.

Disadvantage: Not suitable for large specimen.

Clear the sample by using:

- sodium chloride- hydrogen peroxide- strong alkali or acids- phenol- lactic acid- chloral hydrate- sodium iodide solu-

tion Example:The solution is pre-pared by dissolving sodium thiosulphate in 65 % (aq. v/v) glycerol. Add the sodium iodide and 2 % (v/v) of dimethyl sulfoxide to fi nal solution of refractive index close to 1.5.

Soukup, A. & Tylová, E. (2014). Essential Methods of Plant Sample Preparation for Light Micros-copy. In Žárský, V. & Cvrcˇková, F. (eds.), Plant Cell Morpho-genesis: Methods and Protocols, Meth-ods in Molecular Biology, vol. 1080, DOI 10.1007/978-1-62703643- 6_1. Retrieved on Decem-ber 2, 2016, from from http://link.springer.com/protocol /10.1007%2F978-1- 62703-643-6_1

Section mount

Preserve the struc-tural relationships between individual cells and extracellular components of volu-minous sample that require cutting into adequate size.

Advantage: Allow thin section to be ob-tained for better view under microscope.Disadvantage: It is a laborious method that needs longer time for processing.

The tissue sections then were obtained by using microtome which subsequently mounted on micro-scopic slides to obtain 2–8 mm thick sec-tions.

Bancroft, J.D. & Gamble, M. (2008). Theory and Practice of Histological Tech-niques. Philadelphia, USA: Elsevier Ltd.

Fixativei. Formaldehyde

ii. Formalin-acetic acid-alcohol (FAA)

Advantage: stabilize the tissue arrange-ment via the forma-tion of methylene bridge as it reacts with the tissue com-ponent.

Advantage: pen-etrates tissue quickly, and the ethanol will produce shrinkage ef-fect on the tissue but counterbalance by the acetic acid which has swelling effect

4% formaldehyde in 50 mM phosphate buffer pH 7.2. The can be prepared by dissolving 8% (w/v) of paraformaldehyde (PFA) in distilled wa-ter. Warm the solution up to 60oC in a fume hood. When PFA was dissolved, add equal volume of 100 mM phosphate buffer of proper pH.

of 50% FAA (forma-lin-acetic acid-alco-hol) can be obtained by mixing 50% (v/v) of ethanol, 5% (v/v) of acetic acid, 5% (v/v) of formalin and 40% (v/v) distilled water.

Yeung, E.C.T., Stasol-la, C., Sumner, M.J. & Huang, B.Q. (Eds.). (2015). Plant Micro techniques and Proto-cols. Cham, Switzer-land: Springer.


60

Supporting matrix:i. Paraffi n fi rst

method developed (Johansen 1940)

ii. Glycol-methacry-late (GMA) resin second method developed (Feder and O’Brien 1968)

Advantage:- Allow rapid

examination of wall pattern during tissue development

- Possible usage of rather large specimen (more than 1cm3).

Disadvantage:- Extent of damage

due to the processing method making it less reliable for study of cellular detail.

Advantage:- polymerized plastic

need not to be removed before staining as it is non-crystalline, homogenous and transparent, thus reducing the tissue damage.

The fi xed sample rehydrated at room temperature in 70%, 85%, 95% and 100% ethanol (30 min each step), vitrifi ed with a decreasing gradient from 100% ethanol and increasing gradi-ent to 100% xylene. After that, the speci-mens were infi ltrated and embedded in the paraffi n.

The specimens were dehydrated through a graded ethanol series (70%, 95%,100%, 2h each) and infi ltrated in a mix-ture of glycol- meth-acrylate resin and alcohol 95% at room temperature, and then embedded in pure activated and polym-erized resin.

Gamborg, O.L. & Phillips, G.C. (Eds.). (1995). Plant Cell, Tissue and Organ Culture: FundamentalMethods. Heidel-bergh, Germany: Springer.

Chiarini-Garcia, H. & Melo, R. C. N. (Eds.). (2011). LightMicroscopy: Methods and Protocols. New York, USA: Springer.

Powder microsc opy

Use wet mount technique when the constituents of the herbal medicine are unknown

Advantage:-Allow the discovery of starch grain and calcium oxalate crys-tal that may present in certain plant parts.

During pre-treating, the cleaned sample is pulverized and the powder should pass through a No. 60 sieve where to obtain fi ne granules. The powdered material is then mounted on the slides followed by addition of 1-3 drops of testing agents and stir to distribute it evenly. Place the cover slip over the sample and remove any excess liquid by blotting around its edges gently with fi lter paper

Kagithoju,S., Godishala, V., Pamulaparthi, A., Marka, R., & Nanna, R.S. (2013).Pharmacognostic and Phytochemi-cal Investigations in Strychnos potatorum Linn.F. Journal of Phar-macognosy and Phy-tochemistry 2(4):46- 51.


61

Table 2: Preparatory critical methods for scanning electron microscope (SEM) analysis.

Sample Types Sample processing Examples References

Plant Hydrated samples

The plant sample being cut into small section and the cut faces is seal with glue (conductive carbon glue or two-component epoxy glue). Then the pieces will be mounted on SEM stubs with a conductive adhesive tab and the sample holder is cold with ice close to 0°C prior to insertion into the SEM in order to slow down the desiccation. The sample view under SEM with or without metal coating.

Lotus leaf (Nelumbo nucifera) Figure 1 (A), sample viewed under SEM used at acceleration voltages between 10 and 20 kV, without metal coating at ca 5°C shows little shrink-age after 10 minutes observation time. The waxes are intact, but sample drift re-tards high magnifi ca-tions,

Ensikat, Ditsche-Kuru, Barthlott, & Méndez-Vilas, 2010. Scanning electron microscopy of plant surfaces: simple but sophisticated meth-ods for preparation and exami-nation. Microscopy: Science, technology, applications and education, 1(13), 248-255.

Frozen hydrated samples

The samples being frozen (cryo-fi xation) in their na-tive hydrated state coated and visualized on a cold stage attached to the SEM.

Freeze fi xation involved plunging fresh, hydrated samples into liquid cryogen.

The frozen samples are dissect under liquid nitrogen with scalpel then mount onto the stub using freezing medium and immediately returned to liquid nitrogen.

Figure 1 (B), The sample (wheat surfaces) were attached to the stub and frozen by sample-holder contact with the pre- chamber stage (170 ºC) in an atmosphere of dry nitrogen and viewed using Philips SEM 5052 coupled to a Hexland cryo- prepa-ration system at a temperature of140 ºC to 120 ºC was used at an accelerat-ing voltage of 4 kV.

Pathan, A., Bond, J., & Gaskin, R. (2008). Sample preparation for scanning electron micros-copy of plant surfaces—horses for courses. Micron, 39(8), 1049-1061. doi:http://dx.doi.org/10.1016/j.micron.2008.05.006. Re-trieved on January 12, 2017 from http://www.sciencedi-rect.com/science/article/pii/S0968432808001236

Refshauge, S., Watt, M., Mc-Cully, M. E., & Huang, C. X. (2006). Frozen in time: a new method using cryo-scanning electron microscopy to visual-ize root–fungal interactions. New Phytologist, 172(2), 369-374. Doi: 10.1111/j.1469-8137.2006.01825.x. Retrieved on January 12, 2017 from http://onlinelibrary.wiley.com/doi/10.1 111/j.1469-8137.2006.01825.x/full

Dried / dehy-drated samples

The plant sample will be dry or rehydrated in about 2 hour’s prior fi xation. Then the sample will be freeze-fracturing with liquid nitrogen before being cut into small section and the cut will be fi xed with 2% paraformaldehyde and 2.5% glutaral dehyde fi xative

The dried samples (broccoli) shown in Figure 1(C) were processed and then mounted on the aluminium stubs using conductive silver and chromium coated prior to visu-alization under a

Pathan, A., Bond, J., & Gaskin, R. (2008). Sample preparation for scanning electron microscopy of plant surfaces—horses for courses. Micron, 39(8), 1049-1061. doi: http://dx.doi.org/10.1016/j.micron.2008.05.006. Retrieved on January 12, 2017 from http://www.sciencedirect.com


62

in a buffer solution of 0.05 M cacodylate and 0.001 M CaCl, pH 7.2 (without ad-dition of osmium tetroxide - OsO4) for 1 h in a volume 10 times greater than the volume of the sample. Pro-cessed sample will be dried through CPD or freeze-drying after dehydration procedure with acetone. Then mounting on stub and coating with metal for view-ing.

JEOL JSM- 6700F fi eld emissionscanning electronmicroscope (JEOL Ltd., Japan). The images were taken using SEI detec-tor (upper detector) which could capture image in high resolu-tion and the condi-tion used were a n accelerating voltage 3- 10kV, illuminating current 2.6 nA with a working distance 8–15 mm.

/science/article/pii/S0968432808001236 Spricigo, P. C., Trento, J. P., Breslin, J. D., Soares, V. F., Ferraz,L. F. D. M., & Ferreira, M. D. (2015). Methods of preparing fl ower stem samples for scan-ning electron microscopy. Or-namental Horticulture, 21(1), 17-26.

Cells or tissue (animal or hu-man)

Hydrated samples

The samples processes under standard protocol which are fi xed with 3% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer containing 1% sucrose and then rinsed in the same buffer.

For staining samples such as with uranyl acetate and osmium tetroxide, the fi xed samples are rinse extensive-ly in water, treat with 1% tannic acid for 5 min, rinse in water, and stain with ura-nyl acetate (0.1–2% solu-tion, pH 3.5) for 15–30 min and incubating for 10–30 min in 1% osmium tetroxide in water respectively.

Fixation steps are applied at cellular level using glutar-aldehyde and post fi xation with osmium. The samples should be cut into pieces prior to post fi xation and wash with buffer to avoid chemical reactions between glutaraldehyde and osmium. In order to better preserve mem branes and their con-trast, several additives to the fi xatives have been pro posed and applied, such as Ca2+ or Li+ ions, fer rocya-nide, tannic acid, and picric acid.

Figure 1 (D), Chinese hamster ovary (CHO) cell untreated samples were processed with and without stain-ing procedure to the cell prior to visual-ize the cell culture growth. Different microscopes were used which were JEOL6400SEM, FEI XL30 ESEM, and FEI XL30 ESEM-FEG in both low us-ing 0.1 torr and high vacuum modes at voltage, 12 kV

Thiberge, S., Nechushtan, A., Sprinzak, D., Gileadi, O., Be-har, V., Zik, O., Chowers, Y., Michaeli, S., Schlessinger, J., and Moses, E. (2004). Scan-ning electron microscopy of cells and tissues under fully hydrated conditions. Proceed-ings of the National Academy of Sciences of the United States of America, 101(10), 3346-3351. doi: 10.1073 pnas.0400088101. Retrieved on January 12, 2017 from http://www.pnas.org/con-tent/101/10/3 346.full

Wisse, E., Braet, F., Duimel, H., Vreuls, C., Koek, G., Olde Damink, S., van den Broek, M. A., De Geest, B., Dejong, C. H., and Tateno, C. (2010). Fixation methods for electron microscopy of human and other liver. World J Gastroen-terol, 16(23), 2851-2866. doi: 10.3748/wjg.v16.i23.2851. Retrieved on January 12, 2017 from https://www.ncbi.nlm.nih.gov/pmc/arti cles/PMC2887580/


63

Bacteria Dried samples

Solid specimens are fi xed in buffered fi xative such as 2-3% glutaraldehyde for pe-riods ranging from 5 min to 24 h and then post fi xation osmium tetroxide or Ruthe-nium red, if the specimens not based on the protein. Freeze fracturing (in liquid nitrogen) is applied after fi xed specimens completely dehydrated in absolute etha-nol in dehydration steps.

- Kalab, M., Yang, A.-F., & Chabot, D. (2008). Conven-tional scanning electron mi-croscopy of bacteria. Infocus Magazine, 10, 42-61.

Hydrated sample

The bacteria fi rst and grown on an agar and then the sample being fi xed with agar cross-linking after approximately 30-45 min. Next step, the sample will be dehydrated in an oven at 37ºC for 12 hours, followed with classical dehydration procedure with ethanol. Finally drying at 37ºC for about 1 hour.

Escherichia coli (E. coli) DH5α strain bacteria being pro-cessed and coated with a thin gold fi lm. SEM analyses were performed on a JSM 6390 electron micro-scope, The accelerat-ing voltage was ad-justed to 15 kV, I ~ 65 Ma (Figure 1 (E)).

Piroeva, I., Atanassova-Vladimirova, S., Dimowa, L., Sbirkova, H., Radoslavov, G., Hristov, P., & Shivachev, B. (2013). A simple and rapid scanning electron microscope preparative technique for observation of biological samples: application on bac-teria and DNA samples. Bulg. Chem. Commun, 45, 510-515.

Frozen hydrated

The sample being preserved by freezing method before being process. Sample being collected in the falcon tubes and immediately be frozen in a bath of dry ice and iso-propanol.

A drop of bacterial (Figure 1 (F)) suspen-sion was mounted onto a gold- coated glass slide and air-dried before the im-age collection in a JEOL-JEM 6380 LV SEM (JEOL Ltd), operating at an ac-celerating voltage of 20 kV.

Philip C. Bennett, Annette Summers Engel, and Jennifer A. Roberts (2006) Count-ing and imaging bacteria on mineral surfaces: In Methods of Investigating Microbial- Mineral Interactions, CMS Workshop Lectures, Vol. 14, J. Patricia A. Maurice and Lesley A. Warren eds., The Clay Mineral Society, Chan-tilly, VA, 37-78.

Diaz-Visurraga, J., Cárdenas, G., & García, A. (2010). Mor-phological changes induced in bacteria as evaluated by electron microscopy. Micros-copy: Science, Technology, Applications and Education, edited by: A. Méndez-Vilas and J. Díaz,307-315.

Sample with heavy mucous fi lms

Washing procedure is re-quired prior to the fi xation procedure to clear the mu-cous layer that can obstruct the clarity of the surface ultrastructure. The

SEM images (Figure 1 (G)) were collected with an AMRAY 1000A scanning elec-tron microscope oper-ated at an accel

Little, B., Wagner, P., Ray, R., Pope, R., & Scheetz, R. (1991). Biofi lms: an ESEM evaluation of artifacts intro-duced during SEM prepara-tion. Journal of Industrial Mi


64

procedure involves washing the sample with a suitable physiological saline solution accompanied with a very gentle agitation for every change of the saline solu-tion. Biofi lms must be coat-ed with a conductive fi lm of metal before the specimen can be viewed.

Biofi lm specimen on the copper surfaces were re-moved from the growth medium and fi xed with 4% glutaraldehyde buffered so-dium cacodylate (0.1 M, pH 7.2) at 4 ~ for a minimum of 4 h. Then the biofi lms were gripped thoroughly with distilled water and water was removed from the biofi lm by soaked in graded series ethanol. After that the specimens were taken from ethanol to amyl acetate and followed with critical-point dried from liquid CO2. Dried specimens were sput-ter-coated with gold (20 nm thickness) using a Polaron (Line Lexington, PA) Series II Sputtering System, Type E5100.

erating voltage of 30 kev and a vacuum of 10-5 torr.

crobiology & Biotechnology, 8(4), 213-221. DOI: 10.1007/BF01576058. Retrieved on January 12, 2017 from http://link.springer.com/ar-ticle/10.100 7/BF01576058

Mogana Das Murtey and Pat-chamuthu Ramasamy (2016). Sample Preparations for Scan-ning Electron Microscopy – Life Sciences, Modern Elec-tron Microscopy in Physical and Life Sciences, Dr. Milos Janecek (Ed.), InTech, DOI: 10.5772/61720. Retrieved on January 29, 2017 from http://www.intechopen.com/books/mo dern-electron-microscopy-in-physical-and-life-sciences/sample- preparations-for-scan-ning-electron- microscopy-life-sciences

After that, transitional solvent procedure with propylene oxide (3 changes) for 20 minutes each change, infi ltration of resin with propylene oxide: resin mixture which gradually increasing concentration of resin for overnight of maximum for 3 days. Lastly, embedding with pure resin mixture for 2-4 hour and curing at 60ºC for 1-3 days (Bozzola & Russell, 1999).

TEM has been apply in many scientifi c research including the investigation of cell changes upon treated by the natural product. The damage of E. coli outer membrane as observed under the TEM has been found in the study done by Diao et al., (2014) regarding the antibacterial activity of essential oil from Amomum kravanh. The severe morphological alterations appeared in the cell wall and membrane which signifi cantly results in cell permeability that leads to cell lyses followed by loss of intracellular dense materials at the surface of treated cells. SEM allows observation if there is any abnormal colony arrangement as well as the roughness of the outer surface. This is evidence by the effect of artonin E, the fl avonoid compound derived from the stem bark of Artocarpus communis on the Staphylococcus aureus strains. This study which was carried out by Zajmi et al., (2015) discovered the walls of the treated colonies were shredded and broken led to distorted shape and focally thickened outer membrane, indicating severe damage. The cytoplasm was distributed asymmetrically with eventual budding or septum formation. The membrane integrity was also disturbed by the artonin E where the treated S. aureus has increased roughness of the cell wall.

TEM magnifi es the inner region; enable the identifi cation of any presence of bodies, granules,


65

vacuoles and membrane encapsulated bacterial cells. TEM images of Staphylococcus aureus strains treated with artonin E showed the disintegration of bacteria membrane at polar ends as well as cytoplasm leakage. Artonin E treatment for 24 h showed accumulation of the DNA dense material of the S. aureus and thickening of the membrane, whereas obvious lysed membrane was observed. The cells were characterized by disaggregation of the cytoplasmic matrix, separation of cell membrane and rearrangement into two equal sections. The cytoplasm shows granularity with regions where the DNA aggregations are evident. The bacteria treated with artonin E showed granular deposits, intracytoplamic coagulation, cytoplasmic retraction, and relative decrease in the DNA material in respect to the cell volume. (Example of different sample preparation for TEM shown in Table 3).

Table 3: Preparatory critical methods for transmission electron microscope (TEM) analysis.

Sample Types ofsamples Preparations Examples References

Cells Hydrated samples

Fixation of the samples fol-low the general protocol as mentioned. After embed ding procedure done, the

Figure 2 (A), sperm ample prepared with the gel block. Heads are well preserved,

Cortadellas, N., Garcia, A., & Fernández, E. (2012). Trans-mission Electron Microscopy in Cell Biology: sample prep

samples undergo sectioning to semi-thin sections or ul-tra-thin section before being visualized under TEM.

intact acrosomes are visible. Neck, mid-piece and tail of the central sperm are un-damaged. Transmis-sion electron micros-copy, 2500 X.

aration techniques and image information.Relucenti, M., Petruziello, L., Familiari, G., & Heyn, R. (2010). A simple and reliable method to prepare semen for transmission electron micros-copy.

Frozen hydrated samples

After tissue removal, the tissue directly fi xed in 2.5% glutaraldehyde or 2% glu-taraldehyde with 2% para-formaldehyde in either 1 mM or 0.08 mM cacodylate buffer or in amphibian Dul-becco’s phosphate buffered saline for 2 h at room tem-perature before post fi xation in 1% OsO4 in either 1 mM or 0.08 mM cacodylate buf-fer. Decalcifi ed takes place after fi xation by incubating at 4 ºC for two days in a solution of 4% ethylene-diamine tetraacetic acid (EDTA in 0.1 M cacodylate buffer. Post-fi xation staining was carried out in 1% aque-ous OsO4. Samples were dehydrated in an ethanol series, block stained with a saturated

The sample such as Utricular macula (Figure 2 (B)) was fi xed and then dis-sected prior freezing. The freezing method done include high pressure freezing, the samples were loaded into 200 um deep alu-minium planchettes which fi lled a cryo-protect/fi ller and cov ered with the fl at side of second planchette pressed fi rmly, and slam freezing, the sample was brought rapidly into contact with (“slammed” against) the polishedsurface of a copper block cooled by liq-uid nitrogen.

Bullen, A., Taylor, R., Kachar, B., Moores, C., Fleck, R., & Forge, A. (2014). Inner ear tissue preservation by rapid freezing: improving fi xation by high- pressure freezing and hybrid methods. Hearing research, 315, 49-60. doi: 10.1016/j.heares. 2014.06.006.Retrieved on January 29, 2017 from https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC4152001/


66

solution of uranyl acetate in 70% ethanol overnight at 4 °C before completion of dehydration and embedding in plastic

The samples embed-ded were cut using diamond knife and collected on uncoated copper grids which then being coated with carbon. The samples were ex-amined under TEM operating at 80kV and 160kV.

Bacteria Frozen Hydrated samples

The bacteria are pellet by centrifugation and 10ug of fraction are apply to the 0.3mm diameter fl at surface of a microtome specimen holder and immediately projected into liquid ethane. After 30 second the pellet are expose to the air before freezing less than 1, then the specimen is retain under liquid nitrogen until use.

The specimen, Esch-erichia coli (Figure 2 (C)) sectioned with diamond knife in cryo- ultramicrotome operating at 120K. They were transferred at low temperature onto a carbon coated 700-hexagonal-mesh supporting grid, where they are

Dubochet, J., McDowall, A., Menge, B., Schmid, E., & Lickfeld, K. G. (1983). Electron microscopy of frozen-hydrated bacteria.Journal of Bacteriology, 155(1), 381-390.

pressed between two polished copper surfaces. The speci-men was observed at around 110 K in a Philips 400 electron microscope operat-ing at 80 kV in the bright-fi eld imaging or electron diffraction mode.

CONCLUSIONS

The rule of thumb to extrapolate and isolate novel bioactive molecules is that it is important to ascertain initially a sensitive and reproducible selection system that allows the experimenter the identifi cation of the peptide or biocompounds of interest. A good selection system is the key to fi nd new biologically active compounds that can be used as pharmacological tools for elucidating patho-physiological mechanisms and effi cacies of new therapeutic agents. The biological assays used to follow the activity of the different fractions obtained during the purifi cation steps include a wide range of in vivo and in vitro experiments like hemolysis, cytotoxicity, antimicrobial, anti-tumoral and antiviral activities and electrophysiological experiment (Shaw, 1997). In tandem to this advised critical steps, Cannell (1998), enumerate that there are fi ve main steps to follow for the isolation and characterization of a these bioactive substance: This include (a) the source selection, (b) extraction and fractionation, (c) separation and isolation of the molecules of interest and (d) the structural and functional characterization.

Geochemical signature natural products can be identifi ed and classifi ed by means of microscopic imaging analytical techniques. The issues of tangibility, adulterations and undetection by the naked eye can be validated via high resolution microscopic imaging and detection. In


67

pertinent, microscopy plays an important part in identity testing, authentication and suggestive methodologies in geochemical signature natural products (therapeutic bioactive agents) studies. Microscopy is one of the oldest and most effective techniques employed to assess identity and quality of botanicals. It often times is able to detect adulterants and contaminations including fi llers, extracts and incorrect plant parts. The microscopy technique is applicable to crude raw botanicals from powdered to whole form and is a preferred technique

ACKNOWLEDGEMENT

The authors would like to acknowledge the School of Health Sciences for allowing us to write this review as part and parcel for the fi nal year project and we would like also to acknowledge En. Hasbullah B. Abdul Samad for the technical guidance provided.

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INSTRUCTIONS TO CONTRIBUTORS

Offi cial Journal of the Microscopy Society (Singapore)

Submission of a manuscript implies: (1) that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); (2) that it is not under consideration for publication elsewhere; and (3) that its publication has been approved by all the authors as well as by the responsible authorities at the institute where the work has been carried out. Copyright: when any paper is accepted for publication, the authors agree to automatic transfer of the copyright to the publisher, and that the manuscript will not be published elsewhere in any language without the consent of the copyright holder.

Instructions to contributors

The journal publishes review articles, original research papers, short technical notes, letters to the Editors and short communications on the applications of microscopical techniques and specimen preparation procedures using all forms of microscopes and microanalysers.

Papers must be written in English and should be submitted electronically to the Editor- in-Chief: Samuel SW Tay Department of Anatomy, National University of Singapore Blk MD10, 4 Medical Drive, Singapore 117594 Telephone (65) 6516 3210 Fax (65) 67787643 E-mail: [email protected]

When an article is accepted for publication, the copyright on it passes immediately into the possession of the printer. The copyright covers the exclusive and unlimited rights to reproduce and distribute the article in any form of reproduction (printing, electronic media or any other form); it also covers translation rights for all languages and countries.

No manuscript processing fees, page charges or charges for halftone illustrations will be levied. Authors must prepare manuscripts in accordance with the journal’s accepted practice. Authors submitting diskettes are requested to follow the technical instructions printed in each issue of the journal.

Manuscripts should be divided into the following sections: title page; Abstract; Materials and Methods; Results; Discussion; ACKNOWLEDGEMENT; References, Fig. legends and tables. The original manuscript must be typed double spaced with wide margins and on one side of the paper only; duplicate copies must be photocopied on both sides of the paper to save on postage. In order to reduce mailing costs, when a manuscript is rejected only the original illustrations will be returned. Genus and species names should be marked with a single straight underline for italics. Words the author wishes to emphasize in the body of the text may also be set in italics and should be marked in the same way. The approximate desired positions of fi gures and tables should be marked in the margin of the manuscript. For literature citations in the text the name/year system should be used.

Brief accounts of particularly interesting results will be printed out of turn as “Short communications”. These should not exceed two or at the most three printed pages.

1. The title page must give: fi rst name(s) and surname(s) of author(s); title of the paper; initials(s) of given name(s) and last name(s) of author(s) with full addressees) of institute(s)- any footnotes referring to the title- address to which proofs should be sent and telephone and FAX numbers and e-mail address of the corresponding author.

2. Abstract. Each paper should be preceded by a summary not exceeding 200 words.

3. The list of References should include only works citied in the text. “Unpublished works” and “personal communications” may be referred to in the text but should not be included in the reference list. References should be listed at the end of the text as follows: a) Single author - list alphabetically and then chronologically. b) Author and one co-author - list alphabetically by fi rst author and then by co-author and then chronologically. c) First author and more than one co-author - list alphabetically by fi rst author and then chronologically, because

only the fi rst author’s name and “et al.” followed by the year of publication will be used in the text. d) In the event that more than one paper by the same author or team of authors published in the same year


77

is cited, the letters a, b, c, etc., should follow the year - e.g., Williamson (1990a) - in both the text and the reference list.

Entries in the reference list should be set out as follows:

Journals: name(s) of author(s) followed by initials (do not use “and”)- year of publication; complete title; journal as abbreviated in Index Medicus; volume number, fi rst and last page numbers.

Example: Andersson H, Bacchi T, Hoechl M, Richtel C (1998) Autofl uorescence of living cells. J. Microsc. 119:1-7

Books: name(s) of author(s) followed by initials; year of publication; complete title (in English); edition and volume if appropriate; publisher; place of publication.

Examples: Books: Klir G.J, Yuan B (1995) Fuzzy sets and fuzzy logic: Theory and application. Prentice Hall Englewood, New Jersey.

Book- chapters: Bach L, Mayer E (1987) Physics of water and ice: implications for cryofi xation. In: Steinbrecht RA, Zierold K (eds). Cryotechniques in biological electron microscopy - Springer-Verlag, Berlin, pp 1-34.

4. Figures. The illustrations must be submitted in triplicate. Figures should be restricted in number to the minimum needed to clarify the text. Information given in the legends must not be repeated in the text, nor should the same data be presented in both graph and table form. Illustrations that have already been published elsewhere are not usually accepted. All fi gures, whether photographs, graphs or diagrams, must be numbered in a single continuous sequence in the order in which they are cited in the text. Each Fig. must be mounted on a separate sheet. The author’s name, the number of the Fig. and the top of the illustration should be indicated on the back or on the sheet it is mounted on.

Figures can be grouped into a plate with spaces of not more than I mm between the individual illustrations. All fi gures must he mounted on regular bond paper and not on cardboard. Layouts or single fi gures should either match the width of a single column (8.6cm) or the printing area (17.6 x 23.6cm). The publisher reserves the right to reduce or enlarge illustrations. a) Line drawings: Good-quality prints are needed. The inscriptions must be clearly legible. Letters 2mm

high are recommended. Computer drawings are acceptable provided they are of comparable quality to line drawings.

b) Halftone illustrations: Well-contrasted photographic prints, trimmed at right angles and in the desired fi nal size must be submitted. Inscriptions should be about 3 mm high.

c) Colour illustrations: There is a page charge if a manuscript contains colour illustrations.

5. Legends. Each Fig. should have a short title followed by a concise description. Magnifi cation of micrographs should be given either in the legend of by a scale bar on the micrograph. Such remarks as: “For explanation, see text” should be avoided. Legends are part of the text and should be appended to it in a list starting on a separate page.

6. Tables. All tables must be numbered consecutively with arabic numerals. Each table must be typed on a separate sheet. The title of each table should provide a brief, self-suffi cient explanation of its content.

7. Proofs. To accelerate publication only one set of proofs will be sent to authors. This will show the fi nal form in which the paper will appear in the journal. Changes made in proof should be limited to the correction of typographical errors. Any others involve time-consuming and expensive work, and the costs will be charged to the author. If necessary, additions may be made at the end of the paper in a “Note added in proof’.

8. Offprints can be ordered at the time proofs are returned to the publisher.

Microscopy Society (Singapore) 1 c/o Department of Anatomy, National University of Singapore

4 Medical Drive, MD10, Singapore 117594 http://www.microscopy.org.sg/

Dear Colleagues & Friends, The Microscopy Society (Singapore) is a non-profit think tank actively focused on the promotion of microscopy, organization of microscopy-related activities and facilitation of knowledge and information exchange among its members. With a membership of approximately 100, the society actively promotes and hosts scientific meetings and workshops, and provides scholarships for members to attend conferences. The society always welcomes new members, and encourages members to actively participate and be involved in organizing microscopy-related activities and events. Benefits of joining MS(S) include:

• Free subscription to the society’s annual scientific journal - Annals of Microscopy • Free participation in the society’s Annual General Meeting, scientific symposia and talks, education

workshops, and other events • Travel scholarships and discounted rates to attend international and regional microscopy-related

scientific conferences • Inclusion in mailing list for special events and forums on microscopy • Many others

Assoc. Professor ONG Wei Yi President

--------------------------------------------------------------------------------------------------------------------------

MEMBERSHIP APPLICATION FORM Mr/Ms/Mdm/Dr* ___________________________________________________ Female/Male* Institution/Company: __________________________________ Position: ________________________ Address: ____________________________________________________________________________ Tel. No.: _________________________ (Office/HP) E-mail: __________________________________ Type of Membership: Ordinary/ Corporate* Membership fees per year: S$ 20.00 (Ordinary Member) S$ 250.00 (Corporate Member) Area of Interest: Life Sciences/ Physical Sciences* Signature: ______________________________________________ Date: ________________________ * Please delete where applicable Please submit the completed form together with the fees, and for cheque, please make payable to MICROSCOPY SOCIETY (SINGAPORE) to: Dr Wu Ya Jun (Hon. Secretary) c/o Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore. 4 Medical Drive,Singapore 117594.

Correlative Aberration-Corrected Microscopy

General Email: [email protected], [email protected] Web Site: www.htiweb.com

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