ultrastructural findings and elemental analysis of quercus

ANNALS OF MICROSCOPY Vol 7, April 2007

3�

Ultrastructural Findings and Elemental Analysis of Quercus infectoria Oliv. Soon LK*�, Hasni E�, Law KS�, Waliullah SS�, Farid CG�, and Syed Mohsin SSJ�

1School of Health Sciences, Health Campus, Universiti Sains Malaysia, Malaysia2Hospital Universiti Sains Malaysia, Health Campus, Universiti Sains Malaysia, Malaysia*Corresponding email: [email protected]

ABSTRACT

Under scanning electron microscope (SEM), the ultra-structural features of the Quercus infectoria oliv. gall showed rough rugae-like surfaces governing the whole integument of the Quercus infectoria oliv. White shining materials surrounding the protruding blunt horn-like lumps intersperse over the rugae-like surface are noted. Cross section of the gall revealed the view of the whitish core and concentric circle of resinous materials constituting the middle layer. Elemental analysis result showed that the Quercus infectoria oliv. samples examined had useful minerals of carbon, oxygen, silica, magnesium, aluminium, potassium and calcium. The amount of elements found in two different place of the horn however varies in their elemental distribution. The trough contains high silicon with low calcium while the horn consists of high calcium with low silicon. This important scientific evidence thus provides information suggestive of its role in postpartum care and reason that the gall being the most favoured as compared to other traditional medicines/herbs utilization.

KEYWORDS: Ultrastructure, elemental analysis, scanning electron microscope, Quercus infectoria oliv. (Manjakani)

INTRODUCTION

Malaysia is a biodiverse country with staggering number of plants/herbs thought to have medicinal potential. In Malaysia, modern medicine plays the predominant role in providing of healthcare (Technical Report of the Director-General of Health Malaysia, 2002). However, there is one common factor i.e. the initiative to consolidate and integrate traditional medicine alongside modern medical practice (Traditional/Complementary Medicine Practice and Training, Malaysia Health, �00�). Herbal therapies have become popular worldwide even in the United States, with one in three Americans using some type of herbal remedy in the past year. A MEDLINE search limited to English-language articles and human studies from �966 to �999, revealed that the most commonly used herbs are Echinacea, St. John’s wort, gingko biloba, garlic, saw palmetto, ginseng, goldenseal, aloe, Siberian ginseng and valerian (Mar & Bent, �999).

Before the advent of synthetic drugs, medicinal herbs were major source of therapeutic agents. But, skepticism of the possibility of integrating the use of these medicinal herbs by health care professionals suppressed its overt practice. The Malay women in Malaysia follow many traditional modalities in health practices, and postpartum practice is one of the unequivocal practices among them. Several methods, including the use of herbal preparation is thought to help in revitalization and full recovery of the reproductive functions. Quercus infectoria oliv. (Manjakani) was claimed to be highly beneficial for the Malay Kelantanese postpartum women and hazardous effects were not reported so far. Further, they would continue to use the medicinal herb and felt that traditional medicines/herbs should be integrated into modern medicine (Soon & Hasni, �005).


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Quercus infectoria oliv. is well-known since ancient times. Early study showed that as part of postpartum care, the Arabs, Persians, Indians, Malays and Chinese have traditionally used Quercus infectoria oliv. after childbirth to treat vaginal discharge and related postpartum infections (Grieve, �93�). Quercus infectoria oliv. or ‘Manjakani’ (in the Malay language) is well acknowledged in traditional medicine as a natural astringent that contains antiseptic materials and anti-oxidants, a truly generous gift from Mother nature of women. Studies indicated that Quercus infectoria oliv. is thought to have a variety of pharmacological properties including being an astringent (Muhamad & Mustafa, �994), anti-diabetic (Hwang et al., �000), anti-tremorine (Dar, Ikram and Fakouhi, �976), local anesthetic, anti-viral potential (Hussein et al., �000), anti-bacterial (Fatima et al., �00�), anti-fungal (Digraki et al., 1999), larvicidal (Redwane et al., �00�) and anti-inflammation (Kaur et al., �004).

Quercus infectoria oliv., the oak gall is not a type of fruit or from any part of a tree but an abnormal growth of the oak tree that reacts to viral infection of its bark that result from the puncture of the bark of the young twigs by the female Gall wasp, Cynips Gallae-tinctoria, who lays its eggs inside. It is indigenous to Asia Minor and Persia. The oak galls, primarily Aleppo galls or Mecca Galls imported from Turkey. The main constituents found in the gall of the Quercus infectoria oliv. are tannin (50 – 70%) and were a major source of gallic and tannic acid (Ikram & Nowshad, �977; Evans, �996). Tannin is a phenolic compound, which is soluble in water, alcohol and acetone. It gives precipitates with protein (Leach, �986). Quercus infectoria oliv. astringent property is thought to act in the restoring of health, to tone and increase vigor of the vagina (Muhamad & Mustafa, �994). The similarity in the antimicrobial activity of both the aqueous and acetone extracts suggests that these extracts may have high total tannin content. The anti-microbial activity seemed to depend on the contents of tannin in the plant extracts (Djipa et al., �000). The high amounts of tannin presented in the oak galls of Quercus infectoria oliv. (Wiart & Kumar, �00�; Evans, �996) implied that tannin might suggestively be the active compound that is responsible for the anti-bacterial activity. Tannin has been shown in literature especially from plant extracts to possess anti-bacterial activity (Irobi et al., �994).

The registration criteria for traditional medicines in Malaysia include limitations for the presence of heavy metals limits for microbial contamination, absence of steroids and other adulterants limits on disintegration time claimed indications prohibition of herbs with known adverse effects, prohibition of endangered animal species, compliance with Good Manufacturing Practice standards, and approved marketing authorization from importing countries (Zin, �004).

Over the last few decades, there is global resurgence in the use of herbal remedies. With the tremendous expansion in the use of traditional medicines/herbs world (Mar & Bent, �999), there is sadly a dearth of information pertaining to the ultra-structural findings and elemental analysis of the Quercus infectoria oliv.

The consumption of traditional medicines/herbs in Malaysia is a booming commercial home industry but the lack of scientific information and recognition with evidence based studies are limiting. Since the Malay women favoured Quercus infectoria oliv. for postpartum care, subsequently, this gall was selected for this study. Further, Quercus infectoria oliv. is not a local product of Malaysia but an imported entity from the Middle Eastern countries, and the fact remains that it is the most commonly utilized herb among the local Malay women. Articles on the ultra-structural description and features of Quercus infectoria oliv., are scare and hard to find and electron microscopic techniques are invaluable tool for analyzing complex structures and elemental detection. Further, poor knowledge and recognition based on scientific data is not readily available especially information related to ultra-structural findings and elemental analysis. Thus, information related to high-resolution microscopical characterization of Malaysian herbs is found wanting including that of Quercus infectoria oliv.


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OBJECTIVE OF THE STUDY

Quercus infectoria oliv. is well-known since ancient times and traditionally used by the Arabs, Persians, Indians, Malays and Chinese as part of postpartum care. However, people are not fully aware of the ultra-structural features and elemental composition of Quercus infectoria oliv. Therefore, this study was conducted to identify the surface ultra-structural features and elemental analysis of Quercus infectoria oliv. (Manjakani) that is of importance and that which can provide supportive evidence.

MATERIALS AND METHODS

1. Plant materialQuercus infectoria oliv. used in this study were obtained from the local market.

2. Ultrastructuralfindings(specimenpreparationandSEMprocess)It is prepared by placing the Quercus infectoria oliv. in the deep freezer Frochetti at -30º C

for �4 hours. Next, it is then freeze-dried at temperature -40º C for �4 hours using vacuum gauge pressure of 5 mTorr. For microscopical study, the Quercus infectoria oliv. was observed under the scanning electron microscope Supra 50 VPSE-SEM LEO with EDX (Energy Dispersive X-ray) microanalysis. Next, cross section of the Quercus infectoria oliv. were observed under the scanning electron microscope.

3. Elemental analysisTo detect the mineral content of Quercus infectoria oliv. , the samples were viewed under

the VPSEM. Together with the images, elemental analysis using EDX was carried out.

4. Stereomicroscope processTo examine the whole integument of the gall of Quercus infectoria oliv., the gall were

viewed under the Stereomicroscope integrated with image analyzer. Next, cross section of the Quercus infectoria oliv. were also examined by the stereomicroscope at room temperature.

RESULTS

The gall of Quercus infectoria oliv. (Manjakani) is rounded hard body of dark brownish in colour. Under the stereomicroscope integrated with image analyzer, revealed rough rugae-like surfaces governing the whole integument of the Quercus infectoria oliv. White shining materials surrounding the protruding blunt horn-like lumps intersperse over the rugae-like surface are noted (see Figure �). Cross section of the gall of Quercus infectoria oliv. showed the view of the whitish core and concentric circle of resinous materials constituting the middle layer (see Figure �).

Under the scanning electron microscope SUPRA 50 VPSE-SEM LEO with EDX (Energy Dispersive X-ray) microanalysis at 8 mm working distance, the blunt horn described features of flaking-like structures. It also showed the presence of abundance of grape-like vesicles held together with resinous materials (see Figure 3). Also present are areas suggestive of centers for the vesicular concregation (see Figure 4).

Variable Pressure Scanning Electron Microscopical study exhibited interesting structural features of Quercus infectoria oliv. (see Figures 3 & 4). The elemental distribution revealed the presence of carbon, oxygen, silica, magnesium, aluminium, potassium and calcium in Quercus infectoria oliv. (see Diagrams � & �).


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Figure �. Stereomicrograph showed protruding blunt horn-like lumps intersperse over the rugae like surface.

Figure �. Stereomicrograph showed the view of the whitish core and concentric circle of resinous materials constituting the middle layer.

Figure 3. Scanning electron micrograph showed image of the blunt horn-like and flaking-like structures at 8 mm working distance.

Figure 4. Scanning electron micrograph showed abundance of grape-like vesicles held together with resinous materials and areas suggestive of centers for vesicular congregation at �3 mm working distance.

Element Weight (%)

Atomic (%)

CO

Mg Al Si K Ca

57.6�37.480.4�0.73�.370.56�.83

65.783�.��0.�30.370.670.�00.63

Diagram �. Elemental distribution within Quercus infectoria oliv. (Area �). Scanning electron micrograph showed image of horn of Quercus infectoria oliv. observed under Supra 50 VPSE-SEM LEO

Element Weight (%)

Atomic (%)

CO

Mg Al Si K Ca

47.�545.�50.49�.5��.85�.��.6�

56.0740.400.�90.80�.450.4�0.57

Diagram �. Elemental distribution within Quercus infectoria oliv. (Area �). Scanning electron micrograph showed image in between horns of Quercus infectoria oliv. Area observed under Supra 50 VPSE-SEM LEO


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DISCUSSION

Horn-like protruberance distributed all over the surface of the Quercus infectoria oliv. (Manjakani) are hard and blunt. This characteristic is specific to Manjakani. Upon cross sectioning, three distinct layers were observed using the Olympus SZ40 Stereomicroscope with image analyzer. The layers were concentric ring-like structures namely the whitish core, a middle concentric circle and an outer section layer depicting different features and contents. The inner core is whitish in color, while the middle layer consists of shining resinous materials. The middle of center core surface was filled with nodular-like structure and almost homogenous in diameter of the middle ring area. The tetra area is filled with gel-like structure connecting and submerging onto it. The outer layer show flaking-like plague materials (see Figure 2).

Supra 50 VPSE-SEM LEO revealed the present of important elements distributed around the Quercus infectoria oliv., however, the amount of elements found in two different place (Area � & �) of the horn vary in their elemental distribution (see Diagram � & �). The trough contains high silicon with low calcium while the horn consists of high calcium with low silicon. Calcium is one of the most important element during recuperation, tissue repair, haematological reactions and many other bodily functions. Nonetheless, the role of silicon is unknown. It is well known that maintaining an adequate calcium intake throughout adulthood into the post-menopause should be encouraged. In a meta-analysis, a calcium intake of at least �000 mg daily reduced hip fracture by 22% (Cumming & Nevitt, 1997). Calcium alone is not sufficient to prevent either the hormone or ageing-related bone loss in post-menopausal women but avoiding a negative calcium balance will prevent accelerating this loss. This information together suggests that Quercus infectoria oliv. may help ensure adequate calcium intake. Little have been revealed even at scanning electron microscopic observation of the Quercus infectoria oliv. associated to the said claimed postpartum utilization. This study showed important scientific evidence thus provides information suggestive of its role in postpartum care and reason that the gall being the most favoured as compared to other traditional medicines/herbs utilization among the Kelantanese Malay women during the postpartum period (Soon & Hasni, �005). The fact that the public has already been consuming it could be indicative of its commercial viability.

CONCLUSION

This study emphasized the ultrastructural features and the elemental composition of Quercus infectoria oliv. It may be concluded here from this study that the Supra 50 VPSE-SEM LEO with EDX microanalysis indicated the presence of carbon, oxygen, silica, magnesium, aluminium, potassium and calcium. The use of electron microscopic techniques in this study proves to be valuable in showing relevant features and elemental distribution of Quercus infectoria oliv. It is therefore pertinent that these techniques can be considered as a novel star-gate towards revelations of many other herbal plants as well as other applications. Important scientific evidence can be derived from these sort of scanning electron microscopic study whereby important scientific information are required to ensure that the consumption of herbal product are safe for health and its suggestive role in postpartum care.

ACKNOWLEDGEMENTS

The authors are grateful to Encik Anizan Bakhtiar and Encik Jamaruddin Mat Asan, School of Health Sciences, Health Campus, Universiti Sains Malaysia for their technical assistance provided during the course of this study.


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ultrastructural findings and elemental analysis of quercus

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