in vitro antioxidant and thrombolytic activities of medicinal plants

78 | P a g e International Standard Serial Number (ISSN): 2319-8141

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International Journal of Universal Pharmacy and Bio Sciences 3(1): January-February 2014

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Pharmaceutical Sciences RESEARCH ARTICLE……!!!

IN VITRO ANTIOXIDANT AND THROMBOLYTIC ACTIVITIES OF

MEDICINAL PLANTS

Swati R. Dhande*, Priya P. Dongare, Aakruti A. Kaikini,

Kalpana A. Patil, Dr. Vilasrao J. Kadam

Bharati Vidypeeth‟s College of Pharmacy, Sector-8, C.B.D. Belapur, Navi Mumbai-400086,

Maharashtra, India.

KEYWORDS:

Antioxidant, Bambusa

bambos, Pogostemon

patchouli, Swertia chirata,

thrombolytic.

For Correspondence:

Swati R. Dhande*

Address: Bharati

Vidypeeth‟s College of

Pharmacy, Sector-8,

C.B.D. Belapur, Navi

Mumbai-400086,

Maharashtra, India.

Email:

bvppharmacology@gmai

l.com

ABSTRACT Thrombosis is one of the disorders caused by oxidative injury. A

causative relationship exists between thrombus formation in the vessel

and oxidative stress. Oxidants influence the balance of coagulation

system towards platelet aggregation and thrombus formation.

Therapeutic approaches towards antithrombotic therapy by means of

antioxidants are promising in both experimental and clinical designs.

Thus the present study covers in vitro evaluation of antioxidant and

thrombolytic activities of three medicinal plants in order to provide an

edge for investigation of in vivo thrombolytic activity of respective

plants. Over production of lipid peroxides is the result of oxidant

induced injury that may cause disturbance of cell homeostasis. Level

of MDA formation is directly proportional to and is the marker

indicator of extent of lipid peroxidation. Inhibition of pro-oxidant

(Ferrous sulphate and Sodium nitroprusside) induced MDA formation

after addition of HEPP, MEBB and EESC was found to be

comparable to Gallic acid (standard). Thrombolytic activity was

determined using in vitro clot lysis assay method. The crude extracts

were found to have significant (p<0.01) thrombolytic activity at

varying doses with maximum effect at the dose of 1000µg/ml.

Streptokinase was used as standard.

mailto:[email protected]

mailto:[email protected]



INTRODUCTION:

Oxidative stress is now recognized to be associated with more than 200 diseases, as well as with the normal

aging process.[1-3]

Diseases like Coronary heart disease and hyperlipidemia are associated with the

increased intracellular generation of reactive oxygen species leading to tissue injury with a variety of

pathological processes like ischemia, inflammation, atherosclerosis, and thrombosis. Recent evidence

indicates that reactive oxygen species (ROS) play an important role in the control of both blood

coagulation and thrombosis.[4, 5]

Redox mechanisms control platelet function [6]

and platelet activation is

accompanied by a burst of oxygen consumption and ROS generation. Hence compounds, which can

scavenge the excess of free radicals formed or inhibit their production or protect membranes from

peroxidation, are of wide therapeutic value in disorders like ischemic stroke and cardiovascular

diseases.[7,8]

Pogostemon patchouli, Bambusa bambos and Swertia chirata are the Asian plants used for the treatment of

various diseases by different tribes. A great deal of research has been carried out on these plants with

regards to their usefulness in traditional medicine.Several antioxidant therapies have shown

neuroprotection in experimental models of brain ischemia.These antioxidants pertain to major classes that

include inhibitors of free radical production, free radical scavengers, and boosters of free radical

degradation. However, only few of these agents have been assessed in combination with thrombolytic

therapy.[9, 10]

Present study is a scientific approach to evaluate the antioxidant and thrombolytic properties

of these Asian plants.

Pogostemon patchouli (Lamiaceae) commonly known as „Patchouli‟, is one of the Chinese herbal

medicines. In Asian countries, such as Japan and China, this herb has been used traditionally as an

energizer, tonic, febrifuge, antiseptic and insecticide since ancient time. The plant is also of economic

importance due to patchouli essential oil. Patchouli oil is obtained mainly from distillation of leaves and

found to posses variety of pharmacological activities including radical-scavenging[11]

, analgesic and anti-

inflammatory,[12-15]

antiemetic, antiallergic,[16]

immunomodulatory[17]

antimicrobial actions,[18]

anti-

IFV[19]

and antimutagenic.[20]

Bambusa bambos Druce. (Graminae) is a species of clumping bamboo commonly known as “Indian thorny

bamboo” in English and “Vanshi” in Sanskrit. It mainly occurs throughout India, Srilanka, Malaya, Peru

and Myanmar. According to Ayurveda text, the plant is claimed to be medhoghna (removing or destroying

excessive fat). Charakha prescribed decoction of leaves or seeds in treatment of excessive fat. Fruit and

seeds act on medhadhatu and are useful in fat metabolism and obesity. The other traditional uses of the



plant are as emmenogouge, anti-inflammatory, astringent, anti-spasmodic, tonic and to check cattle in

diarrhoea.[21]

The herb Swertia chirata commonly known as „Chirata‟ is well reputed for its multifarious therapeutic

values since the era of „Atharvaveda‟.The plant Swertia chirata (gentianaceae)aboriginal to Himalayas in

India, Nepal and Bhutan, have been used for millennia, to cure variety of ailments and diseases. The chief

bioactive constituents of plant are xanthones, flavanoids, iridoids, secoiridoids glycosides, which plays

momentous role in its biological activities like antidiabetic, anti-inflammatory, hepatoprotective,

antioxidant, antipyretic, antimalarial, analgesic, anticarcinogenic, antibacterial, antiviral, gastroprotective,

antileishmanial, anthelmentic etc.[22]

Materials and methods

Collection of Plant material

The leaves of Pogostemon patchouli and Bambusa bamboswere collected from V.G. Vase Kelkar College

and Keshavshrusshti, Mumbai, India respectively and were taxonomically identified and authenticated by

Dr. H.M. Pandit, Department of Botany, Guru Nanak Khalsa College, Mumbai. The voucher specimen of

both Pogostemonpatchouli and Bambusa bambos (accession no: pd/150912, ak/170912 respectively) were

deposited at the Herbarium unit for future reference.

Dried aerial parts of Swertia chirata were purchased from Yucca Enterprises, Wadala, Mumbai, in the

month of July. The aerial parts were authenticated by Dr.Vinayak Pandit, Department of Botany, Piramal

Life Sciences, Goregaon, Mumbai and a voucher specimen (accession number:PHL/6524) was deposited

for further reference.

Preparation of plant extracts

Shade dried leaves of Pogostemon patchouli were grind to coarse powder. Powder was subjected to

extraction using n-hexane solvent for 23 hr in soxhlet apparatus. Same way, coarse powder obtained from

dried leaves of Bambusa bambos was extracted with methanol solvent using soxhlet apparatus for 72 hr.

Dried powder obtained from Swertia chirata was subjected to cold maceration with 12% ethanol for 48 hr

with continuous stirring. Later all the extracts were concentrated using rotary evaporator under reduced

pressure and low temperature and then preserved in desiccator.

Chemicals

Thiobarbituric acid (TBA), Sodium nitroprusside (SNP), Ferroussulphate (FeSO4), malondehyde

bis(dimethyl acetal) (MDA) were obtained from SD fine chemicals, Mumbai. All solutions were prepared



just prior to use. Sodium dodecyl sulphate (SDS) and acetic acid were of LR grade. Lyophilized

Streptokinase (15, 00,000 I.U) was obtained as a gift sample from DongkookPharma. Co. Ltd.

Animals

Female Sprague Dawley rats, weighing 180-200g were procured from Haffkine Institute, Mumbai.

Animals were housed in environmentally controlled room (temperature 23-27 C̊, 50-70% humidity with 12

hr light/dark cycle). Animals were provided with food and water ad libitium.Animals of control group were

used for ex vivo TBARS assay. All experiments in this study were carried out with the prior approval of the

Institutional Animal Ethics Committee (IAEC/PR/2012/03) strictly adhering to the guidelines of

Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted

by the Animal Welfare Division of Government of India.

Evaluation of in vitro antioxidant activity

Production of TBARS from animal tissue

Thiobarbituric acid reactive acid substance formation was estimated using the modified method of Ohkawa

et al. (1979). Rats were sacrificed by carbon dioxide overdose. Livers were immediately excised, washed

with ice cold saline and placed on ice bath. Livers were homogenized in ice-cold 100 mm Tris buffer pH

7.4 to obtain 10% liver tissue homogenate. The homogenates were centrifuged for 10 min at 1000g to yield

a pellet that was discarded and supernatant was separated. The supernatant was used for the assay. The

homogenates (100 µl) were incubated with or without 50 µl of the pro-oxidants (FeSO4 and sodium

nitroprusside) and 50 µl of the plant extracts together with an appropriate volume of distilled water to give

a total volume of 300 µl at 37ºC for 1 hr. The colour reaction was carried out by addition of 200, 250, 500

µl each of 8.1% SDS, acetic acid pH 3.4 and 0.6 %TBA, respectively. Final volume was made to 4 ml with

distilled water. The mixtures were incubated at 95-100ºC for 1 hr. Absorbance was measured at 532 nm.

Serial dilutions of 0.03 mM standard MDA were prepared and the absorbance was read at 532nm after

cooling. Observations for basal control (to determine normal level of MDA in rat liver tissue homogenate),

FeSO4 and SNP control (to determine maximum level of MDA formed by respective pro-oxidants) values

of MDA were also recorded. MDA content of test samples was calculated by extrapolation method using

standard graph. The results were expressed in µmol/g liver wt.[1]

In Vitro Thrombolytic activity:

Phosphate buffered saline (PBS) (5 ml) was added to the commercially available lyophilized streptokinase

vial (15, 00,000 I.U.) and mixed properly. 0.5 ml blood was withdrawn from rats by retro orbital plexus.

Blood was collected in pre weighed sterile microcentrifuge tube (0.5ml/tube) and incubated at 37°C for 45



min. Observations/readings were taken in triplicate. After clot formation, serum was completely removed

(aspirated out without disturbing the clot formed) and each tube having clot was again weighed to

determine the clot weight (clot weight = weight of clot containing tube – weight of tube alone). To each

microcentrifuge tube containing pre-weighed clot, 100 μl of n-hexane extract of Pogostemon patchouli

(HEPP), methanolic extract of Bambosa bambus (MEBB) and ethanolic extract of Swertia chirata (EESC)

(0.01-1 mg/ml) were added separately. 100 μl of Streptokinase was used as positive control and 100 μl of

sterile distilled water was used as negative non-thrombolytic control. All tubes were then incubated at 37ºC

for 90 min and observed for clot lysis. After incubation, fluid released was removed and tubes were again

weighed to observe difference in weight after clot disruption. Difference obtained in weight taken before

and after clot lysis was expressed as percentage of clot lysis and calculated using the following formula.[23,

7]

% 𝑐𝑙𝑜𝑡𝑙𝑦𝑠𝑖𝑠 =weight of clot after lysis

weight of clot before lysis× 100

Statistical analysis

The values were expressed as mean ± SEM. Statistical analysis and comparison between the groups for was

performed by one way analysis of variance (ANOVA) followed by Tukey‟s test. Results showed

significant (p<0.01) thrombolytic activity.

Result and discussion

Evaluation of in vitro antioxidant activity

There was a significant increase in the formation of TBARS in SNP and FeSO4-induced oxidative stress as

compared to the basal. Results were compared with Gallic acid. At the highest concentration (1000µg/ml),

SNP induced MDA formation was reduced by HEPP, MEBB, EESC and Gallic acid to 76.38±2.92,

57.14±0.55,72.33±2.83 and 34.31±2.29µM/g liver tissue respectively when compared to SNP control value

(279.32±1.5793 µMDA/g). The percentage inhibition of MDA formation was found to be 70.78±1.01,

77.26±0.20, 67.81±1.52 and 87.12±0.08 by HEPP, MEBB, EESC and Gallic acid respectively.



Figure 1: Comparison of Effect of HEPP, MEBB, EESC, andGallic acid on SNP induced elevation of MDA

levels in rat liver homogenate. Mean basal value and SNP control values were 26.86±0.06 and 279.32±1.5793

µMDA/g of tissue respectively.

0 200 400 600 800 10000

20

40

60

80HEPP

MEBB

EESC

Gallic acid

Concentration (g/ml)

%In

hib

itio

n o

f F

eS

O4

in

du

ced

form

ati

on

of

MD

A

Figure 2: Comparison of Effect of HEPP, EESC, MEBB and Gallic acid on FeSO4 induced elevation of MDA

levels in rat liver homogenate. Mean basal value and FeSO4control values were 26.86±0.06 and 227.64±2.5303

µMDA/g of tissue respectively.

In FeSO4 induced MDA formation, the level of MDA was reduced to 86.27±3.48, 67.3±2.96, 53.78±0.13

and 40.13±2.78 µM/g liver tissue by HEPP, MEBB, EESC, and Gallic acid respectively when compared to

FeSO4 control value (227.64±2.5303 µMDA/g). The percentage inhibition of MDA formation was found to

be 60.41±1.464, 70.59±0.100, 72.20±1.203 and 74.42±0.06 by HEPP, MEBB, EESC, and Gallic acid

respectively.

0 200 400 600 800 10000

20

40

60

80

100

MEBB

HEPP

EESC

Gallic acid

Concentration (g/ml)

%In

hib

itio

n o

f S

NP

in

du

ced

MD

A f

orm

ati

on



Increase in the formation of TBARS in FeSO4 induced oxidative stress suggests cellular damage due to

overload of iron. Iron overload results in the formation of lipid peroxidation products.[24]

The possible

mechanism of iron toxicity includes free radical mediated peroxidative reactions, which are readily

catalyzed by iron. The protections offered by these plants extracts suggest that they may be useful in the

treatment of disease resulting from free radical induced damage.

SNP induced cellular toxicity is associated with overproduction of reactive nitrogen species (RNS) which

are as reactive as ROS producing damage to cellular contents. The results demonstrate the ability of all the

tested plant extracts to protect the cell against oxidative stress induced by various pro-oxidants and

suggests their use in the treatment of various diseases which may be linked with their antioxidant activity.

In Vitro Thrombolytic activity assay

100 200 400 600 800 10000

20

40

60

80HEPP

MEBB

EESC

plant extract concentrations (g/ml)

Clo

t ly

sis

(%)

Figure 3: Comparison of Thrombolytic activity of HEPP, MEBB, EESC and Streptokinase. Results are

expressed as mean±SEM.

The best way to study thrombolytic drugs is through in vitro clot lysis model.[25, 26]

Figure 3 showed the

effect of the extracts and Streptokinase (standard control) on clot lysis activity. When 100µl of sterile

distilled water was added to the clots (negative control) negligible clot lysis was observed. Whereas, tubes

to which streptokinase was added, significant clot lysis could be visually seen.

The percentage (%) clot lysis obtained after treating the clots with different concentrations of extracts was

statistically significant (p<0.01) when compared with negative control (0.8767±0.88%). The plant extracts

showed significant (p<0.01) clot lysis activity with maximum clot lysis value of 62.85±5.74%,



63.69±2.98% and 56.58±3.42% by HEPP, MEBB and EESC respectively, at concentration of 1000 µg/ml.

Percentage (%) clot lysis value for Streptokinase (standard control) was 77.13±2.12%.

Conclusion

In conclusion, the extracts of Swertia chirata, Bambusa bambos and Pogostemon patchouli showed

considerable antioxidant and significant thrombolytic activity in vitro. The mechanism of action of these

three plants is still unknown. This is only a preliminary study and to make a final comment the extracts

should be thoroughly investigated phytochemically and pharmacologically to exploit their medicinal and

pharmaceutical potentialities. Thus, these extracts may be incorporated in herbal preparations as

thrombolytic agents for the improvement of patients in clinical practice.

References:

1. Olalye MT, Rocha JBT, (2007), Commonly used tropical medicinal plants exhibit distinct in vitro

antioxidant activities against hepatotoxins in rat liver. ExpToxicol Pathol.58, 433–8.

2. Ghasanfari G, Minaie B, Yasa N, Leilu AN, Azadeh M, Shekoufeh N, et al., (2006), Biochemical and

histopathological evidences for beneficial effects of SaturejaKhuzestanicaJamzad essential oil on the

mouse model of inflammatory bowel diseases. ToxicolMech Method. 16, 365–72.

3. Sanvicens N, Violeta G, Angel M, Thomas GC, (2006),The radical scarvenger CR-6 proteccts sh-sy5y

neuroblastoma cells from oxidative stress induced apoptosis effects on survival pathways. J

Neurochem.98, 735–42.

4. Chakrabarti S and Freedman JE, (2010), Review: Nutriceuticals as Antithrombotic Agents.

Cardiovascular Therapeutics. 28, 227-35.

5. Gorlach A, (2005), Redox regulation of the coagulation cascade. Antioxid Redox Signal. 7,1398–404.

6. Essex DW. (2009), Redox control of platelet function. Antioxid Redox Signa.l11,1191–225.

7. Chowdhury N.S, Alam MB. Haque ASMT, Zahan R, MazumderMEH and Haque ME, (2011), In vitro

Free Radical Scavenging and Thrombolytic Activities of Bangladeshi Aquatic Plant

AponogetonundulatusRoxb. Glob Pharmacol. 5 (1), 27-32.

8. Diaz, M.N., F. Balz, A.V. Joseph and F.K. John, (1997), Antioxidants and atherosclerotic heart disease.

New Eng. J. Med, 337, 408-16.

9. Amaro S, Chamarrow A, (2011),Translational Stroke Research of the Combination of Thrombolysis

and Antioxidant Therapy. Stroke. 42, 1495-99.

10. Margaill I, Plotkine M, Lerouet D, (2005), Antioxidant strategies in the treatment of stroke. Free

RadicBiol Med. 39, 429–43.



11. Kim HW, Cho SJ, Kim BY, Cho SI, Kim YK,(2010), Pogostemon cablin as ROS scavenger in oxidant-

induced cell death of human neuroglioma cells. Evidence Based Complementary Alternative

Medicine.7(2), 239–47.

12. Li YC, Xian YF, Ip SP, Su ZR, Su JY, He JJ. et al.,(2011), Anti-inflammatory activity of patchouli

alcohol isolated from PogostemonisHerba in animal models. Fitoterapia. 82(8), 1295-301.

13. Xian YF, Suo J, Huang XD, Hou SZ, Chen JN, Ye MR, et al.,(2007), A pharmacological study on anti-

inflammatory effects of refined Huodan recipe. Chinese Journal of Experimental Traditional Medical

Formulae. 13, 54-6.

14. Zhao SC, Jia Q, Liao FL. (2007), The anti-inflammatory and analgesic pharmacological study of

Patchouli extract. Chin Tradit Plant Med. 29, 285–7.

15. Lu TC, Liao JC, Huang TH, Lin YC, Liu CY, Chiu YJ, et al.(2011), Analgesic and anti Inflammatory

activities of the methanol extract from Pogostemon cablin. Evidence. Based Complementary

Alternative Medicine.1-9

16. Suo J, Xian YF, Huang XD, Hou SZ, Chen JN, Ye MR, et al., (2007), A pharmacological study on the

anti-allergy effects of refined Houdan recipe. Chinese Journal of Experimental Traditional Medical

Formulae. 13:47–9.

17. Qi SS, Hu LP, Chen WN, Sun HB, Ma XD,(2009), Immunological regulation effects of essential oil in

leaves of Cablin Patchouli herbal on mice. Chin Arch Tradit Chin Med.27,774–6.

18. Liu XR, Fan R, Zhang YY, Zhu MJ, (2009), Study on antimicrobial activities of extracts from

Pogestemoncablin (Blanco) Benth. Food Science and Technology. 24, 220–7.

19. Kawamura Y, Kiyohara H, Nagai T, Hiramoto T, Yamada H, (2010), Anti-influenza virus active

sesquiterpene from leaves of Pogostemon cablin. Annual Meetingof the Pharmaceutical Society of

Japan. 130(2), 17-9.

20. Miyazawa M, Okuno Y, Nakamura S, Kosaka H, (2000),Antimutagenic Activity of Flavonoids from

Pogostemon cablin. Journal of Agricultural and Food Chemistry.48(3), 642-7.

21. Kaikini AA, Dhande SR, Kadam VJ, (2013), Overview of Indian medicinal Tree: Bambusa bambos

(Druce), Int. Res. J. Pharm. 4(8), 53-56.

22. Patil K, Dhande S, Kadam V, (2013), Thearautic Swertia chirata: An Overview. RJPP. 5(4), 199-207.

23. Bhuiya AM et al.,(2013), In vitro thrombolytic and antioxidant activity Study of Abromaaugusta

(Ulatkambal). The experiment. 14(2), 888-93.



24. Houglum K, Filip M, Witztum JL, Chojkier M. (1990), Malondialdehyde and 4-hydroxynonenal

protein adducts in plasma and liver of rats with iron overload. J Clin Invest. 86, 1991–8.

25. Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM and Daginawala HF, (2006),

Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thrombosis

journal, 4(14), 1-4.

26. Strief TW, (2006), In vitro simulation of therapeutic thrombolysis with microtiter plate clot-lysis assay.

ClinAppl Thrombosis/ Hemostatis.12, 21-32.

in vitro antioxidant and thrombolytic activities of medicinal plants

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