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Research Article
In vitro Clot Lysis Activity of Different Extracts of Mangifera sylvatica Roxb. Leaves

Rashaduz Zaman, Mohammad Parvez, Md. Jakaria, Mohammed Abu Sayeed and Minhajul Islam

Research Journal of Medicinal Plants, 2015, 9(3), 135-140.

Abstract

The present study was aimed to evaluate the in vitro thrombolytic activity of different extracts of Mangifera sylvatica Roxb. leaves. An in vitro thrombolytic model was used to check the clot lysis effect of seven different extracts (benzene, hexane, petroleum ether, chloroform, methanol, ethanol and ethyl acetate) of M. sylvatica along with streptokinase as a positive control and water as a negative control. In the in vitro thrombolytic model, benzene, hexane, petroleum ether, chloroform, ethanol and ethyl acetate extracts showed 17.164, 17.384, 22.876, 28.984, 29.206 and 25.226% clot lysis, respectively. Among the extracts methanol extract showed significant percent of the clot lysis (46.934%) with reference to streptokinase (80.514%). From our study it was found that M. sylvatica possesses thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and active component (s) of M. sylvatica for clot lysis are yet to be discovered. Once discovered, M. sylvatica could be suggested as a thrombolytic agent in the treatment of patients suffering from atherothrombotic diseases.

ASCI-ID: 82-290

Mucklow, 1995).

Herbal medicines are assumed to be of great importance in the primary healthcare of individuals and communities in many developing countries (Ghosh, 2003). Herbal products are often perceived as safe because they are "natural" (Demrow et al., 1995). Epidemiologic studies proved that foods possessing anti-thrombotic effect could reduce risk of thrombosis. Herbs showing thrombolytic activity have been studied and some significant observations have been reported (Basta et al., 2004). In Bangladesh, in recent years, there has been increasing research on traditional ayurvedic herbal medicines on the basis of their known effectiveness for the treatment of ailments for which they have been traditionally applied (Zaman et al., 2015).

Mangifera sylvatica Roxb. is a plant in the ‘Anacardiaceae’ family. It is found in Bangladesh, Cambodia, China, India, Myanmar, Nepal and Thailand (en.wikipedia.org). The fruit is obliquely ovate, 8-10 cm long, much compressed distally forming a hook and has scantly whitish-yellow pulp which is almost fibreless. Few chemical entities isolated from M. sylvatica are friedelin-1, friedelan-3β-ol-23, Cycloartenol-9, Obtusifoldienol-66, β-sitosterol-24 (Anjaneyulu and Radhika, 2000).

As, there is no scientific report available concerning the thrombolytic activity of leaves extract of M. sylvatica, thus we aimed to evaluate the thrombolytic activity of various extracts of leaves of M. sylvatica. Phytochemical screening was also done to find out the potential chemicals within the plant leaves.

MATERIALS AND METHODS

Plant materials: The leaves of M. sylvatica were collected from Chittagong hill tracts area specifically from the area of Department of Forestry, University of Chittagong in May, 2014 and it was authenticated by Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh.

Preparation of extracts: The leaves of the plant were air dried at room temperature before grinding them to powdered form with the help of mechanical grinder (NOWAKE, Japan). Dried and powdered leaves were soaked separately in different solvents having different polarity (i.e., benzene, hexane, petroleum ether (40-60°C), chloroform, methanol, ethanol and ethyl acetate). Each extract was first filtered through muslin cloth to clarify and then through a Whatman No. 1 filter paper. The filtrate was evaporated under reduced pressure in vacuum evaporator. The dried crude extracts were sterilized overnight by UV radiation and then stored at room temperature in amber color glass vials until used for test of thrombolytic activity.

Reagents and chemicals: All the chemicals and reagents (i.e., benzene, hexane, petroleum ether, chloroform, ethanol and ethyl acetate) were of analytical grade and were provided by the Department of Pharmacy, International Islamic University Chittagong. Streptokinase (1.5 million unit/vial; Sanofi-aventis Bangladesh Limited) were used as positive control and water as negative control for in vitro thrombolytic test.

Ethical consideration: The study protocol was approved by the P and D Committee (pharmacy and drug committee-institutional ethics committee), Department of Pharmacy, International Islamic University Chittagong, Bangladesh. Blood samples were collected from the students of the Department of Pharmacy, International Islamic University Chittagong. A written consent was taken from all the volunteers.

Phytochemical evaluation: The leaves powder was dissolved in distilled water and screened for the presence of various chemical constituents like alkaloids, carbohydrates, proteins, cardiac glycosides, steroids, saponins, flavonoids, terpenoid, tannins and phenols (Chowdhury et al., 2014; Musa et al., 2009; Kokate, 1997; Ugochukwu et al., 2013; Yadav and Agarwala, 2011).

Preparation of test solution: Hundred milligram extract was suspended in 10 mL distilled water and the suspension was shaken vigorously on a vortex mixer. The suspension was kept overnight and decanted to remove the soluble supernatant. Hundred microliter of this aqueous preparation of herbs was added to the microcentrifuge tubes containing the clots to check thrombolytic activity.

In vitro thrombolytic test: The thrombolytic activity was evaluated by the method prescribed earlier (Prasad et al., 2006). In our study, 100 μL of each fractional extract was used as experimental drug. Five milliliter of blood samples were collected from volunteer and distributed as 0.5 mL of blood into seven separate pre-weighed (W1) sterile microcentrifuge tubes. The blood samples were collected from ten healthy different volunteers, who did not have contraceptive or anticoagulant therapy for the last 7 days. The blood specimen was incubated for 45 min at 37°C. After clotting of blood, serum was decanted and removed. Then weight of clotted blood (ΔW) was taken by subtracting the pre-weight (W1) from the weight of clot containing tube (W2) as, ΔW = W2-W1. The equation for calculating weight of clot is given below:

Then 100 μL extracts of each fraction of M. sylvatica were added to the clot containing tubes. Similarly, 100 μL of streptokinase was added to clot of standard tube and 100 μL of water was added to the clot of blank tube, those were used as positive and negative control, respectively. Then all the tubes were incubated at 37°C for 90 min and weighed again for getting the weight variation among the pre weight and final weight that was achieved for clot lyses (thrombolysis). The experiment was repeated three times with same blood sample of 10 volunteers. The percentage of clot lysis was calculated using the following equation:

Statistical analysis: The significance between percentage of clot lysis by streptokinase and herbal extracts by means of weight difference was tested by one-way ANOVA followed by Dunnett’s test analysis. Data is expressed as Mean±SE with 95% confidence interval. The statistical analysis was carried out by GraphPad Prism ver. 5.04.

RESULTS

The phytochemical screening of the plant revealed the presence of alkaloids, carbohydrates, proteins, cardiac glycosides, steroid, phenols, tannins, saponins, flavonoids, terpenoids. Reducing sugar was not present in the extract (Table 1).

Addition of 100 μL SK (streptokinase), a positive control to the clots along with 90 min of incubation at 37°C, showed 80.514% clot lysis. Clots when treated with 100 μL water (negative control) showed only negligible clot lysis (3.098%). The mean difference in clot lysis percentage between positive and negative control was very significant (p value<0.001). After treatment of clots with 100 μL of different extracts of M. sylvatica, the highest percentage of clot lysis was in methanol extract with 46.934%, which mean percentage of the clot lysis compared with the negative control was very significant (p-value<0.001). The study also revealed that benzene fraction, ethanol extract, ethyl acetate extract, petroleum ether extract, chloroform extract and hexane extract showed 17.164, 29.206, 25.226, 22.876, 28.984 and 17.384% of clot lysis, respectively.

Table 1:Phytochemical screening of leaves extract of Mangifera sylvatica Roxb.
+: Present, -: Absent

Table 2:Effect of different crude extract on in vitro clot lysis along with negative and positive control
**Significant at p<0.001, *significant at p>0.05, values compared with negative control, percentage of the clot lysis is represented as Mean± SEM of three independent values

Statistical representation of the effective clot lysis percentage by different fractions, positive thrombolytic control (streptokinase) and negative control (water) is tabulated in Table 2.

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DISCUSSION

From the beginning of civilization, for the treatment of many diseases human are dependable on plants, nowadays phytopharmacological investigation has created a new field of plant derivative drugs discovery, which are effective in remedial of certain diseases and renewed the attention in herbal medicines. It is estimated that about 30% of the pharmaceuticals are prepared from plants derivatives (Leta et al., 2002; Gillman et al., 1995). Numbers of pharmaceuticals approved by the Food and Drug Administration (FDA) currently have origins to plant sources. A number of plant sources especially several fruits and vegetables have been studied for their supplements having anti-coagulant, anti-platelet and fibrinolytic activity and there is evidence that consuming such food leads to prevention of coronary events and stroke (Emran et al., 2015; Ramjan et al., 2014). From preliminary phytochemical evaluation it’s revealed that this plant extracts contained several chemical constituents and these types of chemical constituents directly responsible for biological effects. In our present in vitro preliminary clot lysis test confirmed that, seven different extracts of M. sylvatica leaves showed the thrombolytic activity. The maximum clot lysis activity was mostly observed in methanol extract that means methanol soluble compounds are mainly responsible for the thrombolytic activity. In addition, this finding may indicate the possibility of developing novel thrombolytic compounds. Further studies are needed to quantify the amount of chemical constituents present in this plant extract and further studies also help to isolate, characterize the compounds responsible for thrombolytic activity. In near future it may be implemented as a thrombolytic agent for the improvement of patients suffering from atherothrombotic diseases.

ACKNOWLEDGMENTS

The authors acknowledge to Department of Pharmacy, International Islamic University Chittagong (IIUC) for laboratory facilities. We thank Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong for plant identification. We are also grateful to all the volunteers who donated the blood samples for this study.

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