In vitro thrombolytic activity of Dhamasa (Fagonia arabica Linn.), Kushta (Saussurea lappa Decne.), and Guduchi (Tinospora cordifolia Thunb.).

INTRODUCTION: Thrombotic disorders are among the major fatal conditions affecting the society. Treatment modalities used for such disorders are either surgical interventions or use of drugs such as urokinase, streptokinase (SK), or tissue plasminogen activators to dissolve the blood clots. These modalities have their own limitations and side effects apart from being expensive. There is a need for safer and cost effective antithrombolytic agents. AIM: To evaluate in vitro thrombolytic property of Dhamasa (Fagonia arabica Linn.), Kushta (Saussurea lappa Decne.), and Guduchi (Tinospora cordifolia Thunb.) plant extract.
MATERIALS AND METHODS: Venous blood drawn from 20 healthy volunteers was allowed to form clots which was weighed and treated with the extract of test plant materials to disrupt the clots. Weight of clot after and before treatment provided a percentage of clot lysis. SK was used as a positive and water as a negative control. STATISTICAL ANALYSIS USED: The significance between % clot lysis of five groups by means of weight difference was tested by the one-way ANOVA.
RESULTS: Clot lysis observed were 68.06%, 14.85%, 25.01%, 92.54%, and 3.00% for Dhamasa, Kushta, Guduchi, SK, and distilled water, respectively.
CONCLUSION: Herbal extracts possess thrombolytic properties and lyse blood clots in vitro.

Introduction

Thrombosis is defined as “hemostasis in the wrong place,” and is a major cause of morbidity and mortality. Arterial thrombosis is a common cause of myocardial infarction, ischemic stroke, and limb gangrene whereas venous thrombosis leads to deep vein thrombosis which can be complicated by the post thrombotic syndrome, and pulmonary embolism, chronic thrombo-embolism, pulmonary hypertension.[1] Number of other condition that can arise according to the location of the thrombus and the organs affected. The major risk factors for such thrombotic disorders are acquired disorders of hyper-coagulation, others being the exogenous factors such as surgery, hospitalization, immobility, trauma, pregnancy and the puerperium, and endogenous factors such as cancer, obesity.[2]

Current treatment modalities of thrombotic disorders include surgical interventions or use of drugs such as alteplase, anistreplase, streptokinase (SK), urokinase, and tissue plasminogen activators.[3] These modalities are costly as well as have serious side-effects which may be life threatening such as intracranial haemorrhage,[4] spontaneous pulmonary haemorrhage,[5] and angioedema.[6] Moreover, these drugs are not used in patients who have undergone surgery or those with a history of nervous lesions, gastrointestinal bleeding, or hypertension.[7] There is a need for safer and cost effective anti-thrombolytic agents. In order to find the blood thinning agents this work was conceived as it is known that herbal products are often perceived as safe because they are “natural.”[8] This study aims to investigate the multiple solvent extracts of the three medicinal plants viz., Dhamasa (Fagonia arabica Linn.), Kushta (Saussurea lappa Decne.), and Guduchi (Tinospora cordifolia Thunb.) for their clot lysis (thrombolytic activity) by using thrombolytic in vitro model.

Materials and Methods

The whole dry plant of Dhamasa, dry bark of Kushta, and dry stem of Guduchi were collected from the local market and were identified and authenticated by experts of the Department of Dravyaguna, National Institute of Ayurveda, Jaipur. The plants were cleaned, powdered and dried up in the hot air oven to remove the moisture content. The multiple solvent (methanol: Isopropyl alcohol: Acetone 100 ml each) extraction procedure was used to prepare extract by using Soxhlet apparatus. The extract was dried in the hot air oven and weighed to find out the percentage of extract formed from the dried powder.

About 100 mg of the extract thus formed was suspended in 10 ml distilled water (DW) and the suspension was shaken vigorously on a vortex mixer. The suspension was kept overnight and decanted to remove the soluble supernatant. Supernatant was filtered through a 0.22 µ syringe filter and was used to check clot lysis. An in vitro thrombolytic model was used to check the clot lysing effect of aforesaid three plants. SK was used as a positive control and DW as a negative control.

Clot lysis

Experiment for clot lysis was carried as reported earlier.[9] In brief, 2.5 ml venous blood drawn from healthy volunteers was distributed in five different pre-weighed sterile micro-centrifuge tubes (0.5 ml/tube) and incubated at 37°C for 45 min. 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).

As a standard control, 100 μl of SK and as a non-thrombolytic control, 100 μl of DW along with 100 μl of each samples were separately added to the micro-centrifuge tubes. All the tubes were then incubated at 37°C for 90 min and observed for clot lysis. After incubation, fluid released was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis. The experiment was repeated 20 times with the blood samples of 20 volunteers.

Statistical analysis

Data are expressed as mean ± standard error of the mean. The significance between % clot lysis by SK, DW, test plants by means of weight difference was tested by the one-way ANOVA by using Instat GraphPad version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Addition of 100 μl SK, the standard control to the clots along with 90 min of incubation at 37°C, showed 92.54% ±1.25% clot lysis. Clots when treated with 100 μl sterile DW (negative control) showed only negligible clot lysis (3.00% ±0.597%). The mean difference in clot lysis percentage between positive and negative control was very significant (P < 0.001). After treatment of clots with 100 μl of Dhamasa (F. arabica), Guduchi (T. cordifolia), Kushta (S. lappa) clot lysis, i.e. 68.06% ±3.53%, 25.01% ±2.11%, 14.85% ±1.37%, respectively, was obtained. When compared with the negative control (water) the mean clot lysis % difference of Dhamasa and Guduchi was significant (P < 0.001 in both) but mean clot lysis % difference of Kushta was not found to be significant (P < 0.01). When compared with the positive control (SK) the mean clot lysis % difference of all the three test plants was significant (P < 0.001 in all). Statistical representation of the effective clot lysis percentage by test plants, positive thrombolytic control (SK), and negative control (sterile DW) is tabulated in Table 1. Between group comparisons of percentage clot lysis is tabulated in Table 2.

Table 1

Thrombolytic activity of test drugs

Table 2

Between group comparisons of percentage clot lysis

Discussion

In earlier study, thrombolytic effect of Dhamasa has been reported so it was selected to confirm the reported findings.[10] Guduchi has been mentioned as best in “Shonitavibandhprashmana” (one that removes obstruction in blood) in Agrya Prakarana by Acharya Charaka.[11] Kushta has been mentioned by Sushruta during description of Rakta mokshana (blood-letting). Sushruta says that in the process of Rakta mokshana if the bleeding does not occur due to clot formation then area should be rubbed by the powdered form of some drugs. Kushta is one among the drug mentioned to facilitate bloodletting.[12] Textual references suggest that Guduchi and Kushta have thrombolytic property and so they were screened for their thrombolytic activity in the present study.

Herbal preparations are used since ancient times to maintain health and to prevent and treat various ailments. Advancement in field of phytochemistry have paved path for identification and isolation of plant compounds for curing diseases. Presently, about 30% of the pharmaceuticals are prepared from plants worldwide.[13] Researches are going on extensively to find new alternative herbal drugs in various areas. Treatment of hyper coagulable state still remain a great challenge and to combat vascular diseases number of studies have been conducted by various researchers to find out the herbs and natural food sources and their supplements having antithrombotic effect.[7913] SK is a widely used thrombolytic drug by the modern science but it can cause serious and life threatening side effects.[14] SK has its own complications like, bleeding which may be fatal with intracranial haemorrhage, it is also ineffective in individuals with anti-streptococcal and anti-prothrombin antibodies, and in patients who had multiple SK injections.[1516] All available thrombolytic agents still have significant shortcomings, including the need for large doses to be maximally effective, limited fibrin specificity and bleeding tendency.

Coagulation factor or natural anticoagulant factor levels influence the risk of venous thrombosis along with other risk factors such as ageing, obesity, protein C deficiency, etc. Oxidative stress, which is defined as an imbalance between pro-oxidant and antioxidant systems, can be both a cause and consequence of many vascular complications and serve as one of the biomarkers for these conditions.[17] F. arabica has been found to be beneficial in reducing oxidative stress by virtue of its antioxidant potential.[18] Different parts of F. arabica have also been used to cure various ailments, namely hematological, neurological, endocrinological, and inflammatory disorders.[19] T. cordifolia[20] and Saussurea lappa are also found to have good anti-oxidant activity.[21] These plants have properties which can help in preventing vascular diseases. The results of this study shows mild to moderate thrombolytic activity of test plants and so it gives an opportunity to explore their use in field of hypercoagulable state. These plants have Tikta/Katu (bitter/pungent) predominant Rasa (taste) and Ushna Virya (hot potency). Katu Rasa has property of “Shonitasanghata Bhinnati” (dissociates blood clots).[22] Tikta Rasa has Lekhana and Kleda, Meda, Shleshma Upshoshana properties which may help in lysis of formed thrombus in blood vessels.[23] It may be hypothesized that thrombolytic activity of these drugs might be due to above mentioned properties. In earlier studies also herbal medicines such as Hemidesmus indicus (L.) R. Br.,[24] Allium sativum L.,[25] Zingiber officinale Roscoe.,[26] Ocimum sanctum L., Curcuma longa L., and Azadirachta indica A. Juss[27] have been shown to exert thrombolytic or fibrinolytic effects. Positive results give hope to develop drugs in future but there is need of extensive research to find out active constituents so that development of alternative novel thrombolytic drugs can be done.

Conclusion

Dhamasa showed a significant percentage of clot lysis which is comparable with SK, a well-known thrombolytic drug. Guduchi and Kushta also showed mild thrombolytic activity. This study indicates possibility of finding novel thrombolytic drugs. However, there is need of thorough phytochemical and pharmacological research to discover their therapeutic potential. Once proved on scientific grounds these herbal preparations may be incorporated as thrombolytic agent for the improvement of the patients suffering from atherothrombotic diseases.

Financial support and sponsorship

National Institute of Ayurveda, Jaipur, Rajasthan, India.

Conflicts of interest

There are no conflicts of interest.