|Ahead of print publication
Impact of Śodhana (Ayurvedic purification process) on thrombolytic effect of Semecarpus anacardium Linn. f. Nut
Dattatray D Sarvade1, Kamini B Bhingardive2, Mohanlal M Jaiswal3
1 Department of Dravyaguna, IPGT and RA, Gujarat Ayurveda University, Jamnagar, Gujarat, India
2 CGHS, Ministry of Health & Family Welfare, Government of India, New Delhi, India
3 Department of Dravyaguna, National Institute of Ayurveda, Jaipur, Rajasthan, India
Dattatray D Sarvade,
Department of Dravyaguna, IPGT and RA, Gujarat Ayurveda University, Jamnagar - 361 008, Gujarat
Source of Support: None, Conflict of Interest: None
Aims and Objectives: In vitro evaluation and comparison of thrombolytic activity of raw and purified bhallātaka (Semecarpus anacardium Linn.f.) nuts by means of % of clot lysis. Materials and Methods: An in vitro thrombolytic model was used to check the clot lysis effect of two aqueous preparations of herbal extracts viz. raw and purified bhallātaka nuts along with Streptokinase as a positive control and distilled water as a negative control. Results: Among the drugs studied both raw and purified bhallātaka nuts showed significant percentage of clot lysis viz. 59.82% and 66.59% respectively with reference to Streptokinase whose percentage was 87.31%. Clot lysis percentage of purified bhallātaka was highest as compared to raw bhallātaka and was comparable to positive control, i.e. streptokinase. Conclusion: Extremely significant difference (P < 0.0001) between mean clot lysis percentage of tested drug samples was found as compared to positive control streptokinase and negative control distilled water. Both raw and purified bhallātaka nuts showed extremely significant mean clot lysis percentage as compared to negative control distilled water (P < 0.001).
Keywords: Ayurvedic purification process, Semecarpus anacardium Linn. f., thrombolytic effect.
| Introduction|| |
Many tracts of Ayurveda literature deal with thrombus related disorders. It is due to this that we come across various terms related to blood disorders such as śonitasanghātabhinatti (Property of breaking blood clots), grathitaraktapitta(Blood disorder/clotted blood) etc.
According to biomedicine, a blood clot (thrombus) in the circulatory system due to failure of haemostasis causes vascular blockage and while recovering leads to serious consequences in atherothrombotic diseases such as myocardial or cerebral infarction, at times leading to death. MI is a major cause of morbidity and mortality worldwide. More than three million people each year are estimated to have an acute STEMI (ST Elevation Myocardial Infarction), and more than four million have a NSTEMI (Non ST Elevation Myocardial Infarction). The immediate goal of acute ischaemic stroke therapy is to salvage the ischaemic penumbra through recanalization of the occluded cerebral blood vessel. This is currently achieved through thrombolytics, which are pharmacological agents that can break up a clot blocking the flow of blood. Thrombolytic agents that include tissue plasminogen activator (t-PA), Urokinase (UK), streptokinase (SK) etc., are used all over the world for the treatment of these diseases. In India, though SK and UK are widely used due to lower cost,, as compared to other thrombolytic drugs, their use is associated with bleeding complications, hemorrhagic stroke allergy and rarely anaphylaxis.
Moreover, as a result of immunogenicity, multiple treatments with SK in a given patient are restricted. Because of the shortcomings of the available thrombolytic drugs, attempts are underway to develop improved recombinant variants of these drugs.,,,,
Considerable efforts have been directed towards the discovery and development of natural products from various plant and animal sources which have antiplatelet,, anticoagulant,, antithrombotic, and thrombolytic activity. Epidemiologic studies have provided evidence that foods with experimentally proven antithrombotic effect could reduce risk of thrombosis.
Herbs showing thrombolytic activity have been studied and some significant observations have been reported. The aim of present study was to assess and compare raw and purified bhallātaka (Semecarpus anacardium Linn. f.) for their clot lysis property (thrombolytic activity) by using an in vitro procedure. Picchila guṇayukta and kaphayukta state of rakta, is referred to as grathita (clotted). Ayurveda classical texts indicate bhallātaka for kaphajaroga and vibandha (obstructive condition) and have recommended it as the best for these conditions. This is because bhallātaka is hot in potency and thereby acts on clotted blood. Though bhallātaka is hot in potency and poisonous, Caraka opines that it acts as amṛitakalpa if used wisely. Since ancient times, it has been used after śodhana. That is why among drugs possessing kaṭu rasa, bhallātaka has been preferred for the study though semi-poisonous.
| Materials and Methods|| |
Bhallātaka nuts were procured from pharmacy of National institute of Ayurveda, Jaipur. (Authenticated by department of Botany, Rajasthan University Jaipur with authentication no. RUBL211522 dated 11/04/2015)
Precautions taken during śodhana process
Coconut oil was applied to all exposed body parts of those handling bhallātaka. Chemical resistant gloves were worn to avoid contact with the poisonous oil/resin of the drug. Śodhana process was carried out in open air and masks were used to avoid and minimise inhalation of the poisonous fumes of the drug during śodhana.
Bhallātaka nuts which were immersed in water (good quality Bhallātaka seeds get immersed in water and only such seeds were used) weighing 800 g of such seeds were randomly taken and dried under shade. The thalamus portions of the nuts were removed with the help of a steel cutter. They were then shifted to a tray containing brick powder and rubbed thoroughly. Nuts were allowed for seven days to get embedded in brick powder spread over the tray. On the eighth day, the nuts were washed thoroughly with hot water to remove the brick powder. The bhallātaka nuts were then boiled in godugdha (Cow's milk) for about 45 min. Later, the samples were dried properly to remove moisture and stored in an air tight glass container for further studies.
An in vitro thrombolytic model was used to check the clot lysing effect of two drugs viz. raw and purified bhallātaka. Streptokinase was used as a positive control and distilled water as a negative control. 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.
- Distilled water – As a non thrombolytic negative control
- Mixture of fractional extracts of bhallātaka nuts (aśodhita/ impure form)
- Mixture of fractional extracts of bhallātaka nuts (śodhita/ detoxified form)
- Streptokinase (inj. STPase; Cadila pharmaceuticals, Ahmedabad, India) – As a positive control
- Blood Samples -Whole blood (2 ml) drawn from healthy human volunteers (n = 20) without a history of oral contraceptives, anticoagulant and antiplatelet drug therapy. (Blood was collected by venous puncture.) [Protocol was approved by Institutional Ethics Committee, National institute of Ayurveda Jaipur vide letter F10 (5)/EC/2014/2703 dt. 25/06/2014].
Groups Divided for Experimental Trial
- Group 1: 100 μl distilled water – non thrombolytic negative control
- Group 2: 100 μl of aqueous preparation of mixture of fractional extracts of bhallātaka nuts (aśodhita/ impure form)
- Group 3: 100 μl of aqueous preparation of mixture of fractional extracts of bhallātaka nuts (śodhita/ detoxified form)
- Group 4: 100 μl (30,000 IU) of Streptokinase- As a positive control.
| Methods|| |
Time period (season) of experimental trial
The trial was conducted at the Pharmacognosy and Pharmaceutical lab, Dept. of Dravyaguna and Pathology lab, Dept. of Roga and Vikruti Vigyana, National Institute of Ayurveda, Jaipur between November 2015 and January 2016.
To the commercially available lyophilized SK vial inj. STPase (Cadila pharmaceuticals, Ahmedabad, India) of 15,00,000 IU., 5 ml sterile distilled water was added and mixed properly. This suspension was used as a stock from which 100 μl (30,000 IU) was used for in vitro thrombolysis.
Whole blood (2 ml) was drawn from healthy human volunteers (n = 20) aged 20 to 50 yrs. without a history of oral contraceptive, anticoagulant and antiplatelet drug therapy (blood was collected by venous puncture). 500 μl of blood was transferred to each of the four previously weighed microcentrifuge tubes to form clots.
Fractional extraction procedure was used to prepare the extracts from plants in order to extract all reported principles from plant. We used both cold and hot extraction procedure using different solvents. Ethanol and aqueous extracts were prepared by using cold extraction procedure while other solvents i.e. hexane, petroleum ether, toluene, benzene, chloroform, dichloromethane, acetone, ethyl acetate, n-butyl alcohol and methanol were used for hot extraction as per their increasing polarity from non-polar to polar. Soxhlet apparatus was used for hot continuous extraction. Mixture of fractional extracts prepared from equal amount of each extract was used for experiment. Raw and purified bhallātaka nuts without thalamus were used for extract preparation.
Mixture of fractional extracts weighing 100 mg 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, which was filtered through a 0.22 micron syringe filter. 100 μl of this aqueous preparation of nuts was added to the microcentrifuge tubes containing the clots to check thrombolytic activity.
Experiments for clot lysis were carried out as reported earlier by Prasad et al. In brief, 2 ml venous blood drawn from healthy volunteers was distributed in four different pre weighed sterile microcentrifuge tubes (0.5 ml/tube) and incubated at 37°C for 45 minutes. After clot formation, serum was completely removed without disturbing the clot 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 aqueous preparation of mixture of fractional extracts of raw and purified bhallātaka were added separately. As a positive control, 100 μl of SK and as a negative non thrombolytic control, 100 μl of distilled water was separately added to the control tubes so numbered. All the tubes were then incubated at 37°C for 90 minutes 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.
% of clot lysis = (wt. of released clot/clot wt.) ×100
The significance between % clot lysis by Streptokinase and herbal extract by means of weight difference was compared with distilled water and each other by using repeated measures one-way ANOVA with post-hoc Tukey's test. Data are expressed as mean ± standard deviation. A P value < 0.05 was considered to be statistically significant. GraphPad InStat for windows version 3.06, La Jolla, CA, USA was used for all statistical analyses.
| Results|| |
The mean percentage of clot lysis for two drugs was determined and finally it was concluded that among the two trial drugs, both raw and purified bhallātaka nuts have a significant percentage of clot lysis the value being 59.82% and 66.59% respectively. It was comparable with the streptokinase which is a well known thrombolytic drug having mean percentage of clot lysis as 87.31%. The mean percentage for distilled water was 2.85% [Table 1] and [Figure 1].
|Table 1: Percentage of clot lysis of Drugs along with standard deviation|
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| Discussion|| |
Among the two trial drugs, clot lysis percentage of purified bhallātaka was highest and can be compared to that of streptokinase since its mean clot lytic percentage value was 66.59%. Effect of raw bhallātaka was also high having a clot lytic percentage of 59.82%. Both the drugs selected for the study displayed remarkable thrombolytic activity which can be compared to that of streptokinase. Repeated measures one-way ANOVA showed extremely significant difference between mean clot lysis percentage of the four drug samples tested (P < 0.0001). To compare mean clot lysis percentage of the four drug samples with each other, we had applied post hoc Tukey's multiple comparison test which showed that both the drugs had extremely significant difference in mean clot lysis percentage as compared to negative control distilled water. (P < 0.001) [Table 2] and [Figure 2].
|Table 2: Repeated measures one-way ANOVA with post hoc Tukey's multiple comparisons test data depicting P values|
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|Figure 2: Clot lysis of blood samples of volunteers by sample drugs and positive and negative control|
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Raw bhallātaka is classified as an upaviṣa. Purified bhallātaka is safe as it is processed form of raw bhallātaka. Purified bhallātaka has been reported to be safe by various databases. In Ayurveda, it is clearly mentioned that the drug bhallātaka should be used after śodhana.
Purified bhallātaka has higher clot lysis percentage 66.59% than compared to raw bhallātaka 59.82%, and it is also safe to be used in human beings.
After overall comparison, it can be said that purified bhallātaka is the best thrombolytic agent as reported by our study having a clot lysis percentage of 66.59%. The second choice could be raw bhallātaka as it has a mean clot lytic percentage as 59.82% and may not be as safe as purified bhallātaka, so it's use should be restricted to specific indications such as thrombotic diseases in patients of HIV/AIDS, Cancer where risk to benefit ratio is more favourable.
Further studies must be done to evaluate their effects in combination with other drugs. Also in vivo studies and clinical trials should be carried out to confirm their thrombolytic effects in the body. It can be concluded that both purified and raw bhallātaka have good thrombolytic effects and can be used as thrombolytic drugs after successful in vivo studies and clinical trials. Though raw bhallātaka is toxic in nature, it has good thrombolytic effect and hence it's use should be restricted.
As mentioned earlier, streptokinase has many side effects and hence is not considered safe. Both purified and raw bhallātaka can be developed as thrombolytic drugs in future as they are relatively safe. Their results in combination can be evaluated. We need extensive research for screening active chemical constituents responsible for thrombolytic effect of these two drugs so as to find out new alternative drugs for treating thrombotic disorders.
| Conclusion|| |
On the basis of beneficial effect of bhallātaka nuts in the literature and our own results of the experiments on the extracts of same drugs, bhallātaka nuts (both raw and purified forms) lyse blood clots in vitro. In that, purified bhallātaka has greater potential to dissolve clots as compared to raw one. This study implies that śodhana (Ayurvedic purification process) has a crucial role in enhancing thrombolytic activity of bhallātaka nuts. So śodhana (Ayurvedic purification process) of bhallātaka nuts should be preferred for the purpose of using it in the treatment of patients suffering from Atherothrombotic diseases. However, in vivo clot dissolving property and active component (s) of bhallātaka responsible for clot lysis are yet to be found out. Once found, bhallātaka can be incorporated as a thrombolytic agent for the improvement of the patients suffering from Atherothrombotic diseases.
We are very thankful to Dr. Mita Kotecha, HOD of Dravyaguna Department and Dr. Pawankumar Godatwar, HOD of Roga and Vikruti Vigyan Department from National Institute of Ayurveda for their support and guidance throughout the study.
Financial support and sponsorship
National institute of Ayurveda, Jaipur.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
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