|Year : 2010 | Volume
| Issue : 2 | Page : 100-104
Synergistic activity of Fagonia arabica and Heteropneustes fossilis extracts against myocardial, cerebral infarction, and embolism disorder in mice
Atul Kaushik1, Rakesh Das2
1 Department of Pharmacology, IPS College of pharmacy, Gwalior, Madhya Pradesh, India
2 Department of Pharmaceutical Technology, Jadavpur University, Kolkata-32, West Bengal; Department of Pharmacology, IPS College of pharmacy, Gwalior, Madhya Pradesh, India
|Date of Submission||24-Mar-2010|
|Date of Decision||19-Apr-2010|
|Date of Acceptance||21-May-2010|
|Date of Web Publication||2-Aug-2010|
Department of Pharmaceutical Technology, Jadavpur University, Kolkata-32, West Bengal; Department of Pharmacology, IPS College of pharmacy, Gwalior, Madhya Pradesh
| Abstract|| |
Objective : Extract of Fagonia arabica, (family Zygophyllaceae) has thrombolytic activity against the clotted blood in blood vessels and the extract of Heteropneustes fossilis shows the cardiotonic activity. Therefore, combinatory pharmacology shows the synergistic activity in tied mice for a long duration. Materials and Methods : The cardiotonic activity of Heteropneustes fossilis fish extract was examined on a frog intact heart and then pharmacologically performed on 20 mice without plant extract as well as with a combination of plant extract, which gave a remarkable synergistic activity in an in-vivo experiment on mice kept tied for the duration of 12, 18, and 24 hours and injected within one minute after untying. Result and Discusion : The plant extract was compared with streptokinase as well as a non-thrombolytic agent (control). A study showed a percentage of clot lysis, which was 65.5% for plant extract, but streptokinase had 71%. The study was done in 11 healthy volunteers representing a mean value and SD of 65% ± 2.01% and 71.67% ± 0.71% of the plant extract and streptokinase, respectively, in contrast to the non-thrombolytic (control), that is, 0.86% ± 0.08%. Conclusion : Injection of plant and fish extract acts both synergistically in the blood clotted mice and in mice suffering from myocardial or cerebral infarction and embolized mice.
Keywords: Cardiotonic activity, clot lysis, extracts, thrombolytic activity, viability
|How to cite this article:|
Kaushik A, Das R. Synergistic activity of Fagonia arabica and Heteropneustes fossilis extracts against myocardial, cerebral infarction, and embolism disorder in mice. J Pharm Bioall Sci 2010;2:100-4
|How to cite this URL:|
Kaushik A, Das R. Synergistic activity of Fagonia arabica and Heteropneustes fossilis extracts against myocardial, cerebral infarction, and embolism disorder in mice. J Pharm Bioall Sci [serial online] 2010 [cited 2015 Mar 1];2:100-4. Available from: http://www.jpbsonline.org/text.asp?2010/2/2/100/67011
The cardiovascular drugs should have not only cardiac tonic activity, but also thrombolytic activity. At present, we are still all the time trying to focus on herbal drugs, especially the herbal drug combination. However, our study is a combination of herbal Fagonia arabica as well as aquatic animal extract, and especially pharmacological activity, from the extract of piscine venom, that is, Heteropneustes fossilis .
Fagonia Arabica belongs to the Zygophyllaceae family  and is known as 'Kharasan' thorn in English and by a common name of 'Dhamasa' in India. It is a green shrub, one to three feet in height, found on calcareous rocks distributed throughout the Mediterranean region of South Africa, Afghanistan, and India (Rajasthan, northwest Punjab, and Western India).
Atherothrombotic diseases such as myocardial or cerebral infarction are serious consequences of a thrombus formed in blood vessels. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs have certain limitations that cause serious and sometimes fatal consequences.  Thrombolytic agents that include tissue plasminogen activator (t-PA), Urokinase (UK), streptokinase (SK), and so on, are used all over the world for the treatment of these diseases. In India, although SK and UK are widely used on account of their lower cost , as compared to other thrombolytic drugs, their use is associated with the hyper risk of hemorrhage,  severe anaphylactic reaction, and it lacks specificity. Herbal preparations have been used since ancient times for the treatment of several diseases. Herbs and their components, possessing antithrombotic activity, have been reported earlier, however, herbs that could be used for thrombolysis have not been reported so far. Considerable efforts have been directed toward the discovery and development of natural products from various plant and animal sources that have antiplatelet, , anticoagulant, , antithrombotic,  and thrombolytic activity. Using an in vitro thrombolytic model, Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica, and Bacopa monnieri Linn showed 19.3, 14.5, 20.3, 17.8, 75.6, and 41.8% clot lysis respectively. Among the herbs studied Fagonia Arabica showed a significant percentage of clot lysis (75.6%) with reference to Streptokinase (86.2%).
The most potentiative effects of H. fossilis venoms are on the cardiovascular system. All piscine venoms produce profound cardiovascular changes, both in vitro and in vivo, including the release of nitric oxide from endothelial cells, smooth muscle contractions, and differing effects on the atria. Once again, the activities of H. fossilis venoms are very similar, usually consisting of a depolarizing action on both nerve and muscle cells. H. fossilis venoms have potent cytolytic activity, and it seems probable that this activity is the mechanism behind many of their cardiovascular and neuromuscular effects.
| Materials and Methods|| |
Plant extract, Venom extract, microspatula, GeneMate digital balance, microcentrifuge tubes, Streptokinase, incubator, microspatula, Ringer's solution, methanol: isopropyl alcohol: acetone, water for injection, distilled water, Dounce homogenizer, kymograph apparatus.
Extraction of venom
The fishes (20 to 30 cm in length, weighing about 50 to 100 g) were collected from the local freshwater tanks (ponds) located within West Bengal. The specimens were euthanized using MS-222 at a concentration of 300 mg/L in fresh water. All further preparations were carried out either on ice or under refrigeration at 4°C. Spines and caudal fin tissue were removed from each specimen, rinsed in physiological saline and gently scraped with a microspatula, to remove any excess epidermal secretions, and they were weighed to the nearest 0.001 g using a GeneMate digital balance. The spines were minced and then further homogenized in a 2 mL Dounce homogenizer along with either marine (Plotosidae) or freshwater (other families) euteleost physiological saline at a volume of 2 ml/g of tissue. The homogenate was then centrifuged at 6,000 rpm, at 4°C, for 20 minutes and the supernatant collected. The supernatant served as the crude venom extract. Control extracts prepared from caudal fin tissue were prepared in the same manner.
Extraction of plant
The multiple solvent (methanol: isopropyl alcohol l: acetone) extraction procedure was used to prepare the extract. Extract of 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, and was filtered through a 0.22 - micron syringe filter. One hundred microliters of this aqueous preparation of herbs was added to the microcentrifuge tubes containing the clots to check the thrombolytic activity.
Experiments for clot lysis were carried out as reported earlier. In brief, 4 ml of venous blood drawn from healthy human volunteers was distributed in eight different pre-weighed sterile microcentrifuge tubes (0.5 ml/tube) and incubated at 37°C for 45 minutes. After clot formation, the serum was completely removed, without disturbing the clot and each tube having the clot was again weighed to determine the clot weight (clot weight = weight of clot containing tube - weight of tube alone).
[Table 1] depicts eight microcentrifuge tubes, which are represented in μg. The weights of the clot containing tubes and clot weight (in μg) are also represented. [Figure 1] shows the percentage of clot lysis of Streptokinase (A) for positive control, Nonthrombolytic (B) for negative control, and Aqueous Extract of plant (C), in the histogram.
To each microcentrifuge tube containing the pre-weighed clot, 100 μl of aqueous extract of herb, Fagonia arabica was added. As a positive control, 100 μl of streptokinase (SK), and as a negative non-thrombolytic control, 100 μl of distilled water were separately added to the numbered control tube. The tube was then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, the fluid released was removed and the tubes were again weighed to observe the difference in weight after clot disruption. The difference obtained in weight taken before and after clot lysis was expressed as the percentage of clot lysis. The experiment was repeated 11 times with the blood samples of 11 volunteers, as shown in [Table 2]. [Figure 2] is the graphical representation of the percentage of clot and number of human volunteers.
Spines and caudal fin tissue were removed from each specimen, rinsed in physiological saline, and gently scraped with a microspatula in order to remove any excess epidermal secretions, and weighed to the nearest 0.001 g using an electronic digital balance. The spines were minced and further homogenized in a 2 ml Dounce homogenizer along with freshwater (other families) euteleost physiological saline at a volume of 2 mL/g of tissue. The homogenate was then centrifuged at 6,000 rpm at 4°C for 20 minutes and the supernatant was collected. The supernatant served as the crude venom extract. Control extracts prepared from caudal fin tissue were prepared in the same manner.
From the above-mentioned fish extract (Venom), a 5 μg/ml concentration was made and about 0.5 ml was poured into the dissected frog's intact heart in order to get the stimulation activity. Ringer solution was also administered into the heart after the restoration of the extract's activity toward normal. The kymograph in [Figure 3] shows the activity of fish extract compared to normal beats, with 1% prepared Ringer's solution.
Arrangement was made such that 20 albino mice were taken (1039/ac/07/CPCSEA) and all of them were tied with threads in both lower limbs, quite tightly (from the upper portion to the lower extreme), so that blood flow could be interrupted. After 12, 18, and 24 hours of intact tying, five mice were intraperitoneally administered (with water for injection) with 5 μg/ml solution of fish extract, five mice with 5 μg/ml of plant extract, in I.P., 5 mice with both plant as well as fish extract, and five mice without any drugs, that is,, control study. These give the reports of viability.
| Results|| |
The selected eight numbers of microcentrifuge tubes, with a constant weight of 710 μg, filled with 0.5 ml/tube of blood in each of the eight tubes, showed a mean weight with SD of 326.21 + 0.54 μg. Furthermore, after drying in the above-mentioned conditions in three different categories of microcentrifuge tubes A, B, and C, the average weights with SD of streptokinase (+ve control), Non-thrombolytic (-ve control), and aqueous plant extract were 94.48 + 0.45 μg, 329.02 + 1.28 μg, and 112.2+ 0.48 μg, respectively, and also the respective percentage of clot lysis was 71, 0.86, and 65.5%, respectively. Its graphical representation is mentioned in [Figure 1] between the percentage of clot lysis versus A, B, and C of an individual human volunteer (first human volunteer among 11).
From [Table 2], the percentage of clot lysis was estimated for 11 volunteers, in which the evaluation of the percentage of clot lysis of the first volunteer was done in [Table 1]. In such a fashion that the percentage of clot lysis of the other 10 volunteers could be estimated in three categories, Streptokinase (+ve control), Non-thrombolytic (-ve control), and aqueous plant extract in microcentrifuge tubes and their mean as well as the SD shows 71.67 ± 0.71%, 0.86 ± 0.08%, and 65 ± 2.01%, respectively.
- According to the data mentioned in [Table 3], five out of 20 mice were tested for their viability, without any drug extracts, which was shown in a nonmobile state of rest or after loss of their movement after 12 and 18 hours of untying their lower limbs, but after 24 hours the untied mice died within 10, 15, 23, 17, and 12 minutes. Five mice were tested (i.e, from 6 - 10 in number) for their viability test with plant extract and showed that all of them were able to run, except one which could be able to move freely after 12 hours of untying. But after 18 hours of untying all the 5 mices in that group could seems to move freely and after 24 hours of untying all the 5 mices were able to move slightly. Five mice out of 20 (i.e, 11-15 in number) with only fish extract showed slight movement after 12 hours of untying, and after 18 hours of untying four mice died within 22, 20, 20, 20 minutes of untying respectively among 5 mices, and rest one shows slight movement which died within five minutes past 24 hours of untying . However, in the combinatory administration of plant and fish extracts, five out of 20 mice were seen to run after 12 and 18 hours, but after 24 hours they could freely move without any disturbance. Finally the viability results without any drug extract and with only fish extract administration showed that 100% died. With only plant extract and with plants and fish extract administration 0% died.
| Discussion|| |
The activity of plant extract that evolved from Fagonia Arabica performed the thrombolytic action and the activity of fish extract from Heteropneutesis fossilis showed cardiotonic activities. Hence, while dealing with Atherothrombotic diseases such as myocardial or cerebral infarction, where serious consequences of the thrombus formed in blood vessels (embolism), the combination therapy of both (plant extract and fish extract) were very essential as per synergistic action . In this experiment, the combination study has been focused on mice, although, it can also be performed on mammalian species or humans too. The in-vitro study of percentage of clot lysis, however, was performed with blood of 11 human volunteers.
According to [Table 1], it was noticed that after the drying of blood, 0.5 ml in each microcentrifuge tube, the remaining residual weight of 326.21 ± 0.54 μg (average with SD), that is, clotted weight, was less than the initial weight, which was around 515 μg (average with SD), where reduction of weight was due to the removal of serum from the total blood. Blood clot weight in microcentrifuge tubes (in μg) A, B, and C after reducing serum for Streptokinase (+ve control), aqueous plant extract, and Non-thrombolytic (−ve control) of 100 μl, showed the result in percentage of blood clot lysis in descending order, that is, 71, 65.5 and 0.86, respectively. Here, it was noticed that the percentage of clot lysis of Streptokinase and plant extract was not so different, which indicated that the plant extract to be used in the experiment had good thrombolytic activity and its availability and economic factor were also comfortable. As the size, shape, and weight of the microcentrifuge tube was also similar, the concentration of the plant extract and the environmental condition of the incubator were the same, therefore, the percentage of blood clot lysis of the other 11 human volunteers was very close in the in-vitro test, as the inclusion criteria was the same for all 11 human volunteers, such as, no volunteer had taken the anti-coagulant or other drugs prior to four days of test, all volunteers were healthy, no volunteers had donated blood three months prior to the test, and no volunteers in the test had any form of addiction.
Going through [Figure 2], it was observed that the fourth volunteer showed a great diversion, that is, 77.9 % from the mean line, which was 71.76% in the graph plotted for Streptokinase. On the other hand the third volunteer showed a little diversion, that is, 59.9% from the mean line, which was 65.5% in the graph plotted for the aqueous plant extract.
As mentioned in [Table 3], which depicts the experiment done in vivo on 20 selected mices with intraperitoneal injection (IP) of plain water (as control), plant extract, fish extract, and their combination in tied mices. And the observation was noted after 12 hours, 18 hours, 24 hours intervals of untying the mice.
On going through [Table 3], it was observed that when IP (Intra-peritoneal) injection with plain water (as control) was given to five out of 20 mice within a one-minute interval of untying the thread, after 12 hours and after 18 hours of being in a tied condition, the mice remained in a static position. However, after 24 hours of being in a tied condition all the mice died within 23 minutes. Thus, it showed that the formation of Atherothrombotic condition, myocardial and/or cerebral infraction, and finally an embolic condition arose.
With plant extract IP injection, after 12 hours in a tied condition, four mice were able to run within five minutes and the remaining mices was also able to move freely, which could be due to some other unknown reasons . However, all mice were able to move freely after an IP injection after 18 hours in a tied condition. After IP injection after 24 hours in a tied condition all the mice were in condition to move slightly. Thus, in 24 hours of tied condition the clotting density might increase in the vascular tissue and also the heart did not have any strengthening capability to give pressure of flow to the clotted areas.
With fish extract IP injection after 12 hours in a tied condition, all the five mice could move slightly, but after 18 hours in a tied condition, four mice died within 22 minutes and one could move slightly. However, after 24 hours the remaining mice also died within 5 minutes of survival . Thus, it could be due to cardiotonic activity where the heart contracted and worked hard, but the vascular zone is already blocked with clots, and therefore it had no action. Due to rapid forceful contractions of the heart few vessels opened, but still they remained blocked in many vascular positions, which could lead to high tension toward the vascular walls.
However, the combination of fish extract (Cardiotonic activity) and plant extract (Thrombolytic activity) is seen to work much better than the plant extract alone from the reported in vitro study of Prasad S et al.  Thus it works synergistically in a combination therapy, in mice. The plant extract (Thrombolytic activity) opens the vessels and the fish extract (Cardiotonic activity) opens the loosely clotted areas as well as maintains a constant blood flow, keeping the uniform dissolution of declotted blood in the occluded vessels.
Due to the combinatory synergistic effects of Fagonia Arabica and the controlled calibrated dose of Heteropneustes fossilis, the side effect is reported to be much less than that with the synthetic drug combination, and it is also economical, with the best pharmacological activity.
As this study is basically animal-oriented, it can also be carried out in human as an in-vivo study, which has not been done yet, due to failure to get the permission from the ethical committee, within this short period of time. It is one of the limitations which can be fulfilled in future.
| Conclusion|| |
As the plant extract of Fagonia Arabica serves as a thrombolytic activity and the fish extract of Heteropneutesis fossilis serves as a cardiotonic activity, both act synergistically in the mice with blood clots, and mice suffering from myocardial, cerebral infarction, are comparatively better with Fagonia Arabica alone.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]