Phytochemical investigation and In vitro Thrombolytic activity of Terminalia pallida Brandis leaves
Sarvan Kumar Guguloth1,2, Narender Malothu1*, Umasankar Kulandaivelu1,
Koteswar Rao GSN1, Anka Rao Areti1, Sadhana Noothi3
1,2KL College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur, AP.
2Department of Pharmacology, Vijaya College of Pharmacy, Munaganoor, Hayathnagar, Ranga Reddy - 501511.
3B. V. Raju Institute of Technology, Narasapuram, Medak.
*Corresponding Author E-mail: narendermalothu@gmail.com
ABSTRACT:
In the present work, the preliminary phytochemical study and in vitro thrombolytic activity was conducted for Terminalia pallida Brandis leaves extract. In vitro thrombolytic was evaluated by clot lysis effect of different solvent extracts of Terminalia pallida Brandis leaves. In this study, streptokinase and distilled water were used as positive control and negative control, respectively. In vitro thrombolytic model of methanolic extract showed maximum clot lysis (95.43%±0.697) at test concentration (800µg/ml) in 72 hrs. Hydro alcoholic extract also showed potential clot lysis (90.43%±0.748) at similar test conditions. In addition, other plant extracts such as petroleum ether, ethyl acetate, chloroform extract showed moderate activity at 800 µg/ml concentration i.e., 65.56%±0.984, 70.16%±0.325 and 72.98%±0.985 clot lysis, respectively in 72 hrs. Streptokinase (SK) and water showed clot lysis maximum of 97.26%±0.974 and 41.33%±0.843 respectively. The present study reveals that the methanolic and hydro alcoholic extracts of Terminalia pallida Brandis leaves possesses potential thrombolytic activity.
KEYWORDS: Terminalia pallida Brandis leaves, Thrombolytic, Clot lysis, Streptokinase.
INTRODUCTION:
The herbal agents may serve as a major source for medicine for their use in identification, prevention, cure and treatment of various diseases in future5.
Development of blood clots in the body may cause serious consequence and leads to mortality and morbidity, Worldwide6. Agglomeration of blood develops through a cascade of consecutive or serial events; in this process formation of thrombin is the last step, where soluble fibrinogen covert into insoluble fibrin7. Homeostasis regulates the functions of blood circulatory system after injurious or rupturing of the vesseles8. In general, formation of blood clot develops in the circulatory system due to failure of homeostasis process, which may cause vascular blockage and while recovering leads to serious fatal consequences in athero thrombotic diseases such as myocardial infarction, cerebral infarction, at times leading to death9. Natural thrombolytic agents such as tissue plasminogen activator, urokinase, streptokinase etc., are being using to cure vascular diseases10. Although there was several synthetic thrombolytic agents are available for commercial use, they have certain limitations such that developing severe and occasionally fatal adverse drug reactions like hemorrhage, severe anaphylactic reactions11. Thrombus was developed in the blood vessels because of imbalance or lack of homeostasis function leads to blockage in blood vessels and serious effects such as cerebral and myocardial infarction, results in death12. The first step of formation of thrombus in arterial system due to injury of blood vessels, adhesion of platelets to the vascular endothelial membrane and aggregation is the main cause of these vascular diseases13. To minimize the thrombosis associated disorders, low cost thrombolytic drugs are urgently required that do not show any adverse drug reactions like antigenicity and intracranial bleeding14. Various research studies have been in progress in order to find out the medicinal herbs and natural resources with effective anti-thrombotic activity15. Plant based herbal drug formulations, if consumed in therapeutic dose; it can lead to curing various medical ailments. It shows that to establish new therapeutic remedies for various clot related disorders, medicinal plants can be a good source16. In recent study reveals that herbal agents potentially replaces currently using drugs with more efficacious and safer.
Terminalia pallida Brandis is plant of Combretacea family, commonly found in tropical and subtropical countries. It is called with different local names in southern region of India (i.e., Tellakaraka and Velmakaraka in Telugu; Vallaikkadukkay in Tamil and white gallnut tree in English (Latheef et al., 2008)17. Various parts of the plant has medicinally useful phytoconstituents i.e., fruits has antidiabetic properties (Anonymus, 1976)18 and its leaves are being used in various herbal drugs formulations, pharmaceuticals and animal husbandry (Johansen, 1940)19. Powdered form of fruits concoction is being used to treat the hemorrhoids (Savithramma, 2011)20. In the view of wide variety of medicinal importance of target plant the current study aimed to investigate the preliminary screening for phytochemical and assessment of in vitro thrombolytic activity of leaf extracts. The researchers should pay attention that the tribal people of India use many herbal plant extracts to treat various kinds of disorders and the present study was carried out in an experimental model to substantiate the folklore claim21.
MATERIALS AND METHODS:
Collection of plant leaves:
As this plant is widely distributed in southern region of India, the leaves were collected from Tirumala hills (Chittoor, Andhra Pradesh, India) and authentication of plant was done by Dr. K. Madhava Shetty, Taxonomist (Sri Venkateswara University, Tirupati, Andhra Pradesh, India). A copy of sample (0821) was preserved for future reference. All the solvents and chemicals were utilized in this work were procured from SD Fine Chemicals, Mumbai and Merck India, Mumbai.
Preparation of extracts:
An appropriate amount of plant extracts were used for investigation of phytochemical screening22. The leaves were dried at room temperature (in air) and dried leaves were powdered, weighed (250gm) for solvent extraction. The powdered plant material was macerated (for 24-72 hrs) and subsequently extracted with 1 liter of different solvents like petroleum ether, chloroform, ethyl acetate, methanol and hydro alcohol. Resulted leaf extracts were concentrated using a rotary evaporator (in vacuo) and were stored in cold conditions for further investigations.
Preliminary phytochemical investigation:
Different solvents macerates were employed for phytochemical investigation to assess the occurrence of various phytochemical constituents in each solvent extracts. Various qualitative protocols were employed to investigate different phyto-chemicals such as alkaloids, carbohydrates, glycosides, flavonoids, tannins, saponins and steroids (Harborne, 1998)23.
Preparation of standard drug streptokinase (SK):
Lyophilized streptokinase (marketed sample) vial of 1500000 IUs was obtained for the study and to this 5ml of aseptic pure water was added and mixed thoroughly. The resulting solution was used as a reference standard and about withdrawn 100μl (30,000 IU) for in vitro thrombolytic assay24,25.
Collection of blood sample:
Approximately 4ml of whole blood withdrawn from Wistar albino rat and the animal should be free from diseased condition (Institutional Animal Ethical Committee Certificate 1292/ac/09/CPCSEA/47/A). 500 µl of blood was poured to all the 10 pre measured weight of alpine tubes to form clots.
Bio assay:
Clot lysis activity of crude leaf extract:
Initially about 500µl of blood was transferred in previously weight measured sterile micro centrifuge tubes and incubated at 37ºC for 90 min, to acquire blood clot formation. After centrifugation, the serum was collected without distributing the blood clot and measures the weight of tubes to find accurate weight of blood clot26.
(Measurement of clot weight = Weight of tube containing clot - Weight of empty tube)
Each sterile micro centrifuge tube consisting of blood clot was labeled properly and 100µl of leaf crude extract was added to the tubes. 100µl of distilled water were separately added to the numbered control tubes as a negative non thrombolytic control and as a standard positive control, 100µl of streptokinase added to tubes and all the micro centrifuged tubes were then incubated at 37ºC for 90 min and observed for clot lysis. After incubation, obtained serum 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 denoted as percentage of clot lysis27.
Weight of released clot
% of Clot lysis = ----------------------------X 100
Clot weight
Blood clot lysis study:
Different concentrations ranges (200, 400, 600 and 800 µg/ml) of crude leaf extract of Terminalia pallida Brandis were performed at different incubation time intervals which includes (24, 48 and 72 hrs) at 37ºC to assess the maximum clot lysis28.
RESULTS AND DISCUSSIONS:
Preliminary phytochemical screening:
The leaf extraction of Terminalia pallida Brandis was performed in petroleum ether (0.54% w/w), ethyl acetate (2.6% w/w), chloroform (2.6% w/w), methanol (2.3% w/w) and hydro alcoholic (1.06% w/w). In the preliminary phytochemical investigation various qualitative tests were conducted for all the leaf extracts in order to assess the presence of different phytoconstituents. In these studies extracts showed the presence of alkaloids, flavonoids, saponins, tannins, proteins, steroids and glycosides, whereas for carbohydrates and mucilage tests it showed negative results (Table 1).
Table 1: Preliminary phytochemical screening of Terminalia pallida Brandis leaves
Phytochemical test |
Petroleum ether |
Ethyl acetate |
Chloroform |
Methanol |
Hydro alcohol |
Alkaloids |
+ |
+ |
+ |
- |
+ |
Flavonoids |
- |
- |
- |
+ |
+ |
Saponins |
- |
+ |
- |
+ |
+ |
Tanins |
- |
+ |
- |
+ |
+ |
Steriods |
+ |
+ |
- |
- |
- |
Glycosides |
+ |
- |
+ |
- |
- |
Carbohydrates |
- |
- |
- |
- |
- |
Proteins |
- |
+ |
+ |
+ |
+ |
Aminoacids |
- |
+ |
+ |
+ |
+ |
Mucilage |
- |
- |
- |
- |
- |
Note: ‘+’ indicates presence of compounds, ‘-’ indicates absence of compounds.
In vitro thrombolytic assay:
In the current research study, an in vitro thrombolytic model of methanolic extract showed maximum of 95.43%±0.697 clot lysis (at 800µg/ml) in 72 hrs. While hydro alcoholic extract also showed potential thrombolytic activity 90.43%±0.748 (at 800µg/ml concentration in 72hrs of incubation). Whereas, petroleum ether, ethyl acetate, chloroform extract showed moderate activity at 800µg/ml concentration i.e., 65.56%±0.984, 70.16%±0.325 and 72.98%±0.985 clot lysis, respectively in 72 hrs of incubation. It was found that the methanol, hydro alcoholic and chloroform extracts of Terminalia pallida Brandis leaves possesses potential thrombolytic activity (Table 2). In this study, streptokinase (SK) and water showed clot lysis maximum 97.26%±0.974 and 41.33%±0.843 respectively (Table 3). It was found that the methanol, hydroalcoholic and chloroform extracts of Terminalia pallida Brandis leaves possesses potential thrombolytic activity (Figure 1).
Table 2: In vitro thrombolytic activity of different extracts of Terminalia pallida Brandis leaves
Conc. of crude leaf extract (µg/ml) |
Incubation time (hrs) |
Clot lysis in percentage, Mean ± SEM |
||||
Pet. ether extract |
Ethyl acetate extract |
Chloroform extract |
Methanol extract |
Hydro alcoholic extract |
||
200 |
24 |
6.36%±0.651 |
7.63%±0.547 |
9.36%±0.685 |
10.78%±0.784 |
8.32%±0.587 |
48 |
22.45%±0.234 |
24.54%±0658 |
26.64%±0.541 |
29.43%±0.478 |
24.43%±0.584 |
|
72 |
38.46%±0.453 |
39.27%±0.247 |
41.28%±0.257 |
48.23%±0.568 |
43.23%±0.587 |
|
400 |
24 |
9.39%±0.654 |
10.23%±0.581 |
11.32%±0.265 |
17.55%±0.569 |
13.55%±0.658 |
48 |
24.89%±0.871 |
29.42%±0.256 |
31.24%±0.236 |
38.28%±0.258 |
30.28%±0.258 |
|
72 |
42.77%±0.658 |
43.86%±0.214 |
46.67%±0.214 |
62.56%±0.958 |
46.56%±0.365 |
|
600 |
24 |
12.23%±0.547 |
13.23%±0.561 |
13.23%±0.654 |
21.54%±0.687 |
16.54%±0.958 |
48 |
27.88%±0.659 |
32.39%±0.514 |
36.90%±0.589 |
58.28%±0.874 |
43.28%±0.947 |
|
72 |
51.12%±0.589 |
48.68%±0.214 |
49.86%±0.647 |
80.97%±0.658 |
68.97%±0.654 |
|
800 |
24 |
15.36%±0.845 |
16.66%±0.254 |
16.78%±0.478 |
22.65%±0.478 |
22.65%±0.417 |
48 |
32.58%±0.547 |
39.81%±0.258 |
41.18%±0.852 |
66.96%±0.987 |
58.96%±0.587 |
|
72 |
65.56%±0.984 |
70.16%±0.325 |
72.98%±0.985 |
95.43%±0.697 |
90.43%±0.748 |
Note: Clot lysis is expressed in percentage, Mean±SEM; Sample volume = 3 (n=3), SEM= Standard error mean
Table 3: In vitro thrombolytic activity of water control and standard
S. No. |
Incubation time (hrs) |
Clot lysis (%) |
Standard |
24 |
78.2±0.947 |
48 |
92.01±0.647 |
|
72 |
98.2±0.974 |
|
Control |
24 |
8.86±0.541 |
48 |
27.67±0.752 |
|
72 |
41.33±0.843 |
Note: Control = Water; Standard = Streptokinase
Figure 1: Clot lysis data of crude leaf extract (800µg/ml) at 72 hrs incubation time
CONCLUSION:
In the present study leaf extraction of Terminalia pallida Brandis were done successfully by using different solvents and performed the preliminary phytochemical investigation. The extracts were shown the presence of alkaloids, flavonoids, saponins, tannins, proteins, steroids and glycosides. In vitro studies revealed that the methanolic (95.43%±0.697) and hydro alcoholic (90.43%±0.748) leaf extracts were exhibited potential thrombolytic properties (at 800µg/ml) in 72 hrs. In vivo clot dissolving properties and active components responsible for thrombolytic activity of Terminalia pallida Brandis leaves are yet to be discovered.
CONFLIT OF INTREST:
The authors declare that there is no conflict of interest.
ACKNOWLEDGEMENTS:
The authors are thankful to Vijaya College of Pharmacy, Munaganoor, Hyathnagar, Ranga Reddy-501511, and KL College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur, Andhra Pradesh for providing laboratory facilities to carry out this research study.
REFERENCES:
1. Rajeswari S, Vidhya R. Evaluation of In vitro thrombolytic and antiproteinase activities of Wedelia trilobata (Linn.). Innov J Life Sci. 2017; 5(3): 6-9.
2. Umesh MK, Sanjeevkumar CB, Hanumantappa BN, Ramesh LL. Evaluation of in vitro anti-thrombolytic activity and cytotoxicity potential of Typha Angustifolia leaves extracts. Int J Pharm Pharm Sci. 2014; 6(5): 81-85.
3. Kadam SD, Sreedhar C, Chandrasekhar KB. Anti-inflammatory effects of roots of Terminalia pallida and Boswellia ovalifoliolata. World J Pharm Pharm Sci. 2015; 4(2), 508-515.
4. Baliah NT, Astalakshmi A. Phytochemical analysis and antibacterial activity of extracts from Terminalia chebula Retz. Int J Curr Microbiol App Sci. 2014; 3(3): 992-999.
5. Sofowora A. Medicinal plants and traditional medicine in Africa Ibadan. Spectrum Books Limited. 1993; 191-289.
6. Aashwini A. Gholkar, YP. Nikam, KK. Zambare, KV. Reddy, Akash DG. Potential Anticoagulant Herbal Plants: A Review. Asian J Res Pharm Sci. 2020; 10(1):51-55.
7. Kishore K. In-vitro and in-vivo screening methods for antithrombotic agents. Am J Phytomed Clin Ther. 2013; 1(5): 497-506.
9. Fatima T, Chadni SH,Mou KN, Imrul Hasif KM, Ahmed T, Akter, M. In vitro thrombolytic activity and phytochemical evaluation of leaf extracts of four medicinal plants of Asteraceae family. J Pharmacogn Phytochem. 2017; 6(4): 1166-1169.
10. Vaseem AA, Siddiqui HH, Singh SS. Anti thrombotic and thrombolytic activity of Terminalia belerica fruit extracts. Res J Pharm Biol Chem Sci. 2012; 3(2): 471-478.
11. Ali R MD. Preliminary phytochemical screening and in vitro thrombolytic potential of the methanolic extract of Enhydra fluctuans Lour leaves. Int J Phar. Medix India. 2013; 1 (2): 270-280.
12. Rajapakse N, Jung WK, Mendis E, Moon SH, Kim SK. A novel anticoagulant purified from fish protein hydrolysate inhibits factor XIIa and plate aggregation. Life Sci. 2005; 76: 2607-2619.
13. Jorgensen L. The role of platelets in the initial stages of atherosclerosis. J Thrombo Haemost. 2006; (7): 1443-1449.
14. Simkhada JR, Cho SS, Mander P, Choi YH, Yoo JC. Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model. Thrombosis Res. 2012; 129(2): 176-182.
15. Ullas PD, Joshi H, Alsheena N. Anti-anxiety effect of Aerva lanata (L.) using mice model. Research J Pharm Tech. 2020; 13(2):565-568.
16. Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM, Daginawala HF. Effect of Fagonia arabica (Dhamasa) on in vitro thrombolysis. BMC Complement Altern Med. 2007; 7(36): 1-6.
17. Latheef SK. A data on endemic plants at tirumala hills in India, Bio-Information. 2008; 2(6): 260-2.
18. Anonymous, The wealth of India, Raw materials, X, S-W, publication and information. Directorate CSIR, New Delhi. 1976.
19. Johansen DA. Plant micro-technique. McGraw Hill Book Co., New York. 1940; 183.
20. Savithramma N. Studies of Boswellia ovalifoliolata and henry -an endemic and endangered medicinal plant. The Bioscan. 2010; 5(3): 59-62.
21. Mahajan KG, Tamilvanan S, Sawarkar HS, Thenge RR, Adhao VS, Gangane PS. Preliminary Phytochemical and Anti-pyretic Screening of Crude extract of the leaf of Clerodendrum colebrookianum. Research J Pharmacog Phytochem. 2009; 1(3): 191-193.
22. Bembde NS, Meshram PV, Patil MKJS. The Preliminary Phytochemical Analysis of Ethanolic extract of Tylophora indica. Res J Pharmacog Phytochem. 2020; 12(1): 01-06.
23. Harborne, JB. Phytochemical method. A guide to modern techniques of plant analysis. J B Harborne.1998; 2: 1-32.
24. Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM, Daginawala HF. Effect of Fagonia arabica (Dhamasa) on in vitro thrombolysis, BMC Complement Altern Med. 2007; 7(36): 1-6.
25. 25.Daginawala HF, Prasad S, Kashyap RS, Deopujari JY, Purohit HJ and Taori GM. Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thromb J. 2006; 4(14): 1-4
26. Mallik J, Banik RK. In vitro studies on antimicrobial and thrombolytic activity of Swietenia macrophylla King. J Pharm Res. 2012; 2(5): 45 – 48
27. Shahriar.M. Phytochemical screenings and thrombolytic activity of the leaf extracts of Adhatoda vasic. The Experiment. 2013; 7(4): 438-441.
28. Elumalai A, Eswariah CM, Chowdary V, Kumar R, Anusha M, Naresh K, Screening of thrombolytic activity of Bougainvillea glabra leaves extract by in-vitro. Asian J Pharm Sci. 2012; 2(4): 134-136.
Received on 31.03.2020 Modified on 21.04.2020
Accepted on 28.05.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(2):879-882.
DOI: 10.5958/0974-360X.2021.00156.6