INTRODUCTION:

Plants produce a diverse range of secondary metabolites that have recently gained renewed interest for their antimicrobial and antioxidant properties. These may be produced constitutively or in response to external stress.

These compounds are advantageous because of their structural diversity and unique bioactivity which are more favourable than synthetic chemicals. Due to diversity in chemical nature of natural compounds from plant extracts and have opportunities to use as new drugs for treatment of diseases¹. Discovering of novel drugs having biological activities is an continuous emerging need against infectious diseases². However, most of the microorganisms are developing resistances against certain drugs and identification of new agents with antioxidant and antimicrobial activities is one of the challenging in recent years³. Although some of the synthetic drugs are available but plant derived drugs are to be considered as safe without or less side effects when compared to synthetic ones^4,5. Many plants are rich sources of these antimicrobial agents and they are of secondary metabolites^6-9. As infectious agents are getting resistance exploitation of natural sources and usage of plant extracted bioactive compounds is highly interesting in current years.

Cosmos bipinnatus, is famously known as the garden cosmos of Asteraceae family. It is an herbaceous, flowering and half-hardy yearly medium-sized medicinal plant. C. bipinnatus is self-sowing¹⁰. The plant ranges from 0.6 to 1.8 m in height with an open and sprawling habit¹¹. It has varietal colours in full sun and flowering with partial shade. It is used to against stomach disorders pain and headaches and also against bed bugs and lice indicating its insecticidal property. Apart from ecology and morphological descriptions on this plant species, there is no scientific information to validate its medicinal potentials¹². Wild populations of this plant rarely persist. It is having pharmacological activity and used as anti-inflammatory, jaundice, splenomegaly, antioxidant and also used as antimicrobial agents¹³^.Only very few studies were reported on C. bipinnatus. Thus, the purpose of the study was to find qualitative phytochemical screening to ascertain the presence of bioactive metabolites such as tannins, alkaloids, triterpenoids, saponins and steroids and to estimate the total phenolic and flavonoid content of ethanol, hexane and ethyl acetate solvents extract of different parts root, leaf, stem, and flower to explore the underlying curative potentials of the plant in the form of antioxidant, antibacterial, antifungal activities.

MATERIALS AND METHODS:

Materials:

Chemical reagent DPPH was purchased from Sigma (Sigma-Aldrich India). Gallic acid standard solution (Loba Chemie, Mumbai). All other reagents used were of analytical grade.

Collection and identification of the plant:

The plant sample has been collected from Chityal mandal, Warangal district, Telangana state.The plant specimen was identified as Cosmos bipinnatus with the help of Professor Pandal, Department of Botany, Andhra University, Visakhapatnam. The sample was deposited in Botany Department Herbarium (AUV) Andhra University got voucher specimen number 23338. Different parts like root, stem, leaf and flower were separated, and shade dried for a period of 60 days and sample were grinded into the fine powder and stored in glass bottle at 4^°C until further use.

Solvent extraction of different parts of C. bipinnatus:

The dried different parts of C. bipinnatus in the form of powder (1kg) has been taken and subjected to soxhlet solvent extraction process by taking 4L of ethyl alcohol, hexane and ethyl acetate for 24 hours. The crude extracts of C. bipinnatus thus obtained were stored in brown bottles and preserved in a freezer at -20ºC until further use.

Preliminary phytochemical screening of C. bipinnatus:

The preliminary phytochemical screening tests for the presence of carbohydrates, proteins, tannins, phenols, saponins, flavonoids, steroids, glycosides, terpenoids and alkaloids were carried out for the ethanol, hexane and ethyl acetate crude extracts of Cosmos bipinnatus¹⁴.

Quantitative phytochemical analysis:

Determination of total phenolic content of C. bipinnatus:

The total phenolic content of each part of plant extract was determined by the Folin- Ciocalteau reagent method with some modifications¹⁵. All measurements were done in triplicates. The total phenol content was expressed in terms of Gallic acid equivalent (mg/g)¹⁶.

Evaluation of total flavonoid content of C. bipinnatus:

The total flavonoid content of crude extract was determined by the aluminium chloride colorimetric method. 50μL of crude extract were made up to 1mL with methanol, and all other chemicals according to the procedure were added and mixture was allowed to stand for 15 min, and absorbance was measured at 510nm. The total flavonoid content was calculated from a calibration curve, and the result was expressed as mg quercetin equivalent/g dry weight¹⁷.

Evaluation of antioxidant activity of C. bipinnatus

FRAP assay:

Ferric reductive Antioxidant power assay is a quantitative assay for measuring antioxidant potential present in the sample. Different concentrations of the extracts were prepared and results were measured with reference to standard the reducing power of the samples were comparable with the reference standard¹⁸.

DPPH free radical method:

1, 1-Diphenyl-2-picrylhydrazyl (DPPH) is a free radical, in presence of an antioxidant it gets reduced and becomes colorless or pale yellow color in the solution. DPPH is a well-known radical and a scavenger for other radicals hence it can be used to measure the reduction rate of the chemical reaction. Add 50µl of extract to 1.0 ml of 0.1ml of DPPH compound in NaOH solution. Incubate for 30 minutes in dark at 37 degrees centigrade. Measure absorbance at 517nm^19.

Anti-microbial activity minimum inhibitory concentration (MIC) of C. Bipinnatus:

The antibacterial activity was carried out by employing 24h cultures with given compounds by using the Agar-Well diffusion method. The medium was sterilized by autoclaving at 120°C (15 psi) for about 30min. Standard (Ciprofloxacin) with 5μg/ml was used as a positive control. The activity diameter of the zone of inhibition was measured using the HiMedia antibiotic zone scale. The procedure is same for culturing of both Bacteria and Fungi. The difference is medium where Potato Dextrose is used for fungi and temperature for incubation is 28+2^oC²⁰

RESULTS AND DISCUSSION:

Discovering of drugs and screening of medicinal plants for their biological activities is an interesting and helpful to produce new drugs against various diseases for treatment. The dried material of different parts of C. bipinnatus have shown better yield when they were subjected to Soxhlet extraction followed by rota evaporation. The ethyl alcohol extracted leaf has shown yield of (6.7%) compared to the stem (5.3%) and flower (3.8%). The root was shown less yield with the ethyl alcohol. Similarly flower yield is high with ethyl acetate (5.3%) compared to leaf (5.2%) stem (4.7%) and root (1.1%). The hexane extracted leaf has better yield (4.9%) compared to stem (2.8%) and flower (4.2%) and root (1.3%). The results of preliminary phytochemical analysis by the three solvent extracts (ethanol, ethyl acetate and hexane) of different plant parts such as, root, stem, leaf and flower were presented in Table 1.

Table 1: Phytochemical analysis of the different parts of Cosmos bipinnatus in different solvents

Name of the Phyto chemical

Name of the test

Leaf

Stem

Flower

Root

Phenol and alkoloid

Dragendroff’s test

Mayer’s test

Steroid

Salkowski test

+=+

Liebermann buchards

+ =+

Terpenoids

Salkowski test

Liebermann buchards

Tannins

Ferric chloride test

Gelatin test

Flavonoids

Ferric chloride test

Lead acetate test

Saponines

Saponine test

Glycosides

Kellar-killianis test

Bornhaggers test

Carbohydrates

Molisch test

Fehlings test

Proteins

Biuret test

Aminoacids

Ninhydrin Test

Ethyl alcohol; EA: Ethyl acetate; H: Hexane; (-): absent; (+): present

The phytochemicals such as alkaloids, steroids, terpenoids, tannins, flavonoids, saponins, glycosides, carbohydrates, proteins and amino acids were observed in qualitative analysis. From the table, it is clear that carbohydrates, phenols and tannins, flavonoids and saponins were present in some parts of the plant. Flavonoids were abundantly found in the leaves and the root of C. bipinnatus. Similar results have been found in the research paper recently published by Sundaram Sowmya et al. It is stated that flavonoids were abundantly found in the leaf part of the plant Cayratia trifolia when they have conducted qualitative analysis on different parts of this plant²¹. Steroids were absent only in the leaves and flowers of C. bipinnatus while terpenoids were completely absent in the flowers of the plant. Alkaloids were absent in the leaves extracted from all the three solvents and root sample extracted using ethyl acetate and stem sample extracted using ethanol. Proteins were absent in all the parts of the plant extracted using all three solvents namely ethanol, ethyl acetate and hexane. Tannins were absent in the entirely in the flower in addition to the stem sample taken using the solvent hexane. Saponins were present in all the parts like leaf, stem and flower but are not found in the root extract of the plant. The detailed description of the various compounds present in the parts of the plant extracted using three different solvents. The results of total phenolic content of three different solvent extracted plant samples including the leaf, stem, root and flower were represented in the Figure 1.

For the plant samples extracted by using ethanol and hexane as the solvents, the concentration of phenol content was highest for the stem (740.32±0.52µg/mg GAE and 790.08±0.17µg/mgGAE) respectively. For the plant samples extracted by using ethyl acetate as the solvent, the concentration of phenols is highest for the root which was 661.04±1.03µg/mgGAE. Comparatively, in our study the phenolic content was found to be highest for the stem fraction that was extracted by using hexane as the solvent. Similar results have been obtained by Sengul, Memnune, et al., it was reported that the total phenolic content of Crocus sativus was found out to be 422.9µg GAE per mg dry weight basis²². In addition to the total phenolic content the results of total flavonoid content were presented in figure 2.

Figure 1: Total phenolic content of different parts of solvent extracted C. bipinnatus

Figure 2: Total flavonoid content of different parts of solvent extracted C. bipinnatus

For the plant samples extracted by using ethanol as the solvents, the total flavonoid content was found to be highest for the leaf 81µg/mg. Comparatively, the total flavonoid content of the leaves of Meyna spinosa plant was found out to be 58.5µg quercetin equivalents/mg²³. For the plant samples extracted by using ethyl acetate as the solvent, the total flavonoid content was highest for the root which is 178.6µg/mg compared to the flower that is 125µg/mg. In this study the total flavonoid content was found to be highest for the root part extracted using ethyl acetate as the solvent. Polyphenolic compounds such as flavonoids and anthocyanins are of great interest for their radical scavenging activity. Intake of dietary antioxidants that act as radical scavengers is expected to be effective in preventing many diseases. The results of antioxidant activity by using the FRAP method was presented in figure 3.

Figure 3: Ferric acid reducing activity of different parts of solvent extracts of C. bipinnatus

The antioxidant activity of ethanol extracted root sample was highest when compared to ethanol and ethyl acetate fractions of root. The antioxidant activity of various parts of the plant Cosmos bipinnatus was tested using the DPPH assay at different concentrations (20, 60 and 120µg/ml). The percentages of inhibition for various extracts of the plant with different organic solvents were presented in figures 4,5 and 6.

Figure 4: Percentage of Inhibition of DPPH at 20µg/ml of different parts of solvent extracts of C. bipinnatus

Figure 5: Percentage of Inhibition of DPPH at 60µg/ml of different parts of solvent extracts of C. bipinnatus

Figure 6: Percentage of Inhibition of DPPH at 60µg/ml of different parts of solvent extracts of C. bipinnatus

At a concentration of 20µg/ml, the percentage of inhibition with respect to the solvent ethanol was lowest for the flower 52.87% compared to the percentage of inhibition with respect to the solvent ethyl acetate 59.09% and hexane was highest for the leaf 62.3%^24,25,26. Taking all the solvents into consideration, the percentage of inhibition was highest for the leaf extracted using ethyl acetate. Similarly, at the concentration 60µg/ml, the percentage of inhibition with respect to the ethanol extract of flower was highest 91.42% compared to ethyl acetate of the root extract 92.16%. The percentage of inhibition with respect to the leaf in hexane extract was highest for compared to root 91.13% (Figure 4). The results of percentage of inhibition with respect to 120µg/ml of DPPH were shown in figure 5. The ethanol extracted root fraction has shown highest 91% compared to the percentage of inhibition with respect to the solvent ethyl acetate of root 87.7%. The percentage of inhibition with respect hexane fraction of root was highest 89% compared to ethyl acetate. Whereas the percentage of inhibition was highest for the flower extract of ethanol fraction compared to other solvent extracts. Similarly, in a study conducted by Ikhram ilahi et al. the percenat scavenging of stable DPPH free radical at a concentration of 60mg was found out to be 52.83% for the plant Withania somnifera²⁷. The anti-microbial activity of all the extracts was determined by using both bacterial and fungal strains. The zone of inhibition was found and the results were presented in table 2. The fungal strains like Aspergillus niger and Trichophyton mentagrophytes were used^28,29. The bacterial strains Staphylococcus aureus used as gram positive and dermatophyte Trichophyton mentagrophytes was also used. The zone of inhibition is calculated with the help of high media scale.

The antimicrobials from natural sources have drawn attention of many researchers to put an effort in order to identify novel compounds which can effectively act as suitable antimicrobials so as to replace the synthetic drugs. The phytochemicals derived from plant origin can serve as an effective prototype to develop less toxic and more effective drugs to control the growth of microorganisms^30,31,32. Such compounds have significant therapeutic applications to combat the major human pathogens like bacteria, fungi and viruses. Several studies have been carried out with the extracts of various plant parts to identify the antimicrobial property as well as to discover new antimicrobials. The results of the current study states the therapeutic values of the C. Bipinnatus plant parts used in traditional medicine³³. The antimicrobial activity reported in the current investigation is a scientific evidence for the traditional therapies and contains ample number of active compounds that can inhibit the growth of microorganisms. The presence of secondary metabolites in the extracts of different plant parts of C. Bipinnatus confirms the utilization of this medicinal herb in traditional medicine^34,35.

Table 2: Antimicrobial activity of different parts of solvent extracts of C. Bipinnatus against various microorganisms

Name of the organism	Zone of inhibition mm (Mean± SD)
Name of the organism	Solvent	Leaf	Stem	Flower	Root
Aspergillus niger MTCC 961	Ethanol	3± 0.7	21± 0.8	10 ± 0.3	14± 0.5
	Ethyl acetate	16± 0.1	20± 0.4	5± 0.6	4± 0.8
	Hexane	11± 0.3	24± 0.6	19± 0.2	7± 0.3
Staphylococcus aureus MTCC 3160	Ethanol	3± 0.2	3± 0.1	2± 0.2	5± 0.4
	Ethyl acetate	8± 0.3	17± 0.2	8± 0.1	7± 0.2
	Hexane	10± 0.4	22± 0.1	16± 0.2	6 ± 0.3
Klebsiella pneumonia MTCC 9024	Ethanol	10± 0.3	18± 0.2	15± 0.2	12± 0.1
	Ethyl acetate	12± 0.4	14± 0.1	12± 0.5	7 ± 0.1
	Hexane	8± 0.5	12± 0.4	8± 0.4	6± 0.2
Trichophyton mentagrophytes MTCC 7687	Ethanol	14 ± 0.7	22± 0.1	9± 0.6	16± 0.3
	Ethyl acetate	13± 0.1	18± 0.3	21± 0.4	26± 0.2
	Hexane	10± 0.1	19± 0.1	17± 0.5	9± 0.1

CONCLUSION:

The phytochemical analysis of all the plant parts extracted using various organic solvents was done and the results were tabulated. The phytochemical study has shown the presence of several secondary metabolites like carbohydrates, phenols and tannins, flavonoids and saponins were present in some parts of the plant. Flavonoids were abundantly found in the leaves and the root of C. bipinnatus. The total phenolic and flavanoid content was observed in many parts of the plants. The antioxidant activity and antimicrobial activities of different parts of Cosmos bipinnatus in different solvents have shown good antioxidant and antimicrobial activities. The antimicrobial activity of extracts obtained from different parts of C. bipinnatus explains the reason behind the traditional use. Further separation of phyto chemical fractions for identification of biological active molecules can help to find solution against various diseases.

ACKNOWLEDGEMENT:

The authors also thankful to the Management of GITAM (Deemed to be University) for their constant support. The authors are thankful to Prof. Pandal, Dr. Prakash Rao Department of Botany Andhra University for identification of the plant.

CONFLICT OF INTREST:

Authors declared no conflict of interest

REFERENCES:

1. Ujjwal N. Ruchi S. Rashmi KM. Raju KC. Lipid Content and in vitro Antimicrobial Activity of Oil Seeds of Some Indian Medicinal Plants. Current Research in Bacteriology. 2008; 1 (1):1–6. doi.10.3923/crb.2008.1.6

2. Parekh J. Chanda SV. In vitro antimicrobial activity and phytochemical analysis of some Indian medicinal plants. Turkish Journal of Biology. 2007; 31:53-58.http://dergipark.ord.tr

3. Teke GN. Lunga PK. Wabo HK. Kuiate JR. Vilarem G. Giacinti G, et al. Antimicrobial and antioxidant properties of methanol extract, fractions and compounds from the stem bark of Entada abyssinica Stend ex A. Satabie. BMC Complementary Medicine and therapies. 2011;11(1):57. https://doi.org/10.1186/1472-6882-11-57

4. Rai MK. Deepak A. Wadegaonkar P. (2003). Plant derived-antimycotics: Potential of Asteraceous plants, In: Plant-derived antimycotics:Current Trends and Future prospects, Haworth press, N-York, Londin, Oxford, pp 165-185.

5. Iniaghe OM, Malomo SO. Adebayo JO. Proximate composition and phytochemical constituents of leaves of some Acalypha species. Pakistan Journal of Nutrition, 2009:8(3):256-258.doi:10.3923/pjn.2009.256.258

6. Lee HS. Growth-Inhibitory Effects of Various Medicinal Plants against Lactic Bacteria and Harmful Intestinal Bacteria. Food Sci Biotechnol. 2000;9(1):52–6.

7. Hill AF. A textbook of useful plants and plant products, Economic Botany. 2nd ed. New York: Mc Graw-Hill Book company lnc; 1952.

8. Block G. The Data Support a Role for Antioxidants in Reducing Cancer Risk. Nutrition Reviews 2009 Apr 27;50(7):207–13.doi/10.1111/j.1753-4887.1992.tb01329.x

9. Diplock A. Antioxidant nutrients-efficiency in disease prevention and safety. Biochemistry. 1995;34(17):16–8.

10. Jang S. Marchal V. Panigrahi KCS. Wenkel S. Soppe W. Deng XW et al Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response. EMBO J. 2008; 27(8):1277–88. DOI: 10.1038/emboj.2008.68

11. Akihisa T. Yasukawa K.Oinuma H. Kasahara Y. Yamanouchi S. Takido M. et al Triterpene alcohols from the flowers of compositae and their anti-inflammatory effects. Phytochemistry. 1996 ;43(6):1255–60. doi: 10.1016/s0031-9422(96)00343-3.

12. Dubey S. Kumar Singh V. Population Dynamics of Aphis Spiraecola Patch (Homoptera: Aphididae) on Medicinal Plant Cosmos Bipinnatus in Eastern Uttar Pradesh, India. Advances in Life Sciences. 2012; 1(2):54–58. doi: 10.5923/j.als.20110102.10

13. Botsaris AS. Plants used traditionally to treat malaria in Brazil: the archives of Flora Medicinal. Journal of Ethnobiology Ethnomedicine. 2007;3(1):18. https://doi.org/10.1186/1746-4269-3-18

14. Trease GE EW. Pharmacognosy. 15th ed. London: Saunders Publishers; 2002. 42–44. 221–229, 246–249, 304–306, 331–332, 391–393 p.

15. McDonald S. Prenzler PD. Antolovich M. Robards K. Phenolic content and antioxidant activity of olive extracts. Food Chemistry. 2001;73(1):73–84. https://doi.org/10.1016/S0308-8146(00)00288-0

16. Sumitra C. Raj D. In vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties: An overview. African Journal Microbiology Research. 2009; 3(13):981-996.

17. Ibrahim G. Abdulmumin S. Musa KY YA. Anticonvulsant activities of crude flavonoid fraction of the stem bark of Ficus sycomorus (Moraceae). Journal of Pharmacology and Toxicology. 2008;3(5):351-356. doi: 10.3923/jpt.2008.351.356

18. Benzie IFF. Strain JJ. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Anal Biochem .1996;239(1):70–6.doi:10.1006/abio.1996.0292

19. Blois MS. Antioxidant Determinations by the Use of a Stable Free Radical. Nature. 1958;181(4617):1199–1200. https://doi.org/10.1038/1811199a0

20. Doern G V. Brown SD. Antimicrobial susceptibility among community-acquired respiratory tract pathogens in the USA: Data from PROTEKT US 2000-01. Journal of Infection. 2004;48(1):56-65. doi: 10.1016/s0163-4453(03)00123-3.

21. Sowmya S. Perumal PC. Anusooriya P. Vidya B. Pratibha P.Malarvizhi D. et al Comparative preliminary phytochemical analysis various different parts (stem, leaf and fruit) of Cayratia trifolia (L.). Indo American Journal of Pharm Research.2015;5(01). 218-223.

22. Sengul M. Yildiz H. Gungor N. Cetin B. Eser Z. Ercisli S. Total phenolic content, antioxidant and antimicrobial activities of some medicinal plants. Pakistan Journal of Pharmaceutical Sciences. 2009 ;22(1):102–106. PMID:19168430

23. Sen S. De B. Devanna N. Chakraborty R. Total phenolic, total flavonoid content, and antioxidant capacity of the leaves of Meyna spinosa Roxb., an Indian medicinal plant. Chinese Journal of Natural Medicine. 2013;11(2):149-157. doi: 10.1016/S1875-5364(13)60042-4.

24. Rangasamy P. Hansiya V. Maheswari P. Thamburaj S. Geetha N. Phytochemical Analysis and Evaluation of In vitro Antioxidant and Anti-urolithiatic Potential of various fractions of Clitoria ternatea L. Blue Flowered Leaves. Asian Journal of . Pharmeutical Analysis. 2019; 9(2):67-76. doi- 10.5958/2231-5675.2019.00014.0.

25. Hemalatha M. Arirudran B. Thenmozhi A. Mahadeva Rao US. Antimicrobial Effect of Separate Extract of Acetone, Ethyl Acetate, Methanol and Aqueous from Leaf of Milkweed (Calotropis gigantea L.). Asian Journal of Pharmaceutical Research. 2011; 1(4):102-107.

26. Potbhare M. Khobragade D. In Vitro Evaluation of Antioxidant Potential of Ayurvedic Preparations Lauha Bhasma and Mandura Bhasma. Asian Journal of Pharmaceutical Research 2017; 7(2):63-66. doi: 10.5958/2231-5691.2017.00011.9.

27. Ilahi I. Samar S. Khan I AI. In vitro antioxidant activities of four medicinal plants on the basis of DPPH free radical scavenging. Pakistan Journal of Pharmaceutical Sciences. 2013;26(5):949–952.PMID:24035951

28. Valli G. Jeyalakshmi M. Preliminary Phytochemical and Antioxidant Study of Odina woodier Leaf Extract. Asian Journal of Pharmeutical Research. 2012; 2(4): 153-155.

29. Shefali S. Rupali C. Rajan R. Nitin S. Anuradha S. Kamal D. Vikas K. Effect of solvent on yield, phytochemicals and in vitro antioxidant potential of Rhododendron arboreum. Research Journal of Pharmacy and Technology. 2021; 14(1):311-316. doi: 10.5958/0974360X.2021.00057.3

30. Ahmad I, Beg AZ. Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens. Journal of ethnopharmacology. 2001 Feb 1;74(2):113-23. doi: 10.1016/s0378-8741(00)00335-4.

31. Suhas SA. Santosh KS. Kiran AW. In vitro Antioxidant potential and Anticancer activity of Ceratophyllum demersum Linn. extracts on HT-29 human colon cancer cell line. Research Journal of Pharmacy and Technology. 2021; 14(1):28-36. doi: 10.5958/0974-360X.2021.00006.8

32. Latifa Nasser AA.Syed Atheruddin Q. Evaluation of Antibacterial and Antioxidant activities of Tribulus terrestris L. Fruits. Research Journal of Pharmacy and Technology. 2021; 14(1):331-336. doi: 10.5958/0974-360X.2021.00061.5

33. Shaheda N. Bhagya Lakshmi L. In-vitro Analysis of Phytochemical, Anti-oxidant capacity of seed Ethanolic extracts of Sapindus saponaria Vahl and Anti-bacterial activity on Common Dental Pathogens. Research Journal of Pharmacy and Technology. 2021; 14(1):351-355. doi: 10.5958/0974-360X.2021.00064.0

34. Deepthi Y. Sivakkumar T. Srinivas N. Phytochemical Evaluation and in-vitro Antioxidant Potential of Whole Plant of Hyptis suaveolens. Research Journal of Pharmacy and Technology. 2021; 14(1):409-412. doi: 10.5958/0974-360X.2021.00074.3

35. Vrutti P. Ravi AM. Devang BS. Evaluation of in-vitro Thrombolytic activity of methanolic extract of Prunus avium L. Asian Journal of Research in Pharmaceutical Sciences. 2021; 11(1):41-44. doi: 10.5958/2231-5659.2021.00007.2

Received on 24.09.2020 Modified on 19.06.2021

Research J. Pharm.and Tech 2022; 15(4):1455-1460.

DOI: 10.52711/0974-360X.2022.00241