Wound healing and coagulant activity of crude extract metabolites from fungal endophytes

Abstract

Bleeding from a wound as a result of physical injury is a life-threatening condition. In pursuing new drug structures, the effect of crude fungal extracts fungal isolated from Jatropha multifida on wound healing and coagulation of mouse whole blood was investigated. Jatropha multifida leaves were sterilized, cut into small segments, and then incubated in Potato Dextrose Agar for seven days. Four isolates were purified and their morphologies were characterized. Identification of isolates was confirmed by a molecular protocol. Two crude extracts from Phlebiopsis gigantea (OK021602) and Phyllosticta sp (OK021603), which exhibited higher phytochemicals composition, were selected and evaluated using wound excision and coagulation of mouse whole blood, by administering 30 µg/mL, 50 µg/mL and 70 µg/mL crude extracts respectively. The percentage of wound healing in mice was higher (p<0.05) for the crude extracts of Phlebiopsis gigantea (OK021602) as compared to that of Phyllosticta sp (OK021603). The highest percentages of wound contraction were 99% at 70 µg/mL, and 53% at 70 µg/mL for Phlebiopsis gigantea (OK021602) and Phyllosticta sp (OK021603), respectively as compared to the control group which had 42% wound contraction at day 15 post-treatment. The results of the present study clearly indicate that Jatropha multifida leaves harbor endophytic fungi that produce pharmacologically important bioactive secondary metabolites with wound and hemostatic effects; therefore, further exploration is inevitable, particularly for the purification and identification of specific chemical structures of bioactive compounds.

Keywords

• Fungal endophyte
• Wound healing
• Crude extract
• Jatropha multifida
• Coagulation

References

1. Anani, K., Adjrah, Y., Améyapoh, Y., Karou, S.D., Agbonon, A., de Souza, C., & Gbeassor, M. (2016). Antimicrobial, antiinflammatory and antioxidant activities of Jatropha multifida L. (Euphorbiaceae). Pharmacognosy Research, 8, 142–146. https://doi.org/10.4103/0974-8490.172657
2. Araújo, K., Brito, V., Veloso, T., Leite, T., Pereira, O., Mizubuti, E., & Queiroz, M. (2018). Diversity of culturable endophytic fungi of Hevea guianensis: A latex producer native tree from the Brazilian Amazon. African Journal of Microbiology Research, 12, 953–964. https://doi.org/10.5897/AJMR2018.8980
3. Bishanga, D.R., Charles, J., Tibaijuka, G., Mutayoba, R., Drake, M., Kim, Y.-M., Plotkin, M., Rusibamayila, N., & Rawlins, B. (2018). Improvement in the active management of the third stage of labor for the prevention of postpartum hemorrhage in Tanzania: a cross-sectional study. BMC Pregnancy and Childbirth, 18(1), 1–10. https://doi.org/10.1186/S12884-018-1873-3
4. Blanco, A., & Blanco, G. (2017). Hemostasis, in: Blanco, A., Blanco, G. (Eds.), Medical Biochemistry. Academic Press, pp. 781–789. https://doi.org/10.1016/B978-0-12-803550-4.00031-8
5. Cannon, J.W., 2018. Hemorrhagic Shock. New England Journal of Medicine, 378, 370–379. https://doi.org/10.1056/NEJMra1705649
6. Chauncey, J.M., & Wieters, J.S. (2020). Tranexamic Acid, in: StatPearls. StatPearls Publishing, Treasure Island (FL).
7. Chioma, N., A, O., Nathaniel Ejikeme, O., & Aqib, F. (2021). Phytochemical analysis and in vitro screening of antifungal activity of Jatropha multifida, Euphorbia hirta, Occimum gratissimum and Mitracarpus scaber leaves extract. GSC Biological and Pharmaceutical Sciences, 14, 98–112. https://doi.org/10.30574/gscbps.2021.14.3.0023
8. Devi, N.N., Prabakaran, J.J., & Wahab, F. (2012). Phytochemical analysis and enzyme analysis of endophytic fungi from Centella asiatica. Asian Pacific Journal of Tropical Biomedicine, 2, S1280--S1284. https://doi.org/10.1016/S2221-1691 (12)60400-6

9. Dhayanithy, G., Subban, K., & Chelliah, J. (2019). Diversity and biological activities of endophytic fungi associated with Catharanthus roseus. BMC Microbiology, 19, 22. https://doi.org/10.1186/s12866-019-1386-x
10. Dougnon, T.V., Klotoé, J.R., Sègbo, J., & Gbaguidi, F. (2012). Evaluation of the phytochemical and hemostatic potential of Jatropha multifida sap. African Journal Pharmacy and Pharmacology, 6. https://doi.org/10.5897/AJPP12.351
11. Ebrahimi, F., Torbati, M., Mahmoudi, J., & Valizadeh, H. (2020). Medicinal plants as potential hemostatic agents. Journal of Pharmacy & Pharmaceutical Sciences, 23, 10–23. https://doi.org/10.18433/jpps30446
12. Elaju, moses, Ongom, peter A., Kijjambu, S.C., Wangoda, R., & Patson, M. (2013). Short term outcome of haemorrhagic shock in trauma at Mulago Hospital, an urban tertiary hospital in Sub Saharan Africa. Tropical Medicine Surgery, 1. https://doi.org/10.4172/2329-9088.1000148
13. Ezike, A.C., Akah, P.A., Okoli, C.O., Udegbunam, S., Okwume, N., Okeke, C., & Iloani, O. (2010). Medicinal plants used in wound care: A study of Prosopis africana (Fabaceae) stem bark. Indian Journal Pharmceutical Sciences, 72, 334–339. https://doi.org/10.4103/0250-474X.70479
14. Fetse, J., Oppong Kyekyeku, J., Dueve, E., & Mensah, K. (2014). Wound Healing activity of total alkaloidal extract of the root bark of Alstonia boonei (Apocynacea). British Journal of Pharmaceutical Research, 4, 2642–2652. https://doi.org/10.9734/BJPR/2014/13952
15. Fitokimia, P., Asai, J., Flavonoid, K., Fenolik, D., Ekstrak, P., Tepal, P., Mahadeva Rao, U. S., Abdurrazak, M., & Mohd, K.S. (2016). Phytochemical screening, total flavonoid and phenolic content assays of various solvent extracts of tepal of Musa paradisiaca. Malaysian Journal of Analytical Sciences, 20, 1181–1190. https://doi.org/10.17576/mjas-2016-2005-25
16. Guo, B., Wang, Y., Sun, X., & Tang, K. (2008). Bioactive natural products from endophytes: A review. Applied Biochemistry Microbiology, 44, 136 142. https://doi.org/10.1134/S0003683808020026
17. Hamzah, T.N.T., Lee, S.Y., Hidayat, A., Terhem, R., Faridah-Hanum, I., & Mohamed, R. (2018). Diversity and characterization of endophytic fungi isolated from the tropical Mangrove species, Rhizophora mucronata, and identification of potential antagonists against the soil-borne fungus, Fusarium solani. Frontiers Microbiology, 9. https://doi.org/10.3389/fmicb.2018.01707
18. Kjer, J., Debbab, A., Aly, A.H., & Proksch, P. (2010). Methods for isolation of marine-derived endophytic fungi and their bioactive secondary products. Nature Protocols, 5(3), 479–490. https://doi.org/10.1038/nprot.2009.233
19. Kumar, A., &Sharma, S. (2008). An evaluation of multipurpose oil seed crop for industrial uses (Jatropha curcas L.): A review. Industrial Crops and Products, 28, 1–10. https://doi.org/10.1016/J.INDCROP.2008.01.001
20. Kumar, B., Vijayakumar, M., Govindarajan, R., & Pushpangadan, P. (2007). Ethnopharmacological approaches to wound healing Exploring medicinal plants of India. Journal of Ethnopharmacology, 114, 103–113. https://doi.org/10.1016/j.jep.2007.08.010
21. Lesperance, M.M., Francis, T.L., & Norton, B.K.B. (2006). Chapter 1 - Postsurgical soft tissue healing, in: Manske, R.C. (Ed.), Postsurgical Orthopedic sports rehabilitation. Mosby, Saint Louis, pp. 3–18. https://doi.org/10.1016/B978-032302702-1.50004-1
22. Manter, D.K., & Vivanco, J.M. (2007). Use of the ITS primers, ITS1F and ITS4, to characterize fungal abundance and diversity in mixed-template samples by qPCR and length heterogeneity analysis. Journal of Microbiological Methods, 71, 7 14. https://doi.org/10.1016/j.mimet.2007.06.016
23. Masson-Meyers, D.S., Andrade, T.A.M., Caetano, G.F., Guimaraes, F.R., Leite, M.N., Leite, S.N., & Frade, M.A.C. (2020). Experimental models and methods for cutaneous wound healing assessment. International Journal of Experimental Pathology, 101, 21–37. https://doi.org/10.1111/iep.12346
24. Mbilu, M., Wanyoike, W., Kangogo, M., Bii, C., Agnes, M., & Kihia, C. (2018). Isolation and characterization of endophytic fungi from medicinal plant Warburgia ugandensis. Journal of Biology 10.
25. Park, H.L., Lee, H.S., Shin, B.C., Liu, J.P., Shang, Q., Yamashita, H., & Lim, B. (2012). Traditional medicine in China, Korea, and Japan: A brief introduction and comparison. Evidence-based Complementary and Alternative Medicine, Article ID 429103, 9 pages. https://doi.org/10.1155/2012/429103
26. Rakotoarivelo, N.H., Rakotoarivony, F., Ramarosandratana, A.V., Jeannoda, V.H., Kuhlman, A.R., Randrianasolo, A., & Bussmann, R.W. (2015). Medicinal plants used to treat the most frequent diseases encountered in Ambalabe rural community, Eastern Madagascar. Journal Ethnobiology Ethnomedicine, 11, 68. https://doi.org/10.1186/s13002-015-0050-2
27. Rampadarath, S., Puchooa, D., Jeewon, R., & Bandhoa, K. (2018). Diversity, seasonal variation and antibacterial activity of endophytic fungi associated with the genus Jatropha in Mauritius. Journal of Biotechnology & Biomaterials, 8(1), 1 8. https://doi.org/10.4172/2155-952X.1000280
28. Rampadarath, S., Puchooa, D., Ranghoo-Sanmukhiya, V.M. (2014). Antimicrobial, phytochemical and larvicidal properties of Jatropha multifida Linn. Asian Pacific Journal of Tropical Medicine, 7, S380-S383. https://doi.org/10.1016/S1995-7645(14)60262-5
29. Rana, K.L., Kour, D., Sheikh, I., Yadav, N., Yadav, A.N, Kumar, V., Singh, B.P, Dhaliwal, H.S, Saxena, A.K. (2019). Biodiversity of endophytic fungi from diverse niches and their biotechnological applications. In: Singh BP (ed) Advances in endophytic fungal research: present status and future challenges. Springer International Publishing, Cham, pp 105–144. https://doi.org/10.1007/978-3-030-03589-1_6
30. Seifert, E. (2014). OriginPro 9.1: Scientific data analysis and graphing software—software review. Journal of Chemical Information and Modeling, 54, 1552 1552. https://doi.org/10.1021/ci500161d
31. Shaheen, A., Hanif, M.A., Rehman, R., Jilani, M.I., & Shikov, A. (2020). Alkanet. Medicinal Plants of South Asia 1–12. https://doi.org/10.1016/B978-0-08-102659-5.00001-X
32. Sharma, A., Khanna, S., Kaur, G., & Singh, I. (2021). Medicinal plants and their components for wound healing applications. Future Journal of Pharmaceutical Sciences, 7(53). https://doi.org/10.1186/S43094-021-00202-W
33. Spotnitz, W. (2007). Active and mechanical hemostatic agents. Surgery, 142, S34-S38. https://doi.org/10.1016/j.surg.2007.06.024
34. Tadych, M., & White, J.F. (2009). Endophytic Microbes, in: Schaechter, M. (Ed.), Encyclopedia of Microbiology (Third Edition). Academic Press, Oxford, pp. 431–442. https://doi.org/10.1016/B978-012373944-5.00328-X
35. Tochie, J.N., Fonkwo, V., Kelly, G., Njinkeu, D., Mbaya, C., & Badjang, T. (2019). Challenges in the management of postpartum haemorrhage in sub-saharan Africa. Acta Scientific Women's Health, 1(1), 25–27.
36. Torres, M., Tadych, M., White, J., & Bills, G. (2011). Isolation and identification of fungal endophytes. pp. 153–164.
37. Victorien, D.T., Jean Robert, K., Jacques, D.T., Julien, S., Jean-Marc, A., Aléodjrodo, E.P., Olufunkè, S., Ferdinand, D., Carlos, D., Frédéric, L., Karim, D., 2012. Hemostatic activity screening and skin toxicity of sap of Jatropha multifida L. (Euphorbiaceae) used in traditional medicine (Benin). Asian Pacific Journal of Tropical Disease, 2, S927--S932. https://doi.org/10.1016/S2222-1808 (12)60293-X
38. Wikee, S., Lombard, L., Crous, P.W., Nakashima, C., Motohashi, K., Chukeatirote, E., Alias, S.A., McKenzie, E.H.C., & Hyde, K.D. (2013). Phyllosticta capitalensis, a widespread endophyte of plants. Fungal Diversity, 60, 91–105. https://doi.org/10.1007/S13225-013-0235-8