Original Article CYTOTOXIC METABOLITES OF ALTERNARIA ALTERNATA, AN ENDOPHYTE OF THE MEDICINAL PLANT BIDENS BIPINNATA RANDA ABDOU 1,2* , MOHAMED DAWOUD 3 1 Department of Pharmacognosy, Faculty of Pharmacy, Umm Al Qura University, Makkah, KSA, 2 Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Cairo, Egypt, 3 Department of Pharmaceutics, Faculty of Pharmacy, Umm Al Qura University, Makkah, KSA Email: randafathi@ymail.com Received: 09 Nov 2019, Revised and Accepted: 22 Jan 2020 ABSTRACT Objective: Endophytes are widely spread in the plant kingdom and represent a very promising source of biologically active natural products. The medicinal plant Bidens bipinnata Lin. (Asteraceae) which is known for its anti-inflammatory, antifungal and antitumor effects has been chosen for the investigation of its endophyte to search for bioactive metabolites. Methods: An endophytic Alternaria alternata species was isolated from the leaves of the plant B. bipinnata Lin. To investigate the metabolic profile of this endophytic fungus it was cultivated in several culture media as static and shaken culture. The antimicrobial and cytotoxic activities of the ethyl acetate extracts of the fungus were examined. Extracts exhibiting highest antimicrobial activities in agar diffusion assay and cytotoxicity against HeLa cancer cell line were subjected to activity-guided chromatographic fractionation for the identification of bioactive metabolites. A cytotoxic assay was performed on the isolated compounds against HeLa cancer cell lines as well as cytostatic activity tests against HUVEC and K-562 cell lines. Results: Chromatographic fractionation resulted in the isolation and identification of alternariol and tentoxin from the extract of the fungus cultivated in medium M5 while sterigmatocystin was isolated in addition to alternariol and tentoxin from the extract of the fungus grown in medium M25. Both alternariol and sterigmatocystin proved to be of moderate cytotoxicity and weak cytostatic activity with alternariol showing higher cytotoxic activity than sterigmatocystin. Highest cytotoxicity against HeLa cell lines was observed for tentoxin with a CC50 of 22.5 µg/ml. Conclusion: This study presents the isolation and identification of the bioactive metabolites alternariol, sterigmatocystin and tentoxin from the endophyte A. alternata in addition to the antifungal activity of the strain extract as well as the cytotoxic and cytostatic activities of the isolated metabolites against HeLa, HUVEC and K-562 cell lines, respectively. Keywords: Alternariol, Sterigmatocystin, Tentoxin, Endophytes, Alternaria alternata, Bidens bipinnata © 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijpps.2020v12i3.36317. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps INTRODUCTION Endophytes are considered as a promising source for bioactive metabolites and only few plant species of the entire plant kingdom on earth have been examined so far [1-3]. Plants having special uses and biological activities are considered as a promising source for study [4]. Accordingly, the medicinal plant Bidens bipinnata has been chosen for the investigation of its endophytic content. B. bipinnata L. is a widely distributed herbaceous plant that was used in traditional medicine for the treatment of inflammation accompanying several diseases such as hepatitis and diabetes [5]. A study has been conducted to investigate the role of total flavonoids of B. bipinnata in suppressing tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO) release in HUVECs cultured with sera from active Henoch-Schonlein purpura (HSP) patients. Results showed that total flavonoids of the plant may inhibit the inflammatory mediators in HUVECs induced by sera from active HSP patients [5]. Furthermore, polyacetylenes from B. bipinnata were reported to exert anti- inflammatory activities on lipopolysaccharide (LPS)-induced interleukin 1 and tumor necrosis factor (TNF-α) production in macrophage (RAW264.7) cells [6]. In a recent study, the plant extract exerted inhibitory effects on human HepG2 cell lines and Hela cell lines with IC50 values of 14.80 μg/ml and 13.50 μg/ml respectively [7]. Since mainly the plant but not its endophytes has been studied so far and as it is a biologically active plant that could represent a promising source of bioactive endophytes according to the plant selection strategies identified by Strobel [4], it has been chosen for the investigation of its endpophytes. In a previous study an endophytic Khuskia oryzae species have been isolated from the stem parts of B. bipinnata and two bioactive oxylipins, as well as the mycotoxin sterigmatocystin, have been isolated from it [8]. This study presents the isolation and identification of biologically active metabolites of another endophytic fungus, Alternaria alternata from the leaves of B. bipinnata. Alternaria fungi are widely spread in nature and have been reported to act as phytopathogens, plant pathogens, parasites, saprophytes and endophytes [9]. Accordingly, a wide variety of biological activities have been reported for Alternaria metabolites such as phytotoxic, cytotoxic, and antimicrobial properties [10-14]. Porritoxin isolated from an endophytic Alternaria species has been studied as a potential cancer chemopreventive agent [15]. Depudecin, an inhibitor of histone deacetylase (HDAC) from A. brassicicola, also showed antitumor potency [16, 17]. Some Alternaria metabolites such as tenuazonic acid and tentoxin have been studied as herbicidal agents [18-20]. Due to the importance of culture medium composition for microbial production of secondary metabolites [21-23], it was necessary to use different culture media for the cultivation of this endophytic fungus. Thus, it was cultivated in three different culture media (M4, M5 and M25) under shaking and stationary conditions to investigate the effect of varying culture media and growth conditions on the production of secondary metabolites. HPLC chromatograms of the ethyl acetate (EtOAc) extracts of the obtained cultures showed different chemical patterns and different antimicrobial and cytotoxic activities in preliminary biological screening tests. MATERIALS AND METHODS The wildly growing plant material was collected near Cairo/Egypt and identified by Dr. Abdel Megid (Head of Department of Botany at the Museum of Agriculture, Cairo/Egypt). Leaves of the plant were cut into small pieces, washed with sterilized water, followed by treatment with 70% ethanol for 1-2 min and air-drying under a International Journal of Pharmacy and Pharmaceutical Sciences Print ISSN: 2656-0097 | Online ISSN: 0975-1491 Vol 12, Issue 3, 2020