Phytochemical and pharmacological evaluation of Pongamia pinnata, Sesbania grandiflora and associated endophytic fungi

Abstract

Sesbania grandiflora (L.) Pers and Pongamia pinnata (L.) Pierre, belonging to the Fabaceae family, have been investigated for isolating new natural compounds with promising pharmacological activities. A total of tweenty five compounds have been isolated from these two plants and endophytic fungi associated with Sesbania grandiflora. The isolated compounds were identified by extensive analysis of high resolution NMR ( 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, HSQC and HMBC) experiments and the molecular weights were determined by ESI mass spectrometry. The stem bark of Sesbania grandiflora afforded fourteen natural compounds namely, Sesbagrandiflorain A, (-) maackiain, medicarpin, sativan, 4-hydroxy-2methoxybenzaldehyde,

nobiletin, kaurenoic acid, betulinic acid, lupeol, β-amyrin, stigmast-4-en-3-one, stigmasta-4, 22-dien-3-one, stigmasterol and linoleic acid. Among them, eight compounds like (-) Maackiain, 4-hydroxy-2methoxybenzaldehyde,

nobiletin, kaurenoic acid, β-amyrin, stigmast-4-en-3-one, stigmasta-4, 22-dien-3-one and linoleic acid were isolated for the first time from Sesbania grandiflora. Five flavonoids and lupeol were isolated from Pongamia pinnata and were identified as pongachromene, kanugin, demethoxykanugin, 5’methoxypongapine

and karanjin. Seven different endophytic fungi were isolated and identified from the plant Sesbania grandiflora, such as Fusarium solani, Fusarium fujikuroi, Fusarium equisetti, Xylaria bambusicola, Cladosporium sp, Clonostachys rosea and Colletotrichum aeschynomenes. Two endophytic fungal extract were also investigated for chemical constituents and anhyrofusarubin, bostrycoidin and myristic acid were isolated from Fusarium solani and 3β, 5α, 9α-trihydroxy-ergosta-7,22diene-6-one,

3β,5α-dihydroxy-6β-acetoxy-ergosta-7,22-diene and 3β, 5α,dihydroxyergosta-7,

22-diene-6-one were isolated from Xylaria bambusicola. All these compounds were reported for the first time from the associated endophytic fungi of Sesbania grandiflora. Different solvent fractions of the crude extract and the pure compound Sesbagrandiflorain A, were subjected to several biological activities like antimicrobial, antioxidant, brine shrimp lethality and thrombolytic activities. Among the different fractions tested against five microorganisms, the ethylacetate soluble fraction showed the highest anti-bacterial and antifungal activity against Bacillus megaterium and Aspergillus niger with zone of inhibition of 17.67 ± 1.53 mm and 12.67 ± 0.58 mm compared to the standard, kanamycin (30.67 ± 1.15mm) and ketoconazole (28.00± 1.00 mm) respectively. The pure compound displayed mild activity against Bacillus megaterium (11.67 ± 0.58 mm). In vitro antioxidant activity was studied by DPPH radical scavenging method using butylated hydroxy anisole (BHA) as the standard. Among all the samples tested, the pure compound Sesbagrandiflorain A and crude methanolic extract showed very significant antioxidant activity with IC 50 value of 28.26 ± 0.35 μg/ml and 54.59 ± 0.09 μg/mL as compared to the standard BHA (9.21 ± 0.10 μg/ml). In brine shrimp lethality assay the ethylacetate soluble fraction and methanolic extract demonstrated significant cytotoxic activity with LC 50 of 0.68 ± 0.10 and 0.66 ± 0.04 μg/ml respectively compared to Tamoxifen (0.30 μg/ml). The dichloromethane soluble fraction exhibited promising thrombolytic activity with % of clot lysis of 54.29% as compared to the standard Streptokinase, 73.57%. Among the different extractives of P. pinnata, the methanolic extract showed highest anti-bacterial activity against Bacillus megaterium and antifungal activity against Aspergillus niger with zone of inhibition of 20.67 ±1.15 mm and 17.00 ±1.00 mm respectively compared to the standards Kanamycin (30.67 ± 1.15 mm) and Ketoconazole (28.00 ± 1.00 mm). The Highest antioxidant activity was demonstrated by pongachromene with IC 50 values of 19.69 µg/ml. (BHA, 8.70 ± 0.29 µg/ml). Very mild cytotoxic activities were found for different extracts of P. pinnata in the brine shrimp lethality assay. The isolated endophytic fungi from Sesbania grandiflora were evaluated for their antioxidant activity, cytotoxicity and antimicrobial activity. Among the seven samples tested, SGFRE-1 showed highest antibacterial activity with zone of inhibition of 22.67 mm against Bacillus megaterium as compared to the standard antibiotic kanamycin (30.67 mm) and highest antifungal activity with zone of inhibition of 19.33 mm each against A. niger and A. flavus as compared to the standard antifungal Ketoconazole (28.00 mm). Moderate antioxidant and cytotoxic activities were observed by SGFE-1 with IC 50 value of 54.06 ± 0.20 µg/ml and LC 50 value of 1.90 µg/ml as compared to the standards BHA (9.30 ± 0.06) and tamoxifen 0.34 µg/ml respectively.