Isolation and biological evaluation of demethylcassiarin B from Senna siamea Lam.

Abstract In search of potential natural products for the amelioration of obesity, phytochemical investigation of leaves of Senna siamea Lam. was carried out. It led to the isolation of demethycassiarin B (1), 6,8-dihydroxy-3-methyl-1H isochromenone (2), and 6-hydroxymellein (3). The structures were elucidated by a detailed analysis of spectral data. Compound 1 was found to possess a similar structural skeleton as that of cassiarin B except for a methoxyl substitution. Detailed HPLC and HPTLC analysis of methanolic extract for the presence of compound 1 and cassiarin B were performed. Cassiarin B was not detected in extract. Compounds 2 and 3 have been reported for the first time from the genus Senna. The isolated compounds were evaluated for their antiadipogenic activity in murine 3T3L1 cells. Graphical abstract


Introduction
Natural products play a pivotal role in drug discovery and development.Nearly 50% of the approved drugs in last 30 years derived from nature (Veeresham 2012).They are an exclusive source for new lead molecules (Mali and Bhatia 2021) and provide dependable functional foods and health supplements to support the well-being of mankind.It is of utmost importance to standardize and characterize the chemical constituents of the medicinal plants with certainty to provide consistent quality products (David et al. 2015).Since the past century, there is a remarkable advancement in the techniques to isolate and characterize new molecules and extracts.Basic techniques like extraction, liquid-liquid partitioning, and fractionation are inevitable in modern laboratories where the usage of solvents can lead to the formation of artifacts.
Senna siamea Lam.(Fabaceae) is a medium-sized, evergreen tree native to Southeast Asia, whose leaves are used in the preparation of a famous Thai curry named 'Khi Lek'.Its leaves have been used by the local healers of Thailand and West Africa to treat diabetes, periodic fever, and malaria (Laleye et al. 2015;Neamsuvan et al. 2015).Methanolic extract of S. siamea leaves reduces hyperglycemia by 39.3 À 54.3% in streptozotocin-induced diabetes in rats (Kumar et al. 2010) and reduces in vitro lipid accumulation in 3T3LI to 64.7 ± 2.2% at 50 mg/mL.DCM extract inhibits pancreatic lipase activity by 57.62 ± 6.34% at 250 mg/mL.Further, cassiamin A isolated from S. siamea inhibited pancreatic lipase with an IC 50 value of 41.8 ± 1.2 lM (Kumar et al. 2013).S. siamea is reported to have a diverse class of phytochemicals including anthraquinones, triterpenoids, steroids, flavonoids, chromones, and Dioxophenalene type of molecules that have been majorly reported from leaves (Kamagat e et al. 2014).Owing to the antiobesity potential of S. siamea it is pertinent to explore the phytoconstituents for antiobesity activity (Kumar et al. 2010).

Results and discussion
Phytochemical investigation of MeOH extract lead to the isolation of Compound 1.It was isolated as yellow amorphous powder and showed a molecular ion peak with m/z value of 300 (M þ H) þ .The molecular formula was deduced as C 17 H 17 NO 4 by HRESIMS m/z 300.1295 (M þ H) þ calcd.300.1235).The 13 C and DEPT NMR spectra of 1 at 100 MHz in CD3OD (Supplementary information, Figures S9-S16, Tables S2 and S3) revealed 17 signals due to two carbonyls (dc 169.6, 178.9 ppm), six sp 2 quaternary carbons (dc 114.7, 140.5, 146.7, 153.5, 159.9, and 172.8 ppm), four sp 2 methines (dc 101.4,105.4,108.2 and 121.0 ppm), three methylene (dc 26.5, 33.2, 51.5 ppm), and two methyl (dc 22.7 and 23.5 ppm).One methylene (dc 51.5 ppm; d H 4.22 ppm) attached to either nitrogen or an oxygen atom.The 1 H-1 H COSY and HMBC correlation of H 2 -14/C-14 to H 2 -13/C13 and H 2 -15/C-15 revealed connectivity of C13 to C15.The connectivity from C-13 to C-4 and C-11 through a nitrogen atom was inferred by correlations for H2-13 to C-4 and C-11.Two dimensional NMR data of 1 including the 1 H-1 H COSY, HSQC, NOESY and HMBC spectra corroborated well with those of cassiarin B (4) (Morita et al. 2007) suggesting compound 1 to have similar skeleton as that of 4 except C-17 methyl group.Based on spectral data described above compound 1 was characterized and named as demethylcassiarin B.
In the previous studies, isolation of cassiarin B from S. siamea was reported using the conventional acid-base neutralization method (Morita et al. 2007;Deguchi et al. 2012).In the structure of cassiarin B (4), N-substituted butanoate could undergo esterification in the presence of acid leading to the formation of methyl butanoate (Maltese et al. 2009).To investigate this possibility, we have synthesized cassiarin B using an earlier reported method (Kanputhorn et al. 2010) and characterized using NMR spectroscopy.Comparative HPLC and HPTLC analysis of methanolic extract prepared by soxhlet extraction and maceration method was done for detecting the presence of compound 1 and 4 in these extracts.Compound 1 could be identified in both these extracts while compounds 4 could not be identified.The presence of demethylcassiarin B was confirmed in HPLC chromatogram by spiking experiment (10 mL of 1 mg/mL of demethylcassiarin B solution) and calculation of the increase in relative peak area.Cassiarin B was not detected in both these extracts.
Further compounds 2 and 3 were isolated during isolation of phytoconstituents.The structures were elucidated as 6,8-dihydroxy-3-methyl-1H isochromenone and 6hydroxymellein, respectively by comparison of the observed spectral data with that reported in the literature (Kendall et al. 1989) and 2 D-NMR spectral data analysis.To the best of our knowledge, both these isochromones are reported for the first time from the genus Senna.All the isolated compounds were evaluated for their cytotoxicity and antiadipogenic potential in vitro using 3T3L1 preadipocytes at 1 mM, 10 mM, and 100 mM concentration.There was no significant reduction in cell viability at 1 mM and 10 mM in case of all the three compounds (1-3).Compound 1 showed moderate activity with 85.6 ± 3.6% lipid accumulation as compared to control at 10 mM concentration.Compounds 2 and 3 did not show any inhibition to lipid accumulation (Supplementary information Figure S8).

Experimental
Experimental details are provided as supplementary information.

Conclusion
In the present study demethycassiarin B was isolated along with two compounds i.e. 6,8-dihydroxy-3-methyl-1H isochromenone and 6-hydroxymellein from genus senna for the first time.Out of all the tested molecules for antiadipogenic potential, Demethylcassiarin B was found to be active in vitro with an IC50 of 29.9 mM, which supports its antiobesity potential.