ANew Triterpenoid Saponin and Antimicrobial Activity of Ethanolic Extract from Sapindus mukorossi Gaertn .

A new acetylated triterpenoid saponin elucidated as hederagenin-3-O-ββ-D-xylopyranosyl (2→ 1)-[3-O-acetyl-αα-L-arabinopyranosyl-28-O-αα-L-rhamnopyranosylester has been isolated from pericarps of Sapindus mukorossi Gaertn. e structure of the compound was determined by means of chemical and spectral analysis including advanced 2DNMR studies.e ethanolic extract from pericarps of the plant showed signi�cant in vitro antimicrobial activity against various test organisms by Agar well diffusion method.


Introduction
Sapindus mukorossi Gaertn. of family Sapindaceae is wellknown soap nut tree, distributed in tropical and subtropical regions of Asia.Fruits are popular ingredient in Ayurvedic shampoos and cleansers.e plant is an important remedy for relieving cough, detoxi�cation, emetic, contraceptive, treatment of excessive salivation, epilepsy, and chlorosis [1][2][3].Previous studies on the plants of this genus led to the isolation of triterpenoids, saponins [4][5][6][7] fatty acids [8], and �avonoids [9].e present paper illustrate the isolation and structure elucidation of a new acetylated triterpenoid saponin (1) (Figure 1) for the �rst time from this species.

Result and Discussion
Acetylated triterpenoid saponin was isolated from -butanol (BuOH) extract of the pericarps of S. mukorossi.e -BuOH extract was subjected to repeated column chromatography and the fractions were eluted with methanol in chloroform.e compound 1 was puri�ed from among number of reported compounds with (50 mg) yield.e compound 1 was crystallized as an amorphous solid.It revealed an [M + H] + ion peak at m/z 925.10684 in HR-ESIMS in agreement with molecular formula C 48 H 76 O 17 and melting point is 202-206 ∘ C. e IR spectrum exhibited absorptions at 3450 (OH) and 1731 (C=O of ester) cm −1 .Positive results with Liebermann-Burchard and Molisch's reagents make obvious its triterpenoidal glycosidic nature. 13C NMR and DEPT spectra exhibited 48 carbon signals for eight methyl, thirteen methylene, ninteen methane, and eight quaternary carbons.Acid hydrolysis of 1 afforded triterpenoid as the aglycone, Dglucuse, D-xylose, and L-rhamnose, respectively.e sugar moieties were established by the comparison of spectral data from previously reported compounds isolated from this plant and also con�rmed by authentic samples, respectively [10,11].e down�eld 13 C-NMR chemical shi at  C 81.5 and the up�eld 13 C-NMR chemical shi at  C 178.8 suggested that 1 was a triterpenoid saponin with glycosidic linkages at C-3 through an ether bond and at C-28 through an ester bond.e anomeric proton signals were at  H 5. 16   with C-3 ( C 81.5), H-1 ( H 5.0) of arabinose with C-2 ( C 72.2) of xylose, and H-1 of rhamnose ( H 4.8) with C-28 ( C 178.8) of the aglycone as shown in Table 1.Since the chemical shi of C-3 of aglycone is linked to the H-1 of xylose was same as that C-2 of xylose was linked to the H-1 of arabinose and C-28 was directed linked to the H-1 of rhamnose, their linkages of sugars could be �nally determined from the NOE experiment.e cross-peak between the H-1 ( H 5.1) of xylose and H-1 ( H 5.1) of arabinose indicated that the xylose moiety of C-2 was linked to C-1 of arabinose, respectively.A broad signal at  5.24 (H-12) in the 1 H NMR is due to the presence of an olefenic proton which was further supported by 13 C NMR values at  123.60 (C-12) and 145.02 (C-13).In the 13 C NMR spectrum, the values at  64.2 (C-23) and 81.53 (C-3) were corroborated to a methylene carbon linked to hydroxyl group and a tertiary carbon linked to sugar moiety, respectively.Moreover, a down�eld signal at  178.8 (C-28) was clearly indicative to a carbonyl carbon [12].e index of hydrogen de�ciency for C 48 H 76 O 17 (= 11) showed the presence of eight rings (pentacyclic rings and three sugar rings) and three double bonds (an olefenic and two carbonyl).e 1 H NMR value at  2.15 and 13 C NMR signals at  173.48 (−COO) and 22.05 (Me) indicated the presence of acetoxyl group attached to arabinose ring as found in the previous literature [11].From above discussion, it is quite obvious that two sugar moieties (xyl → ara) were linked to C-3 and the third (rham) was attached to C-28 of pentacyclic triterpenoidal skeleton.All protons and carbons of each sugar unit were assigned on the basis of extensive NMR experiments including HMQC, COSY, NOE, and HMBC.e LCMS of 1 was found very informative in support of the structural con�rmation which showed a quasimolecular ion peak at m/z 925 (100%) along with other fragment ions at m/z 882, 750, 618, and 471 due to the loss of sugar fragments.Hence, compound 1 was elucidated ashederagenin Antimicrobial activity of ethanolic extract from pericarps of S. mukorossi was determined by the agar well diffusion method.e extract showed signi�cant inhibitory activity against Esherichia faecalis, Pseudomonas aeuroginosa, Staphylococcus aureus, �lcaligenes denitri�cans, Klabsiella pneumoniae, Bacillus cereus, Pseudomonas alcaligenes, Micrococcus luteus, and Bacillus subtillis.e ethanolic extract showed most signi�cant activity against Pseudomonas aeruginosa, Esherichia faecalis, and Bacillus cereus by inhibition zone diameter (IZD) value of 20, 19, and 17 mm, respectively, at minimum inhibitory concentration (MIC) of 100 g/mL, whereas the IZD values of 15, 17, and 15 mm, respectively, were shown at MIC value of 50 g/mL compared with positive control.

Experimental
3.1.General.Melting point was recorded on the Per�t melting point apparatus.IR spectra were recorded on Perkin-Elmer, spectrum RX I FT-IR spectrometer (KBr discs).NMR spectra were obtained on Bruker, 400, Ultra shield NMR spectrometer (300 MHz for 1 H and 125 MHz for 13 C, DMSOd 6 as solvent, and TMS as internal standard).MS were recorded on LCMS Q-TOF Micro mass spectrometer and LCMS-LCQ, Finnigam, MAT mass spectrometer.Column chromatography was performed on silica gel (Merck 60-120 mesh, 15 × 100 cm).in layer chromatography was carried out on silica gel (Merck 10-40 m) precoated plates which were visualised by spraying with 7% H 2 SO 4 as a universal spray reagent.

Antimicrobial Assay
Evaluation of antimicrobial activity of ethanolic extract was carried out by the agar well diffusion method [13,14].e extract was reconstituted in 20% DMSO for the bioassay analysis [15].Different culture media were bioassayed for inhibition zone diameter (IZD) and minimum inhibitory concentration (MIC) determination using Kanamycin as a positive control.In this method, pure isolate of each microbe was subcultured on the recommended speci�c media for each microorganism at 37 ∘ C for 24 hrs.One hundred microlitre (100 L) of inoculum of each test organism was spread onto the speci�c media plates so as to achieve a con�uent growth.e agar plates were allowed to dry and wells or cups of 8 mm were made with a sterile borer in the inoculated agar plates and the lower portion of each well was sealed with a little speci�c molten agar medium.e plates were allowed to stand for 10 minutes for diffusion of the extract to take place and incubated at 37 ∘ C for 24 h [16][17][18] and the zone of inhibitions was measured.MIC was determined as the least concentration of extract inhibiting the growth of the test organisms during 24 h.e results of antimicrobial activity are shown in Table 2.It is new due to the attachment of rhamnose at C-28 position which has not been reported earlier in the previous literature.Consequently, this compound is a novel acetylated triterpenoid saponin.Further the ethanolic extract of S. mukorossi showed signi�cant activity against the test organism as compared with positive control.e biological potential for the compound has to be further investigated.
T 1: 13 C, 1 H and HMBC data of compound 1 in DMSO d 6 .