Limonoid and Steroidal Saponin from Azadirachta indica

A new limonoid, 17-(5-methoxy-2-oxofuran-3-yl)-28-deoxonimbolide (1), and a new C21 steroidal saponin, 2α,4α-dihydroxy-pregn-5-en-16-one-3α-O-d-glucopyranoside (2), together with 11 known compounds were isolated from the methanol extract of the leaves of Azadirachta indica. The structures were elucidated by means of spectroscopic analysis and putative biosynthetic origins. All the compounds were evaluated for their antibacterial activities against six bacterial strains.


Introduction
Herbal medicines are widely used and formed as an integral part of primary health care in many countries [1][2][3], and some are used to treat fungal diseases, which may constitute a reservoir of antifungal substances. In recent years, a number of antifungal agents are currently used in antifungal therapies with clinical practice, such as griseofulvin and terbinafine [4,5]. These antifungals are isolated from fungi or are synthetized, and searching for new antifungal substances from plants is still challenging [6].
Compound 2 was assigned a molecular formula of C 27 H 42 O 9 , by its HR-EI-MS peak at m/z 510.2833 ([M] ? calcd. for 510.2829), in combination with 1 H, 13 C NMR and DEPT data, corresponding to 7 degrees of unsaturation. IR absorptions bands at 3428 and 1631 cm -1 revealed the existence of OH and C=C groups. Signals of two high-field quaternary carbons (d C 38.5, 42.9) in the 13 C NMR spectrum, along with two singlet methyls (d H 0.74, 1.29) and a triplet methyl (d H 1.03) (Table 1) in the 1 H NMR spectrum, suggested that compound 2 was a pregnane derivative [15].  (Fig. 2). The HMBC correlations of d  (Fig. 2). Thus, the structure of 2 was elucidated as shown, and named as 2a,4a-dihydroxy-pregn-5-en-16-one-3a-O-D-glu-copyranoside. All the compounds were evaluated for their antibacterial activities against Escherichia coli ATCC 11775, Enterococcus faecalis ATCC 10541, Klebsiella pneumonia ATCC 13883, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25922, and Salmonella enterica ATCC 13076. Norfloxacin was used as the positive control. The results showed that compounds 1 and 2 exhibited strong antibacterial activities against some bacterial strains, equivalent to norfloxacin with MIC values of 0.78 lg/mL ( Table 2). And the other results (MIC values) are summarized in Table 2.

General Experimental Procedures
Optical rotations were obtained with a Jasco P-1020 Automatic Digital Polariscope. UV spectrum was measured with a Shi madzu UV2401PC spectrometer. IR

Extraction and Isolation
The

Acidic Hydrolysis of 2 and GC analysis
Compound 2 (2 mg) was hydrolyzed with 2 M HCl/dioxane (1:1, 10 mL) under reflux for 3 h. The reaction mixture was partitioned between CHCl 3 and H 2 O. The aqueous layer was neutralized with 2 M NaOH and then dried to give a sugar. The sugar was dissolved in anhydrous pyridineand (1 mL) and reacted with L-cysteine methyl ester hydrochloride (1.5 mg) stirred at 60°C for 1.5 h. Then trimethylsilylimidazole (1.0 mL) was added to the reaction mixture, and it was kept at 60°C for 30 min. The mixture (4 lL) was subjected to GC analysis, run on an HP 5890 gas chromatograph (Agilent) with a quartz capillary column (30 mm 9 0.32 mm 9 0.25 lm): H 2 flame ionization detector, column temp 180-280°C at 3°C/min, carrier gas N 2 (1 mL/min), injector and detector temp 250°C, split ratio 1:50. Peak of the hydrolysate was detected by comparison with retention time of authentic samples of D-glucose after treatment with trimethyl-chlorsilan (TMCS) in pyridine. The absolute configurations of the sugar residue was determined to be D-glucose (t R 19.01 min).

Antimicrobial Assays
The antibacterial assay of compounds 1-13 was evaluated against E. coli ATCC 11775, E. faecalis ATCC 10541, K. pneumonia ATCC 13883, P. aeruginosa ATCC 27853, S. aureus ATCC 25922, and S. enterica ATCC 13076. All the bacterial strains were obtained from the American Type Culture Collection (Rockville, USA). The antibacterial assay was carried out as described in the literature [23]. The preparation of bacterial inocula was done by using 18 hold overnight bacterial cultures prepared in Nutrient Agar. A few colonies of bacteria were collected aseptically with a sterile loop and introduced into 10 mL of sterile 0.90 % saline solution. The concentration of the suspension was then standardized by adjusting the optical density to 0.10 at 600 nm, corresponding to bacterial cell suspension of 108 colony-forming units/mL (CFU/mL) [24]. This cell suspension was diluted 100 times to obtain 106 CFU/mL before use. The compounds were dissolved in DMSO and then added to bacteria suspension to obtain the final concentration of 5 % (v/v) DMSO or less. Serial twofold dilutions from 200 lg/mL of the compounds were performed in 96-well micro-titer plates. Each well contained 100 lL of sample of each concentration. Then each well was infunded 100 lL of the bacterial suspension. The final concentration range of the test compounds was 100-0.781 lg/mL, and the plates were incubated at 37°C for 24 h. After incubation, the wells were examined for growth of microorganisms by measuring optical density (OD) value of the wells. Each experiment was repeated three times and Norfloxacin, bacteria suspension of 5 % (v/v) DMSO were used as a positive control and a blank control, respectively. By comparing to OD values, we can point out MIC values of these compounds among the selected concentration range.