Isolation of Antidiabetic Principle from Bougainvillea spectabilis Willd (Nyctaginaceae) Stem Bark

Purpose: To isolate and identify the constituents of Bougainvillea spectabilis Willd (Nyctaginaceae) stem bark. Methods: The methanol extract of Bougainvillea spectabilis stem bark powder was suspended in water and extracted with dichloromethane (CH 2 Cl 2 ), ethyl acetate (EtOAc), and butanol (BuOH) successively. The ethyl acetate fraction was loaded in a column packed with silica gel and eluted with a gradient of chloroform (CHCl 3 ): methanol (MeOH), and water yielded five fractions (A - E). Chemical constituents were isolated by repeated column chromatography of these fractions. Results: Column chromatography of fractions B and C afforded four compounds identified as pinitol, β-sitosterol, quercetin and quercetin-3-O-α-L-rhamnopyranoside. For the first time, pinitol, β-sitosterol, quercetin and quercetin-3-O-α-L-rhamnopyranoside were isolated from the stem bark of B. spectabilis Willd. Conclusion: An antidiabetic principle, pinitol, was successfully isolated from the stem bark of B. spectabilis Willd.


INTRODUCTION
Diabetes is one of the major causes of premature illness and death worldwide. The prevalence of diabetes has reached epidemic proportions. World Health Organization predicts that developing countries will bear the brunt of this epidemic in the present 21st century. Currently available treatments for diabetes are expensive and not easily accessible in developing countries such as India.
Therefore, WHO has recommended continuous search for new antidiabetic agents from plants and other natural resources [1]. Herbal products are gaining popularity in developing countries due to their lesser side effects and easy availability [1,2].
The potent antihyperglycemic activity of its leaf, root and bark extracts have been reported [10,11].

EXPERIMENTAL General experimental procedures
Spectrophotometric (Shimadzu UV 1800) evaluation of each compound was determined in MeOH and after addition of different shift reagents such as aluminum chloride (AlCl 3 ), AlCl 3 / hydrochloric acid (HCl), sodium acetate (CH 3 COONa), CH 3 COONa/ boric acid (H 3 BO 4 ) and sodium methoxide (NaOMe). IR spectra were recorded on a Jasco FTIR-4000 spectrometer in KBr pellets and are expressed in cm -1 . The 1 H and 13 C NMR spectra were obtained with a Brucker Avance 400 MHz spectrometer and chemical shifts (ppm) were related to tetramethylsilane (TMS, CH 3 ) 4 Si as internal standard. Elemental analysis was carried on CHNSO analyser (Thermofinnigan-Flash EA 1112 series). Electrospray ionization (ESI) mass spectra were recorded on Micromass Quattro II while melting point was determined by differential scanning calorimeter (DSC 60, Shimadzu, Japan). Open column chromatography was carried out on silica gel, Sephadex LH-20 and octadecylsilyl (ODS) (Amersham Pharmacia Biotech Co., UK) as packing material and Whatmann no. 1 filter paper and TLC on silica gel 60 F 254 sheets (Merck Co., Germany). All other chemicals used were of analytical reagent grade (Ranbaxy Fine Chemicals Ltd, India).

Plant material
The stem bark of B. spectabilis Willd

Extraction and isolation
Stem bark of B. spectabilis was air-dried under a shade and pulverized in electric grinder. The powdered bark (1.2 kg) was soaked in methanol, placed on a shaker for 24 h, filtered and concentrated at 45 ˚C. The weight of the crude extract obtained was 122.5 g (≈10 %w/w yield). Phytochemical investigations of stem bark extract was carried out for the presence of alkaloids, flavonoids, tannins, glycosides, anthraquinones, saponins, reducing sugars, triterpenes and steroids according to standard quantitative and qualitative methods [12].
The MeOH extract (100 g) was suspended in H 2 O (1 L) and extracted with hexane (1 L ×3) to give a hexane-soluble fraction (16 g). The aqueous layer was further extracted with CH 2 Cl 2 (1 L ×3), EtOAc (1 L ×3), and BuOH (1 L ×3) successively. The yield of ethyl acetate fraction was 67 g which was higher than that of the other fractions, and hence was selected for column chromatography.
The column was packed with silica gel and eluted with a gradient of CHCl 3 : MeOH, and H 2 O to give five fractions, A -E, with yields of 1.2, 7.9, 15.4, 1.8 and, 0.9 g, respectively. Repeated column chromatography of fractions B and C afforded the compounds 1-4 ( Fig. 1). The purity of the compounds was determined by TLC plates using iodine vapors as visualizing agent.

RESULTS
The stem bark extract showed the presence of glycosides, saponins, alkaloids, steroids and tannins. The compounds (1-4) were isolated by column chromatography of the EtOAc fractions

DISCUSSION
Compound 1 was isolated as a white powder having m.p. of 182 -185 ˚C with a molecular formula of C 7 H 14 O 6 based on mass spectral (m/z= 194) and 13 C NMR data. 1 H and 13 C NMR spectra showed signals for pinitol. The 1 H NMR spectrum exhibited two doublets at δ 3.92 and 3.78 (2.4 Hz each) for H-1 and H-5. The presence of one doublet at δ 3.72 (9.2 Hz) for H-2 and two triplet at δ 3.62 and 3.29 (9.6 Hz each) were ascribed to H-4 and H-3, respectively. One singlet at δ 3.65 indicates protons of OMe group. 13 C NMR spectrum showed signals for 7 carbons including oxygenated carbon at δ 60.75 (OMe), 72.04 (C-4), 72.56 (C-2), 73.47 (C-6), 73.76 (C-3), 74.33(C-5), and 85.93 (C-1). The spectral data obtained were identical to pinitol previously described in the literature. . The UV spectrum of the ethanol solution of 3 exhibited two major absorption bands at 374 nm and 256 nm, which confirmed the flavonol structure. Degradation of 3 in the presence of MeONa and hypsochromic shifts with AlCl 3 /HCl and AcONa/H 3 BO 4 supported the presence of 3, 3, 4' trihydroxy system. Bathochromic shifts with AcONa are related to 7-hydroxyl and the bathochromic shift with AlCl 3 /HCl to 5-hydroxyl. MS spectrum showed molecular ion peak at m/z 302 which was also observed as a base peak which is a characteristic feature of flavones. The other prominent peaks were observed at 285, 274, 257, 245, 229, 217, 200, 153, 137 and 69. 1 H NMR of this compound showed five signals for methyne groups at δ 6.25, 6.43, 7.65, 6.89, and 7.74. The 13 C NMR spectrum showed 15 signals which were assigned as five methyne and ten quaternary carbons. The spectral data obtained were identical to those of quercetin as previously described in the literature.