Abstract: The dichloomethane/methanol extract of the roots of Anthocleista schweinfurthii Gilg. has provided a new steroid, schweinfurthiin 1, two known compounds, bauerenone 2 and bauerenol 3 which were found to be highly promising α-glucosidase inhibitors. Along with these, two known xanthones, 1-hydroxy-3, 7, 8-trimethoxyxanthone 4 and 1, 8-dihydroxy-3, 7-dimethoxyxanthone 5 were also isolated.
INTRODUCTION
Anthocleista (Gentianaceae) is a genus of trees which grow in the tropical rain forest areas of Africa. These plants are used in traditional medicine for the treatment of various diseases. A. djalonensis is known for its antipyretic, analgesic and purgative actions (Keay et al., 1964). On the other hand A. grandiflora is known for its antimalarial activity (Watt and Breyer, 1962). Most importantly, the roots decoction of A. djalonensis and related species such as A. vogelli and A. kerstingii, have been used in the treatment of diabetes mellitus. Herbalists claim a high percentage of cures in their diabetic patients, treated with plants of genus Anthocleista (Amofo, 1977) A. schweinfurthii is used in Matachouom, Cameroon, to treat diabetes, malaria and stomach diseases. In order to establis the link between the traditional use and the chemical constituents of this species, we carried out the phytochemical and pharmacological investigation of A. schweinfurthii and we report here in the isolation and structure determination of a new steroid 1, four known compounds including two α-glucosidase inhibitors 2, 3 and two xanthones 4 and 5 from this natural source.
Glucosidases have drawn the attention of the scientific community for its wide role in the living biological systems. Glucosidases are involved in several biological processes, intestinal digestion, the biosynthesis of glycoproteins and the lyposomal catabolism of the glycoconjugates (Asano et al., 1997). Glucosidases inhibitors are of considerable current interest in view of potential aspects in the treatment of the AIDS (Acquired Immunodeficiency Virus), glucosidase inhibitors are of current considerable interest, because of its anti-HIV (Human Immunodeficiency Virus) activity shown by the natural competitive inhibitors nojirimycin (Josie et al., 1992). Intestinal α-glucosidases are involved in the final step of the carbohydrates digestion and convert them into monosaccharides, which are absorbed from the intestine thus its inhibitors could therefore suppress the postprandial hyperglycemia and can be used for the treatment of the type II diabetes (Sou et al., 2000). α-Glucosidases inhibitors have been also used as inhibitors of tumor metastasis, antiobesity drugs, fungistatic compounds, insects antifeedants, antiviral and immune modulators (El Ashry et al., 2000).
We have focused to study on the discovery of effective inhibitors because of the multi dimensional scope of this enzyme. The acarbose is a very widely prescribed drug in the management of the type II diabetes.
Compounds 2 and 3 were found to be highly promising α-glucosidase inhibitors when compared with the standard inhibitors deoxynijorimicin and acarbose in the studies.
MATERIALS AND METHODS
General
This study was conducted in the HEJ Research Institute of Chemistry, University
of Karachi, Pakistan from may to September 2005. IR Spectra were recorded on
a Nicolet Magma 750 spectrophotometer as KBr disc. Optical rotation was determined
on a JASCO DIP-181 polarimeter. 1H-NMR spectra were recorded on Avance
400 MHZ Bruker NMR machine, while 13C-NMR spectra were recorded at
100 MHZ. Chemical shifts are given in δ (ppm), taking TMS as reference
and relative to the solvent used. NMR spectra were recorded in CDCl3.
EIMS and HREIMS were recorded on a JEOL HX110 MS. Silica gel (70-230 and 230-400
mesh) (Merck) was used for column chromatography. Melting points were recorded
on BUCHI 535 melting points apparatus.
Plant Material
The stem bark of Anthocleista schweinfurthii Gilg. was collected
from Dschang, Menoua Division, Western province of Cameroon, in February 2005.
Plant material was identified by Dr. Onana, National Herbarium, Yaoundé,
Cameroon and a voucher specimen No. WCS 2489b/26859/Ya. was deposited.
Extraction and Isolation
The air-dried and powdered plant material (800 g) was macerated in 4 L mixture
of dichloromethane and methanol (1:1) for 3 days. Removal of the solvent in
a rotary evaporator provided an organic extract (14 g). This extract was dissolved
in methanol (500 mL) and re-extracted with petroleum ether (1 L) to obtain fraction
A (700 mg). The resulting organic phase was then concentrated and dissolved
in water (500 mL). This aqueous part was extracted with dichloromethane (1 L),
ethyl acetate (1 L) and butanol (500 mL) to yield fractions B (6 g), C (2 g)
and D (5 g). Fraction B was concentrated and subjected to column chromatography
on silica gel (230-400 mesh), using a mixture of n-hexane-acetone, followed
by dichloromethane-acetone as eluent. Fractions of 100 mL each were collected
and combined on the basis of their TLC profiles. The fraction eluted with n-hexane-acetone,
(3:1) (500 mg) was further purified by column chromatography on silica gel (70-230
mesh) to yield bauerenone 2 (32 mg) and bauerenol 3 (16 mg). The combined fractions
(100 mg), eluted with n-hexane-acetone (95:5), was purified by column chromatography
on silica gel (70-230) with n-hexane-chloroform as eluent to yield schweinfurthiin
1 (18 mg). Fraction eluted with dichloromethane-acetone (9:1) (300 mg) was further
purified by column chromatography on silica gel (70-230 mesh) to yield 1-hydroxy
-3,7,8- trimethoxyxanthone 4 (8 mg) and 1,8-dihydroxy-3,7-dimethoxyxanthone
5 (6 mg).
Enzyme Inhibition Assay
α-Glucosidase (E.C.3.2.1.20) enzyme inhibition assay has been performed
according to the slightly modified method of Matsui et al. (1996). α-Glucosidase
(E.C.3.2.1.20) from Saccharomyces species, purchased from Wako Pure Chemical
Industries Ltd. (Wako 076-02841). The enzyme inhibition was measured spectrophotometrically
through continuous monitoring of the nitropenyl produced by the hydrolysis of
the substrate p-nitrophenyl α-D glucopyranoside (PNP-G) (0.7 mM) and 500
milli units mL-1 of the used enzyme. Whole enzymatic reaction was
performed at 37°C for 30 min. The increment in absorption at 400 nm, due
to the hydrolysis of PNP-G by α-glucosidase, was monitored continuously
on microplate spectrophotometer (Spectra Max, Molecular Devices, USA). Phosphate
saline buffer at pH 6.9 was used, which contains 50 mM sodium phosphate containing
100 mM NaCl. 1-Deoxynojirimycin (0.425 mM) and Acarbose (0.78 mM) were used
as positive controls.
Schweinfurthiin 1, (campest-5-en-3β-ol tridecanoate)
White crystals from acetone mp: 76-77°C (uncorrected).
Rf: 0.4 (Hex-EtOAc, 92:8).
(α)D27 = +2.4, c: 50, CHCl3
IR (KBr): 1720 cm-1.
1H and 13C NMR (in CDCl3 400 and 100 MHZ, respectively,
Table 1):
MS (EI, 70 eV): m/z (%): 393 (82), 241 (41) 229 (100), 129 (8), 71 (30);
HREIMS: (calculatded for C41H72O2). 597.0186
found: 597.0154.
Bauerenone 2
White crystals from acetone mp: 230-231°C
IR (KBr): 1712, 1470, 1390, 1380 cm-1; C30H48O,
MS (EI, 70 eV) m/z (%): 409 (11), 271 (13), 257 (17), 245 (100), 218 (12), 205
(22). Molecular formula C30H48O
13C- and 1H-NMR in agreement with literature.
Bauerenol 3
White crystals from acetone, mp: 192-193°C (uncorrected).
MS (EI, 70 eV) m/z (%): 411 (21), 393 (6), 273 (3), 259 (18), 247 (100), 229
(47), 207 (18). Molecular formula C30H50O.
13C-and 1H-NMR in agreement with literature.
Compound 1 was isolated as white crystals, mp: 76-77°C [αD]27
2.4, c: 50, CHCL3, The molecular formula was determined to be C41H72O2
from the High Resolution EI mass spectroscopy (HREIMS, m/z 597.0514). Mass fragments
at m/z 393, 229, 241, 129, 73 and 71 were characteristics of steroids (Diekman
and Djerassi, 1967).
| Table 1: | 1H- and 13C -NMR data of schweinfurthiin 1 recorded in CDCl3 |
| δ: Chemical shifft, m: Multiplet, d: Doublet, t: Triplet, s: Singlet, J: coupling constant, br: Broad, Hz: Hertz | |
Moreover several fragments were observed exhibiting a uniform difference of
14 mass units, revealing an aliphatic chain in the molecule (Misra et al.,
1991; Akihisa et al., 1989). This suggested that compound 1 was a steroid
with an aliphatic side chain. The IR spectrum of 1 showed carbonyl absorption
at 1720 cm-1. The 1H- NMR spectrum of 1 (Table
1) displayed signal dues to four tertiary methyl groups at δ 0.82 (Me-28),
0.93 (Me -26/27) and 1.02 (Me-21). Two singlets appeared at δ 0.75 and
0.97, corresponding to Me-18 and Me-19 respectively. A triplet at δ 0.87
was due to Me-13’ of the side chain while the other one at δ 2.29
was attributed to the methylene H-2’ protons. A broad signal at δ
1.23 was attributed to many methylene protons. H-6 appeared as a broad doublet
at δ 5.39 (J = 2.4 Hz). The doublet of doublets observed at δ 4.50
was attributed to C-3 proton characteristic of cholest-5-ene-3α H (Funel
et al., 2004; Goad, 1991; Thompson, 1972). The 13C- NMR spectrum
(Table 1) showed the carbonyl carbon signal at δ 173.6,
while C-3 resonated at δ 80.8, along with seven methyl carbons, 4 quaternary
carbons and 29 methylene carbons. In the HMBC spectrum, correlations of H-3
with C-1, C-2, C-4 and C-1´; H-2´and H-3´ with C-1´ and H-12´ with C-13´ were
observed (Fig. 1). Based on the data here described and on
comparison with data of similar compounds (Rösecke and Konig, 2000), compound
1 was assigned structure 1 trivially named schweinfurthiin. Lubsy et al.
(1984) has previously reported the same compound as a synthetic derivative from
crystallization of soybean sterols.
| Fig. 1: | Key HMBC correlations in compound 1 |
| Table 2: | The α-glucosidase inhibitory activity of compounds 1-4 |
| *Standard drug; NA: Not Active | |
They gave neither physical nor spectral data of the compound. To best of our knowledge, its isolation and complete characterization (physical and spectral) from a natural source is here reported for the first time. Thus, it is a new compound.
Compound 2 was isolated as white crystals, mp: 230-231°C and gave a positive Liberman Buchar test. The IR spectrum in KBr revealed the presence of carbonyl group (1712 cm-1); a double bond (1470 cm-1) and germinal dimethyl group (1380-1390 cm-1). The molecular formula of compound 2 was deduced to be C30H48O on the basis of a molecular ion peak at m/z 424.3795 as shown by the HREIMS. Mass fragments at m/z 409, 271, 257, 245, 218 and 205 were observed. The 1H-NMR spectrum in CDCl3 suggested the presence of six tertiary methyl proton each singlets at δ 0.92, 0.98, 1.01, 1.02, 1.02 and 1.09. Two secondary methyl groups were observed at δ 0.89 and 1.04 as doublets. These data suggested the compound in hand to be a pentacyclic triterpenoid. The multiplet at δ 5.45 was attributed to H-7 and the doublet doublet of doublets at δ 2.73, was attributed to one of the C-2 methylene proton. In the 13C-NMR spectrum of 2, 30 carbon signals were observed. The carbonyl carbon appeared at δ 216.9. The overall spectral behavior was identical to the reported compound, bauerenone 2 (Campello et al., 1975).
Compound 3 was obtained as white crystals, mp: 192-193°C. The EI mass spectrum showed the M+ at m/z 426 for C30H50O. Fragment ions at m/z 411, 393, 273, 259 and 207. The 1H-NMR spectrum of 3 exhibited eight methyl groups. In addition, the germinal proton to hydroxyl group was observed at δ 3.23. The hydroxyl bearing carbon appeared at δ 79.3. These spectroscopic data were identical to those reported by Khan et al. (1979) for bauerenol 3.
The compounds Bauerenone and Bauerenol showed approximately very similar IC50 values 9.8 and 5.8, respectively (Table 2). These results indicate that the basic skeleton of the compounds are responsible for the binding to the targeted enzymes, the action may be through some sorts of hydrophobic interaction with the binding sites of the enzyme.
ACKNOWLEDGMENTS
Mbouangouere R.N. is grateful to the Organisation of Islamic Conference Standing Committee on Scientific and Technological Cooperation (COMSTECH) and HEJ Research Institute of Chemistry (International Center for Chemical Science), for their financial support. Authors will like to thank Dr. Onana for plant identification.