Abstract: Ten compounds were isolated from the CH2Cl2/MeOH (1/1) stem bark extract of Piptadenia africana, a western Cameroonian plant species. These compounds included three new lactone derivatives piptadenol A-C. The known compounds include 5,6-dimethoxy-7-hydroxyflavone, antiquol B, sistosterol, β-amyrine, betulinic acid, cholesterol and 24(S)-stigmat-5,22-dien-3β-O-glucopyranoside. The latter was found to be highly promising α-glucosidase inhibitor. These compounds were characterized using physical and spectroscopic methods. The plant extract and 24(S)-stigmat-5,22-dien-3 β-O glucopyranoside showed antibacterial activity.
INTRODUCTION
The genus Piptadenia or Piptadeniastrum belongs to the family Leguminoseae. This genus possesses several species including: P. peregrina, P. macrocarpa, P. excelsus, P. rigida, P. vidiflora, P. paraguayensi and P. Africana (Hook.f). Brenan. (Letouzey, 1969). These species are distributed in tropical Africa, from Senegal to Angola and across the Congo region to Uganda (Letouzey, 1969). Only the last species is found in Cameroon (Letouzey, 1985). P. africana is a tree, the leaves are very small and numerous, contiguous, about 3-8 mm long and 0.8-1.5 mm broad, fruits are broadly linear, elongated, 17-36 cm long and 2-3 cm broad. They are found in mixed deciduous and evergreen forest, it often stands as a single tree on farmlands (Letouzey, 1969). The leaves and fruits are used by traditional healers for aphrodisiac, tonic, enema urethritis and abortificient effect (Iwu, 1993); while the stems bark are use for gastric pain, fever and caught by local population of Noun division, Cameroon. Previous investigation on several species of this genus yielded mainly alkaloids. Phytochemical study of the seeds of P. peregrina yielded bufotenine (Stromberg, 1954), work on P. macrocarpa and P. excelsa revealed the presence of the base from tryptamine type (Iacobucci and Ruveda, 1964) which was absent in P. rigida, P. vidiflora and P. paraguayensis (Fellows and Bell, 1971). P. africana was suggested not to contain alkaloids (Paris et al., 1967). In the course of our phytochemical and pharmacological study on this species (Mbouangouere et al., 2007), we carried out the investigation of the stem bark of Piptadenia africana (Hook. f.) and we report herein the isolation and characterisation, of three new compounds 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). Glucosidase inhibitors are of considerable current interest in view of potential aspects in the treatment of AIDS (Acquired Immunodeficiency Syndrum), because of its anti-HIV (Human Immunodeficiency Virus) activity shown by the natural competitive inhibitors deoxynojirimycin (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-glucosidase 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 work in 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.
MATERIALS AND METHODS
General
IR spectra were recorded on a Nicolet Magma 750 spectrophotometer with KBr
disc and CHCl3. 1H-NMR spectra were recorded on advance
300 and 500 MHZ; while 13C-NMR spectra were recorded on 100 MHZ.
Chemical shifts are given in δ (ppm) with TMS as reference. NMR was taken
in pyridine and CDCl3. EIMS and HRMS were recorded on a JEOL MS Route.
Silica gel (70-32, 230-400 mesh) (Merck) was used for column chromatography.
Melting points were recorded in BUCHI 535 melting points apparatus. GC-MS (Masslab
Trio 1000), column DB5-MS, 60 m, 0.25 mm internal diameter, 0.25 μm film
thickness; injector, 325°C; initial oven temperature, 100°C (5 mn),
10°C/mn rate up to 240°C, 3°C/mn rate up to 300°C, 1°C/mn
rate up to 325°C (10 mn).
Plant Material
The stem bark of Piptadenia africana was collected at Matachouom,
Noun Division, Western province of Cameroon in February 2005. Plant identification
was performed by Dr. Onana, National Herbarium, Yaoundé Cameroon where
a voucher specimen (N°5000/12115/HNC) has been deposited. This work was
conducted in the H.E.J. Research Institute of Chemistry, (International Center
for Chemical Science), University of Karachi, Pakistan from April to September
2005.
Extraction and Isolation
The air-dried and powdered plant material (3 kg) was macerated in a mixture
of dichloromethane and methanol (1:1) for 3 days. Removal of the solvent with
rotary evaporator provided an organic extract (250 g). This extract was dissolved
in methanol and re-extracted with petroleum ether to yield extract A (23 g).
The residu was then concentrated and dissolved in water. This water part was
partitioned with dichloromethane and ethyl acetate to yield extract B (22 g)
and C (75 g). Extract A was concentrated and subjected to column chromatography,
using a mixture of n-hexane-chloroform as eluent. Fractions of 200 mL each were
collected and combined on the basis of their TLC profiles. The fraction eluted
with n-hexane-chloroform (7:3) (450 mg) was further purified by column chromatography
with silica gel (70-32 mesh) to yield compound 3 (4 mg) and antiquol (5 mg).
Extract B was concentrated and subjected to column chromatography, using a mixture
of n-hexane-acetone with increasing polarity as eluent. Fractions of 200 mL
each were collected and combined on the basis of their TLC profiles. The fraction
eluted with n-hexane-acetone (8:2) (410 mg) was further purified by column chromatography
with silica gel (70-32 mesh) to yield compound 2 (4 mg) and cholesterol (6 mg).
Extract C was concentrated and subjected to column chromatography, using a mixture
of n-hexane-acetone with increasing polarity. Fractions of 500 mL each were
collected and combined on the basis of their TLC profiles. The main fraction
eluted with n-hexane-acetone (8:2) (350 mg) was further purified by column chromatography
on silica gel (70-32 mesh) to yield betulenic acid (6 mg), 5,6-dimethoxy-7-hydroxyflavone
(4 mg), and β-amyrin (13 mg). The combined fractions (800 mg) eluted with
n-hexane-acetone (1:1) revealed the presence of sitosterol. Further purification
of this fraction by chromatography on silica gel column with dichloromethane-acetone
as eluent afforded sitosterol (30 mg) and compound 1 (6 mg). Fraction eluted
with n-hexane-acetone (7:3) (22 mg) yielded 24(S)-stigmat-5,22-dien-3β-O-glucopyranoside
4 (18 mg).
Piptadenol A 1 (27-Hydroxymethyl-25-Oxaheptacosan-27,1-Olide)
Amorphous solid, crystallized in methanol mp: 205°C (uncorrected). [α]27D
+1.6°(c 0.06, CHCl3/MeOH). IR (υKBrmax
cm-1): 3342, 1738 cm-1; EIMS m/z 440, 382, 339 307, 146,
116, 101, 58; HREIMS found m/z 440.7014 (calc. 440.7038 for C27H52O4).
13C and 1H NMR see Table 1 and 2,
respectively.
Piptadenol B 2 (29-Hydroxymethyl-27-Oxanonacosan-29,1-Olide)
Amorphous solid, mp: 196°C (uncorrected), [α]27D
+2.6°(c 0.01, CHCl3/MeOH). IR (υKBrmax
cm-1): 3347, 1730 cm-1; EIMS m/z 468, 440, 366, 348, 185,
98; HREIMS found m/z 468.7531, (calc. 468.7574 for C29H56O4);
13C and 1H NMR see Table 1 and 2,
respectively.
| Table 1: | 1H-NMR data of compounds 1, 2 (in pyridine 300 MHZ) and 3 in CDCl3 (500 MHZ) |
| δ: Chemical shift, m: Multiplet, t: Triplet, dd: Doublet of doublet, J: Coupling constant, br: Broad, Hz: Hertz | |
| Table 2: | 13C-NMR data of compounds 1, 2 (in pyridine) and 3 (in CDCl3) (100 MHZ) |
Piptadenol C 3 (2’-Hydroxylethyl 4-Oxo-Tetracontanoate)
White solid, crystallized from acetone, mp: 73°C (uncorrected). [α]27D
+0.24°(c 0.01, CHCl3/MeOH). IR (υKBrmax
cm-1): 3324, 2917, 2849, 1730, 1463); EIMS m/z 748, 720, 157, 143,
113, 95, 71, 57; HREIMS found m/z 749.2921 (calc. 749.2934 for C49H96O4).
13C and 1H NMR see Table 1 and 2,
respectively.
Alkaline hydrolysis of 3: To 5 mL of 15 m mol L-1 NaOCH3, prepared by dissolving metallic sodium in dry methanol, 2 mg of 3 was added. The mixture was kept under reflux for 1 h, before water was added and the organic compounds extracted with CH2Cl2. The mixture was analysed by GC-MS.
Microorganisms
The microorganisms used in this study included six clinical isolates of
bacteria: Bacillus subtilis, Escherichia coli, Shigella flexneri,
Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi
collected from the Dr Panjwani Center for Molecular Medecine and drug Research,
University of Karachi in Pakistan. The bacterial isolates were grown at 37°C
and maintained on nutrient agar Slant.
Antibacterial Activity
The preliminary screening test was performed by the agar well diffusion
technique as per Atta-ur-Rahman et al. (2001) with slight modifications.
Stock solution of the extracts (crude dichloromethane/methanol (1/1) extract
and one pure compound) were prepared in 5% v/v aqueous Dimethyl Sulfoxyde (DMSO)
at concentrations of 3 mg mL-1 (for crude extract and 1 mg mL-1
for pure compound) resulting in homogeneous solution of each one. The inocula
of micro-organisms were prepared from 24 h old broth cultures. The absorbance
was read at 530 nm and adjusted with sterile distilled water to match that of
a 0.5 Mc Farland Standard solution from the prepared stock solutions. Other
dilutions with sterile distilled water were prepared to give a final concentration
of 106 cfu mL-1. Bottles containing 19.8 mL of sterile
Mueller Hinton Agar (MHA) were maintained in a steam bath set at 40°C to
prevent solidification and then inoculated aseptically with 200 μL of bacteria
suspension followed by thorough mixing. Sterile Petri dishes (diameter, 90 mm)
were filled to 20 mL final of each bottle to give a solid plate. Wells of 6
mm of diameter were bored aseptically on the solid plates and 10 μL of
molten MHA were previously introduced into each well to ceal the bottom of the
well. After this, 50 μL of stock solution of extracts (crude extract or
pure compound) were finally introduced into each well and allowed for 2 h at
+ 4°C for extract to diffuse. The Petri dishes were then incubated at 37°C
for 24 h. The final disc charges were 150 μg (for crude extract) and 50
μg (for pure compound) per well. The antibacterial activity was recorded
by measuring the clear zone of growth inhibition on agar surface around the
well. All the experiments were carried out in triplicate. Imipenum (reference
drug) at 50 μg per well was used as positive control and 5% v/v aqueous
DMSO as a negative control.
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 nitrophenyl produced by the hydrolysis
of the substrate p-nitrophenyl α-D-glucopyranoside (PNP-G) (0.7 mM) and
500 milli units/mL of the enzyme used. 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, which
contains 50 mM sodium phosphate and 100 mM NaCl was used. 1-Deoxynojirimycin
(0.425 mM) and Acarbose (0.78 mM) were used as positive controls.
RESULTS AND DISCUSSION
The CH2Cl2/MeOH (1/1) extract of the stem bark of P. africana was defatted with petroleum ether followed by extraction with dichloromethane and ethyl acetate. Purification of these fractions afforded ten compounds. Piptadenol A (1), piptadenol B (2), piptadenol C (3), 24(S)-stigmat-5,22-dien-3β-O-glucopyranoside (4) in addition to 5,6-dimethoxy-7-hydroxyflavone, antiquol B, sistosterol, β-amyrine, betulenic acid and cholesterol. Compound 1 was obtained as amorphous solid from methanol mp: 205°C. It molecular formula was deduced to be C27H52O4 on the basis of a molecular ion peak [M+] at m/z 440, from EIMS analysis. This was in agreement with the HREIMS analyses where the molecular ion was observed at m/z 440.7014. This accounts for two unsaturations including a carbonyl and a ring. Additionally, fragments could be observed at m/z 382[M-H2O]+, 339[382-CH3CO]+ and 58[HOCH2CHCH2]+. The IR spectrum of compound 1 showed the presence of a carbonyl group at 1738 cm-1, hydroxyl group at 3342 cm-1. The 1H-NMR spectrum (Table 1) revealed the presence of seven hydrogen atoms geminal to oxygen atom, at δ 3.80 (2H, t, J = 6.5, H-24), 4.10 (2H, d, J = 5.4, H-2’), 4.60 (1H, dd, J = 6.3, 11.8, H-3’a), 4.70 (1H, dd, J = 6.2, 12.4, H-3’b) and the multiplet at δ 4.40 (1H, m, H-1’). Moreover we could easily observe distinguish methylene proton at δ 2.30 (2H, t, J = 7.4, H-2), downfield because of the neighbouring carbonyl (Plasman et al., 1999). Furthermore other methylene protons were observed at δ 1.70 (2H, m, H-4), 1.60 (2H, m, H-3) and 1.50 (2H, m, H-23). A broad multiplet was observed between 1.30 and 1.40 ppm integrating for thirty six cyclic methylene protons. These data were close to those of macrolactone molecules (Plasman et al., 1999). The 13C-NMR spectrum (Table2) confirmed this suggestion and showed no methyl carbon. Analysis of these data with the help of DEPT 90 and 135 lead to their classification into a carbonyl carbon at δ 173.7 (C-1), four carbon connected to oxygen atom at δ 70.9 (C-1’), 66.7 (C-3’), 64.3 (C-2’) and 62.1 (C-24). In addition eighteen signals were found from δ 26.5 to δ 29.9 accounting for long chain methylene carbon. The carbon at δ 34.4 (C-2) and 33.8 (C-4) were deduced to be those of the shielded methylene proton at δ 2.30 and 1.70 respectively from HMQC experiments. COSY 1H-1H spectrum (Fig. 1) shows correlation between H-3 and H-2, in the other hand H-23 showed direct correlation with H-22, H-3’a/b and H-27. Analogously HMBC (Fig. 1) experiment showed correlation of H-2 with C-1and C-4, H-3 with C-1, C-2 and C-4, H-3’ with C-1’, C-2’. By comparing all these data with those of similar compound (Plasman et al., 1999), structure of 1 was assigned. The compound is a new natural product, trivially named Piptadenol A.
Compound 2 was obtained as amorphous solid from methanol mp: 196°C. The molecular formula of Compound 2 was deduced to be C29H56O4 on the basis of a molecular ion peak at m/z 468 as shown by the EIMS. This was in agreement with the HREIMS analyses where the molecular ion was observed at m/z 468.7531. The mass spectra showed ion peaks at m/z 468[M]+, 440[M-2CH2]+, 366[M-HOCH2CHCH2O]+, 348[366-H2O]+.
| Fig. 1: | Structures of the isolated |
The IR showed the carbonyl group at μ 1730 cm-1, one hydroxyl group at 3347 cm-1. The 1H-NMR spectrum (Table 1) revealed four groups of proton geminal to oxygen at 3.80, (2H, t, 6.5, H-26), 4.10 (2H, d, 5.4, H-2’), 4.38 (1H, m, H-1’), 4.60 (1H, dd, 6.2, 11.8, H-3’a) and 4.70 (1H, dd, 6.2, 12.4, H-3’b). A down field methylene proton was found at δ 2.30 (2H, t, 7.4, H-2). Three methylene protons could be observed at δ 1.70 (2H, m, H-4), 1.60 (2H, m, H-3), 1.50 (2H, m, H-25). A broad multiplet at δ 1.10-1.30 was attributed to fourthy cyclic methylene protons. No methyl signal was found in the spectra. The 13C-NMR spectrum (Table 2) revealed the presence of a carbonyl at δ 173.8 (C-1). In addition, four carbons atoms connected to oxygen were found at δ 70.9 (C-1’), δ 66.8 (C-3’), δ 64.3 (C-2’) and δ 62.2 (C-26). Twenty signals were found between 26.5-29.9 ppm for cyclic methylene carbons. No methyl carbon was observed. These data suggested that compound 2 has 2 more methylene groups than compound 1. From the COSY experiment, correlation between H-3 and H-2 were observed, furthermore H-25 showed direct correlation with H-24. Analogously HMBC experiment showed correlation of H-2 with C-1, C-3 and C-4, H-3 with C-1, C-2 and C-4, H-3’ showed correlation with C-1’ and C-2’. Thus, compound 2 was assigned structure. Compound 2 is a new natural product and was trivially named Piptadenol B.
Compound 3 was isolated as white solid, mp: 73°C. Its cross formula was deduced to be C49H96O4 as observed from a molecular ion peak at m/z 749[M+H]+ on the EIMS and confirmed by the HRMS analyses. This molecular formula accounts for two unsaturations and equivalent to two carbonyl groups from the 13C-NMR spectra. In the EIMS spectrum, fragments at m/z 720[M-2CH2]+ and 57[OCH2CHOH]+ were observed. In the other hand, a large number of fragment ions were observed exhibiting a uniform difference of 14 mass units, thus suggesting aliphatic long chain. More intense clusters of peaks corresponding to CnH2n-1 (m/z 83, 97, 115, 125, 139 and 153), in comparison to those corresponding to CnH 2n+1 (m/z 85, 99, 113, 127, 141 and 155) also supported the acyclic olefinic nature of the compound (Silverstein and Basler, 1967). The 1H-NMR of the compound (Table 1) revealed six groups of protons. Four methylene protons geminal to oxygen at δ 4.10 (2H, br (t), H-2’) and 4.15 (2H, br (t), H-1’). Two deshielded protons at δ 2.30 (2H, t, 7.0, H-3). Other distinguish methylene protons were found at δ 1.60 (2H, br(t), H-2) and 1.20 (2H, br(t), H-5). The broad multiplet at δ 1.22-1.30 (66 H) was attributed to a long chain methylene protons. The methyl proton appeared at δ 0.80 (3H, t, 6.3, H-47). The 13C-NMR data of 3 (Table 2) determined from DEPT (45, 90 and 135) revealed the presence of two carbonyl carbons at δ 173.9 (C-1) and 177.4 (C-4), two carbons attached to oxygen at δ 68.4 (C-2’) and 65.0 (C-1’). A broad band at δ 29.6 was attributed to fourthy methylene carbons C-6-C-38. Moreover the COSY experiment showed correlations between H-1’and H-2’, H-2 and H-3. There was also correlation between H-5 and H-6. The HMBC spectrum exhibited correlation between H-2 and C-1, C-3 and C-4. H-3 showed correlation with C-2, C-4, C-5 and C-1. H-1’ showed correlation with C-1. By comparing these data with those previously published (Misra et al., 1991) compound 3 was assigned structure, has shown in Fig. 2 which is a new natural product trivially named piptadenol C.
| Fig. 2: | Key HMBC and COSY correlations in compounds 1-3 |
The structure of 3 was further confirmed by the GC-MS analysis, which after alkaline hydrolysis yielded ethylene glycol and methylester heptatetracontanoic acid as the major products, based on the peaks at m/z 62 and 719, respectively Compound 4 was obtained as white solid and was identified as 24(S)-stigmat-5,22-dien-3β-O-glucopyranoside by comparison with the authentic specimen available in our laboratory. The known compounds 5,6-dimethoxy-7-hydroxyflavone (Buschi et al., 1981), antiquol B (Mohan et al., 1990), sistosterol, β-amyrin, betulinic acid (Siddiqui et al., 1988) and cholesterol were identified by comparison of their (1H and 13C) NMR data with those reported in the literature and authentic specimen available in our laboratory.
The CH2Cl2-MeOH (1/1) extract of the stem bark of P. africana and 24(S)-stigmat-5,22-dien-3β-O-glucopyranoside were tested by Agar diffusion assay (Atta-ur-Rahman et al., 2001) against six species of bacteria comprises (Bacillus subtilis, Escherichia coli, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi). As result, good activity was observed with the extract against E. coli, P. aeruginosa and S. typhi; the compound 24(S)-stigmat-5,22-dien-3β-O-glucopyranoside showed potent activity against S. flexnari, S. aureus and S. typhi. This activity was low compared to that of reference drug on the same micro-organism. It was also low compared to other steroid such as 3-O-[beta-D-(6'-nonadeanoate)glucopyranosyl]-beta-sitosterol isolated from Arctotis arctotoide, while stigmaterol was not active (Sultana and Afavan, 2007). This indicated that the activity may be due to the aglycone instead of the basic squeleton.
The compound 24(S)-stigmat-5,22-dien-3β-O-glucopyranoside showed very potent and significant α-glucosidase inhibition compared with the standards drugs, while the other compounds were not tested due to their little amount.
CONCLUSIONS
The result from this work is of great interest: No alkaloid was found on the isolated as previously mentioned (Paris et al., 1967). The isolates included triterpenes, flovonoids ester derivatives and macrolactones. These isolated constituted the first report from this genus. This may constituted a chemotaxonomic marker for further investigations of plants species. The antibacterial activity showed by the extract as well as the α-glucosidase inhibition of the compound may confirmed that the plant could be use in this purpose.
ACKNOWLEDGMENTS
Mbouangouere R.N. is grateful to The H.E.J.R.I.C (International Center for Chemical Science) for the grant he provided to conduct this work and to COMSTECH (Organisation of Islamic Conference Standing Committee on Scientific and Technological Cooperation) for its financial support. Authors will like to thank Dr. Onana for plant identification and Mss. Samreen Hussein and Shamsun Khan for the biological activities.