Piptadenol A-C and α-Glucosidase Inhibitor From Piptadenia africana
Roukayatou N. Mbouangouere,
Mohamed I. Choudhary,
Pierre C. Djemgou,
Bonaventure T. Ngadjui
Research Journal of Phytochemistry,
2008, 2(1), 27-34.
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.
Table 1 and 2,
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,
|| 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
|| 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,
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.
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.
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.220.127.116.11) enzyme inhibition assay has been performed
according to the slightly modified method of Matsui et al. (1996). α-Glucosidase
(E.C.18.104.22.168) 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-3a), 4.70 (1H, dd, J = 6.2, 12.4, H-3b) 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-3a/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]+,
|| 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-3a) and 4.70 (1H, dd, 6.2, 12.4, H-3b). 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-1and 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.
|| 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.
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.
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." class="btn btn-success" target="_blank">View Fulltext
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