Abstract: Pongamia pinnata (L.) Pierre (Fabacae) has been used in traditional medicine for treatment of diabetes. The aim of the research was to study the antihyperglycaemic activity of cycloart-23-ene-3β, 25-diol (code name compound B2) isolated by column chromatography method from stem bark of Pongamia pinnata in alloxan induced diabetic mice. The structure of compound B2 was elucidated by spectroscopical data. Diabetes was induced in mice by alloxan (80 mg kg-1, i.v.). Compound B2 was administered orally. Serum glucose level was determined at 0, 2, 4, 6 and 24 h. The onset was at 2nd h; peak effect at 6th h and the antihyperglycaemic effect was sustained until 24th h. Results obtained in the present study indicated antihyperglycaemic activity of cycloart-23-ene-3β,25-diol (B2).
INTRODUCTION
Pongamia pinnata (Linn.) Pierre (family Fabacae , synonym; P. glabra Vent., Derris indica (Lam.) Bennet, Cystisus pinnatus Lam.) popularly known as Karanj or Dittouri in Hindi and Indian beech, Pongam oil tree, Hongay oil tree in English (Krishnamurthi, 1998). It is a handsome flowering tree with drooping branches, having shining green leaves laden with lilac or pinkish white flowers and greyish green or brown bark (Joy et al., 1998). Different parts of the plant have been used in traditional medicine for the treatment of tumors, piles, skin diseases, wounds, bronchitis, whooping cough, rheumatic joints, ulcers and quench dipsia in diabetes (Kirtikar and Basu, 1987). Flowers were prescribed in glycosuria and remedy for diabetes (Chatterjee, 1992; Krishnamurthi, 1998). The traditional practitioners of Indian system of medicine. Ayurveda and Siddha boil the flowers of plant in water, cool and administer the decoction including marc for treatment of diabetes. Bark is useful as anthelmintic, elexteric and used for treatment of hemorrhoids, beriberi, ophthalmopathy, vaginopathy and diabetes (Joy et al., 1998). The phytochemicals like flavonoids, alkaloids, triterpenoids reported in flowers are also present in the bark (Asolkar et al., 1992).
Previous phytochemical investigation of this plant indicated the presence of pongamone A-E (Li et al., 2006); isopongaglabol, pongaflavonol (Yin et al., 2006a); dihydropyranoflavones (Yin et al., 2006b); lanceolatin B (Alam, 2004); pongaflavone, karanjin, pongapin, pongachromene, 3,7-dimethoxy-3’, 4’-methylenedioxy flavone, millettocalyxin C; 3,3’,4’, 7-tetramethoxyflavone (Yin et al., 2004); pyranochalcones, β-sitosterol, steroids, terpenoids, triterpenes, volatile oils (Carcache-Blanco et al., 2003); two triterpenes i.e., cycloart-23-ene-3β, 25 diol and friedelin; dipeptide aurantinamide acetate (Joy et al., 1998; Chatterjee, 1992); flavonoids, furanoflavones (Tanaka et al., 1992); glabrachalcone (Pathak et al., 1983) isopongaglabol and 6-methoxyiso-pongaglabol (Talapatra et al., 1982). Pongamol and karanjin isolated from fruits of P. pinnata were reported to have antihyperglycaemic activity (Tamrakar et al., 2008).
Recently, we have reported the antihyperglycaemic activity of alcoholic (Badole and Bodhankar, 2008) and petroleum ether extract (Badole and Bodhankar, 2009) of P. pinnata (L.) in alloxan-induced diabetic mice and increased oral glucose tolerance in non-diabetic as well as diabetic mice. Maximum antihyperglycaemic activity was observed in petroleum ether extract (25, 50, 100, 200 and 400 mg kg-1, p.o.) compared to alcoholic extract (100, 200 and 400 mg kg-1, p.o.). LD50 of petroleum ether extract of P. pinnata was found to be more than 5000 mg kg-1 p.o., (Badole and Bodhankar, 2009). The objective of the present investigation was to isolate and determine structure of active antihyperglycaemic compound from stem bark of Pongamia pinnata. There was paucity of data on the nature and activity of compound B2 present the bark of Pongamia pinnata.
MATERIALS AND METHODS
General Experimental Procedure
IR spectra were recorded on a JASCO FT/IR-5300 infrared spectrophotometer.
The 1H-NMR and 13C-NMR spectra were recorded on a VARION,
Model: Mercury plus (England). ESITOF-MS were recorded on a Micromass (Water
U.K.) Model: Q-Tof micro (YA-105). HPTLC λmax spectra were recorded
on a Linomat IV, JASCO spectrophotometer. Silica gel (Spectrochem Pvt. Ltd.
India, 100-200 mesh) was used for column chromatography. Precoated silica gel
plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC.
Collection and Authentication of Plant
Pongamia pinnata (L.) Pierre bark was collected during May-June 2006
from hilly area of Bhandara, Bhandara District, Maharashtra State, India. The
plant was identified and authenticated at Agharkar Research Institute, Pune,
India and the voucher specimen was deposited at that Institute (voucher specimen
sample No. AHMA-23892).
Extraction and Isolation
Petroleum ether extract of stem bark of P. pinnata was prepared according
to previously reported method by Badole and Bodhankar (2009). Petroleum ether
extract (30 g) was subjected to column grade silica gel (1500 g) borosil glass
column chromatography (height, 120 cm; diameter, 7 cm) eluting with mobile phase
containing n-hexane: chloroform: ethyl acetate (8:2:2). Polarity of mobile phase
was increased by their polarity order and 11 fractions (50 mL each) were collected.
All fractions were analyzed by HPTLC and fractions showed similar compounds
were pooled together and labeled alphabetically A to K and tested for antihyperglycaemic
activity.
Only fraction B showed significant antihyperglycaemic effect hence fraction B was further processed for isolation by preparative TLC. From fraction B the compounds obtained and their quantity were B1 (8.4 mg), B2 (27.9 mg), B3 (10.5 mg), B4 (61.4 mg), B5 (9.9 mg), B6 (19.2 mg), B7 (20.2 mg), B8 (21.5 mg), B9 (25.5 mg), B10 (49.20 mg) and B11 (98.00 mg). The compounds from fraction B were further evaluated for antihyperglycaemic activity. The compound B2 showed significant antihyperglycaemic activity compared to remaining compounds. Hence compound B2 was further analyzed by HPTLC for determination of Rf and λmax. The chemical structure of isolated compound B2 was elucidated by FT-IR, 1H-NMR, 13C- NMR and ESITOF-MS spectroscopy.
B2 Compound
Yellow colored semisolid; HPTLC: silica gel 0.25 mm (mobile phase- n hexane:
chloroform: ethyl acetate, 8:2:2) Rf= 0.3, λmax:
340 nm; melting point 196-198°C (not corrected); IR λmax
(KBr) cm-1: 3441.32, 3040.23, 2930.14. 1H-NMR (400 MHZ,
CDCl3): δ 1.25-1.415 (16H, s, CH2-1, CH2-2,
CH2-6, CH2-7, CH2-11, CH2-12, CH2-15,
CH2-16), δ 1.33 (3H, s, CH-5, CH-8, CH-17), δ 3.5 (H, s,
CH-3), δ 0.86 (2H, m, CH2-19), δ 1.61-2.01 (6H, m, CH2-20,
CH2-21, CH2-22), δ 3.032 (2H, m, -CH-23, -CH-24),
δ 2 (2H, s, OH-3, OH-25), δ 1.04 (12H, s, CH3-18, CH3-26,
CH3-27, CH3-28), δ 0.97 (3H, s, CH-29), δ 0.81
(3H, s, CH-30); 13C NMR (400 MHZ, CDCl3): (Table
1); ESITOF-MS (positive mode) m/z : 443.38 [M +H]+ , 428 (M+-methyl),
410 (M+- methyl-H2O), 316.52 (M+-side chain)
[calc. for C30H50O2 442.38].
| Table 1: | 13C-NMR (400 MHZ in CDCl3) spectral data of compound B2 |
Preliminary Phytochemical Screening
The preliminary phytochemical analysis for compound B2 was carried out for
the alkaloid (Mayer’s, Hager’s, Dragendorff’s and Wagner’s
test), flavonoids (Shinoda test), triterpenes (Liberman-Burchard test) and volatile
oils (Kokate, 1991).
Drugs and Chemicals
Glyburide (Ranbaxy Pharma. Ltd., India), alloxan monohydrate (Spectrochem,
India), glucose estimation kit (glucose oxidase/peroxidase kit) (Accurex Biomedical
Pvt., Ltd., India), tween 80 (Research-Lab., India), petroleum ether, chloroform,
n-hexane, ethyl acetate, ethanol (Merck, India) of GR grade were purchased from
the respective vendors.
Animals and Research Protocol Approval
Swiss albino mice (25-30 g) of either sex were purchased from National Toxicology
Centre, Pune, India. Animals were maintained at a temperature of 25±1°C
and relative humidity of 45 to 55% under 12 h light: 12 h dark cycle. The animals
had free access to standard food pellets (Chakan Oil Mills, Pune, India) and
water was available ad libitum. The experimental protocol was approved
by the Institutional Animal Ethics Committee (IAEC) constituted in accordance
with the rules and guidelines of the Committee for the Purpose of Control and
Supervision on Experimental Animals (CPCSEA), India.
Induction of Diabetes
Diabetes was induced in Swiss albino mice by a single intravenous injection
of aqueous alloxan monohydrate (80 mg kg-1) solution and serum glucose
was determined. Mice showing serum glucose level above 300 mg dL-1
(diabetic) was selected for the study (Badole and Bodhankar, 2009).
Antihyperglycaemic Activity of Fractions A to K and Compounds B1 to B11
in Alloxan Induced Diabetic Mice
The selected non-fasted mice were divided into following groups (n = 6)
viz; Group I- vehicle (tween 80, 2%; 10 mL kg-1), Group II- standard
drug, glyburide (10 mg kg-1), Group III to Group XIII- Fractions
A to K (25 mg kg-1, p.o.) respectively. Serum glucose was determined
at 0, 2, 4, 6 and 24 h after fractions administration. Isolated compounds from
fraction B were selected further antihyperglycaemic study and processed by dividing
animals in following groups i.e. Group I- vehicle (tween 80, 2%; 10 mL kg-1),
Group II- glyburide (10 mg kg-1), Group III to Group XIII- B1 to
B11 (10 mg kg-1, p.o.), respectively.
Statistical Analysis
Data was expressed as Mean±SEM and statistical analysis was carried
out by one-way ANOVA with post hoc Tukey test performed using GraphPad InStat
version 3.00 for Windows VistaTM BASIC, GraphPad Software, San Diego
California USA. The p<0.05 was considered significant.
RESULTS AND DISCUSSION
Eleven fractions were labeled alphabetically A to K and evaluated for antihyperglycaemic activity. Only fraction B showed significant antihyperglycaemic activity (Table 2). Fraction B was further processed for preparative TLC. Eleven compounds were obtained and further tested for antihyperglycaemic activity. The compound B2 (Fig. 1) showed significant antihyperglycaemic activity compared to remaining compounds (Table 3).
Compound B2 was obtained as a yellow colored semisolid substance (27.9 mg).
The yield was 0.93%. Melting point of compound B2 was 196-198°C and showed
positive results in Lieberman-Buchard test for triterpenes. HPTLC of isolated
sample showed a single peak with its Rf value 0.3 and absorption
maxima (λmax) at 340 nm, which is a characteristic absorption
frequency of cycloart-23-ene-3β, 25-diol (Djerassi and McCrindle, 1962).
The IR spectrum showed bands at 3441.32 cm-1 indicated presence of
hydroxyl groups, cyclopropyl ring at 3040.23 cm-1 and aliphatic stretch
at 2930.14 cm-1. The 1H-NMR (CDCl3, 400 MHZ)
spectrum of B2 compound showed a two proton downfield signal at integral value
δ 2 which indicated presence of two chelated hydroxyls at the 3rd and 25th
positions. The presence of cyclopropyl ring was confirmed by the presence of
characteristic integral value δ 0.86 for non-equivalent protons at C-19.
The multiple integral value of δ 3.032 at C-23 and C-24 for the vinylic
two protons indicated the presence of double bond in the structure. 13C
NMR (CDCl3, 400 MHZ) spectrum revealed that double bond is present
between positions between C-23 at integral value δ 139.93 and C-24 at δ
124.34. The ESITOF-MS indicated a molecular ion with a large peak at [M+]
m/z 443.38 suggesting a possible formula of C30H50O2.
Further fragments were observed at, m/z 428 (M+- CH3),
410 (M+- CH3-H2O), 316 (M+- side
chain).
| Table 2: | Antihyperglycaemic activity of fractions (A to K) on serum glucose level in alloxan induced non-fasted diabetic mice |
| Values are expressed as Mean±SEM; n = 6 in each group; Statistical analysis by one-way ANOVA followed by post hoc Tukey’s test using Graphpad Instat software; *p<0.05; ***p<0.001 compared to vehicle treated group (Tween 80, 2%; 10 mL kg-1) | |
| Fig. 1: | Structure of cycloart-23-ene-3β, 25-diol (compound B2) |
| Table 3: | Antihyperglycaemic activity of compounds (B1 to B11) from fraction B on serum glucose level in alloxan induced non-fasted diabetic mice |
| Values are expressed as Mean±SEM, n = 6 in each group; Statistical analysis by one-way ANOVA followed by post hoc Tukey’s test using Graphpad Instat software; **p<0.01; ***p<0.001 compared to vehicle treated group (Tween 80, 2%; 10 mL kg-1) | |
The peak observed at m/z 316 indicated the loss of entire substituent chain at C-17 as well as revealed that the second hydroxyl group and double bond were present in the side chain. On the basis of physical properties and spectroscopic data like IR, 1H-NMR, 13C-NMR and ESITOF-MS the compound seems to be triterpene compound identical to previously reported cycloart-23-ene-3β, 25-diol (Djerassi and McCrindle, 1962; Teresa et al., 1987; Madureira et al., 2003).
Single dose administration of fraction B (25 mg kg-1, p.o.) as well as glyburide (10 mg kg-1, p.o.) significantly (p<0.001) reduced serum glucose level at 2nd, 4th and 6th h after administration. The reduction in serum glucose from basal value (before drug administration in each group) at 6th h after fraction B (25 mg kg-1) and glyburide (10 mg kg-1) was 267.27 and 180.80 mg dL-1, respectively (Table 2). The onset of antihyperglycaemic effect of fraction B (25 mg kg-1) and glyburide (10 mg kg-1) was observed at 2nd h; peak effect at 6th h but the antihyperglycaemic effect waned at 24th h (Table 2). The other fractions (A, C, D, E, F, G, H, I, J and K) did not show significant antihyperglycaemic activity (Table 2).
The fraction B was further fractionated into 11 compounds by preparative TLC. The isolated compound B2 (10 mg kg-1, p.o.) was administered in alloxan (80 mg kg-1, i.v.) induced diabetic mice. The reduction in serum glucose from basal value (before drug administration in each group) at 6th h after administration of compound B2 (10 mg kg-1, p.o.) and B8 (10 mg kg-1, p.o.) was 292.55 and 114.57 mg dL-1, respectively (Table 3). The onset of antihyperglycaemic effect of compound B2 and B8 was observed at 2nd h; peak effect at 6th h. The antihyperglycaemic effect of compound B2 (10 mg kg-1) was sustained at 24th h but the antihyperglycaemic effect of compound B8 (10 mg kg-1) waned at 24th h (Table 3). The other compounds (B1, B3, B4, B5, B6, B7, B9, B10 and B11) did not show significantly antihyperglycaemic activity (Table 3).
CONCLUSION
Compound B2 (cycloart-23-ene-3β, 25-diol) was isolated from the petroleum ether extract of stem bark of Pongamia pinnata. Compound B2 (cycloart-23-ene-3β, 25-diol) when administered orally to alloxan induced diabetic mice reduced the blood sugar. The onset was 2h, peak 6h and after which the antihyperglycaemic effect gradually waned. It is concluded that cycloart-23-ene-3β, 25-diol is the active antihyperglycaemic compound of stem bark of Pongamia pinnata. The precise site(s) and the molecular and cellular mechanism(s) of this compound remain to be investigated.
ACKNOWLEDGMENTS
The researchers would like to acknowledge Dr. S. S. Kadam, Vice-Chancellor and Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune, India, for providing necessary facilities to carry out the study. We are thankful to the All India Council of Technical and Education (AICTE), India for financial support by awarding National Doctoral Fellowship for the research work. The authors are thankful to Department of Chemistry, Indian Institute of Technology, Mumbai, India for providing 1H-NMR, 13C-NMR and ESITOFMS spectra of isolated compound of P. pinnata. We are especially thankful to Nilesh K. Wagh for helping for structure elucidation of B2 compound.