Abstract: The aim of this study is to identify and characterize the bioactive principles from the woody stem of Abelmoschus manihot. It has wide folk medicinal use. For isolation of the compounds, the dried woody stem’s powder of Abelmoschus manihot was subjected to hot extraction with petroleum ether, this extract was saponified with alcoholic KOH and subjected to chromatography. Two compounds (PEA-2 and PEA-3) were isolated and purified by chloroform. Mass spectrum of PEA-2 and PEA-3 showed a parent molecular ion [M+] peak at mlz 412 which corresponds to the molecular formula C29H48O and 414 corresponds to C29H50O. In lH-NMR spectrum of PEA-2, H-3 proton appeared as a triplet of a double doublet (tdd) at S 3.62 and H-6 olefinic proton showed a multiplet at S 5.14. Two olefenic protons appeared downfield at S 4.16 (m) and S 4.14 (m) and in the 1H-NMR data of PEA-3, H-3 proton appeared at S 3.51 as a triplet of a double doublet with a J value of 4.5 and 1.1 MHZ and H-6 olefinic proton showed a multiplet at S 5.10. From the physical, chemical and spectral characteristics, PEA-2 and PEA-3 were concluded as stigmasterol and γ-sitosterol.
INTRODUCTION
Research studies leading to extraction, isolation and biological study of plant constituents have now formed the major field of study. Abelmoschus manihot, Malvaceae is a large annual erect hairy plant, 1.2-1.8 m high. It is Native to China, introduced into India, near Calcutta and in coastal areas of Maharashtra. The plants mucilage contains polysaccharides and proteins (Kiritikar and Basu, 1994). The flower contain quercetin-3-robinoside, quercetin-3’-glucoside, hyperin, myrecetin and anthocyanins. The saturated acids and liquid acids such as linoleic and oleic acids were isolated from the seed fat and unsaponifiable matters. The different chromatographic methods have been developed on the flavones present in the plant (Liang et al., 2007; Lai et al., 2006; Yi et al., 2008). The ethanol extract of flower was screened for antiviral activity and it was observed that the hyperoside shown significant anti HBV activity (Lin-Lin et al., 2007). The flavones present in the plant showed preventive effect in the injury (Liu et al., 2009; Wen and Chang, 2007). The leaves were tested on bone loss in ovarectomised rats and it was observed that it was able to prevent the ovariectomy-induced femoral osteopenia (Puel et al., 2005). The modulatory effect of Total Flavone of Abelmoschus manihot (TFA) on NMDA-activated current (INMDA) was investigated in cultured rat hippocampal neurons using the whole-cell patch-clamp technique. TFA rapidly and reversibly inhibited the INMDA in a concentration-dependent manner (Xin-Ping et al., 2006).
But these studies are not enough for identifying and characterizing the bioactive compounds in this plant. The purpose of this study is to identify and characterize the bioactive principles from the root bark of Abelmoschus manihot.
MATERIALS AND METHODS
Preparation of Plant Material
The woody stem of Abelmoschus manihot were collected during the
month of May-June, 2007 from the Toranmal Hills of Maharashtra. The plant was
taxonomically identified by Professor Dr. D.A. Patil, HOD Botany Dept, SSVPS
College, Dhule, North Maharashtra University, Jalgaon. The dried woody stem
powder (4 kg) was subjected to hot extraction with petroleum ether by Soxhlet
extractor and after evaporation of the solvent 26 g extract was found. The extract
then saponified with alcoholic KOH, to remove fatty material yields 12 g of
unsaponified matter.
Isolation and Purification of Compounds
A small quantity of unsaponifiable matter was dissolved in chloroform and
this solution was spotted on TLC plates. Then the TLC plates were run by specific
solvent system and were viewed individually under UV light and also with the
vanillin-H2SO4, reagent. Through several pilot experiments,
it was found that the compounds of unsaponifiable fraction were separated by
the solvent system of petroleum ether and ethyl acetate in the proportion of
7:3. The unsaponifiable fraction, 8 g, was subjected to column chromatography
on a silica gel (60-120 mesh) with gradient elution using petroleum ether: ethyl
acetate (Stahl, 1969).
Two fractions were found homogeneous on TLC plate by using petroleum ether: ethyl acetate (7:3), petroleum ether: chloroform (10:1), n hexane: ethyl acetate (9:1) and petroleum ether: methanol (9:1) solvent systems. These fractions were crystallized (Bahl and Bahl, 1992) and named as PEA-2 (Pet Ether Abelmoschus-2) and PEA-3 (Pet Ether Abelmoschus-3), respectively.
Test for Alcohol
Four gram of ceric ammonium nitrate was dissolved in 10 mL of 2 N HNO3,
on mild heating. A few crystals of PEA-2 and PEA-3 were dissolved in 0.5 mL
of dioxane. The solution was added to 0.5 mL of ceric ammonium nitrate reagent
and diluted to 1 mL with dioxane and shaken well. Both PEA-2 and PEA-3 developed
yellow to red color indicating the presence of an alcoholic hydroxyl group (Harborne,
1998).
Test for Steroid
Salkowski Reaction: A few crystals of PEA-2 and PEA-3 were dissolved in
chloroform and a few drops of concentrated sulfuric acid were added to the solution.
For both PEA-2 and PEA-3, a reddish color was seen in the upper chloroform layer
(Harborne, 1998).
Liebermann-Burchard Reaction
A few crystals of PEA-2 and PEA-3 were dissolved in chloroform and a few
drops of concentrated sulfuric acid were added to it followed by the addition
of 2-3 drops of acetic anhydride. Solution for both PEA-2 and PEA-3 turned violet
blue and finally green (Harborne, 1998).
Spectroscopic Characterization
Different spectroscopic methods were used to elucidate the structure of
PEA-2 and PEA-3. Among the spectroscopic techniques IR, 'H-NMR and GMS were
carried out. The infra red spectrum was recorded on FTIR 8400 s (Shimadzu) at
NMU, Jalgaon, MS, India. 1HNMR spectra were recorded on a Varian-500
MHZ NMR spectrometer (Shimadzu) at Cadila Pharmaceuticals, Ahmedabad, India.
The 1HNMR spectra were recorded using CDC13, as solvent
with tetramethylsilane (TMS) as an internal standard. Mass spectrum was recorded
at high resolution on a mass spectrometer (Perkin Elmer Autosystem XL with Turbomass)
at Sophisticated Instrumentation Centre for Applied Research and Technology,
Anand, Gujarat, India and the data are given in mlz values.
RESULTS
From the positive tests for steroids and alcohols given by the PEA-2 and PEA-3, they were assumed to be a sterol. The melting point of PEA-2 and PEA-3 were 176 and 137°C. The UV ëmax value of PEA-2 and PEA-3 is 257 and 251 nm, respectively. Mass spectrum of PEA-2 and PEA-3 showed a parent molecular ion (M+) peak at mlz 412 and 414, respectively which corresponds to the molecular formula C29H48O (Fig. 1) and C29H50O (Fig. 2).
| Fig. 1: | The chemical structure of PEA-2 (Stigmasterol) |
| Fig. 2: | The chemical structure of PEA-3 (γ-sitosterol) |
| Table 1: | Spectroscopic data of PEA-2 (Stigmasterol) |
| Table 2: | Spectroscopic data of PEA-3 (γ-sitosterol) |
In the IR spectrum of PEA-2 and PEA-3, an intensely broad band at 3320 and 3319 cm-1 showed presence of OH stretching and in the 1H-NMR spectrum of PEA-2, H-3 proton appeared as a triplet of a double doublet (tdd) at S 3.62 and H-6 olefinic proton showed a multiplet at S 5.14. Two olefenic protons appeared downfield at S 4.16 (m) and S 4.14 (m) and of PEA-3, H-3 proton appeared at S 3.51 as a triplet of a double doublet H-6 olefinic proton showed a multiplet at S 5.10 and six methyl proton appeared at S 1.26, S 1.17, S 1.03, S 1.00, S 0.91 and S 0.90 for methyl group (Table 1, 2). From the above observations, PEA-2 and PEA-3 was found be stigmasterol and γ-sitosterol, respectively.
DISCUSSION
In IR spectrum of PEA-2, a very intensely broad band at 3320 cm-1 and moderately intense band at 1220 and 680 cm-1 were observed for the O-H bond vibrations of hydroxyl group. The out of plane C-H vibrations of the unsaturated part was observed at 890 cm-1. The corresponding C-C vibrations was shown around 1648 cm-1 as weakly intense band. The stretching and bending vibrations of methyl part were noticed by the intense band 2946 cm-1 and medium intensity band at 1450 cm-1. The vibration of the methylenic part was shown by the band at 2854 cm-1 and the medium band at 1460 cm-1. The moderately intense band at 740 cm-1 was attributed to the rocking movement of methylenic part. The corresponding C-C vibration was shown as weak intense band at 1050 cm-1. In lH-NMR spectrum of PEA-2, H-3 proton appeared as a triplet of a double doublet (tdd) at S 3.62 (J = 4.5 and 1.1 MHZ) and H-6 olefinic proton showed a multiplet at S 5.14. Two olefenic protons appeared downfield at S 4.16 (m) and S 4.14 (m) which were identical with the chemical shift of H-22 and H-23, respectively of stigmasterol (Li et al., 2006). Six methyl protons also appeared at S 1.27, S 1.19, S 1.07, S 1.00, S 0.99 and S 0.91 (3H each, s, CH3). These assignments are in good agreement for the structure of stigmasterol (Habib et al., 2007).
Similarly from the IR spectrum of PEA-3, a very intensely broad band at 3319 cm-1 and moderately intense band at 1189 and 670 cm-1 were observed for the O-H bond vibrations of hydroxyl group. The out of plane C-H vibrations of the unsaturated part was observed at 870 cm-1. The corresponding C = C vibrations was shown around 1640 cm-1 as weakly intense band. The stretching and bending vibrations of methyl part were noticed by the intense band 2946 cm-1 and medium intensity band at 1460 cm-1. The vibration of the methylenic part was shown by the band at 2854 cm-1 and the medium band at 1470 cm-1. The moderately intense band at 720 cm-1 was attributed to the rocking movement of methylenic part. The corresponding C-C vibration was shown as weak intense band at 1060 cm-1. The 1H-NMR data of PEA-3 it was seen that H-3 proton appeared at S 3.51 as a triplet of a double doublet with a J value of 4.5 and 1.1 MHZ and H-6 olefinic proton showed a multiplet at S 5.10. Moreover, Six methyl proton appeared at S 1.26, S 1.17, S 1.03, S 1.00, S 0.91 and S 0.90 (3H each, s, CH3). These assignments are in good agreement for the structure of γ-sitosterol (Habib et al., 2007).
In the previous study on Abelmoschus manihot literature revealed that plant contains flavonoids are present in the flowers only, by present study it was confirmed that this plant also contains steroids.
CONCLUSION
From these physical, chemical and spectral evidences PEA-2 and PEA-3 were confirmed as Stigmasterol (Fig. 1) and γ-sitosterol (Fig. 2).
ACKNOWLEDGMENTS
Authors are thankful to the Principal, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, MS, India for the support and encouragement in the study.