IDENTIFICATION OF POTENTIAL PLANT STEROLS FROM
IPOMOEA AQUATICA FORSK FOR ALZHEIMER’S DISEASE BY
GC-MS ANALYSIS
D.Sivaraman *1, P. Panneerselvam 2, P.Muralidharan 3, T.Purushoth Prabhu4, R.Vijaya Kumar 5
*1,3Department of Pharmacology and Toxicology,C.L.Baid Metha College of Pharmacy, Jyothi Nagar,Rajiv Gandhi salai, Thoraipakkam,Chennai - 600097,Tamil Nadu,India. 2,5
Department of Pharmaceutical Chemistry , C.L.Baid Metha College of Pharmacy,Jyothi Nagar,Rajiv Gandhi salai, Thoraipakkam,Chennai - 600097,Tamil Nadu,India. 4
Department of Pharmacognosy , C.L.Baid Metha College of Pharmacy,Jyothi Nagar,Rajiv Gandhi salai, Thoraipakkam,Chennai - 600097,Tamil Nadu,India.
ABSTRACT
Ipomoea aquatica Forsk (IA) belongs to the family Convolvulaceae grows wild and is cultivated throughout Southeast Asia and it is a widely consumed vegetable in these regions and also considered to be as an important folklore medicine for the treatment of various ailments. Only a very few scientific studies have been conducted on its medicinal, pharmacological and ethano botanical aspects of this plant. The current study was carried out to analyze the active phytoconstituents present in the leaf of Ipomoea aquatica. Total of eighteen constituents was identified in the gas chromatography with mass spectroscopic analysis of hydro alcoholic extract of Ipomoea aquatica Forsk (HAEIA) .In which the bio active plant sterol such as stigmasterol and gamma-sitosterol possesses potent Nootropic, anti-inflammatory, antioxidant, hypoglycemic and anti-cancer activity. Keywords: Ipomoea aquatica Forsk; Convolvulaceae; Stigmasterol;Gamma-sitosterol;Gas chromatography. VVoolluummee 33, , IIssssuuee 11,, 666611--666666. . SShhoorrtt CCoommmmuunniiccaattiioonn ISISSSNN 2278 – 4357 Article Received on 21 October 2013, Revised on 23 November 2013, Accepted on 26 December 2013 *Correspondence for Author: D.Sivaraman Department of Pharmacology and Toxicology,C.L.Baid Metha College of Pharmacy, Jyothi Nagar,Rajiv Gandhi salai, Thoraipakkam,Chennai - 600097,Tamil Nadu,India. [email protected]
1.INTRODUCTION
Recent evidence shows that plant sterols shown to cross the blood-brain-barrier and accumulating in regions of brain. From this it was further confirmed the neuro protective effect of naturally occurring plant cholesterol in some potential neurodegenerative disorders like Alzheimer’s disease (AD). Deposition of beta amyloid (Aβ) plagues was considered to
be one of the significant contribution of AD. Scientific investigation of the plant derived
stigmoterol on AD in mice reveals that stigmasterol reduced Aβ generation by decreasing the
enzymes such as β and γ-secretase and also reducing presenilin distribution in lipid rafts
implicated in amyloidogenic amyloid protein precursor (APP) cleavage1.
Research has indicated that stigmasterol may be useful in prevention of certain cancers, including ovarian, prostate, breast, and colon cancers. Studies have also indicated that a diet high in phytoesterols may inhibit the absorption of cholesterol and lower serum cholesterol levels by competing for intestinal absorption. It was demonstrated that it inhibits several pro-inflammatory2.
It is well known that plant sterols compete with cholesterol for space within bile salt micelles in the intestinal lumen thereby reducing cholesterol absorption. The cholesterol lowering properties of plant sterols such as gamma-sitosterol have been known since long back and many clinical studies have confirmed the potency and efficacy of gamma-sitosterol in lowering plasma LDL-C concentrations. Animal studies have also shown reductions in LDL by sitosterol3.
Genus Ipomoea (Convolvulaceae) are used in traditional system of medicine all over the world and species Ipomoea aquatica Forsk widely used as ailment in the treatment of liver diseases4 and constipation5. IA contains several phyto constituents such as vitamins, including A, B, C, E, and “U” (S-methyl methionine) and is used to treat gastric and intestinal disorders 6
The study related to Alzheimer’s disease (A.D) is focused towards the traditional use of neurotropic and rejunuvating agents 7.The recent trends in the neuropharmacological as well as reverse pharmacology research focused towards the molecular and biochemical mechanism which leads to the development of CNS active principles from the herbal source.
Validation of herbal drugs grabs attention in understanding the nature and biological properties of various plant phytoconstituents and also clinical utilization of the same against some life threating and chronic degenerative disorders hence the objective of the present research work is to analyze the HAEIA by Mass spectrometry, coupled with Gas chromatography (GC/MS) technique and to reveal the information about bioactive components of this indigenous Indian medicinal herb to the scientific world.
2. MATERIALS AND METHODS 2.1. Plant material.
The fresh leaves of Ipomoea Aquatica (IA) were collected from (Perambur region of chennai,Tamilnadu,India).The plant was identified and authenticated by Dr.Sasikala Ethirajulu.Captain srinivasa moorthy research Foundation,Chennai,Tamil nadu,India.The specimen voucher was deposited in the Department of Pharmacology and toxicology, C.L.Baid Metha College of Pharmacy, Chennai,Tamil nadu,India.
2.2. Preparation of the Hydro alcoholic Extract of IAF.
The fresh leaf of IAF was collected and washed with running water. It was shade dried at room temperature and 1 kg of the dried leaf was made in to coarse powder. The powder was passed through a 60 No mesh sieve. Air dried Powdered drug was extracted with mixture of Ethanol: water (6:4) (hydro-alcoholic extract) by using soxhlet extraction. Then the extract obtained was filtered, concentrated by rotary vacuum pump to get the solid mass. The weight of extract obtained was 20.6 %.
2.3. Gas Chromatography—Mass Spectroscopy Analysis of HAEIA.
The hydro alcoholic extract of IAF was subjected to MS analysis on the instrument GC-MS SHIMADZU QP2010 with Elite – DB-5M column and the GC-GC-MS solution version 2.53 software. Initially oven temperature was maintained at 70oC for 2.0 minutes and the temperature was gradually increased up to 300oC at 10.0/30.0 min and 4.0 µl of sample was injected for analysis. Helium gas 99.995% of purity was used as a carrier gas as well as an eluent. The flow rate of helium gas was set to 1.5 ml/min. The sample injector temperature was maintained at 260ºC and the split ratio is 20 throughout the experiment periods. The ionization mass spectroscopic analysis was done with 70 eV. The mass spectra were recorded for the mass range 40-1000 m/z for about 30 minutes. Identification of components was based on comparison of their mass spectra. As the compounds separated, on elution through the column, were detected in electronic signals.
As individual compounds eluted from the Gas chromatographic column, they entered the electron ionization detector where they were bombarded with a stream of electrons causing them to break apart into fragments. The fragments were actually charged ions with a certain mass. The m/z ratio obtained was calibrated from the graph obtained which was called as the mass spectrum graph which is the fingerprint of the molecule. The identification of compounds was based on the comparisons of their mass spectra with NIST Library 2008 WILEY, FAME.
3. RESULTS AND DISCUSSION
GC-MS analysis was carried out on the hydro alcoholic extract of Ipomoea Aqutica Forsk (HAEIA) which reveals the presence of 18 compounds. The GC-MS analysis was done using the instrument GC-MS SHIMADZU QP2010 with GCMS solution version 2.53 software. The sample volume was 4.0 µL. The sample of HAEIA was run for 30 minutes. The Chromatogram (Figure.1) shows 9 prominent peaks in the retention time range 16.688 – 27.846.
The first prominent peak at 16.688 retention time with percentage peak area of 10.31% . This largest peak is due to the presence of Hexadecanoic Acid. The Second prominent peak at 17.093 retention time with the peak area of 11.85% is due to the presence of n-Hexadecanoic acid. The third less prominent peak at 18.316 having retention time with the peak area 5.36% denotes the presence of Methyl linoleate. The fourth less prominent peak at 18.376 having retention time with the peak area 4.56% denotes the presence of 9,12,15-Octadecatrienoic acid, methyl ester. The fifth significant peak at 18.460 having retention time with the peak area 7.96% is characteristic of 2-Hexadecen-1-OL, 3,7,11,15-Tetramethyl-, [R-[R*,R*-(E)]]-.The sixth significant peak at 18.608 having retention time with the peak area 1.69% is denotes the presence of octadecanoic acid. The seventh less prominent peak at 27.107 having retention time with the peak area 3.29% is due to the presence of Ergost-5-En-3-Ol, (3.Beta.)The eighth prominent peak at 27.304 having retention time with the peak area 6.02% is due to the presence of stigmasterol. The ninth less prominent peak at 27.846 having retention time with the peak area 9.95% is due to the presence of gamma-sitosterol. The other less prominent peaks at other retention times are given in Table 1. The total ion chromatograph (TIC) showing the peak identities of the compounds identified have been given in Figure 1.
Fig. 1: GC-MS analysis of the hydro alcoholic extract of Ipomoea Aqutica Forsk Leaf.
Table 1 : List of phytoconstituents present in the leaf of Ipomoea Aqutica Forsk
Peak Retention Peak Percentage Name of the Constituent Time Area Area
1 4.045 6107752 15.54 1,2,3-Propanetriol
2 6.451 857539 2.18 Methyl caprylate ; Octanoic acid, methyl ester 3 8.923 1889055 4.81 Sec-Butyl nitrite
4 10.526 1125484 2.86 5-Oxo-Pyrrolidine-2-Carboxylic Acid Methyl Ester 5 12.191 699580 1.78 Octadecanoic acid, 7-hydroxy-, methyl ester
6 13.674 3630824 9.24 1,3,4,5-Tetrahydroxy-Cyclohexanecarboxylic Acid 7 15.742 367392 0.93 Neophytadiene
8 15.829 348642 0.89 2-Pentadecanone, 6,10,14-trimethyl- 9 16.688 4052173 10.31 Hexadecanoic Acid, Methyl Ester 10 17.093 4657881 11.85 n-Hexadecanoic acid
11 18.316 2105097 5.36 Methyl linoleate
12 18.376 1790679 4.56 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z
13 18.460 3126658 7.96 2-Hexadecen-1-Ol, 3,7,11,15-Tetramethyl-, [R-[R*,R*-(E)]]- 14 18.608 662930 1.69 Octadecanoic acid, methyl ester
15 20.367 311330 0.79 Methyl arachisate
16 27.107 1292748 3.29 Ergost-5-En-3-Ol, (3.Beta.)- 17 27.304 2365550 6.02 Stigmasterol
18 27.846 3910273 9.95 Gamma-Sitosterol
4. CONCLUSION
The GCMS analysis of hydro alcoholic extract of Ipomoea Aqutica Forsk reveals the presence of phytoconstituents belonging to the category of sterols,acids, esters, alcohols and
ethers etc. Thus, the medicinal plant Ipomoea Aqutica is found to possess significant phytoconstituents. The presence of such a variety of phytochemicals may be attributed to the medicinal characteristics of this plant.
ACKNOWLEDGEMENT
The authors are grateful to Dr.Grace Rathnam (Principal, C.L.Baid Metha college of Pharmacy) for her technical and secretarial assistance.
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