Int J Pharm Pharm Sci, Vol 7, Issue 12, 299-303Short Communication

GC-MS ANALYSIS OF METHANOLIC EXTRACT OF LEAVES OF RHODODENDRON CAMPANULATUM

SAKSHI PAINULI, NISHANT RAI, NAVIN KUMAR*

Department of Biotechnology, Graphic Era University, Clement Town, Dehradun 248001, Uttarakhand, India
Email: navinkumar.bt@geu.ac.in

Received: 03 Sep 2015 Revised and Accepted: 02 Nov 2015

ABSTRACT

Objective: Rhododendron campanulatum is a native of high altitude and is known for its medicinal properties. The present study is aimed to identify the phytochemical constituents in the leaf extract of R. campanulatum using Gas Chromatography and Mass Spectroscopy (GC-MS).

Methods: The methanolic leaf extract was prepared using Accelerated Solvent Extraction system at room temperature and high pressure. Phytochemical screening of methanolic extract of R. campanulatum was performed using GCMS-QP2010 Plus (Shimadzu, Kyoto, Japan) and the spectrum was interpreted on the basis of the databases of National Institute Standard and Technology (NIST11LIB) and WILEY8LIB.

Results: The GC-MS analysis revealed the presence of 49 phytochemical compounds in the methanolic leaf extract. Baccharis oxide (9.99%), betuligenol (8%), alpha and beta-amyrin (7.38 and 2.64%), geranyl acetate (5.91%), (R)-(-)-14-methyl-8-hexadecyn-1-ol (5.19%) and phthalic acid (5.16%) were identified as major constituents.

Conclusion: The methanolic leaf extract of R. campanulatum contains various phyto-compounds of pharmaceutical and industrial importance.

Keywords: Rhododendron campanulatum,Gas Chromatography-Mass Spectrometry, Phytochemical compounds.

Rhododendron genus is comprised of about 1025 species, which are mostly found at higher altitudes [1, 2]. In India, there are around 80 species and 14 subspecies of Rhododendron, distributed in the Himalayan region at the altitude ranging 1500-5500 meters [3]. Different species of Rhododendron are known for their ethnopharmacological values [4]. R. campanulatum is a very important member of the genus Rhododendron, which is known for its traditional medicinal importance for the different ailments like body ache, sore throat, digestion, skin diseases, rheumatism, syphilis, cold and fever, etc. [4-9]. R. campanulatum is found at altitudes between 2500-4300 m [2]. There are very few scientific studies on phytochemical constituents and therapeutic potential of the R. campanulatum. Analysis through High Performance Thin Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR) have shown the presence of epicatechin, syringic acid, quercetin, chlorogenic acid, gallic acid, proto catechic acid and oleanane triterpenoid in the different extracts of leaves of R. campanulatum [6, 9, 10]. Antibacterial properties have also been shown in the leaf extract of R. campanulatum [8]. In the present study, we attempted to identify the phytochemical constituents in the methanolic leaf extract of R. campanulatum using GC-MS method of analysis.

R. campanulatum leaves were collected from the Govind ghat region of Chamoli District, Uttarakhand, India. Plant sample was authenticated by Department of Botany at H. N. B Garhwal University (Srinagar), Uttarakhand. The Herbarium specimen was deposited in the Department of Botany; H. N. B Garhwal University vide voucher number GUH-0743.

The leaves of R. campanulatum were shade dried at sterilized condition and pulverized with the grinder. The powder was successively extracted with methanol (100%) in the Accelerated Solvent Extraction System, which was equipped with a solvent controller unit (ASE350, Dionex Corporation, Sunnyvale, CA, USA) and further the extract was subjected to lyophilization [11]. The lyophilized leaf extract was then stored at 4 °C for further investigation.

1 µl of prepared sample (1 mg/ml lyophilized extract in methanol) was applied for GC-MS analysis.

GC-MS analysis was performed at University Science Instru-mentation Centre, AIRF, Jawaharlal Nehru University, Delhi. For GC-MS analyses of plant extract, GC-MS QP2010 Plus (Shimadzu, Kyoto, Japan) system was utilized. The system was equipped with an auto injector (AOC-20i), head space sampler (AOC-20s), a mass selective detector with an ion source (220 °C) and an interface (260 °C). Rtx-5 MS capillary column having 30 mm X 0.25 mm of length X diameter and 0.25 µm of film thickness was used for MS analyses. The mass range of 40-650 m/z with 1,000 ev of the threshold was purposed. The injector was set in the split injection mode having 250 °C of temperature. The ratio applied for split mode was 10.0. The starting temperature was adjusted to 80 °C (3 min), which afterwards increased to 280 °C with a ramp rate of 10 °C/min. Helium (>99.99 %) with 40.5 cm/s of linear velocity was employed as a carrier gas. The system was programmed with 16.3 ml/min of total flow rate and 1.21 ml/min of column flow.

Fig. 1: GC-MS chromatogram of the constituents of methanolic leaf extract of R. campanulatum

Components were recognized by their retention time (RT) and elucidation of mass spectra. The spectral fragmentation of unknown components was compared with the known and standard components provided by the databases of WILEY8LIB and NIST11LIB. The I. U. P. A. C name, molecular weight (MW) and chemical structure of the unknown components are mentioned in table 1. The GC-MS chromatogram of the extract of R. campanulatum, revealed the presence of 49 phytochemicals, which manifest the presence of several classes of compounds like alkane, fatty acid, terpenes, organic compounds, ester, steroids and flavonoids (fig. 1).

Table 1: The phytochemical compounds identified in R. campanulatum by GC-MS

Peak	IUPAC name	RT	Area%	Formula	MW	Chemical structure
1.	3-Methoxypropane-1,2-Diol	4.622	2.84	C₄H₁₀O₃	106
2.	Bis(2-Hydroxypropyl) Ether	5.177	0.63	C₆H₁₄O₃	134
3.	D-Limonene	5.309	0.73	C₁₀H₁₆	136
4.	Eucalyptol	5.39	0.29	C₁₀H₁₈O	154
5.	Beta-Linalool	6.482	3.35	C₁₀H₁₈O	154
6.	Phenethyl Alcohol	6.798	1.66	C₈H₁₀O	122.17
7.	Pentadecyl 3-Methyl- 2-Butenoate	7.365	0.76	C₂₀H₃₈O₂	310
8.	Acetic Acid, Phenylmethyl Ester	7.604	2.14	C₉H₁₀O₂	150
9.	1-Isopropyl-4-Methyl-3- Cyclohexen-1-Ol	7.875	0.68	C₁₀H₁₈O	154
10.	Dodecane	8.087	1.85	C₁₂H₂₆	170
11.	Beta-Citronellol	8.581	3.44	C₁₀H₂₀O	156
12.	Geraniol	8.999	3.39	C₁₀H₁₈O	154
13.	Phenylacrylaldehyde	9.350	0.96	C₉H₈O	132
14.	Thymol	9.590	1.13	C₁₀H₁₄O	150
15.	1,6-Octadien-3-Ol, 3,7-Dimethyl-, Formate	9.705	0.55	C₁₁H₁₈O₂	182
16.	3,7-Dimethyl-2,6-Octadienyl Acetate	10.576	1.01	C₁₂H₂₀O₂	196
17.	Geranyl Acetate	10.838	5.91	C₁₂H₂₀O₂	196
18.	Tetradecane	11.027	2.14	C₁₄H₃₀	198
19.	Alpha-Gurjunene	11.373	0.29	C₁₅H₂₄	204
20.	7-Isopropenyl-4a-Methyl-1-Methylenedecahydronaphthalene	12.413	0.55	C₁₅H₂₄	204
21.	1-(4-Methoxyphenyl)-3-Methylpropanol	12.658	1.81	C₁₁H₁₆O₂	180
22.	Lily Aldehyde	12.835	0.56	C₁₄H₂₀O	204
23.	Hedycaryol	13.144	0.41	C₁₅H₂₆O	222
24.	Betuligenol	13.286	8.00	C₁₀H₁₄O₂	166
25.	Iron, Tricarbonyl[N-(Phenyl-2-Pyridinylmethylene)Benzenamine-N,N']	13.557	1.63	C₂₁H₁₄FeN₂O₃	398
26.	Phthalic Acid	13.641	5.16	C₁₂H₁₄O₄	222
27.	4-Methoxy-1,4,4a,5,8,8a-Hexahydro -1-Naphthalenyl Acetate	13.837	0.38	C₁₃H₁₈O₃	222
28.	Ethyl hexopyranoside	13.990	1.52	C₈H₁₆O₆	208
29.	Methyl (3-Oxo-2-Pentylcyclopentyl) Acetate	14.322	1.53	C₁₃H₂₂O₃	226
30.	2-(4,8-Dimethyl-3,7-Cyclodecadien -1-Yl)-2-Propanol	14.442	2.00	C₁₅H₂₆O	222
31.	1-(4-Isopropylphenyl)-2- Methylpropyl Acetate	14.564	0.92	C₁₅H₂₂O₂	234
32.	5-(7a-Isopropenyl-4,5-Dimethyl- Octahydroinden-4-Yl)-3-Methyl- Pent-2-En-1-Ol	14.929	0.58	C₂₀H₃₄O	290
33.	1-Phenyl-3-buten-1-ol	15.225	0.73	C₁₀H₁₂O	148
34.	Alpha-Hexylcinnamyl Aldehyde	15.417	0.73	C₁₅H₂₀O	216
35.	Benzoic Acid, Phenylmethyl Ester	15.668	0.41	C₁₄H₁₂O₂	212
36.	Chlorooctadecane	15.797	1.21	C₁₈H₃₇Cl	288
37.	Tonalid	16.723	0.24	C₁₈H₂₆O	258
38.	1,4-Dioxacyclohexadecane -5,16-Dione	17.295	0.45	C₁₄H₂₄O₄	256
39.	L-(+)-Ascorbic Acid 2,6- Dihexadecanoate	17.485	2.15	C₃₈H₆₈O₈	652
40.	Dibutyl Phthalate	17.606	0.93	C₁₆H₂₂O₄	278
41.	(R)-(-)-14-Methyl-8- Hexadecyn-1-ol	19.220	5.19	C₁₇H₃₂O	252
42.	10,12-Hexadecadien-1-Ol	19.591	2.59	C₁₆H₃₀O	238
43.	Cis-9,Cis-12-Octadecadienoic Acid	19.949	1.58	C₁₈H₃₂O₂	280
44.	Baccharis Oxide	35.597	9.99	C₃₀H₅₀O	426
45.	Stigmast-5-En-3-Ol	36.671	1.43	C₂₉H₅₀O	414
46.	Beta-Amyrin	37.676	2.64	C₃₀H₅₀O	426
47.	Alpha-Amyrin	39.025	7.38	:C₃₀H₅₀O	426
48.	4,4a,6b,8a,11,11,12b,14a- Octamethyl-Docosahydro-Picen-3-Ol	42.621	1.82	C₃₀H₅₂O	428
49.	Flavone 4'-OH,5-OH,7-Di-O-Glucoside	43.600	0.86	C₂₇H₃₀O₁₅	594

Among these 49 phytoconstituents, baccharis oxide showed the highest area (9.99 %) followed by betuligenol (8.00 %), alpha-amyrin (7.38 %) geranyl acetate (5.91 %), phthalic acid (5.16), linalool (3.35%), citronellol (3.44%) and geraniol (3.39%).

Most of the major phytochemical compounds are either pharmacologically active compounds or the compounds useful for various industries. Baccharis oxide is a type of triterpene, known as a precursor of steroids in both plants and animals [12]. Betuligenol also known as Rhododendrol is an inhibitor of melanin synthesis hence is used in cosmetic industries [13, 14]. A few pharmacological investigations on alpha and beta-amyrin have proven its antioxidant, antimicrobial, anti-inflammatory and anticancer properties [15]. Grenayl acetate, an organic monoterpene, is known to possess antioxidant properties and specific fragrance due to which it is used as cleanser in industries [16, 17]. Citronellol is a monoterpene alcohol found in essential oils and is reported to have anti-covulsant property. It is also used in cosmetic industries [18]. Linalool, a terpene alcohol is a natural compound being used in toothpaste and gargling solution due to its anti-inflammatory and antibacterial activities [19, 20]. Geraniol showed anticancer activity against human colon cancer cell lines (Caco-2) at 400µM of concentration [21]. Phthalic acid is used in industries for the preparation of other important chemicals [22]. From above discussion, it is obvious that the compounds of methanolic extract of leaves of R. campanulatum have diverse medicinal properties and different industrial applications. Therefore, the extract can be used for the sourcing of these compounds for various applications.

ACKNOWLEDGEMENT

We are grateful to the Graphic Era University, Dehradun for supporting this research and providing us a platform for the execution of our research idea. We are also thankful to Dr. Kshipra Mishra, Sc 'F' and Dr. Raj Kumar Tulswani, Sc 'D', Manimaran Manickam, S. R. F, Department of Biochemical Sciences, Defense Institute of Physiology and Allied Sciences, DRDO, Delhi-54, for their assistance in the extraction through ASE.

CONFLICT OF INTERESTS

The author hereby declares no conflict of interest regarding the manuscript and experimentation done

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