RESEARCH ARTICLE
Open Access Full Text Article
GC-MS analysis of ethanolic extract of
Nothapodytes nimmoniana
(Graham) Mabb. leaves
Kavitha S
1, Packia Lincy M
2, Mary Jelastin Kala S
3, Mohan V R
2*
1 Department of Chemistry, MDT Hindu College, Tirunelveli, Tamil Nadu, India.
2Ethnopharmacology Unit, Research Department of Botany, V. O. Chidambaram College, Tuticorin-628008,
Tamil Nadu, India.
3Department of Chemistry, St. Xavier’s College, Palayamkottai, Tamil Nadu, India.
*For correspondence e-mail: [email protected]
Article Info: Received 17 Mar 2015; Revised: 28 Apr 2015; Accepted 26 May 2015
ABSTRACT
Nothapodytes nimmoniana (Graham) Mabb belonging to family Icacinaceae is commonly known as Amruta and found in Maharashtra, Goa, Kerala, Karnataka, Assam and Tamil Nadu in India. It is one of important medicinal plant used in various types of cancer, HIV, malaria, antibacterial, inflammatory and anti-fungal activity have been reported. The present investigation was carried out to determine the phytocompounds of ethanol extract of Nothapodytes nimmoniana leaves. GC-MS analysis of ethanol extract was performed using a Perkin-Elmer GC clarus 500 system and Gas Chromatograph interfaced to a Mass Spectrometer (GC-MS) equipped with an Elite-1,fused silica capillary column (30mm×0.25mm 10×1µMdf, composed of 100% Di methyl poly siloxene). Interpretation on mass spectrum GC-MS was conducted using the database of National Institute standard and Technology (NIST). Fourteen compounds were identified. The prevailing compounds in the ethanol extract of Nothapodytes nimmoniana leaves were 3 -O-Methyl-d-glucose (24.20%), phytol (20.07%), n-Hexadecanoic acid (17.87%), 1,2-Benzenedicarboxylic acid, diisooctyl ester (9.64%), Propane, 1,1,3-triethoxy (4.67%), 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- (7.6%), 9,12-Octadecadienoic acid (Z,Z)- (4.38%), E-2-Tetradecen-1-ol (1.69%), Acetic acid, 3-hydroxy-6-isopropenyl-4,8a -dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-yl ester (1.6%) and Ocimene (1.13%).
Keywords: Nothapodytes nimmoniana, 3-O-Methyl-d-glucose, Antiarthritic, Antioxidants.
1. INTRODUCTION
GC-MS is a technique used for screening, identification and quantification of many susceptible compounds in plant extracts. Gas chromatography (GC) is needed to separate drugs that might be present in the sample. The retention time (RT) is an -
identifying characteristic of a drug. The detector for the GC is the mass spectroscopy (MS). The fragmentation pattern for a drug is unique and therefore is an identifying characteristic of a drug. The identification of a drug by its retention time (GC) and fragmentation pattern (MS), along with sample
Malaya
Journal of
Biosciences
specific information afforded to make GC-MS the foremost confirmation method for analyzing herbal extract [1]. In recent years GC-MS studies have been increasingly applied for the analysis of medicinal plants as this technique has proved to be a valuable method for the analysis of non-polar components and volatile essential oil, fatty acid, lipids and alkaloids [2].
Nothapodytes nimmoniana (Graham) Mabb. formerly known as Nothopodytes foetida Sleymer and Mappia foetida Miers (Icacinaceae), is a small tree, naturally distributed in many parts of the Western Ghats, South India, some parts of the Himalayan foot hills, Srilanka, Myanmar and Thailand [3]. This tree is a rich source of camptothecin, isoquinoline alkaloid, which is currently being used for treating colorectal and ovarian cancer [4,5,6]. The objective of the present study is to identify the possible phytoconstituents present in the ethanol extract of Nothapodytes nimmoniana leaves using GC-MS study.
2. MATERIALS AND METHODS
2.1. Collection of plant sample
Leaves from N. Nimmoniana (Graham) Mabb. was collected from Kollankodu, Kanyakumari, Tamil Nadu. The plant was identified with help of local flora and authenticated in Botanical survey of India, Southern circle, Coimbatore, Tamil Nadu. The voucher specimens preserved in the Ethnopharmacology Unit, Research Department of Botany, V.O.Chidambaram College, Tuticorin, Tamil Nadu for further references.
2.2. Plant sample extraction
The leaves were cleaned, shade dried and pulverized to powder in a mechanical grinder. Required quantity of powder was weighed and transferred to Stoppard flask and treated with ethanol until the powder is fully immersed. The flask shaken every hour for the first 6 hours and then it was kept aside and again shaken after 24 hours. This process was repeated for 3 days and then the extract was filtered. The extract was collected and evaporated to dryness by using a vacuum distillation unit. The final residue obtained was then subjected to GC- MS analysis.
2.3. GC - MS Analysis
GC-MS analysis of leaves extract were performed using a Perkin - Elmer GC Clarus 500 system and Gas Chromatograph interfaced to a Mass
Spectrometer (GC-MS) equipped with a Elite - 1, fused silica capillary column (30 mm x 0.25 mm 10 x 1 µMdF, composed of 100% Di methyl poly siloxene). For GC-MS detection an electron ionization system with ionizing energy of 70ev was used. Helium gas (99.999%) was used as the carrier gas at constant flow rate of 1ml/min and an injection volume of 2µ1 was employed (split ratio of 10:1); injector temperature 2500 C; ion-source temperature 2800 C. The oven temperature programmed from 1100 C (isothermal for 2 min) with an increase of 100 C/min to 2000 C, then 50 C/min to 2800 C, ending with a 9 min isothermal at 2800 C, mass spectra were taken at 70ev; a scan interval of 0.5 seconds and fragments from 45 to 450Da, total GC running time was 36 minutes. The relative % amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a Turbo mass.
Interpretation on mass spectrum GC-MS was conducted using the database of National Institute of Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name molecular weight and structure of the components of the test materials were ascertained.
3. RESULTS
The compounds present in the ethanol extract of leaves of Nothapodytes nimmoniana were identified by GC-MS analysis (Figure.1). Twenty four compounds were identified in N. nimmoniana leaves extract. The active principles with their retention time (RT), molecular formula (MF), molecular weight (MW) concentration % and mass spectrum in the ethanol extract of N. nimmoniana leaves are present in Table.1.
The prevailing compounds in the ethanol extract of leaves were 3-O-Methyl-d-glucose (24.20%), phytol (20.07%), n-Hexadecanoic acid (17.87%), 1,2-Benzenedicarboxylic acid, diisooctyl ester (9.64%), Propane, 1,1,3-triethoxy (4.67%), 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- (7.6%), 9,12-Octadecadienoic acid (Z,Z)- (4.38%), E-2-Tetradecen-1-ol (1.69%), Acetic acid, 3-hydroxy-6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-
IICPT, Thanjavur, 25-MAR-2014 + 17:19:27
Sample No. 543
4.00
9.00
14.00
19.00
24.00
29.00
34.00
Time
0
100
%
GCMS Analysis 1285
Scan EI+
TIC
2.87e9
19.43
12.38
10.74
5.33
2.40
14.50
13.82
14.70
16.14
18.48
28.44
29.81
33.01
Figure 1. GC-MS chromatogram of the ethanol extract of N. nimmoniana leaves
Table 1. Phytocomponents detected in N. nimmoniana leaves.
No RT Name of the compound Molecular
Formula MW
Peak Area %
Structure
1. 2.40 Propane, 1,1,3-triethoxy- C9H20O3 176 4.67
2. 5.33 Ocimene C10H16 136 1.13
3. 10.74 E-2-Tetradecen-1-ol C14H28O 212 1.69
4. 10.99 Z,Z-2,5-Pentadecadien-1-ol C15H28O 224 0.56
5. 11.17 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C20H40O 296 0.80
7. 13.74 3-O-Methyl-d-glucose C7H14O6 194 24.20
8. 13.82 Phytol C20H40O 296 20.07
9. 14.40 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 280 4.38 10. 14.50 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- C18H30O2 278 7.60
11. 16.14 Hexadecanal, 2-methyl- C17H34O 254 0.65
12. 18.48
3,trsns-(1,1-dimethylethyl)-4,trans-methoxycyclohexanol C11H22O2 186 0.57
13. 18.79 Octadecane, 6-methyl- C19H40 268 0.16
14. 19.43 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 9.64
15. 23.01 Squalene C30H50 410 0.15
16. 26.28 1H-Benzocyclohepten-7-ol,
2,3,4,4a,5,6,7,8-octahydro-1,1,4a,7-tetramethyl-, cis- C15H26O 222 0.18
17. 27.15 Vitamin E C29H50O2 430 0.25
18. 28.44 5,8,11,14-Eicosatetraenoic acid, methyl ester,
(all-Z)- C21H34O2 318 0.73
20. 29.81 Stigmasterol, 22,23-dihydro- C29H50O 414 1.59
21. 30.35
2,2,6-Trimethyl-1-(2-methyl-cyclobut-2-enyl)-hepta-4,6-dien-3-one C15H22O 218 0.46
22. 31.13 2H-Pyran, 2-(7-heptadecynyloxy)tetrahydro- C22H40O2 336 0.42
23. 31.97 Urs-12-en-24-oic acid, 3-oxo-, methyl ester,
(+)- C31H48O3 468 0.34
24. 33.01
Acetic acid, 3-hydroxy-6-isopropenyl-4,8a-
dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-yl ester
C17H26O3 278 1.69
Table 2. Activity of phytocomponents identified in the ethanol extract of N. nimmoniana leaves
No. RT Name of the
compound
Molecular Formula
Compound Nature
**Activity
1. 5.33 Ocimene C10H16 Monoterpene
Antiacne, Analgesic, Anesthetic, Antiseptic, Antispasmodic, Cancer preventive, Fungicide,
Herbicide, Nematicide Stimulant, Insect repellent, Insectifuge, Decongestant, Emetic,
Pesticide, Antifeedent
2. 11.17
3,7,11,15- Tetramethyl-2-hexadecen-1-ol
C20H40O Terpene alcohol Antimicrobial, Anti-inflammatory
3. 12.38 n-Hexadecanoic
acid C16H32O2 Palmitic acid
Antioxidant, Hypocholesterolemic, Nematicide, Pesticide, Lubricant, Antiandrogenic, Flavor,
Hemolytic 5-Alpha reductase inhibitor
4. 13.74
3-O-Methyl-d-glucose C7H14O6
Sugar moiety Preservative
5. 13.82 Phytol C20H40O Diterpene Antimicrobial, Anti-inflammatory, Anticancer Diuretic
6. 14.40
9,12-Octadecadienoic
acid (Z,Z)-
C18H32O2 Linoleic acid
Anti-inflammatory, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Nematicide Insectifuge, Antihistaminic, Antieczemic, Antiacne, 5-Alpha reductase inhibitor, Antiandrogenic, Antiarthritic, Anticoronary,
Insectifuge
7. 14.50
9,12,15-Octadecatrienoic
acid, (Z,Z,Z)-
C18H30O2 Linolenic acid
Anti-inflammatory, Hypocholesterolemic Cancer preventive, Hepatoprotective, Nematicide Insectifuge, Antihistaminic Antieczemic,
Antiacne, 5-Alpha reductase inhibitor, Antiandrogenic, Antiarthritic, Anticoronary,
Insectifuge
8. 16.14 Hexadecanal,
2-methyl- C17H34O
Aldehyde compound
9. 19.43
1,2-Benzenedicarboxyl
ic acid, diisooctyl ester
C24H38O4 Plasticizer compound Antimicrobial, Anti-fouling
10. 23.01 Squalene C30H50 Triterpene
Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immunostimulant, Chemo
preventive, Lipoxygenase-inhibitor, Pesticide
11. 26.28
1H-
Benzocyclohepten-7-ol,
2,3,4,4a,5,6,7,8- octahydro- 1,1,4a,7-tetramethyl-, cis-
C15H26O
Sesquiterpene alcohol
tumor, Analgesic, Antibacterial, Anti-inflammatory, Sedative, Fungicide
12. 27.15 Vitamin E C29H50O2
Vitamin compound
Antiageing, Analgesic, Antidiabetic, Anti-inflammatory, Antioxidant, Antidermatitic,
Antileukemic, Antitumor, Anticancer, Hepatoprotective, Hypocholesterolemic, Antiulcerogenic, Vasodilator, Antispasmodic,
Antibronchitic, Anticoronary
13. 28.44
5,8,11,14-Eicosatetraenoic acid, methyl ester,
(all-Z)-
C21H34O2 Unsaturated fatty acid ester Cardio protective
14. 28.84
Cholestan-3-ol, 2-methylene-,
(3á,5à)-
C28H48O
Steroid Antiarthritic, Hepatoprotective, Antiasthma, Anti-inflammatory, Diuretic, Cancer preventive
15. 29.81 Stigmasterol,
22,23-dihydro- C29H50O Steroid
Antiarthritic, Hepatoprotective, Antiasthma, Anti-inflammatory, Diuretic, Cancer preventive
16. 33.01
Acetic acid, 3- hydroxy-6-
isopropenyl-4,8a- dimethyl- 1,2,3,5,6,7,8,8a-octahydronaphthal
en-2-yl ester
C17H26O3 Acetic acid compound
Antimicrobial
*Activity Source: Dr Duke’s Phytochemical and Ethnobotanical databases
4. DISCUSSION
Authentication of medicinal plants as genetic and chemical level is a critical step in the use of these botanical materials for both research purposes and commercial preparations. For any living organisms, identity is very important in order to distinguish itself from other organisms within the population and other populations. In plant taxonomy, during this molecular era, the morphological characters also play a vital role in plant systematic study and used as a tool for the classification of a taxon. In recent times, in addition morphological markers, anatomical, cytological, biochemical and molecular markers are also being used to classify the organisms. Gas chromatography –Mass spectroscopy (GC-MS) is a -
the regulatory and tissue rebuilding mechanisms related to estrogen effects, as well as acting as an intermediate in the biosynthesis of androgens, estrogens and corticoids. It is also used as the precursor of vitamin D3 [12,13]. Vitamin E is thought to be important chain breaking antioxidant, which plays an important role in various stages of carcinogenesis through its contribution and immunocompetence, membrane and DNA repair and decreasing oxidative DNA damage [14]. In vitro studies showed that vitamin E can prevent oxidation of DNA by inhibiting activated neutrophils. Vitamin E can protect the conjugated double bond of β-carotene from oxidation [15]. 1,2 Benzene dicarboxylic acid diisoctyl ester also known as diisooctyl phthalate (DIOP). It is report on Human health Hazard Assessment on DIOP, the bulk of labelled phthalates ingested by humans were eliminated in urine within the first 24 hours and there was no significant tissue accumulation of DIOP [16]. This constituent may therefore not be injurious to health. Phytol was also found to give good as well as preventive and therapeutic results against arthritis. The results show that reactive oxygen species- promoting substances such as phytol constitute a promising novel class of pharmaceuticals for the treatment of rheumatoid arthritis and possibly other chronic inflammatory diseases [17]. Phytol was observes to have antibacterial activities against Staphylococcus aureus by causing damage to cell membrane as a result there is a leakage of potassium ions from bacterial cells. Phytol is a key acyclic diterpene alcohol that is a precursor for vitamin E and K1. It is used along with simple or corn syrup as a hardener in candies [18,19]. Phytol and its isomer 3,7,11,15- tetramethyl 1,2-hexadecen-1 –ol present in N. nimmoniana leaf possibly contribute to the therapeutic effect of the plant in asthma patients [17]. The above said components found in the leaf of N. nimmoniana which are being used for the pharmacological work. In addition to this, the results of the GC-MS profile can be used as pharmacognostical tool for the identification of the plant [20].
5. CONCLUSION
GC-MS analysis showed the existence of various compounds with different chemical structures. The presence of various bioactive compounds confirms the application of N. nimmoniana leaves for various ailments by traditional practitioners. However, isolation of individual phytochemical constituents may proceed to find a novel drug.
Acknowledgement
We would like to thank Mr. S. Kumaravel, Senior Scientist, Indian Institute of Crop Processing Technology (Ministry of food Processing Industries, Government of India) Tanjavore, Tamilnadu for providing all the facilities and support used to carry out the work.
Conflict of Interest
The authors declare that they have no conflicts of interest.
References
1. Mythili K, Umamaheswara Reddy C, Chamundeeswari D and Manna PK (2013). GC-MS analysis of phytocomponents and in vitro inhibitory effects of Calanthe triplicata. Journal of Natural Products. 6: 141- 146. 2. Milne A. (1993). Inhalational and local
anaesthetics reduce tactile and thermal responses in Mimosa pudica L. Journal of Global Pharmaceutical Technology. 1190-1193.
3. Suhas S, Ramesha BT, Ravikanth G, Rajesh P Gunaga, Vasudeva R, Ganeshaiah KN and Uma Shaanker R (2007). Current Science. 92: 8. 4. Liberbaum RC, Ratain MJ, Miller MA, Hargis
JB, Hollis DR, Rosner GL, O’Brien, SM, Brewster L, Green MR and Schilsky RL (1995). Phase I study of paclitaxel and topotecan in patients with advanced tumors; a cancer and leukemia group B study. Journal of Clinical Oncology. 13: 2230-2237.
5. Romaanelli SP, Perego G, Prateri N, Carenini M, Tortoreto Zunino F (1998). In vitro and in vivo interaction between cisplatin and topotecan in ovarian carcinoma systems. Cancer Chemotherapy and Pharmacology. 41: 385-390.
6. Vladu B, Woynarowski JM, Manikumar G, Wani MC, Von Hoff DD, Wadkins RM (2000). 7- and -10 Substituted Compothecins; dependence of topoisomerase I-DNA cleavable complex formation and stability on the 7 and -10 substituents. Molecular Pharmacology. 57: 243-251.
8. Johnson M, Mariswamy Y and Gnaraj WF (2011). Chromatographic fingerprint analysis of steroids in Aerva lanata L. by HPTLC technique. Asian Pacific Journal of Tropical Biomedicine. 1: 428-433.
9. Balamurugan K, Nishanthini A and Mohan VR (2012). GC-MS analysis of Polycarpea corymbosa (L.) Lam whole plant. Asian Pacific Journal of Tropical Biomedicine. 2: S1289-S1292.
10. Lalitha Rani S, Kalpana Devi V, Tresina Soris P, Maruthu Pandian A and Mohan VR (2011). Ethnomedicinal plants used by Kanikkars of Agastiarmalai Biosphere Reserve, Western Ghats. Journal of Ectobiotechnology. 3: 16-25. 11. Rao CV, Newmark HL and Reddy BS (1998). Chemopreventive effect of squalene on colon cancer. Carcinogenesis. 1998; 19: 287-297. 12. Sundararaman P, Djerassi C (1977). A
Convenient synthesis of progesterone from stigmasterol. Journal of Organic Chemistry. 42: 3633-3634.
13. Kametani T and Furuyama H (1987). "Synthesis of vitamin D3 and related compounds". Medicinal Research Reviews. 7: 147–171.
14. Machlin, L.J and Bendich, A. (1987). FASEB. J., 6:441-444.
15. Kimmick GC, Bell RM and Bostock RM (1997). Vitamin E and breast cancer: A review. Nutrition Cancer. 27: 109.
16. NICNAS (2008) Phthalate hazard compendium: a summary of physicochemical and human health hazard data for 24 ortho-phthalate chemicals. Sydney, National Industrial Chemicals Notification and Assessment Scheme.
17. Ogunlesi M, Okiei W, Osibote AE (2009). Analysis of the essential oil from the dried leaves of Euphorbia hirta Linn. (Euphorbiaceae), a potential medication for asthma. African Journal of Biotechnology. 8: 7042-7050.
18. Inoue Y, Hada TA, Shiraishi K, Hirore, H, Hamashima and Kobayashi S (2005). Biphasic effects of Geranylgeraniol, Terpenone and Phytol on the growth of Staphylococcus aureus. Antimicrobial Agents Chemotherapy. 49: 1770-1774.
19. Sathiyabalan. G, Packia Lincy. M, Muthukumarasamy. S and Mohan VR (2014). GC-MS analysis of bioactive components of Petiveria alliacea L. whole plant (Phytolaccaceae). International Journal of Pharma Research Health Science. 2: 387-392.