Int J Curr Pharm Res, Vol 9, Issue 3, 98-101Original Article


GC-MS ANALYSIS OF METHANOLIC STEM EXTRACT OF GYNOCHTHODES RIDSDALEI, RAZAFIM AND B. BREMER, AN ENDEMIC, ENDANGERED MEDICINAL PLANT OF SOUTHERN WESTERN GHATS

RENJI R. NAIR, A. GANGAPRASAD*

Plant Tissue Culture and Molecular Biology Lab, Department of Botany, University of Kerala, Thiruvananthapuram, Kerala, India, 695581
Email: agangaprasad@yahoo.com

Received: 27 Dec 2016, Revised and Accepted: 27 Mar 2017

ABSTRACT

Objective: The present research study was undertaken to determine the presence of bioactive components present in the methanolic stem extract of Gynochthodes ridsdalei using Gas Chromatography–Mass Spectrometry (GC-MS) analysis.

Methods: the Fresh stem of Gynochthodes ridsdalei collected from the forest areas of Ponmudi region of Thiruvananthapuram district of Kerala state, India was used. The mass spectrum GC-MS of the crude methanolic extract was estimated using the database of National Institute of Standard and Technology (NIST).

Results: The active principles with their retention time, peak area, molecular formula, molecular weight, structure and category of the compound were predicted. The analysis revealed the presence of 52 bioactive components. Most of the identified compounds are basically biological important. The components were identified by comparing their retention time and peak area with that of literature and by interpretation of mass spectra. The phyto components screened were of biological importance. Some of them were sterols, anthraquinones, vitamins etc.

Conclusion: The result reveals the existence of various bioactive compounds and validates the earlier reports of therapeutic importance of the plant. Gynochthodes ridsdalei is recommended as a plant of phytochemical and pharmaceutical importance.

Keywords: Gynochthodes ridsdalei, Morinda reticulata, endangered, southern Western Ghats, gas chromatography

© 2017The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2017v9i3.19665

INTRODUCTION

The use of medicinal plants has gained considerable importance in our day to day life since ancient times. Traditional medicine is the sum total of knowledge, skills and practices based on the theories, beliefs, experiences indigenous to different cultures that are used to maintain health as well as to diagnose, improve or treat physical and mental illness. The therapeutic use of some plants against critical human illnesses predates recorded history and represents the most significant direct antecedent to modern medicine [1]. Medicinal plants are rich resources of ingredients which can be used in drug development and synthesis. Many higher plants are a major source of secondary metabolites which are used for many medicinal purposes. Gynochthodes ridsdalei (Syn: Morinda reticulata) is a large woody climbing shrub with coriaceous reticulate leaves belonging to the family Rubiaceae. The plant is endemic to southern Western Ghats [2]. It forms an important component in a variety of herbal formulation in traditional medicine [3]. Plants belonging to family Rubiaceae are known to contain a substantial amount of anthraquinones especially in the roots [4] and are characterised by brightly coloured anthraquinones that have been used in the past for various dyeing purpose. The screening of plant extracts is an innovative method to find therapeutically important compounds which will help to develop novel drugs [5]. Gas Chromatography–Mass Spectrometry (GC-MS) analysis is used for the direct analysis of bioactive components in traditional medicine and for separation and analysis of multi-component mixtures such as essential oils, hydrocarbons etc [6].

MATERIALS AND METHODS

Plant material

Fresh stem of Gynochthodes ridsdalei collected from the forest areas of Ponmudi region of Thiruvananthapuram district of Kerala state, India was used. The taxonomical identification of the plant was done using authentic literature [7, 8]. A voucher specimen was deposited at the Herbarium of Department of Botany, University of Kerala, Kariavattom (KUBH No. 8095).

Preparation of plant extract

The collected stem was chopped and shade dried under room temperature for 7 d and then milled into coarse powder by the mechanical grinder. About 10 gm of the powdered stem sample was subjected to Soxhlet extraction using 200 ml methanol. The extract was concentrated using rotary evaporator (Superfit rotavap) under reduced pressure and stored in the refrigerator until further use. Two microliters of the extract were employed in GC–MS analysis for analysis of different compounds.

GC–MS analysis

The analysis of the extract was performed using GC–MS (Model: GC MS-QP 2010, Shimadzu, Tokyo, Japan) equipped with a VF 5 ms fused silica capillary column of 30 m length, 0.25 mm diameter and 0.25 µm film thickness. For GC–MS detection, electron ionization energy of 70eV was used. The carrier gas was helium (99.9%) and used at constant flow rate of 1.2 ml/min. Injector and mass transfer line temperature were set at 200 ° C and 255 ° C respectively. The oven temperature was set from 70 to 300 ° C at 10 °C/min for 9 min. One microliter of the sample was injected in a split mode with a scan range of 40-1000 m/z. The total running time of GC–MS was 35 min. The relative percentage amount of each component was calculated by comparing its average peak area normalization [9].

Identification of the components

Elucidation of mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) and Wiley Spectra Libraries. The spectrum of the unknown component was compared with the spectrum of known components, which was stored in the NIST library source [10]. The name, molecular weight and molecular mass of the identified compounds were further confirmed by comparison of their retention indices with literature data. For quantitative analysis, compounds concentrations (as % content) were calculated by integrating their corresponding chromatographic peak area.

RESULTS AND DISCUSSION

The bioactive components present in the methanolic stem extract of G ridsdalei were identified by GC–MS analysis. The gas chromatogram shows the relative concentrations of various compounds getting eluted as a function of retention time (fig. 1). Identification of the compounds was accomplished by comparing their mass spectra and retention indices with those given in the literature and those authentic samples. The active principles with their retention time (RT), molecular formula, molecular weight (MW), concentration (%), nature of the compound and their biological activities are presented in (table 1) and are listed by their order of retention times. The heights of the peak indicate the relative concentrations of the compounds present in G ridsdalei.

Fig. 1: GC-MS Chromatogram of methanolic stem extract of Gynochthodes ridsdalei


Table 1: Phytocomponents identified in the methanolic stem extract of G. ridsdalei by GC-MS

S.No. Retention time Peak area% Name of the compound Molecularformula Molecularweight Nature of compound Uses
1 7.303 1.16 1,3-Benzenediol, 5-chloro- C6H8O4 144.1253 Phenol(Resorcinol) Diazodyes, Dermatology
2 8.618 0.83 5-HydroxymethylfurfuraBenzene methanol, 3-fluoro- C6H6O3 126.1100 Organic compound Baking industry
3 9.889 0.80 2-Methoxy-4-vinylphenol3-Methoxyacetophenone C7H7NO4 169.136 Phenol Flavoring agent
4 10.416 5.09 Phenol, 2,6-dimethoxy-3-Amino-2,6-dimethoxypyridine C8H10O3 154.1632 SyringolPhenol Smoky aroma in foods
5 13.135 1.60 1,Butanol, 3-methyl, formate C6H12O2 116 Alcoholic compound Antimicrobial
6 14.688 0.39 Phenol, 2,6-dimethoxy-4-(2-propeny D-3-Hydroxy-4-methoxycinnamic acid C11 H14 O3 194.230 PhenolEugenol Perfumarydentistry
7 15.126 4.34 4-((IE)-3-Hydroxy-l-propenyl)-2-me thoxyphenol C10H22O3 C10H12O3 Coniferyl alcohol Fungal growth inhibitor
8 15.468 1.00 3,5-Dimethoxy-4-hydroxyphenylacetic acid C8H8O3 152.147 Acid Synthesis of atenolol
9 16.032 1.02 2,5-Diethoxyaniline C6H11N 121.18 Aniline Precursor to crystal violet dye
10 16.099 0.56 6-Octen-l-ol C10H20 O 156.27 Citronellol (monoterpenoid) Insect repellents, perfumary
11 16.448 0.42 3-Methyl-l-penten-4-yn-3-ol C6H10O 80.128 Tertiary hexanol Hypnotic/sedative
12 17.340 2.58 n-Hexadecanoic acid C16H32O2 256.4241 Palmitic acid Antioxidant hypocholesterolemic, nematicide
13 17.436 2.29 Scopoletin C10H8O4 192.16 Coumarin Used in food making
14 17.711 2.69 Squalene C30H50 410 Triterpene Antimicrobial, antioxidant, antitumour
15 18.172 2.24 1-Butanol,3-Methyl, Formate C6H12O2 116 Alcoholic Antimicrobial
16 18.810 2.35 9,10-Anthracenedione, C14H8O2 208.22 Anthraquinone Dyes
17 18.862 1.98 Phytol
  • C20H40O
 296 Diterpene Anticancer, antimicrobial,anti-inflammatory
18 18.944 1.01 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 280.4455 Linoleic acid (carboxylic acid) Antioxidant effect, paints, varnishes
19 18.989 1.72 Oleic Acid cis-13-Octadecenoic acid cis-Vaccenic acid C18H34O2 282.4614 Fatty acid Reducing blood pressure
20 19.189 1.48 Octadecanoic acid C18H36O2 284.4772 Stearic acid Dietary supplements
12 19.650 0.57 1-Hydroxy-2-methylanthraquinonel-Hydroxy-4-methylanthraquinone C15H10O3 238.242 Anthraquinone Dyes, MedicinalImportance
22 20.742 1.76 9,10-Anthracenedione, 2-hydroxy-l-methoxy C19H18O7 358.342 Anthraquinone Dyes, Medicinal importance
23 20.905 0.64 9-Octadecenamide, (Z)— C18H35NO 281.4766 Amide For depression, sleep disorders
24 21.418 0.40 1,2,4-Benzenetricarboxylic acid, 5-methyl-, trimethyl ester- C12H12O6 252.2201 Trimellitic acid Unknown
25 21.715 4.03 2-(Hydroxymethyl) anthraquinones1,4,7-Trimethyl-2-azafluorene4-Propylxanthen-9-one C15H10O3 238.238 Anthraquinone Dyes, Medicinal importance
26 21.804 0.65 9,10-Anthracenedione, 1,5-dimethoxybenzopyrenol C14H10N2O2 238.241 Anthraquinone Dyes, Medicinal importance
27 22.124 4.42 1-Hydroxy-4-methylanthraquinone3-Phenoxy-2H-chromen-2-one2,6-Diaminoanthraquinone C15H10O3 238.242 Anthraquinone Dyes, Medicinal importance
28 22.324 1.32 1-Hydroxy-4-methylanthraquinone9,10-Anthracenediol, 2-ethyl- C15H10O3 238.242 Anthraquinone Dyes, Medicinal importance
29 22.488 0.35 Docosanoic acid C22H44O2 340.5836 Behenic acid) carboxylic acid Detergents, floor polishes
30 22.636 2.20 9,10-Anthracenedione, l-hydroxy-2-(hydroxymethyl)- C15H10O4 254.238 Anthraquinone Dyes, Medicinal importance
31 22.777 1.31 9,10-Anthracenedione,1,8-dihydrox y-3-methyl- C18H18N2O4 326.346 Anthraquinone Dyes, Medicinal importance
32 23.238 1.44 4-Ethenyl-2-methoxyphenol C9H10O2 150.18 Aromatic compound Flavouring agent
33 23.342 2.09 Benzoic acid, heptadecyl ester C23H28 N O3 380.48 Aromatic carboxylic acid Food preservative
34 23.439 0.59 Fumaric acid, cis-hex-3-enyl tetra decyl Ester C4H4O4 116.07 Unsaturated fatty acid Food industry
35 23.632 1.69 Octadecanoic acid, 2,3-dihydroxypr opyl ester C21H42O4 358.5558 Glycerol ester of stearic acid Food additive
36 24.107 7.31 13-Docosenamide, (Z)- C22H43NO 337.5829 Erucid acid (Fatty acid) Lubricant, biodiesel fuel precursor
37 24.865 0.62 22-Tricosenoic acidTriacontyl acetate CH3(CH2)21COOH 354.61 Fatty acid Oil paints, lubricants
38 25.073 0.67 4,5-Dibenzopyrene C20H12 302.35 Aromatichydrocarbon Naturally emittedcoal tar
39 25.964 0.69 gamma.-Tocopherobeta.-Tocopherol C28H48O2 416.680 Vitamin E Antioxidant
41 27.301 2.49 Campesterol5-Cholestene-3-ol,24-methyl- C28H48O 400.69 Phytosterol Lowering cholesterol
42 27.569 3.46 Stigmasterol C29H48O 412.6908 Unsaturatd phytosterol Food making
43 28.096 7.59 gamma.-Sitosterolbeta.-SitosterolStigmast-7-en-3-ol, C29H50O 414.71 Sterol Lowers blood cholesterol
44 28.497 2.22 Cholest-4-en-3-onePregn-4-ene-3, 20-dione, (8. alpha, 10. alpha.)- C27H46O 386.65 Sterol Food making
45 28.661 1.74 Tetradecanoic acid C14H28O2 228.37 Fatty acid Antioxidant, Cancer preventive
46 28.817 2.23 4,22-Stigmastadiene-3-one Spinasterone C39H46O 410.686 Phytosterol Precursor of vitamin D3
47 29.010 0.65 Cyclohex-2-enone, 2 C6H8O 96.13 Ketone Synthesis of pharmaceuticals and fragrances
48 29.463 6.71 Stigmast-5-en-3-one C29H50O 414.72 Beta-Phytosterol Food preparations
49 29.567 0.91 Imidazolidine C3H8N2 72.109 Diamine Muscle relaxant
50 31.447 1.04 Cholestan-3-one C27H48 372.68 Triterpene Food industry
51 31.494 2.83 Stigmasterol C29H48O 412 Sterol Precursor of vitamin D3
52 32.561 1.00 Gibbane-1,10-diarboxyli acid C19H22O6 182.22 Heterocylic acid Fungicides, making dyes

The analysis revealed the presence of 52 photo components. Major compounds detected were sterols, anthraquinones, terpenes, vitamins etc. Sitosterol (7.59%) showed highest peak (dominant component), followed by docos enamide. Among the identified compounds, the diterpene alcohol, phytol is vital in the dispensation of glucose and can trigger enzymes within the body that have strong positive effects on insulin level. This means that phytol in the human diet could perhaps help reinstate the metabolic activities of those with type-2 diabetes [11, 12]. It is also a constituent of chlorophyll in plants and precursor for the manufacture of synthetic forms of vitamin E [13].

Stigmasterol is an unsaturated phytosterol occurring in the plant fats or oils. Stigmasterol is also found in various vegetables, legumes, nuts, seeds etc. Stigmasterol is used as a precursor in the manufacture of semisynthetic progesterone, a valuable human hormone that plays an important physiological role in 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 [14]. It is also used as the precursor of vitamin D3. Recently squalene possesses chemo preventive activity against colon carcinogenesis [15, 16].

CONCLUSION

This is the first report on the analysis of bioactive components present in G. ridsdalei. The result reveals the existence of various bioactive compounds and validates the earlier reports of therapeutic importance of the plant. G. ridsdalei is recommended as a plant of phytochemical and pharmaceutical importance. Further studies can be done to isolate the active principle of the methanolic extract as well as to elucidate the effect of extract for various diseases.

ACKNOWLEDGEMENT

The authors express their sincere gratitude to the Head, Department of Botany, University of Kerala for providing necessary facilities for doing this work and to the University of Kerala for financial support in the form of Junior Research Fellowship is acknowledged by the first author.

CONFLICT OF INTERESTS

Declare none

REFERENCES

  1. Dutt R, Garg V, Madan AK. Can plants growing in diverse hostile environments provide a vital source of anticancer drugs? J Cancer Ther 2014;10:13-37.
  2. Mohanan N, Sivadasan M. Flora of Agasthyamala, Bishen Singh Mahendra Pal Singh. Dehradun; 2002. p. 333.
  3. Zhang A, Sun H, Wang X. Recent advances in natural products from plants for treatment of liver diseases. Eur J Med Chem 2013;63:570-57.
  4. Han YS, van der Heijden R, Verpoorte R. Biosynthesis of anthraquinone in cell cultures of the Rubiaceae. Plant Cell Tissue Organ Culture 2001;67:201–20.
  5. Santos CC, Salvadori MS, Mota VG, Costa LM, de Almeida AA, de Oliveira GA, et al. Antinociceptive and antioxidant activities of phytol in vivo and in-vitro models. Neurosci J 2013;11. http://dx.doi.org/10.1155/2013/949452
  6. Gamble JS. Flora of Presidency of Madras. Vol II. Bishen Singh Mahendra Pal Singh, Dehradun; 1921. p. 650-2.
  7. Ijinu TP, Anish N, Shiju H, George V, Pushpangadan P. Home gardens for nutritional and primary health security of rural poor of South Kerala. Indian J Traditional Knowledge 2011;10:413-28.
  8. Razafimandimbison SG, Bremer B. Nomenclatural changes and taxonomic notes in the tribe Morindeae (Rubiaceae). Adansonia 2011;33:283-309.
  9. Sharma MD, Rautela I, Gahlot M, Sharma N, Koshy EP. GC–MS analysis of photo components in juice sample of Indian cane: Saccharum barberi. Int J Pharm Sci Res 2015;6:5147-53.
  10. Stein SE. National Institute of Standards and Technology (NIST), Mass Spectral Database and Software. Version 3.02. Gaithersburg, USA; 1990.
  11. Mohammad TG, Mohammed HE, Ali J, Seyedhossein H, Mohammad M. Antimicrobial activity, toxicity and stability of phytol as a novel surface disinfectant. Environ Health Eng Manage J 2015;2:13-6.
  12. Peter O, Malin H, Lars I Hellgren, Rikard H. Phytol: a chlorophyll component with anti-inflammatory and metabolic properties. Recent Advances in Redox-Active Plant and Microbial Products; 2014. p. 345-59.
  13. Netscher T. Synthesis of vitamin E. Vitamins Hormones 2007;76:155-202.
  14. Rao CV, Newmark HL, Reddy BS. Chemopreventive effect of squalene on colon cancer. Carcinogens 1998;19:287-97.
  15. . Mohan VR, Sudha T, Chidambarampillai S. GC-MS analysis of bioactive components of aerial parts of Kirganelia Reticulata poir (Euphorbiaceae). J Curr Chem Pharm Sci 2013;3:4.
  16. Alagammal M, Tresina P, Sand Mohan VR. GC-MS determination of bioactive components of Polygala javana dc. Int J Curr Pharm Res 2012;4:42-4.

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