Identification of Bioactive Phytocomponents of Hydroalcoholic Extract of Enhalus acoroides by Gas Chromatography- Mass Spectrometry Analysis

S. Navaith Ahmed, P. Kalaivani, P. Amudha, B. Usharani*

Department of Biochemistry, Vels Institute of Science, Technology and Advanced Studies,

Pallavaram, Chennai, Tamilnadu-600117, India.

*Corresponding Author E-mail: raniushab1@gmail.com

ABSTRACT:

Sea grass are good source of potent drug which has medicinal properties and able to cure human disease. One such marine plant is the Enhalus acoroides which belong to hydrocharitaeceae family. This marine species are widely distributed in the tropics of Indian and western Pacific Ocean and the species were collected from Ramanathapuram district for further process. This study reveals about the bioactive components present in Enhalus acoroides and identified its biological activity by Gas chromatography Mass spectrometry analysis using hydroalcoholic extract. The compounds present in Enhalus acoroides are Benzoic acid, 2- methyl 7- oxa bicyclol heptanes, 1, 3 Nonadiene, silane, ethoxytriethyl. Biological activities of the compounds present in the sample include antioxidant, hypocholesterolemic, antihypertensive, anti-inflammatory, anti-microbial, antiviral and anti-hepatotoxic effect.

KEYWORDS: Cancer, Enhalus acoroides, Gas Chromatography- Mass Spectrometry, Phytocompounds, Biological activity, 2-Butynediamide, Antioxidant.

INTRODUCTION:

All over the globe, non-communicable diseases including cancers, cardiac problem, diabetes and chronic respiratory illness plays a major threat to today’s mankind health and development¹. In 1985, World Health Organization estimated that approximately 65% of the world’s population predominately relied on medicinal herbs for their primary health care illness². The traditional medicines like Herbs and species makes human beings healthier³. Herbs are used from the ancient periods for the treatment of human ailments⁴. In India, around 2500 herbs have been identified for the valuable medicinal values⁵. Due to the folkloric properties of herbs, many developing countries use the plants for therapeutic remedies⁶. Knowledge in phytochemicals of plants is desirable not only for the therapeutic properties, but also helps in finding the economic phytoconstituents for the synthesis of chemical substances for the folkloric problems⁷. Phytochemicals plays a major role in the alternatives for the control of antibiotic resistant human pathogens⁸.

Herbs possess several essential phytochemicals including flavonoids, phenolics, carotenoids and so on which possess antioxidant propeties⁹. Basically sea grass has high antioxidant activity and able to cure various type of cancers. Especially Cymodoceae serrulata has high cytotoxic effect on silver nano particles and capable of curing cervical cancer. It shows high inhibition rate¹⁰. Apart from potent drug application, sea grass may also be used as a fertilizer; nourishment since they are good in lipid, protein and fibre. Caroteniods and chlorophyll acts as an antioxidants and vitamins because they present in higher amount and hence it has a wide application on Pharmacological industry¹¹. Seagrass is one of the essential medicinal plants. Sea grass is the marine plants which serve as a feedent for marine organisms and endangered species¹². Over the Past years it has been proved that sea grass used for remedial purposes which may include stomach problems, skin diseases, fever, muscle pain, wounds¹³. Enhalus acoroides comes under hydrocharitaceae family which is a monotypic marine genus¹⁴. Leaves of Enhalus acoroides are larger and found in the sub tidal region. The extract of Enhalus acoroides show high antioxidant activity¹⁵. GC-MS analysis has various applications in finding the mode of action of medicinal herbs¹⁶. This study reveals about the bio-compounds present in the hydro alcoholic extract of Enhalus acoroides by Gas Chromatography Mass Spectrometry analysis and determined its pharmacological activities.

MATERIALS AND METHODS:

Collection and Authentication of Enhalus acoroides:

Sample has been collected from Devipattinam, Ramanadhapurm District on June, 2019 and authenticated from Dr. P.Jeyaraman, PhD., Director, Plant Anatomy Research Centre, Presidency College.

Preparation of Alcoholic Extract:

The sample was washed with tap water, shadow dried and grinded in herbal grinder. 20 grams of powder sample were transferred separately in to conical flasks. The conical flask containing 1000ml of hydro-alcohol (70%) were added and vigorously shaking for one hour using rotatory shaker and kept for 24 hours. Through whatman No.1 filter paper, the extracts were filtered after 24 hours. Store the filtrate at 4^◦C and the extract was used for GC MS analysis.

GC MS-Analysis:

GC-MS analysis was carried out on Shimadzu 2010 plus comprising a AOC-20i auto sampler and gas chromatograph interfaced to a mass spectrometer instrument employing the following conditions: column RTX 5Ms (Column diameter is 0.32mm, column length is 30m, column thickness 0.50µm), operating in electron impact mode at 70eV; Helium gas (99.999%) was used as carrier gas at a constant flow of 1.73 ml /min and an injection volume of 0.5 µI was employed (split ratio of 10:1) injector temperature 270 ºC; ion-source temperature 200 ºC. The oven temperature was programmed from 40 ºC (isothermal for 2 min), with an increase of 8 ºC/min, to 150ºC, then 8ºC/min to 250ºC, ending with a 20min isothermal at 280ºC. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 40 to 450 Da. Total GC running time is 51.25min. The relative percentage 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 Ver 5.2.00¹⁷.

Identification of components:

By using the database of National Institute Standard and Technology (NIST) interpretation on GCMS was conducted since it has more than 62,000 patterns. The spectrum of the known components was compared with the spectrum of the unknown component stored in the NIST library. The name of the compound, molecular weight and structure of the phytocomponents were ascertained.

RESULTS AND DISCUSSION:

Identification of Bioactive Compounds in Enhalus acoroides by GC MS analysis:

Twenty compounds were identified in hydro alcoholic extract of Enhalus acoroides by GC-MS analysis.. The chromatogram obtained from the hydro alcoholic extract of Enhalus acoroides shown in Figure 1. The active phytocomponents along with their retention time (RT), molecular wirght, molecular formula and concentration (%) are presented in Table 1. Figure 2 shows the structure of identified prevailing compounds which includes Hexadecanoic acid (1.73%), methyl ester, 9-Octadecenoic acid (Z) (5.34%), 9,12,15-Octadecatrienoic acid, methyl ester, e, 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]l, 8,9,9,10,10,11-hexafluoro-4,4-Dimethyl-3,5-dioxatetracyclo and Stigmast-5-en-3-ol, (3.beta.,24S).

Table 1: Identification of Phytochemical compounds in the Hydro alcohohic extract of Enhalus acoroides using GC-MS

Peak	R. Time	Area %	Height %	Molecular Formula	Molecular Weight	Bioactive Compounds
1	9.990	0.18	0.56	C₉H₁₁N₃O₂	193	Benzoic Acid, 2-(3,3-Dimethyl-1-Triazenyl)
2	12.611	0.12	0.47	C₇H₁₂O	112	2-Methyl-7-Oxa-Bicyclo[2.2.1]Heptane
3	13.106	0.37	0.97	C₈H₁₄O	126	4-Hepten-3-one, 5-methyl-, (E)
4	13.225	0.13	0.27	C₄H₄N₂O₂	112	2-Butynediamide
5	13.367	0.20	0.43	C₉H₁₆	124	1,3-Nonadiene, (E)
6	13.548	1.73	4.41	C₁₇H₃₄O₂	270	Hexadecanoic acid, methyl ester
7	13.977	5.34	8.06	C₁₈H₃₄O₂	282	9-Octadecenoic acid (Z)
8	15.200	0.03	0.19	C₁₂H₂₂O	182	3-Dodecen-1-AL
9	15.336	1.12	3.14	C₁₉H₃₄O₂	294	9,12-Octadecadienoic acid, methyl ester, (E,E)
10	15.423	2.56	5.29	C₁₉H₃₂O₂	292	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)
11	15.540	1.33	2.86	C₂₀H₄₀O	296	2-Hexadecen-1-ol, 3,7,11,15 tetrame thyl-, [R-[R,R-(E)]]
12	15.600	1.39	2.04	C₁₀H₂₀O₂	172	Methyl 4-methyloctanoate
13	15.700	0.85	1.85	C₁₂H₁₂F₆O₂	302	8,9,9,10,10,11-HEXAFLUORO-4,4-dimethyl-3,5-dioxatetra cyclo
14	15.858	5.27	6.37	C₂₂H₄₂O₂	338	Laurinsaeure, 9-Decen-1-Ylester
15	15.908	2.52	6.64	C₁₅H₂₆	206	7-Pentadecen-5-yne, (E)
16	15.992	3.69	6.36	C₈H₂₀OSI	160	Silane, ethoxytriethyl
17	16.267	20.79	11.34	C₂₉H₃₉NO₄	465	2-(6-[1-ethyl-4-[4-(1h-pyrrole-2-carbonyl)-2,3,3a,4,5,7a-hexahydro-1h-inden-5-yl]-buta-1,3-dienyl]-5-methyl-tetra
18	16.415	19.12	12.85	C₂₉H₅₀O	414	Stigmast-5-en-3-ol
19	16.608	13.05	12.69	C₂₉H₅₀O	414	Stigmast-5-en-3-ol
20	16.660	20.20	13.22	C₂₉H₅₀O	414	Stigmast-5-en-3-ol

Figure 1: Chromatogram of hydroalcoholic extract of Enhalus acoroides

Table 2: Pharmacological activities of bio-compounds identified in the Hydro alcohohic extract of Enhalus acoroides using GC-MS

Peak	R. Time	Name of the compounds	Pharmacological Activity²²
1	13.548	Hexadecanoic acid, methyl ester	Antioxidant, hypocholesterolemic, Anti androgenic, hemolytic, Alpha reductase inhibitor.
2	13.977	9-Octadecenoic acid (Z)	Antihypertensive, Increase HDL and decrease LDL Cholesterol
	15.336	9,12-Octadecadienoic acid, methyl ester	Antiinflammatory,hypocholesterolemic cancer preventive, hepatoprotective, nematicide, insectifuge, antihistaminic antieczemic, antiacne, 5-Alpha reductase inhibitor, antiandrogenic, antiarthritic, anticoronary, insectifuge
3	15.423	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)	Hypocholesterolemic, Nematicide Antiarthritic, Hepatoprotective, Anti androgenic, Nematicide, 5-Alpha reductase inhibitor, Antihistaminic, Anticoronary, Insectifuge, Antieczemic Anticancer
4	15.540	2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R,R-(E)]]	Antimicrobial, Anti-inflammatory
5	15.700	8,9,9,10,10,11-hexafluoro-4,4-dimethyl-3,5-dioxatetracyclo	Antimicrobial
6	16.415	Stigmast-5-en-3-ol, (3.beta.,24S)	Anti-hepatotoxic, Antiviral, Antioxidant, Cancer¬ preventive, Hypocholesterolemic.

Figure 2: The Structures of Identified Phytochemical Compounds of Hydro alcoholic extract of Enhalus acoroides

In asthma patients 2-Hexadecen-1-ol produce therapeutic effects and also acts as a source of anti-inflammatory and antitumor agents²³. Sermakkani et al.,narrates that hexadecane shows high antioxidant and anticancer activities²⁴. The compound 9, 12, 15-Octadecatrienoic acid possess hepatoprotective, cancer preventive, nematicide, antieczemic, antihistamine, anticoronary, antiarthritic and antiviral properties^{25, 26}. The methanol and hexane extract of Enhalus acoroides has antimicrobial activity. The hydroalcoholic extract shows high antioxidant activity which has ability to cure degenerative diseases²⁷. The compounds like hexadecenoic acid and 12, 15-Octadecatrienoic acid has reduced growth of human and fish pathogens. Thus, the compound hexadecnoic acid has important application on pharmaceutical industry²⁸.

CONCLUSION:

The current investigation of Gas Chromatography- Mass Spectrometry analysis shows that there are 20 various phytocompounds were identified in the hydro alcoholic extract of Enhalus acoroides. This study reveals that Enhalus acoroides has pharmacological activities like antioxidant, anti-inflammatory, anti-androgenic, anti-microbial, anticancer and antihepatotoxic. Due to it adverse pharmacological properties, the hydro alcoholic extract of Enhalus acoroides has the ability to cure many therapeutic diseases. Hence Enhalus acoroides might be used for developing novel drugs, and further study needs to elucidate the novel bioactive compounds and their mechanism of action.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 26.04.2020 Modified on 14.01.2021

Research J. Pharm.and Tech 2021; 14(12):6511-6515.

DOI: 10.52711/0974-360X.2021.01126