Separation and Identification of New Phytocomponents in Methanolic Extract of Leaves of Hairknot Plant (P. daemia) by GC-MS Analysis

Introduction

The use of plants in several countries as a major source of medicines is hereditary and has an important element of the health care system. India has a lengthy history and strong base for the traditional herbal medicinal system and is suitably called as the botanical garden of the world¹. Biological studies are used to extract wide medicinal properties from plants².

As synthetic drugs which are consumed by human beings may have various side effects and may lead to serious health complications. So, herbal medicine was the life saving drug with fewer side effects and minimum cost effort. Generally, the progress of herbal medicine was carried by the preliminary screening of the constituents in plant sample extracts. The plant chemicals that are not necessary for growth and development but have the properties to protect or prevent diseases are called phytochemicals. The plant produces these chemicals to protect them and they have the ability to treat human diseases also³. There are many phytochemicals, which have their own pharmacological importance⁴. The plant Pergularia daemia is known as “Dushtupatige” in Telugu and “Uttaravaruni” in Sanskrit. Pergularia daemia dried leaf is effectively used in treating asthma, dysmenorrheal, rheumatic fever, bronchitis, amenorrhea, and wounds, etc.⁵. It has pharmacological importance like anti-microbial, anti-fungal properties, anti-rheumatic, anti-arthritic, anti-inflammatory, anti-proliferative, and a good anti-oxidant ^6-9. The previous analysis only reported for potent snack venom potency of four constituents ß-sitosterol, ß-amyrin, alpha-amyrin, and Lupeol from leaves of pergularia daemia by GC-MS method¹⁰. The study on ethanolic extract of leaves reports the existence of methyl Ester pentadecoinic acid, 14- methyl- methyl Esterethyl 9-12-15- Octodecotrionate, hexadecanoic acid, and 4-4 chlorobenzyl- 1 – cyclohexoxyl l-5-tosylamino-1, H- 123 and also ß- sitosterol, lupeol, lupeol acetate, alpha-beta amyrin and its acetate in entire plant and flower. While lupeol-3 Betatranscrotonate and oleanolic acid acetate in dried entire plant¹¹. These reports are helpful for our investigation to go for a complete analysis.

The medicinal assets of P.daemia may be due to the occurrence of its phytochemical ingredients which is not yet explored thoroughly in the Andhra Pradesh region of India except other species in the family¹². Moreover, the preliminary studies^13,14 on the phytochemicals of P. daemia also encouraged for the present study. In the present investigation, the Gas Chromatographic-Mass Spectrometric Analysis (GCMS) is attempted for the methanol extract of dried leaves for the exploration of most phytochemicals in a quantitative manner. The plant possesses various medicinal properties as per the report on Indian medicinal plants¹⁵. So the motto of our study is to identify the most phyto-compounds in the methanolic extract of Pergularia daemia leaves along with their concentrations by GC-MS analysis.

Experimental

Plant Material Collection

The Pergularia daemia plant leaves were collected at Nallamalla forest, Prakasam district, Andhra Pradesh in August. The plant was properly identified by a senior Botanist Prof. Dr. Vatsavaya S. Raju, Department of Botany, Kakatiya University, Telangana-506 009.

Preparation of Extracts

The air dried powder of leaf was extracted in a soxhlet extractor with methanol and was air dried in a hot air oven at 40 degrees Celsius then the material was softened by soaking (maceration) in hot water with occasional steering for 16 hours and water extract was filtered. Finally, the solvent extract was evaporated to remove the final traces of the concerned solvent. The extract recovery in the solvent was expressed as a percent of the plant sample dry matter. This extract was utilized for the GC-MS examination to identify the components.

GC-MS (Gas Chromatography-Mass Spectrometry) analysis

To the sample, 500 µL of n-butane was added, and vortex for 1 min and poured into a screw-capped GC glass vial. Next, the sample was exposed to GC-MS analysis for metabolic data at 70 eV with an electron impact ionization (EI⁺) source. The source is GC 7890 and MS of 5977N, Agilent Technologies, palo Alto, CA, USA. The capillary HP-5 MS column with dimensions 30 mX 250 µmi.d. X 0.25 µm film thickness) consisting of 5% phenyl 95% methylpolysiloxane as stationary phase in splitless mode was used for analysis. The investigation was performed by injecting an aliquot of 1µL of the extract into the injection port at 25 ^oC temperature, by the use of Helium as flow gas with an overall flow rate of 1 mL/min. In this study the oven temperature was programmed as- initial oven temp set at 50 ^oC for 2 min then elevated to 150 ^oC at a rate of 50 ^oC/min and maintained for 2 min then finally raised to 300 ^oC at a rate of 15 ^oC/min, where it was held for 20 min.

Data Pre-Processing

The individual components from the peaks obtained in GC-MS spectra were recognized by relating their mass spectra with the spectra of known compounds stored in the spectral catalog of NIST library (version 8.0). The baseline correction, smoothing, noise reduction, and integration were done before the identification.

Results and Discussion

GC-MS is one of the leading techniques to recognize various constituents of crude extracts like branched chain, long chain, hydrocarbons, amides, vitamins, acids, alcohols, esters, etc. So, in this study GC-MS analysis was selected to identify the components of based on retention time, peak area, molecular formula,molecular weight, and nature of compounds were also analyzed from NIST data. The separated components with their proper Retention times (RT), peak area in percentage, molecular formula, and molecular weight (MW) obtained as per GC-MS analysis (Fig. 1) of the methanol extract of Pergularia daemia leaves were presented Table 1.

Table 1: Compounds recognized in methanolic extract of P. daemia leaves in GC-MS

S.No.	Retention time	Peak area %	Name of the compound	Molecular Formula	Molecular Weight	Nature of the compound
1	3.79	0.31	1-Dodecene	C12H24	168	Alkene
2	4.19	0.09	Trans–alpha–Bergamotene,	C15H24	204	Bicyclic compound
3	4.71	0.16	Pentadecane	C15H32	212	Alkane
4	5.57	0.88	Dichloroacetic acid, 4- hexadecylester	C18H34Cl2O2	353.4	Acid ester
5	6.85	0.17	Tetratriacontane	C34H70	478	Alkane
6	7.29	0.04	Heptacosane	C27H56	380	Alkane
7	7.60	1.21	1- octadecene	C18H36	252	Alkene
8	8.12	0.10	6-Octen-1-ol, 3,7-dimethyl-, (R)-	C10H20O	156	Terpenoids
9	8.69	0.12	Nonadecane	C19H40	268	Alkane
10	9.11	0.18	Dibutyl phthalate	C16H22O4	166	Aromatic carboxylic acid ester
11	9.58	1.68	E-15-Heptadecenal	C17H32O	252	Fatty aldehyde
12	10.59	0.42	Heneicosane	C21H44	296	Acyclic alkanes
13	11.07	0.24	Ethyl Oleate	C20H38O2	310	Fatty acid Ester
14	11.42	1.76	1-Docosene	C22H44	308	Alkene
15	12.36	2.18	1-Iodo-2-methylundecane	C12H25I	296	Iodoalkane
16	13.14	1.74	Dichloroacetic acid, heptadecyl ester	C19H36Cl2O2	367	Acid ester
17	14.03	8.46	Pentacosane	C25H52	352	Alkane
18	14.74	2.36	Hexacosane	C26H54	336	Alkane
19	15.57	15.89	Heptacosane	C27H56	380	Alkane
20	16.28	4.25	Squalene	C30H50	410	Triterpene
21	16.92	7.87	Nonacosane	C29H60	408	Alkane
22	17.55	3.13	Tridecane, 7-hexyl-	C19H40	268	Alkane
23	18.35	3.17	Hentriacontane	C31H64	436	Alkane
24	19.9	2.50	9-Hexacosene	C26H52	364	alkene
25	19.48	1.44	(Z)-14-Tricosenyl Formate	C24H46O2	366	ester
26	20.19	1.71	Z-12-Pentacosene	C25H50	350	Alkene
27	20.48	1.49	8-Hexadecenal,14-methyl-(z)-	C17H32O	252	aldehyde
28	21.44	7.74	1-Hexacosanol	C26H54O	382	alcohol
29	21.84	1.47	Beta-Sitosterol acetate	C29H48	396	Steroid
30	22.72	1.82	1-Docosanethiol	C22H46S	342	Fatty Thioalcohol
31	23.42	2.49	Phytol	C20H40O	296	Terpenoid
32	24.33	2.21	Cyclotriacontane	C30H60	420	Cycloalkane
33	25.06	0.97	Tricosane	C23H48	324	Alkane
34	25.99	1.46	Heptanoic acid, phenyl methyl ester	C7H14O2	130	Ester
35	26.72	1.87	Cyclohexane, 1-(1,5-dimethylhexyl)-4-(4-methylpentyl)-	C6H12	84	Alkane
36	27.19	1.03	1,19-Eicosadiene	C20H38	278	Terminal alkene
37	27.77	0.64	Octanoic acid, octadecyl ester	C26H52O2	396	Ester
38	28.30	2.56	Undecanoic acid, phenyl methyl ester	C18H28O2	276	Ester
39	29.38	0.52	2-methyl- 2-docosene	C23H46	322	Alkene
40	29.63	0.43	Propanamide, N-(4-methoxyphenyl )-2,2,3,3,3-pentafluoro-	C10H8F5NO2	193	Amide
41	29.97	0.48	Androst-5,7-dien-3-ol-17-one,acetate	C22H32O4	360	Steroid
42	30.50	0.85	2-Propenamide,3-phenyl-N,N- bis(phenylmethyl)-	C23H21NO	327	Amide
43	31.05	1.57	Butanoic acid, 2-methyl-	C5H10O2	88	Fatty acid
44	31.90	0.39	1,2-Benzenediol, 3,5-bis(1,1-dimethylethyl)-	C14H22O2	222	Catechol
45	32.25	0.54	Pyridine-3-carboxamide, Oxime, N-(2-trifluoromethylphenyl)-	C13H10F3N3O		Vitamin
46	32.65	0.68	Octadecane, 1-(ethenyloxy)-	C20H40O	296	Ether
47	33.00	0.60	2,6-methano-3-benzazocin-8-ol	C17H23NO	257	Phenolic
48	33.58	1.09	Octanoic acid, Hexadecyl ester	C24H48O2	368	Esters
49	34.42	2.61	Dodecanoic acid, phenylmethyl ester	C19H30O2	290	Ester
50	36.21	1.82	2-Benzo [1,3] dioxol-5-yl-8-methoxy-3-nitro-2H-chromene	C17H13NO6	327	chromene
51	36.70	0.61	Androst-5-ene-3,17-diol, 4,4-di…	C19H26O2	286	Steroid

Figure 1: Graphical presentation of GC-MS spectral chromatogram of methanolic leaf extract Click here to View figure

As per the NIST library, the leaf extract contains several hydrocarbons such as alkanes, alkenes; fatty acids, alcohols, esters, ethers, amides, steroids, terpenoids, triterpenes, vitamin, chromenes and various phenolic compounds. Further, as per the knowledge of the author on the reports of P. daemia, the compounds Pentadecane, Tetratriacontane, Dibutyl phthalate, 1-(ethenyloxy)-Octadecane, and Squalene were not reported in any GC-MS analysis; and some are known to be biologically important (Table 2).

Further, the mass spectrum of the major component (Fig. 2) separated at a retention time of 15.57 min with peak area of 15.89% was also studied and reported in Fig.3. After heptacosane the compound pentacosane was separated as major component at 14.03 with peak area 8.46%.

Figure 2: Structure of the major compound identified in GC-MS of leaf extract Click here to View figure

Figure 3: Mass spectrum of the major compound identified in GC-MS of leaf extract. Click here to View figure

The identified compounds were of many biological importance, the details of some of the components were presented in Table 2. GC-MS examination of phytochemicals in plants provides the design of the pharmacological importance of that plant. So, this type of study with GC-MS examination is the leading step towards knowing the type of medicinal values in the concerned medicinal plants and helpful for detailed study.

Table 2: Bioactivities of some phytocompounds identified in methanolic extract of P.daemia leaves by GC-MS Click here to View table

Conclusions

A total of 51 components were identified in the complete GC-MS examination of P.damea leaves extract. The existence of various bioactive compounds reported in this GC-MS analysis for the methanolic extract of leaves of P. daemia confirmed the use of leaf extract for various activities by the traditional practitioner. Always isolation of individual phytochemicals and subjecting them to various biological activities will surely give effective results that open a new area of examination of the components and their pharmacological efficacy. From these results, it could be concluded that “Pergularia daemia” contains various bioactive compounds. Therefore, the plant is recommended as a plant with good phytopharmaceutical rank.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

The authors are thankful to Acharya Nagarjuna University for constant support and encouragement.

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