1. INTRODUCTION:

Medicinal plants serve as nature’s gift to human beings to live disease free health life. Plants and their bioactive compounds are useful in medicinal practices since ancient times. According to WHO, 80% of the World’s population depends on herbal medicines for their primary health care needs [1]. The plants are important and considered as biosynthetic laboratory due to presence of multitude of compounds having physiological effects. Secondary metabolites present in the plants are the significant compounds which gives therapeutic effects. Now-a-days, medicines from nature, particularly from plants have importance as they are safe and having fewer side effects [2].

The novel molecules from the plant sources have been instrumental to develop structurally modified compounds, which is useful for development of modern therapeutic system [3]. The phytochemicals are naturally occurring biochemical in the plants which are responsible for the medicinal activity and have defense mechanism and protection from various diseases [4]. GC –MS techniques have proved ideal technique for the qualitative and quantitative analysis of volatile and semi-volatile compounds [5].

Phyllanthus niruri is an annual herb belongs to family Euphorbiaceae commonly known as Bhoomi amla or Bhooi amla in Indian local language. This herb is found throughout tropical and subtropical regions worldwide. Phyllanthus niruri has been tagged as a ‘wonder plant’ due to its numerous beneficial effects [6]. It has been widely studied for its species classification, phylogeny and morphology of its flowers, phytochemicals and pharmacological properties [7,8]. Figure 1 shows the plant photograph.

Taxonomy: Kingdom: Plantae; Division: Angiospermae; Class: Dicotyledoneae; Order: Tubiflorae; Family: Euphorbeaceae; Genus: Phyllanthus; Species: niruri Linn

Figure 1. Phllanthus niruri Plant

Phyllanthus niruri is a small erect annual herb which is found in every part of India. It is an erected annual herb 30 to 60 centimeter high, with smooth cylindrical stem often branched at the base. The leaves are numerous, distichous, often imbricating, elliptic oblong obtuse. Stipules are present and very acute. The flowers are alone or usually one male and one female in each leaf axil together. The seeds capsules on stalks are 1 to 2 mm long, 2 mm wide, round, smooth wide and having seeds. The seeds get hurled away with bursting of fruits. Seeds are light brown triangular in shape and nearly 1mm long with 5 to 6 ribs on the back [9]. This plant has been reported useful in Indian traditional systems. People have wide spread attention towards this plants day by day due to its medicinal values. The plant parts had showed the presence of active phytochemicals like flavonoids, alkaloids, terpenoids, lignans, polyphenols, tannins , coumarins etc. [10].

The extracts of this herb have shown therapeutic effects in clinical studies. The plant is of medicinal importance for numerous ailments like diabetes, tumors, diarrhea, tuberculosis, cough, diuretics and influenza [11]. It is also known for a variety of uses antihepatotoxic, antihepatitis B, antihyperglycemic, antibacterial, hepatoprotective action, lipid lowering action, antifungal action, analgesic, anti-inflammatory and cardioprotective [12].

2. MATERIALS AND METHODS:

2.1 Collection of Plant material:

The fresh whole plant of Phyllanthus niruri were collected from the field sides of Bela Village, Nagpur, Maharashtra, India. The plant sample were thoroughly washed with clean water to remove dust and dirt and then air dried under shade at room temperature for about 15 days. The various parts viz, the roots, stem, and the leaves were separated. The dried plant samples were ground to fine powder using pestle and mortar and a domestic electric grinder. The material was stored in an airtight brown color glass bottle for further use.

2.2 Extraction of Plant material:

The powdered plant material of Phyllanthus niruri was extracted using methanol (≥99% pure, Merck) solvent by cold extraction (maceration) method. The process was repeated number of times. In each process, 25grams of plant material was dissolved in 250ml. of methanol solvent in air-tight stopper brown bottle. After an interval of nearly 5-6 hours, the bottle was shaken for half an hour using a shaking machine. This process was repeated for 3 days. After extraction, the solution was filtered through Whatman filter paper no. 1. The filtrate was evaporated using rotary evaporator and kept for total dryness at room temperature. The obtained dried crude extract was stored in an airtight glass container. The bottle was stored in a refrigerator at 4⁰C for further analysis.

2.3 Preliminary Phytochemicals Analysis:

The crude extracts were subjected to the qualitative chemical tests for the detection of various phytochemicals present using standard procedures as described by Harbone, Trease and Evans and Sofowora [13,14,15]. The reported classes of phytochemical are listed in Table 1.

1. Alkaloids-2ml. of extract was treated with few drops of Hager’s reagent (picric acid dissolved in benzene). Formation of a yellow precipitate indicated the presence of alkaloids.

2. Flavonoids -1ml. of extract was treated with 10% of 1ml. Pb(OAC)₄. Formation of intense yellow colour indicated the presence of Flavonoids.

3. Phenols- 2ml. of extract was treated with 3 to 4 drops of FeCl₃ solution. The formation of bluish black colour indicated the presence of Phenols.

4. Saponins- 5ml. of extract, a drop of sodium bicarbonate solution was added. The test tube was shaken and allowed to stand for 3 minutes. Formation of honey comb like froth indicated the presence of Saponins.

5. Terpenoids- 2ml. extract, was treated with 2ml (CH₃CO)₂O and 2 to 3 drops of conc. H₂SO₄ was added. Appearance of red colour indicated the presence of Terpenoids.

6. Tannin- 1ml of extract was treated with 2 to 3 drops of FeCl₃ solution. Formation of green precipitate indicated the presence of Tannin.

7. Coumarins-2 ml extract was treated with 3 ml of 10% NaOH solution. Appearance of yellow colouration indicated the presence of Coumarins.

8. Phytosterols (Salkowski test for steroids)- Extract was treated with 2ml. chloroform and then filtered. Filtrate was treated with few drops of H₂SO₄, shaken gently and allowed to stand. Appearance of golden colour (reddish brown) indicated the presence of Phytosterols.

Table 1: Reported phytochemicals in the methanol extract of Phyllanthus niruri

Sr. No.	Phytoconstituents	Methanol Extract
1	Alkaloids	+
2	Flavonoids	+
3	Phenols	+
4	Saponins	+
5	Terpenoids	+
6	Tannin	+
7	Coumarins	+
8	Phytosterols	+
(+ Present) (- Absent)

It is observed that the methanol extract of Phyllanthus niruri contains valuable phytochemicals. Hence, decided to perform GC-MS analysis of the extract.

2.4 GC-MS Analysis:

The GC-MS analysis was carried out using Thermo Scientific TSQ 8000 Gas Chromatograph-Mass Spectrometer. For the analysis, an electron ionization system with energy of 70 ev was used. Helium gas (99.99%) was used as the carrier gas at a constant flow rate of 1ml./minute. In the gas chromatography part, the oven temperature was raised from 60⁰C to 280⁰C (Rate of 10⁰C/min). The total GC running time was about 35 minutes.

The Mass spectra were carried out coupled with Gas chromatography by maintaining 70 ev voltage and keeping the source temperature of 230⁰C. The inlet line temperature was maintained at 280⁰C and mass scan rate was tuned to 50-700. The total MS running time was about 34 minutes.

2.5 Identification of Components:

Interpretation of mass spectrum of GC -MS was conducted using database of National Institute of Standard and Technology (NIST) having more than 62,000 patterns in the library. The spectrum of the unknown components was compared with the spectrum of the known components stored in NIST library. Retention time, molecular weight, molecular formula and percentage composition helps in identification of components.

3. RESULTS AND DISCUSSION:

Preliminary phytochemical screening of crude methanol extracts of Phyllanthus niruri showed the presence of flavonoids, alkaloids, tannins, coumarins, saponins, terpenoids, phenonls, and steroids. Qualitative screening of Phyllanthus niruri extracts confirms the presence of the secondary metabolites in plants.

The obtained GC-MS chromatogram of the methanolic extract is shown in figure 2. It shows nearly 20 peaks indicating presence of many important phytochemical constituents. Table 2 shows the probability of some of the identified compounds present in the methanol extract from the CG-MS chromatogram using NIST library. More than 8 peaks shows the retention time higher than 10 whereas, three compounds shows relative abundance higher than 50.

Figure 2. GC- MS Chromatogram of methanol extract

Table 2. Phytocomponents detected in the methanol extract of whole plant of Phyllanthus niruri using GC-MS

Sr. No.	Retention time	Name of compound	Molecular formula	Peak Area %	Molecular weight
1	6.50	a-Ylangene	C₁₅H₂₄	0.66	204.18
2	6.50	6-Isopropenyl-3-methoxymethoxy-3-methyl-cyclohexene	C₁₂H₂₀O₂	0.66	196.29
3	6.50	1,1,3a-Trimethyl-1a,3a,5,6-tetrahydro-1H-cyclopropa[c] pentalen-4-one	C₁₂H₁₆O	0.66	176. 25
4	19.48	9,12,15-Octadecatrienoic acid, methyl ester,(Z,Z,Z)-	C₁₉H₃₂O₂	0.57	292.45
5	19.48	Methyl,8,11,14-heptadecatrienoate	C₁₈H₃₀O₂	0.57	278.43
6	19.48	Ethyl 9,12,15-Octadecatreinoate	C₂₀H₃₄O₂	0.57	306.49
7	24.38	1-Monolinoleoylglycerol trimethylsilyl ether	C₂₇H₅₄O₄Si₂	0.21	498.89
8	24.38	9,12,15-Octadecatreinoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester,(Z,Z,Z)-	C₂₇H₅₂O₄Si₂	0.21	502.92
9	24.38	8,14,-Seco-3,19-epoxyandrostane-8,14,-dione,17-acetoxy-3a-methoxy-4,4-dimethyl-	C₂₄H₃₆O₆	0.21	420.54
10	25.12	13-Docosenamide,(z)-	C₂₂H₄₃NO	5.15	337.59
11	25.12	trans-13-Docosenamide	C₂₂H₄₃NO	5.15	337.59
12	25.12	Bis(cis-13-docosenamido)Methane	C₄₅H₈₆N₂O₂	5.15	687.19
13	26.33	2,3,O-p-Anisylideneguanosine	C₁₈H₁₉N₅O₆	0.71	401.37
14	26.33	6,7-Epoxypregn-4-ene-9,11,18-triol-3,20-dione,11,18-diacetate	C₂₅H₃₂O₈	0.71	460
15	26.33	2-[5-(2,2-Dimethyl-6-methylene-cyclohexyl)-3-methyl-pent-2-enyl]-1,4-dimethoxy-benzene	C₂₃H₃₄O₂	0.71	342.5
16	26.67	Carissanol dimethyl ether	C₂₂H₂₈O₇	28.30	404.5
17	26.67	3Furanmethanol, à-(3,4-dimethoxyphenyl) tetrahydro-3-hydroxy-4-veratryl-	C₂₂H₂₈O₇	28.30	404.5
18	26.67	1H-1,2,4-Triazol-5-amine, N-(3,4-dimethoxybenzyl)-	C₁₁H₁₄N₄O₂	28.30	234.25
19	26.86	2,3,3',4-'tetramethoxy-à-methyl-5-(prop-1-enyl)stilbene	C₂₂H₂₆O₄	6.31	354.43
20	26.86	4H-Cyclopropa[5',6'] benz [1',2':7,8]azuleno[5,6]oxiren-4-one, 8,8a-bis(acetyloxy)-2a-[(acetyloxy)methyl]-1,1a,1b,1c,2a,3,3a,6a,6b,7,8,8a-dodecahydro-6b-hydroxy-3a-methoxy-1,1,5,7-tetramethyl-,	C₂₇H₃₆O₁₀	6.31	520.56
21	26.86	[1aR-(1aà,1bá,1cá,2aá,3aà,6aà,6bà,7à,8á,8aà)]-Spiro[isoquinoline-1,2'-indene],1,2,3,4,2',3'tetrahyd ro-6'-hydroxy-6,7,3',7'-tetramethoxy-2-methyl-1'-ox o-	C₂₂H₂₅NO₆	6.31	399.43
22	27.62	7-Azadibenz[a,e]azulen-12-one, 5,6,7,7a,12,12a-hexahydro-7-methyl-8,9-bis(methoxy)-12a-hydroxy-2,3-methylenedioxy-	C₂₁H₂₁NO₆	12.81	383.39
23	27.62	2,3,3',4'tetramethoxy-5-(3-methoxyprop-1-enyl-)- à-m ethylstilbene	C₂₃H₂₈O₅	12.81	384.46
24	27.62	Bufa-20,22-dienolide, 14,15-epoxy-3,16-dihydroxy, (3á,5á,15á,16á)-	C₂₄H₃₂O₅	12.81	400.50
25	28.60	Furo[3',4':6,7]naphtho[2,3-d]-1,3-dioxol-6(5aH)-one,5,8,8a,9-tetrahydro-5-(3,4,5-trimethoxyphenyl)-,[5R-(5à,5aá,8aà)]-	C₂₂H₂₂O₇	8.32	398.40
26	28.60	2,3,3',4-'tetramethoxy-à-methyl-5-(prop-1-enyl)stilbene	C₂₂H₂₆O₄	8.32	354.43
27	28.60	1',1-'Dicarboethoxy-1á,2á-dihydro-17á-hydroxy-3'H-cycloprop[1,2]androsta-1,4,6-trien-3-one	C₂₆H₃₄O₆	8.32	442.54
28	28.71	3-[(3,4-Dimethoxy-benzylamino)-methyl]-8a-methyl-5-methylene-decahydro-naphtho[2,3-b]furan-2-one	C₂₄H₃₃NO₄	27.28	399.52
29	28.71	Phenethylamine, 2-methoxy-à-methyl-4,5-(methylenedioxy)-	C₁₁H₁₅NO₃	27.28	209.24
30	28.71	4-Methyl-2,5 Dimethoxyphenethylamine	C₁₁H₁₇NO	27.28	195.25
31	29.12	2,3,3,4-tetramethoxy-5-(3-methoxyprop-1-enly)stilbene	C₂₂H₂₆O₅	0.34	370.43
32	29. 12	9-Desoxo-9-x-acetoxy-3,8,12-tri-O-acetlingol	C₂₈H₄₀O₁₀	0.34	536.61
33	29.12	6,7-Epoxypregn-4-ene-9,11,18-triol-3,20-dione,11,18-diacetate	C₂₅H₃₂O₈	0.34	460
34	30.58	7,8-Epoxylanostan-11-ol,3-acetoxy-	C₃₂H₅₄O₄	0.62	502.78
35	30.58	Rhodopin	C₄₀H₅₈O	0.62	554.90
36	30.58	Propanoic acid,3,3-thiobis-, didodecyl ester	C₃₀H₅₈O₄S	0.62	514.84

Table 3: Activity of a few Phytochemicals identified in the methanol extracts of the whole plant of Phyllanthus niruri by GC-MS

R. T.	Name of Compound	Molecular formula	Molecular weight	Biological Activity [16,17]
6.50	a-Ylangene	C₁₅H₂₄	204.18	Anti-inflammatory,
19.48	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-	C₁₉H₃₂O₂	292.45	Antifungal
25.12	13-Docosenamide,(z)-	C₂₂H₄₃NO	337.59	Antimicrobial
25.12	Bis(cis13-docosenamido) methane	C₄₅H₈₆N₂O₂	687.19	Antimicrobial, antioxidant, anti-inflammatory
27.62	Bufa-20,22-dienolide,14,15-epoxy-3,16-dihydroxy, (3á,5á,15á,16á)-	C₂₄H₃₂O₅	400.50	Cell growth inhibitory activity and antitumor
29.12	6,7-Epoxypregn-4-ene-9,11,18-triol-3,20-dione,11,18-diacetate	C₂₅H₃₂O₈	460	Antimicrobial, Anti-inflammatory, Anticancer, Diuretic Antiasthma, Antiarthitic

4. CONCLUSION:

GC-MS analysis of the methanol extract of Phyllanthus niruri showed the existence of various compounds with different chemical structures. The biological activities of some identified phytocomponents used for antimicrobial, anifungal, antioxidant, anti-inflammatory, anticancer, diuretic antiasthma, antiarthitic. The research findings have shown that whole plant extract of Phyllanthus niruri is extensively rich in secondary metabolites. The whole plant has a high potential for a vast number of bioactive compounds which justified its use for treatment of various ailments by traditional practitioners. These findings have formed a scientific basis to the ethnomedical usage of the plant. However isolation of the individual phytochemical constituents and subjecting it to biological activity and toxicity profile may be significant further for finding of a novel drug or a lead compound.

5. ACKNOWLEDGMENT:

The authors are thankful to Chemistry and Environment Science Department, Institute of Science, Nagpur for providing all the necessary laboratory facilities for experimental work. The authors are also thankful to Sophisticated Analytical Instrument Facility, Punjab University, Chandigarh for CG-MS analysis.

6. REFERENCES:

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Received on 06.09.2019 Modified on 12.10.2019

Research J. Pharm. and Tech. 2020; 13(8):3618-3622.

DOI: 10.5958/0974-360X.2020.00640.X

1. INTRODUCTION:

2. MATERIALS AND METHODS:

2.1 Collection of Plant material:

2.2 Extraction of Plant material:

2.3 Preliminary Phytochemicals Analysis:

Sr. No.

Phytoconstituents

Methanol Extract

1

Alkaloids

+

2

Flavonoids

+

3

Phenols

+

4

Saponins

+

5

Terpenoids

+

6

Tannin

+

7

Coumarins

+

8

Phytosterols

+

(+ Present) (- Absent)

2.4 GC-MS Analysis:

2.5 Identification of Components:

Sr. No.

Retention time

Name of compound

Molecular formula

Peak Area %

Molecular weight

4. CONCLUSION: