INTRODUCTION:

Andrographis paniculata (Burma. f.) Wall. ex Nees (AP) is an important medicinal plant and widely used around the world. It belongs to the family Acanthaceae. Andrographis paniculata is used as a traditional herbal medicine in Bangladesh, China, Hong Kong, India, Pakistan, Philippines, Malaysia, Indonesia and Thailand ^[1,2] and is ethno botanically used for the treatment of snake bite, bug bite, diabetes^[3], dysentery, fever and malaria^[4]. Andrographis paniculata is the most predominantly used plant in the Indian traditional systems of Ayurveda, Siddha, Unani and in other Asian traditional systems. The aerial part of the plant is used in the form of infusion, raw juice, powder and decoction either alone or in combination with other medicinal plants. Andrographis paniculata is given in the form of tablets in Indian system of medicine and injection form in Chinese medicine for the treatment of common flu, swine flu, chikungunya, malaria and other viral fevers.

The aerial parts of Andrographis paniculata are most commonly used; its extracts contain diterpenoids, diterpene glycosides, lactones, flavonoids and flavonoid glycosides. Whole plant leaves and roots are also used as a folklore remedy for different diseases in Asia and Europe^[1,5]. Snake bite is a serious problem in tropical and subtropical countries like India. According to World Health Organization (WHO), poisonous snakes are responsible for at least 5 million human fatalities annually^[6].

Andrographis paniculata L. is an erect annual herb extremely bitter in taste in all parts of the plant body. It is known as Kalmegh or Kalamegha, meaning dark cloud in Ayurveda and Nilavembu in Siddha. Andrographis paniculata grows erect to a height of 30-110 cm in moist, shady places. The slender stem is dark green, squared in cross-section with longitudinal furrows and wings along the angles. The leaves are lanceolate having hairless blades measuring up to 8 cm length by 2.5 cm width. The small flowers are borne in spreading racemes. The fruit is a capsule around 2 cm long and a few mm wide. It contains many yellow-brown seeds^[7]. In under developing countries numerous medicinal plants are used traditionally which are remedial against these disease (Pinn, 2000). It is found in wild throughout the plains of India especially in Tamilnadu, Karnataka, Maharashtra, Orissa and Uttar Pradesh.

MATERIALS AND METHODS:

Plant Material:

Andrographis paniculata was collected from the surrounding of Nagapattinam during August to December 2017.

Preparation of Extract:

The collected plant leaves were washed with water. Washed leaves were dried at room temperature for 10-15 days in shaded place and grinded into coarse powder. Required quantity of the whole plant of Andrographis paniculata was weighed and transferred to flask treated with ethanol [Polar solvent] until the coarse powder was fully immersed, incubated over night and filtered through a Whatmann N0.41 filter paper. The coarse powder was subjected to successive extraction with ethanol. The extracts were collected. The collected extracts were mixed with diethyl ether to separate non polar compounds using separating funnel by three to five times. Alcohol layer and Diethyl ether layer was segregated using separating funnel and collected in separate beakers and subjected to evaporation. The dried substance obtained is used for further analysis^[8,9]. The active compounds were identified by Gas Chromatography-Mass Spectrometer technique.

GC-MS analysis:

The GC-MS analysis was carried out using a Shimadzu-GCMS-QP-2010plus (Auto system XL) Gas chromatograph equipped and coupled to a mass detector SHIMADZU Rts-5MS-5.1 spectrometer, 30m × 0.32 mm× 0.50µm, of capillary column. The injector temperature of instrument was set to an initial temperature of 270˚C, and maintained at this temperature. The oven temperature was maintained at 45 degree centigrade. The oven temperature was raised to 150 degree centigrade (0 min hold) and oven temperature was again raised from 150˚C to 250˚C at the rate of 5˚C/min, and maintained for 10 min at the end. The total sample running time is 30 minutes. Injection port temperature was ensured as 270˚c and Helium flow rate as 5.5lb/in2. The pressure was 11.6Kpa. The samples were injected in split mode as 10:1. Mass spectral scan range was set at 45-450 (m/z). The NIST library is used to identify the compounds present in the sample extract^[10]. The mass spectrum of individual compound is matched with the mass spectrum of compounds in the sample chromatogram showed as peaks and identified the nature of compounds.

RESULTS AND DISCUSSION:

The studies on the Active Compounds^[11,12,13] in the Andrographis paniculata whole plant ethanol extract in organic layer by Gas Chromatography-Mass Spectrometer analysis is clearly showed in the chromatogram. Figure 1.

Table.1: compounds identified in GCMS analysis- Ethanolic extract

S.No	RT	Compound Name	Formula	Peak Area %	Compound Nature	**Activity
1	5.792	Butane, 1,1-diethoxy-3-methyl-	C₉H₂₀O₂	0.28	Ether compound	No activity reported
2	5.827	Pentane, 1,1-diethoxy-	C₉H₂₀O₂	0.36	Ether compound	No activity reported
3	6.011	3,3- Diethoxy – 2 -butanone		0.22	Ketone compound	No activity reported
4	7.824	Propane, 1,1,3-triethoxy-	C₉H₂₀O₃	8.38	Ether compound	No activity reported
5	8.256	1,1,3-Triethoxy butane	C₁₀H₂₂O₃	0.09	Ether compound	No activity reported
6	13.878	6-Chloro-2,2,9,9-tetramethyl -3,7--Decadiyn-5-ol	C₁₄H₂₁ClO	0.12	Chlorine compound	Antimicrobial
7	15.392	Sulfurous acid, butyl pentyl ester	C₉H₂₀O₃S	0.06	Sulfur compound	Antimicrobial Fungicide
8	15.565	1,2-Benenedicarboxyli acid, diethyl ester	C₁₂H₁₄O₄	2.19	Plasticizer compound	Antimicrobial Antifouling
9	17.760	Tetracosane	C₂₄H₅₀	0.26	Alkane compound	No activity reported
10	17.925	Bicyclo[3.1.0]hexane-6-methanol, 2-hydroxy-1,4,4-trimethyl-	C₁₀H₁₈O₂	0.09	Alcoholic compound	Antimicrobial
11	18.165	2, 2, 5, 5-Tetramethyl-3-hexene.	C₁₀H₂₀	0.07	Unsaturated compound	No activity reported
12	18.228	2,6,10-Trimethyl, 14-ethylene-14-pentadecne	C₂₀H₃₈	1.56	Unsaturated compound	No activity reported
13	18.333	2-Pentadecanone, 6,10,14-trimethyl-	C₁₈H₃₆O	0.56	Ketone compound	No activity reported
14	18.500	3,7,11,15-Tetramethyl-2-hexadecen-1-ol-[Phytol]	C₂₀H₄₀O	0.21	Diterpene	Antimicrobial Anti-inflammatory Anticancer Diuretic
15	18.712	Cyclopropanoic acid, 2-[(2butylcyclopropyl) methyl]	C₂₁H₃₈O₂	0.72	Acidic compound	No activity reported
16	19.741	Dibutyl phthalate	C₁₂H₁₄O₄	0.62	Plasticizer compound	Antimicrobial Antifouling
17	19.885	Hexadecanoic acid, ethyl ester-	C₁₈H₃₆O₂	1.80	Palmitic acid ethyl ester	Antioxidant, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Flavor, Hemolytic 5-Alpha reductase inhibitor
18	20.874	Pentafluoropropionic acid, pentadecyl ester	C₁₈H₃₁F₅O₂	0.40	Fluorine compound	Antimicrobial
19	21.178	Phytol isomer	C₂₀H₄₀O	1.23	Diterpene	Antimicrobial, Anti-inflammatory, Anticancer Diuretic
20	21.364	2,8-Dimethyl-2-(4,8,12-Trimethyl tridecyl)-6-Chromanol [Delta-Tocopherol]	C₂₇H₄₆O₂	0.90	Vitamin E	Antimicrobial, Antidiabetic, Antioxidant Anti-inflammatory, Skin care agent, Anticholestrol
21	21.733	(1,3-Dimethyl-2,4,6-trioxo-2,3,4,5,6,7-Hexahydro-1H- Pyrrolo [2,3-D] Pyrimidin-5-yl)-Acetic acid.	C₁₀H₁₁N₃O₅	0.32	Alkaloid	Antimicrobial, Anti-inflammatory Antioxidant
22	22.029	Octadecanoic acid, Ethyl ester.	C₂₀H₄₀O₂	0.79	Stearic acid ethyl ester	No activity reported
23	23.791	1,7-Di(dodec-9-ynyl)-2,2,4,4,6,6-hexamethyl-1,3,5,7-tetraoxa-2,4,6-trisilaheptane.	C₃₀H₆₀O₄Si₃	0.05	Silica compound	No activity reported
24	27.500	3-(4-Methoxycarbonylmethyl-2-oxo-2,5-dihydro -1H-Pyrrol-3-yl)-Propionic acid methyl ester,	C₁₁H₁₅NO₅	0.06	Alkaloid	Antimicrobial Anti-inflammatory Antioxidant
25	28.696	Bis(2-ethylhexyl) phthalate	C₂₄H₃₈O₄	68.99	Plasticizer compound	Antimicrobial Antifouling
26	29.050	2-(1-Piperidino)-5-nitropyridine	C₁₀H₁₃N₃O₂	0.52	Alkaloid	Antimicrobial Anti-inflammatory Antioxidant
27	29.833	3-Isopropoxy-1,1,1,5,5,5-hexamethyl-3- (trimethylsiloxy)trisiloxane	C₁₂H₃₄O₄Si₄	0.16	Silica compound	No activity reported
28	30.143	gamma. -Tocopherol	C₂₇H₄₆O₂	6.45	Vitamin E compound	Antimicrobial, Antidiabetic, Antioxidant Anti-inflammatory, Skin care agent Anticholestrol
29	30.225	2H-1-Benzopyran-6-ol, 3,4-Dihydro- -2,7,8-Trimethyl-2-(4,8,12-trimethyltridecyl)-	C₂₈H₄₈O₂	2.42	Flavonoid fraction	Antimicrobial Anti-inflammatory
30	30.392	2'-Ethyl-3-[(phenoxyacetyl)hydrazono]butyranilide	C₂₀H₂₃N₃O₃	0.12	Aromatic compound	No activity reported

**Dr. Duke’s Phytochemical and Ethnobotanical Database

Table.2: compounds identified in GCMS analysis- Diethyl ether extract

S. No	RT	Compound Name	Formula	Peak Area %	Compound Nature	**Activity
1	13.735	1, 2-Benzene dicarboxylic acid, dimethyl ester.	C₁₀H₁₀O₄	0.29	Plasticizer compound	Antimicrobial Antifouling
2	15.533	Diethyl Phthalate	C₁₂H₁₄O₄	2.05	Plasticizer compound	Antimicrobial Antifouling
3	17.931	9,12-Octadecadienoic acid (Z, Z)-, 2-[( 2-[(Trimethylsilyl)oxy]-1-[[(Trimethylsilyl)oxy] Methyl ethyl ester.	C₂₇H₅₄O₄Si₂	0.55	Linoleic acid compound	Anti-inflammatory, Hypocholesterolemic Cancer preventive, Hepatoprotective, Nematicide Insectifuge, Antihistaminic Antieczemic, Antiacne, 5-Alpha reductase inhibitor Antiandrogenic, Antiarthritic, Anticoronary, Insectifuge
4	18.087	1-Cyclohexyldimethylsilyloxybutane	C₁₂H₂₆OSi	1.02	Aromatic silica compound	No activity reported
5	18.220	3,7,11,15-Tetramethyl-2-hexadecen-1-ol [Phytol]	C₂₀H₄₀O	0.52	Diterpene	Antimicrobial Anti-inflammatory Antioxidant Diuretic
6	18.716	Phthalic acid, butyl undecyl ester	C₂₃H₃₆O₄	0.18	Plasticizer compound	Antimicrobial Antifouling
7	9.732	Dibutyl phthalate	C₁₂H₁₄O₄	1.54	Plasticizer compound	Antimicrobial Antifouling
8	19.867	9-Octadecenoicacid (Z)-2[(Trimethylsilyl)oxy]-1-[[(Trimethylsilyl)oxy] Methyl ethyl ester.	C₂₇H₅₆O₄Si₂	0.58	Oleic acid compound	Anti-inflammatory, Antiandrogenic Cancer preventive, Dermatitigenic Hypocholesterolemic 5-Alpha reductase inhibitor, Anemiagenic Insectifuge, Flavor
9	20.403	9,12-Octadecadienoic acid (Z, Z)-, 2,3-dihydroxypropyl ester	C₂₁H₃₈O₄	66.89	Linoleic acid compound	Anti-inflammatory, Hypocholesterolemic Cancer preventive, Hepatoprotective, Nematicide Insectifuge, Antihistaminic Antieczemic, Antiacne, 5-Alpha reductase inhibitor Antiandrogenic, Antiarthritic, Anticoronary, Insectifuge
10	20.975	9,12-Octadecadienoic acid (Z, Z)-, methyl ester	C₁₉H₃₄O₂	3.33	Linoleic acid methyl ester	Anti-inflammatory, Hypocholesterolemic Cancer preventive, Hepatoprotective, Nematicide Insectifuge, Antihistaminic Antieczemic, Antiacne, 5-Alpha reductase inhibitor Antiandrogenic, Antiarthritic, Anticoronary, Insectifuge
11	21.198	E,E,Z-1,3,12-Nonadecatriene-5,14-diol	C₁₉H₃₄O₂	3.18	Unsaturated alcoholic compound	Antimicrobial
12	21.425	2,2,6,7-Tetramethyl-10-oxa-Tricyclo	C₁₃H₂₂O₂	1.82	Phenolic compound	Antimicrobial Anti-inflammatory Antioxidant
13	21.615	Heneicosanoic acid, methyl ester	C₂₂H₄₄O₂	12.55	Saturated fatty acid ester	No activity reported
14	22.233	Cyclohexane-1,3-dione, 2-allylaminomethylene	C₁₂H₁₇NO₂	1.71	Amino compound	Antimicrobial Anti-inflammatory
15	22.492	1-Dimethyl(3-chloropropyl) silyloxy octane	C₁₃H₂₉ClOSi	0.50	Chlorine compound	Antimicrobial
16	22.550	Ethyl 2-acetamido-3,3,3-trifluoro-2-(2- fluoroanilino) propionate.	C₁₃H₁₄F₄N₂O₃	0.38	Fluro compound	Antimicrobial
17	22.818	1H-PURIN-6-AMINE, [(2-FLUOROPHENYL	C₁₂H₁₀FN₅	0.36	Amino compound	Antimicrobial Anti-inflammatory
18	23.494	Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl	C₃₅H₆₈O₅	0.14	Palmitic acid compound	Antioxidant, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Flavor, Hemolytic 5-Alpha reductase inhibitor
19	23.781	Decyl oleate [9-Octadecenoic acid (Z)-, decyl ester]	C₂₈H₅₄O₂	0.20	Oleic acid ester compound	Anti-inflammatory, Antiandrogenic Cancer preventive, Dermatitigenic Hypocholesterolemic 5-Alpha reductase inhibitor, Anemiagenic Insectifuge, Flavor
20	26.444	Squalene	C₃₀H₅₀	1.14	Triterpene	Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immunostimulant, Chemo preventive, Lipoxygenase-inhibitor, Pesticide
21	26.826	Z,E-2,13-Octadecadien-1-ol	C₁₈H₃₄O	0.15	Unsaturated alcoholic compound	No activity reported
22	28.679	Bis(2-ethylhexyl) phthalate	C₂₄H₃₈O₄	0.91	Plasticizer compound	Antimicrobial Antifouling

The Gas Chromatography-Mass Spectrometer analysis of Diethyl ether extract identified the presence of twenty two compounds and was shown in Table.2 with activities of each active compound.

The results revealed that 9,12-Octadecadienoic acid (Z, Z)-,2,3-dihydroxypropyl ester (66.89%) was found as the one major compound in the Diethyl ether extract with activities like Anti-inflammatory, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Nematicide, Insectifuge, Antihistaminic, Antieczemic, Antiacne, 5-Alpha reductase inhibitor, Antiandrogenic, Antiarthritic, Anticoronary and Insectifuge and rest of the compounds are minor compounds. Squalene and Phytol identified are terpenes with specific activities.

CONCLUSION:

In the present study, Andrographis paniculata in ethanol extract were isolated and separated as polar (ethanol fraction) and non-polar (Diethyl ether fraction) and dried the substance. These substances were investigated using GCMS.

Ethanolic extract of Andrographis paniculata on GCMS study revealed the presence of thirty active chemical constituents. One of the major active compound present in ethanolic extract was Bis (2-ethylhexyl) phthalate. It was a Plasticizer compound and used as Antimicrobial and Anti-fouling activity. Tocopherol identified in the ethanolic extract is ideal for controlling diabetic.

In the Diethyl ether extract nineteen active chemical constituents were identified. One of the major active compound present in aqueous layer was 9, 12-Octadecadienoic acid (Z, Z)-2,3-dihydroxy propyl ester. It had Anti-inflammatory, Insectifuge and Cancer preventive activities.

Other active compounds such as alkaloid, flavonoid, saponin, aromatic compounds were present and having indifferent pharmaceutical activities. The presence of various bioactive compounds justifies the use of whole plant for various ailments by traditional practitioners.

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Received on 31.10.2018 Modified on 22.12.2018

Asian J. Research Chem. 2019; 12(1): 01-06.

DOI: 10.5958/0974-4150.2019.00001.4