INTRODUCTION:

From olden days, the utilization of medicinal plants has plays a significant role in daily life even the development in modern medicine and pharmaceuticals research. In India, About 3000 plants species are identified as threpeutic agent¹. Bioactive metabolites which present in the medicinal plant sources are extremely safe and can exhibit overwhelming biological actions. Several plants are screened for phytochemical compounds, which are used as a home medication to treat various diseases. Generally, these beneficial medicinal plants possess lesser side effects have been applied in a diverse system of Indian medication².

WHO defined that 70–80% of the world’s population utilizing medicinal plants as a preliminary therapeutic agent³. Medicinal activity of the medicinal plants can be primarily demonstrated by their phytochemical and antioxidant constituents⁴.

All living organisms comprise of antioxidant and repairing mechanism to prevent themselves from oxidative damage, but these mechanisms cannot completely protect from damage which may result in oxidative stress. Oxidative stress and free radicals are recognized to be the basic reason for various human diseases⁵. Naturally occurring antioxidant from medicinal plant plays a vital role in preventing and restoring the impairment activated by the ROS and free radicals to the cells as a result of the imbalance among the formation of reactive oxygen species (ROS) and the antioxidants. Normally free radicals such as superoxide radical (O2 ⋅), hydroxyl radical (OH·), peroxide radical (ROO·) and nitric oxide radicals etc are generated in the biological system during excessive metabolism^6,7.

Phytochemicals are bioactive metabolites that are naturally found in plants, which offers high antioxidant potential since it is used as a therapeutic agent for many human diseases. Therefore, these present studies are focused to naturally occurring antioxidants from medicinal plant Acontium heterophyllum⁸. Medicinal plants contain various valuable bioactive compounds namely phenolic compounds, carotenoids, vitamins, terpenoids, essential oils, aromatic compounds, alkaloids, sterols, fatty acids, anthocyanin, tannins that are highly potential to prevent cellular oxidative stress could be separated from plant extracts and then used as a therapeutic agent for the treatment of various disorders. Several bioactive compounds that are detected from the medicinal plants of which some are currently in preclinical trials^9,10.

Aconitum heterophyllum belongs to family Ranunculaceae is traditionally utilized externally as well as internally for treating various human diseases¹¹. Aconitum heterophyllum, commonly known as Indian atees and Atividayam in Tamil, is commonly distributed all over the world and cultivated in tropic fields. Throughout the world, about 300 species of Aconitum are identified, of which around 24 species are found in India¹². In India, Aconitum heterophyllum is utilized in many ayurvedic formulations as the major ingredient. The plant Aconitum heterophyllum roots are ideal for the treatment of various disorders such as rheumatic fever, painful joints, gastroenteritis, diarrhoea, oedema, Bronchial asthma. The plant Aconitum Heterophyllum also explored to have antidiarrheal, hepatoprotective, antipyretic and analgesic, antioxidant, anti-flatulent, anti-periodic and anti-phlegmatic properties^13,14.

MATERIALS AND METHODS:

Plant collection and Authentication:

The roots of Aconitum heterophyllum were collected from the IMPCOPS (Indian Medical practitioners co-operative society, Thiruvanmiyur Chennai, India, and were authenticated by Dr P.T. Kalaichelvan, Professor, Advanced Studies in Botany, University of Madras, Chennai, India. The voucher specimen is available in the herbarium file of the Indian Medical Practitioners Co-operative Society, Thiruvanmiyur, Chennai, India.

Extraction:

Preparation of Aconitum heterophyllum extract:

The roots of Aconitum heterophyllum (1kg) were desiccated in room temperature and powdered into a coarse powder. The powder was separated through a 40-mesh sieve and extracted with ethanol and ethyl acetate in soxhlet apparatus at 60^oC. The filtrate left after ethanol and ethyl acetate extraction was dried successively. The extracts were evaporated under reduced pressure using rota flash evaporator until all the solvent had been detached and the extract was stored in the refrigerator for further studies¹⁵.

Phytochemical screening:

GC–MS Analysis:

Gas Chromatograph:

A Shimadzu GC-2010 Plus gas chromatograph was armed with a straight deactivated 2mm direct injector liner and a 15m Alltech EC-5 column (250μ I.D.,0.25μ film thickness). A split injection was useful for sample administration and the split ratio was set to 10:1. The oven temperature program was programmed to start at 35°C, hold for 2minutes, then ramp at 20°C per minute to 450°C and hold for 5 minutes. The helium carrier gas was fixed to 2ml/minute flow rate.

Mass Spectrum:

A Direct connection with capillary column metal quadrupole mass filter pre-rod mass spectrometer operating in electron ionization (EI) mode with software GCMS solution ver. 2.6 was used for all analyses. Low-resolution mass spectra were acquired at a resolving power of 1000 (20% height definition) and scanning from m/z 25 to m/z 1000 at 0.3 seconds per scan with a 0.2-second inter-scan delay. High-resolution mass spectra were acquired at a resolving power of 5000 (20% height definition) and scanning the magnet from m/z 65 to m/z 1000 at 1 second per scan.

Mass spectrometry library search:

Identification of the components of the compound was matching their recorded spectra with the data bank mass spectra of NIST library V 11 provided by the instrument's software.GC/MS metabolomics Database was used for the similarity search with retention index¹⁵.

Antioxidant Assay

DPPH radical scavenging activity:

DPPH radical scavenging ability was examined by the method of Cuendet et al¹⁶. In varying concentration range of Aconitum heterophyllum root extracts (100 to 500µg/ml) were added to 3.0 ml of methanolic solution of DPPH (0.1mM). In control, the methanol was replaced for root extract. The reaction mixture was incubated for 30 min in the darkroom at 37⁰C and absorbance was read at 517nm by UV-visible spectrophotometer. The percentage of inhibition was determined by the following equation: A0-Ax100/A0, where A0 and A represents the absorbance of the control and test sample, respectively. Ascorbic acid was used as a standard.

Anti-inflammatory assays:

Nitric oxide scavenging activity:

Griess reaction was employed to determine the Nitric oxide formed from sodium nitroprusside¹⁷. The reaction mixture (3ml) containing sodium nitroprusside (10mm) in phosphate buffer saline and the Aconitum heterophyllum root extracts (100, 200, 300, 400and 500μg/ml) was incubated at 25^oC for the period of 150min, after this period of incubation 1.5ml of the reaction mixture was taken and mixed with 1.5ml of the Griess reagent (1% sulphanilamide, 2% orthophosphoric acid and 0.1% Napthylethyline diamine hydrochloride). The absorbance of the chromophore generated was read at 546 nm. Percentage of nitric oxide inhibited was determined by using the formula below

Percentage Inhibition = (A of Control – A of Sample)/A of Control× 100. A- absorbance.

Statistical analysis:

The experiments were repeated thrice for each parameter and the SD was calculated

RESULTS

Gas Chromatography-Mass Spectrometry

The phytochemical compounds present in the ethanol and Ethyl acetate root extract of Aconitum heterophyllum was screened by the GC-MS chromatogram are shown in Figure 1 and 2. The phytochemical compounds of the ethanol and ethyl acetate extract of Aconitum heterophyllum with their retention time (RT), molecular formula, molecular weight (MW) and peak area as a percentage has shown in Table 1 and 2. GC-MS analysis revealed the presence of 22 phytochemical compounds in ethanol extract and 24 phytochemical compounds in ethyl acetate extract. The major constituents identified from ethanol and ethyl acetate extract of Aconitum heterophyllum are Alpha-D-Glucopyranoside beta-D-Fructofuranosyl (2.43%), Benzophenone semicarbazone (1.24%), Dodecanoic acid ethyl ester (1.01%), Ethyl iso-allocholate (0.99%), l-(+)-Ascorbic acid 2,6 dihexadecanoate (14.50%), Phytol (1.58%), Methyl ricinoleate (0.99%), 13-docosenamide (z) (1.20%), 1,2,4-Triazol-3-amine5-(1,3,5-trimethyl-4-pyrazolyl)amino (46.93%).

Antioxidant Assay:

DPPH radical scavenging activity:

The DPPH radical scavenging activities of the root extracts and standard (Ascorbic acid) are shown in Table 3. DPPH is reduced to diphenyl picryl hydrazine with plant extracts in a concentration-dependent manner. The radical scavenging potential of ethanol and ethyl acetate extracts of Aconitum heterophyllum on DPPH radical is 23.05% and 21.05% respectively, at the highest concentration (500 μg/ml). Among the two extracts, ethanol extract had shown the highest DPPH scavenging activity similar to the standard antioxidant Ascorbic acid (25.05%).

Nitric oxide scavenging activity:

The Nitric oxide radical scavenging ability of the extracts and standard (Ascorbic acid) are shown in Table 4. Nitric oxide scavenging power of ethanol and ethyl acetate extracts of Aconitum heterophyllum was concentration-dependent, and the highest inhibitory effect at 48.58% and 46.70% was observed at the concentration of 500 μg /ml. Among the two extracts, ethanol extract had shown the highest Nitric oxide scavenging activity similar to standard antioxidant Ascorbic acid (51.22%)

Fig 1: GC-MS chromatogram of Aconitum heterophyllum ethanolic root extrac

Table 1: Phyto-components identified in Aconitum heterophyllum Ethanolic root extract

Peak#	R.Time	Name of compound	Molecular Formula	Mol. weight	Area%
1	5.139	L-Gala--ido-octose	C₈H₁₆O₈	240	25.06
2	5.585	Butane, 2,3-dichloro-2-methyl-	C₅H₁₀C_l2	140	0.87
3	5.803	Aziridine, 2-methylene-1-(1-methylethyl)	C₆H₁₁N	97	1.12
4	6.094	4,5-Diamino-6-methyl-2-thiopyrimidine	C₅H₈N₄S	156	0.60
5	6.450	1,3-Butanedione, 1-(2-furanyl)	C₈H₈O₃	152	0.69
6	6.745	6-Tridecene, 7-methyl-	C₁₄H₂₈	196	5.10
7	7.133	Pyrrolidine, 2 alpha-[1-pyrrolidinoformyl]	C₉H₁₆N₂O	168	13.80
8	7.317	alpha-L-Galactopyranoside, methyl 6-deoxy	C₇H₁₄O₅	178	1.38
9	7.531	alpha-D-Glucopyranoside, beta-D-Fructofuranosyl	C₁₂H₂₂O₁₁	342	2.43
10	7.750	2-Pipecoline-1-dithiocarbamate	C₇H₁₃NS₂	175	1.32
11	8.704	4-Amino-2,3-xylenol	C₈H₁₁NO	137	1.83
12	11.141	Mannitol, 1,1'-O-1,16-hexadecanediylbis-	C₂₈H₅₈O₁₂	586	2.05
13	12.381	Benzophenone semicarbazone	C₁₄H₁₃N_3O	239	1.24
14	13.732	3-Chloropropionic acid, heptadecyl ester	C₂₀H₃₉ClO₂	346	1.38
15	15.954	1,2-Benzenedicarboxylic acid, diheptyl ester	C₂₂H₃₄O₄	362	0.72
16	16.560	Dodecanoic acid, ethyl ester	C₁₄H₂₈O₂	228	1.01
17	17.286	Ethyl iso-allocholate	C₂₆H₄₄O₅	436	0.99
18	18.315	I-(+)-Ascorbic acid 2,6-dihexadecanoate	C₃₈H₆₈O₈	652	14.50
19	19.522	I-Methionine, N- neopentyloxycarbonyl-, ethyl ester	C₁₃H₂₅NO₄S	291	18.07
20	22.371	Phytol	C₂₀H₄₀O	296	1.58
21	29.275	Bacteriochlorophyll-c stearyl	C₅₂H₇₂MgN₄O₄	840	1.61
22	30.108	Phenylalanine, 4-acetylamino	C₁₁H₁₄N₂O₃	222	0.63
23	30.296	3-pyrrolidin-2-yl-propionic acid	C₇H₁₃NO₂	143	1.68
24	31.580	alpha-D-Glucopyranoside, methyl 2-(acetylamino)-2-deoxy-3-O-(trimethylsilyl)-, cyclic butylboronate	C₁₆H₃₂BNO₆Si	373	0.35

Table 2: Phytocomponents identified in Aconitum heterophyllum ethyl acetate root extract

Peak	R.time	Name of compound	Molecular Formula	Mol. weight	Area%
1	8.649	Phenol, 2,4-di-tert-butyl	C₁₄H₂₂O	206	0.82
2	8.889	2,4-diethyl-6-methyl-1,3,5-trioxane	C₈H₁₆O₃	160	0.09
3	8.992	4-Allyl-2-t-butyl-4-methyl-1,3-oxathiolan-5-one	C₁₁H₁₈O₂S	214	0.07
4	9.595	Z,Z-2,5Pentadecadien-1-ol	C₁₅H₂₈O	224	0.10
5	9.667	Isoindole-1,3(2H)-dione-4,7-ethano-3a,4,7,7a-tetrahydro-2-phenyl-	C₁₆H₁₅NO₂	253	0.01
6	10.655	Pyridine, 3-(phenylmethyl)-	C₁₂H₁₁N	169	0.04
7	12.262	Methyl ricinoleate	C₁₉H₃₆O₃	312	0.99
8	13.317	N-(1-Anilino-2,2,2-trichloroethyl)decanamide	C₁₈H₂₇C_l3N₂O	392	0.01
9	15.834	2,3-O-Benzal-d-mannosan	C₁₃H₁₄O₅	250	0.11
10	17.568	Thiazolidin-4-one, 5-ethyl-2-imino-	C₅H₈N₂OS	144	3.60
11	18.042	2H-Pyran,2-(2 heptadecynyoxy)tetrahydro-	C₂₂H₄₀O₂	336	0.84
12	18.658	3-Propylnorleucine	C₉H₁₉NO₂	173	0.83
13	20.230	1,2,3-Thiadiazole-4-carboxylic acid, (1-amino-2-chloroethenylideneamino) ester	C₅H₅ClN₄O₂S	220	0.73
14	21.291	1,5-Cyclooctadiene,1-t-butyl-	C₁₂H₂₀	164	1.11
15	23.667	2H-Pyran, tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-	C₁₀H₁₈O	154	0.03
16	24.000	2H-Pyrimido[1,2-a] pyrimidine, 1,3,4,6,7,8-hexahydro-1-methyl	C₈H₁₅N₃	153	0.23
17	26.714	Citronellyl Tiglate	C₁₅H₂₆O₂	238	1.14
18	28.865	4,4-Dimethyl-2-allylcyclohexanone	C₁₁H₁₈O	166	1.17
19	28.984	Bis(cic-13-docosenamido) methane	C₄₅H₈₆N₂O₂	686	3.46
20	29.179	2-(p-Methoxyphenyl) ethyl beta.-d-glucopyranoside	C₁₅H₂₂O₇	314	2.35
21	35.025	Azacyclononan-2-one,5,5,8,8-tetramethyl-	C₁₂H₂₃NO	197	0.03
22	35.218	dl-alanyl-l –leucine	C₉H₁₈N₂O₃	202	0.07
23	37.125	5-Bromovaleric acid, oct-3-en-2-yl ester	C₁₃H₂₃BrO₂	290	0.01
24	38.153	N-maleic acid 2,2,6,6-Tetramethyl,-4-piperidylmonoamide	C₁₃H₂₂N₂O₃	254	0.02
25	38.551	4-(3-Hydroxy-2,2,6-trimethyl-7-oxa-bicyclo[4.1.0]hept-1-yl)-but-3-en-2-one	C₁₃H₂₀O₃	224	0.06
26	41.921	Fumaric acid,2-decyl tridecyl ester	C₂₇H₅₀O₄	438	0.97
27	42.483	Batilol	C₂₁H₄₄O₃	344	2.91
28	42.625	13-docosenamide,(z)	C₂₂H₄₃NO	337	1.20
29	43.238	13,14-epoxyursan-3-ol,acetate	C₃₁H₅₀O₃	470	5.19
30	45.131	1,2,4-Triazol-3-amine, 5-(1,3,5-trimethyl-4-pyrazolyl)amino-	C₈H₁₃N₇	207	46.93
31	45.486	Triaraachine	C₆₃H₁₂₂O₆	974	16.50

Figure 2: GC-MS chromatogram of Aconitum heterophyllum ethyl acetate root extract.

Table 3: DPPH Scavenging activity of Aconitum heterophyllum ethanolic and ethyl acetate root extract

Concentration (µg/ml)	Percentage inhibition (%)
Concentration (µg/ml)	EEAH	EAEAH	Ascorbic Acid
100	11.52±1.76	09.12±0.80	14.06±1.12
200	14.40±1.52	12.23±1.34	17.25±1.15
300	17.56±1.22	15.24±1.36	19.54±1.37
400	20.71±2.50	18.36±1.40	22.16±1.36
500	23.05±1.34	21.05±1.23	25.05±1.40

Values are mean ± SD, n = 3; EEAH: Ethanolic root extract of Aconitum heterophyllum: EAEAH: Ethyl Acetate root extract of Aconitum heterophyllum ,

Table 4: No Scavenging activity of Aconitum heterophyllum ethanolic and ethyl acetate root extract

Concentration (µg/ml)	Percentage inhibition (%)
Concentration (µg/ml)	EEAH	EAEAH	Ascorbic Acid
100	38.30±1.24	34.22±1.14	40.12±1.32
200	40.78±1.27	37.78±1.17	43.18±1.36
300	44.68±1.34	42.15±1.20	46.23±1.37
400	46.81±1.37	45.12±1.26	48.81±1.40
500	48.58±1.41	46.70±1.32	51.22±1.44

Values are mean ± SD, n = 3; EEAH: Ethanolic root extract of Aconitum heterophyllum, EAEAH: Ethyl Acetate root extract of Aconitum heterophyllum

DISCUSSION:

Mass spectrometry has been the best tool for screening the qualitative and quantitative information on molecules based on their structural compositions and molecular weight. The phytochemical analysis studied on the ethanol and ethyl acetate extract of Aconitum heterophyllum exposed the existence of efficient compounds that are known for its medicinal value. The GC-MS analysis exhibits the presence of nine compounds in the ethanol and ethyl acetate extract of Aconitum heterophyllum which are responsible for the biological activities such as antioxidant, antibacterial, anti-inflammatory and anti-cancer have been tabulated Table 5 and 6.

Table 5: Major Bioactive compounds of ethanol root extract of Aconitum heterophyllum

S. No	Bioactive compounds	Biological Activity
1.	Alpha-D-Glucopyranoside,beta-D-Fructofuranosyl	Antioxidant¹⁸
2.	Benzophenone semicarbazone	Anticancer agent¹⁹
3.	Dodecanoic acid, ethyl ester	Antibacterial, Antiviral, Antioxidant, Candidicide, Hypercholesterolemic²⁰
4.	Ethyl iso-allocholate	Cytotoxic activity, Anticancer and Anti-inflammatory agent²¹
5.	l-(+)-Ascorbic acid 2,6 dihexadecanoate	Antitumor and Antimicrobial activity²²
6.	Phytol	Anti-inflammatory,Anti-thrombotic, Antimicrobial, and Antitumor²³

Table 6: Major Bioactive compounds of ethyl acetate root extract of Aconitum heterophyllum

S. No	Bioactive compounds	Biological Activity
1.	Methyl ricinoleate	Antioxidant²⁴
2.	13-docosenamide,(z)	Antifungal and Antibacterial activity²⁵
3.	1,2,4-Triazol-3-amine, 5-(1,3,5-trimethyl-4-pyrazolyl)amino	Antitumor activity²⁶

The root extract was reacted with DPPH radical which reduced the radical to diphenyl picryl hydrazine due to its antioxidant potential. The antioxidant potential of the root extracts to scavenge the DPPH free radical by donating hydrogen proton was indicated by stage of discolouration²⁷. The free radical scavenging potential of the root extracts against DPPH was compared with the standard ascorbic acid. It was observed that radical scavenging potential of ethanol and ethyl acetate extracts of Aconitum heterophyllum on DPPH radical at the highest concentration (500μg/ml). The activity of the Aconitum heterophyllum root extracts was comparatively lesser than that of the standards. The results of this study demonstrated that ethanol and ethyl acetate extracts of Aconitum heterophyllum possess antioxidant potential might be related to its phenolic, sterols and terpenoids contents²⁸.

Nitric oxide is an important signalling molecule that played a vital role in the pathological process of numerous inflammatory disorders. Nitric oxide becomes toxic when it reacts with the strong oxidant superoxide anion to form peroxynitrite. The root extract act as a scavenger of nitric oxide by interacts with oxygen that leads to the reduction of nitrite ions formation²⁸. NO scavenging power of ethanol and ethyl acetate extracts of Aconitum heterophyllum was concentration-dependent, and shows the highest inhibitory effect at the highest concentration (500μg/ml). The results of this present study demonstrated that ethanol and ethyl acetate extracts of Aconitum heterophyllum possess antioxidant potential might be related to its phytochemical consituents²⁹.

CONCLUSION:

From these findings, it is concluded that the Aconitum heterophyllum might be used as an alternative source for the investigation of new antioxidant agents.

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Received on 21.10.2020 Modified on 17.07.2021

Research J. Pharm. and Tech 2023; 16(2):703-708.

DOI: 10.52711/0974-360X.2023.00120