Identification, quantification of bioactive constituents, evaluation of antioxidant and in vivo acute toxicity property from the methanol extract of Vernonia cinerea leaf extract

Figures & data

Table 1.  Phytocomponents identified in the V. cinerea leaf extract by GC-MS analysis.

Compound	Nature	Molecular formula	MW	RT(min)	Peak area %
Benzene, 1,4-dichloro-	Aromatic compound	C6H4Cl2	146	4.15	0.57
Caryophyllene	Sesquiterpene	C15H24	204	9.80	0.78
Azulene, 1,2,3,4,5,6,7,8 octahydro-1,4-dimethyl-7-(1-methylethenyl)-, [1S-(1α,4α,7α)]- (Synonym: α-Guaiene)	Sesquiterpene	C15H24	204	9.92	0.24
α-Caryophyllene (Synonym: α-Humulene)	Sesquiterpene	C15H24	204	10.22	0.21
Naphthalene, 1,2,3,5,6,7,8,8ª-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-, [1R-(1α,7α,8aα)]- (Synonym: Valencene)	Sesquiterpene	C15H24	204	10.45	0.17
Azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, [1S-(1α,7α,8aα)]- (Synonym: α-Bulnesene)	Sesquiterpene	C15H24	204	10.77	0.18
2-Isopropylidene-5-methylhex-4-enal	Alkene compound	C10H16O	152	10.95	0.10
Dodecanoic acid	Lauric acid	C12H24O2	200	11.14	0.22
Caprylic anhydride	Caprylic compound	C16H30O3	270	11.26	0.06
Azulene, 1,4-dimethyl-7-(1-methylethyl)-	Hydrocarbon	C15H18	198	11.78	1.13
Tetradecanoic acid	Myristic acid	C14H28O2	228	13.66	0.22
3,7,11,15-Tetramethyl-2-hexadecen-1-ol	Terpene alcohol	C20H40O	296	14.80	2.90
n-Hexadecanoic acid	Palmitic acid	C16H32O2	256	16.60	12.30
Hexadecanoic acid, ethyl ester	Fatty acid ester	C18H36O2	284	16.88	0.74
9,12-Octadecadienoic acid, methyl ester	Linoleic acid	C19H34O2	294	18.45	3.69
9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-	Fatty acid ester	C19H32O2	292	18.54	5.75
Phytol	Diterpene	C20H40O	296	18.86	2.92
9,12,15-Octadecatrienoic acid, (Z,Z,Z)-	Linolenic acid	C18H30O2	278	19.33	22.61
8,11,14-Eicosatrienoic acid, (Z,Z,Z)-	Fatty acid ester	C20H34O2	306	19.58	4.20
11-Eicosenoic acid, methyl ester	Fatty acid ester	C21H40O2	324	21.59	1.51
Phenol, 2,4-bis(1-phenylethyl)-	Phenolic compound	C22H22O	302	23.36	1.16
Methanone, [1,4-dimethyl-7-(1-methylethyl)-2-azulenyl]phenyl-	Ketone compound	C22H22O	302	23.57	1.18
13-Docosenoic acid, methyl ester	Fatty acid ester	C23H44O2	352	24.59	16.26
1,2-Benzenedicarboxylic acid, diisooctyl ester	Plasticizer compound	C24H38O4	390	25.18	17.81
15-Tetracosenoic acid, methyl ester	Fatty acid ester	C25H48O2	380	27.44	0.77
Squalene	Triterpene	C30H50	410	29.53	1.41
1-Monolinoleoylglycerol trimethylsilyl ether	Ether compound	C27H54O4Si2	498	33.73	0.91

MW-Molecular weight of the phytocomponents. RT- Retention time in minutes. Synonyms for the sesquiterpenoids are given in the table.

Figure 1.  GC-MS analysis of the phytoconstituents in the methanol extract of V. cinerea leaf extract showing the retention time (min) of the compounds in X axis and percentage (%) of peak area in the Y axis.

Figure 2.  HPTLC analysis of the phytochemicals present in the V. cinera leaf extract showing Rf values. Detection was at 280 nm. Figrue 2a) Chromatogram of standard phenolic compounds. Figure 2b) Chromatogram of V. cinera leaf extract. The numbers indicate the unidentified compounds.

Table 2.  Elemental present in V. cinerea leaf extract.

Name of the element	Quantity in ppm
Fe	0.050
Mn	0.022
Co	0.018
Pb	0.029
Hg	3.885
Se	4.524

ppm – parts per million.

Table 3.  Antioxidant activity of V. cinerea leaf extract by several in vitro assays

Assay	Sample	Concentration (µg/ml)	% Inhibition ± SD^c	r^2	IC50 (µg/ml)
DPPH radical scavenging	V. cinerea	10	26.3 ± 1.13^a	0.6825	56 ± 6.92
		50	47.7 ± 2.67^a
		100	64.4 ± 5.36^a
		250	74.6 ± 2.39^a
		500	80.9 ± 3.24^a
	Ascorbic acid	250	92.5 ± 0.39^b
ABTS radical cation scavenging	V. cinerea	10	21.9 ± 2.54^a	0.8278	98.8 ± 32.33
		50	38.7 ± 6.94^a
		100	50.9 ± 6.12^a
		250	67.4 ± 4.70^a
		500	80.0 ± 1.89^a
	Ascorbic acid	250	87.1 ± 0.15^b
Hydroxyl radical scavenging	V. cinerea	10	20.8 ± 2.46^a	0.8342	220 ± 27.49
		50	28.7 ± 5.62^a
		100	38.8 ± 6.69^a
		250	52.1 ± 1.80^a
		500	60.4 ± 1.88^a
	Ascorbic acid	250	88.4 ± 1.22^b
Nitric oxide radical scavenging	V. cinerea	10	13.3 ± 1.85^a	0.7638	93.3 ± 13.10
		50	34.7 ± 3.76^a
		100	51.5 ± 2.34^a
		250	69.5 ± 2.81^a
		500	77.6 ± 1.87^a
	Ascorbic acid	250	81.6 ± 0.25^b
Superoxide-radical scavenging	V. cinerea	10	14.5 ± 1.07^a	0.7070	81.3 ± 3.05
		50	35.2 ± 0.09^a
		100	57.2 ± 2.36^a
		250	70.1 ± 0.54^a
		500	76.4 ± 1.92^a
	Ascorbic acid	250	85.9 ± 1.33^b
In vitro anti-lipid peroxidation	V. cinerea	10	08.1 ± 0.44^a	0.9573	558 ± 32.5
		50	11.3 ± 1.21^a
		100	19.9 ± 0.89^a
		250	33.0 ± 0.91^a
		500	46.0 ± 1.91^a
	Vitamin E	250	97.3 ± 0.30^b
Oxidative hemolysis inhibition	V. cinerea	10	40.5 ± 2.63^a	0.7598	46 ± 8.02
		50	52.0 ± 1.83^a
		100	63.3 ± 2.29^a
		250	79.4 ± 0.94^a
		500	81.5 ± 0.61^a
	Ascorbic acid	250	90.7 ± 0.19^b

^cValues are mean ± standard deviation. Difference was considered significant when p value ≤ 0.05 for test sample and ≤ 0.01 for standard (n = 3, ^ap < 0.05, ^bp < 0.001). Results were evaluated by one-way ANOVA followed by Duncan multiple range tests. r² is the regression equation analysis and IC₅₀ is the half maximal inhibitory concentration.

Figure 3.  Histopathological observation of liver and kidney tissues: (A & B) Liver of control (A) and extract treated (B) animals, showing normal architecture and orderly alignment of hepatocytes. (C & D) Kidney of control (C) and extract treated (D) animals showing normal pattern. [Animal treatment details: Refer materials and methods].