Phenolic compounds’ characterization of Artemisia rutifolia spreng from Pakistani flora and their relationships with antioxidant and antimicrobial attributes

Figures & data

Figure 1. Polyphenolic compounds in different solvent extracts of A. rutifolia leaves. (A) Total phenolic compounds expressed as microgram gallic acid equivalent per milligram of plant extract. (B) Total flavonoid compounds expressed as microgram quercetin equivalent per milligram of plant extract.

Table 1. High-performance liquid chromatography (HPLC) study of methanol, chloroform, and hexane extracts of A. rutifolia leaves for identification of polyphenolic compounds.

Phenolic compounds	Regression equation	Correlation coefficient	LOD	LOQ
Gallic acid	y = 639.4x − 9.42	0.99	0.01	0.05
Caffeic acid	y = 1364.2x – 16.98	0.99	0.01	0.02
Chlorogenic acid	y = 879.1x – 3.46	0.99	0.02	0.05
Syringic acid	y = 1489.4x – 35.84	0.99	0.01	0.03
Sinapic acid	y = 2389.0x – 41.55	0.99	0.01	0.02
p-coumaric acid	y = 1703.1x – 4.58	0.99	0.12	0.04
m-coumaric acid	y = 406.2x – 16.59	0.99	0.02	0.06
Ferulic acid	y = 1060.7x – 10.65	0.99	0.01	0.04
Vanillic acid	y = 400.4x – 0.49	0.99	0.03	0.09
Quercetin	y = 1672.3x – 6.04	0.99	0.01	0.03
Myricetin	y = 1849.1x – 44.52	0.99	0.01	0.02

Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicates significant difference (P < 0.05) among solvents (methanol, chloroform, and hexane) used for extraction.

Table 2. High-performance liquid chromatography (HPLC) study of methanol, chloroform, and hexane extracts of A. rutifolia leaves for identification of polyphenolic compounds.

Phenolic compounds	Methanol extract	Chloroform extract	Hexane extract
Concentration (µg/g of dry plant material)
Gallic acid	17 ± 0.1^d	9 ± 0.1^a	2 ± 0.1^c
Caffeic acid	3 ± 0.0	—	—
Chlorogenic acid	2 ± 0.1^c	1 ± 0.0^d	—
Syringic acid	3 ± 0.1^f	2 ± 0.1^c	—
Sinapic acid	6 ± 0.2^d	4 ± 0.2^e	0.1 ± 0.2^e
p-coumaric acid	9 ± 0.0^b	6 ± 0.0^a	5 ± 0.0^d
m-coumaric acid	5 ± 0.0^a	2 ± 0.1^d	2 ± 0.0^b
Ferulic acid	1 ± 0.3^f	1 ± 0.3^f	—
Vanillic acid	1 ± 0.0	—	—
Quercetin	14 ± 0.1^d	7 ± 0.2^e	1 ± 0.3^c
Myricetin	8 ± 0.1^a	3 ± 0.0^b	3 ± 0.1^f

Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicates significant difference (P < 0.05) among solvents (methanol, chloroform, and hexane) used for extraction.

Figure 2. HPLC phenolic profile of (A) methanol, (B) chloroform, and (C) hexane extracts of A. rutifolia leaves. Peaks: (1) myricetin, (2) quercetin, (3) gallic acid, (4) caffeic acid, (5) chlorogenic acid, (6) syringic acid, (7) p-coumaric acid, (8) vanillic acid, (9) m-coumaric acid, (10) ferulic acid, and (11) sinapic acid.

Table 3. Ferric reducing antioxidant power (FRAP) and free radical (DPPH) scavenging activity of A. rutifolia leaf extracts.

A. rutifolia Leaf extracts and positive controls	Ferric reducing antioxidant power (mg GAE/g of plant extract)^a	Free radical (DPPH) scavenging activity, IC50(µg/mL)
Methanol	17 ± 0.2^a	39 ± 0.0^c
Chloroform	12 ± 0.5^c	85 ± 1.7^d
Hexane	10 ± 0.1^d	173 ± 3.3^b
Gallic acid	—	20 ± 1.5^a

(—) = not tested. All data are represented as Mean ± SD of triplicate determinations. Mean with different superscript letters in the same column indicates significant (P < 0.05) difference among the tested solvent extracts and positive controls.

Figure 3. Inhibition (%) of free radicals (DPPH) by A. rutifolia leaf extracts.

Figure 4. Ferric reducing–antioxidant power (FRAP) of A. rutifolia leaf extracts.

Table 4. Antimicrobial activity of A. rutifolia leaf extracts estimated by disc diffusion assay (mm).

		Solvent extracts			Positive controls
Microbial Strains	Dose(mg/mL)	Methanol extract	Chloroform extract	Hexane extract	Rifampicin (10 µg/mL)	Terbinafine (10 µg/mL)
		Diameter of inhibition zone (mm)
E. coli	3.5	8.0 ± 0.2^a	8.0 ± 0.0^a	7.4 ± 0.4^d	21.6 ± 1.4^a
	7	8.9 ± 0.3^b	8.0 ± 0.2^b	7.6 ± 0.1^c
	14	10.1 ± 0.1^c	9.6 ± 0.0^c	8.5 ± 0.1^e
	28	13.3 ± 0.0^e	11.8 ± 0.0^c	9.9 ± 0.0^a
P. multocida	3.5	9.6 ± 0.1^b	8.7 ± 0.3^a	8.2 ± 0.1^d	23.3 ± 1.7^a
	7	12.0 ± 0.6^d	11.9 ± 0.0^b	10.1 ± 0.1^c
	14	14.7 ± 0.1^b	15.6 ± 0.4^c	12.6 ± 1.3^b
	28	18.5 ± 0.4^e	17.1 ± 0.2^e	14.1 ± 0.6^f
B. subtilis	3.5	11.3 ± 0.0^c	10.4 ± 0.1^b	9.9 ± 0.4^b	24.6 ± 0.5^a
	7	12.6 ± 0.0^a	13.2 ± 0.5^d	11.2 ± 1.1^b
	14	16.9 ± 0.3^c	15.6 ± 0.1^e	12.6 ± 0.8^a
	28	19.3 ± 1.2^d	18.2 ± 0.1^d	15.4 ± 0.0^e
S. aureus	3.5	11.2 ± 0.7^e	10.9 ± 0.2^c	9.5 ± 0.2^f	30.1 ± 0.0^b
	7	14.1 ± 0.1^a	12.6 ± 0.4^b	10.2 ± 0.1^b
	14	15.3 ± 0.1^d	16.2 ± 0.3^d	13.9 ± 0.5^e
	28	16.2 ± 0.1^e	20.2 ± 1.2^b	17.6 ± 0.8^d
A. flavus	3.5	11.4 ± 1.2^b	11.0 ± 0.4^c	9.8 ± 0.1^a		25.2 ± 0.2^b
	7	13.2 ± 0.9^d	13.6 ± 0.2^d	10.4 ± 0.1^e
	14	13.8 ± 0.0^c	15.2 ± 0.0^e	12.5 ± 0.2^c
	28	20.6 ± 0.1^f	18.4 ± 0.0^b	19.9 ± 1.3^f
A. niger	3.5	12.9 ± 0.2^d	12.0 ± 0.4^d	10.6 ± 0.7^e		31.8 ± 1.2^c
	7	15.4 ± 0.1^a	14.3 ± 1.1^c	12.4 ± 0.4^b
	14	17.7 ± 0.5^b	15.4 ± 0.9^d	13.9 ± 0.2^f
	28	23.4 ± 0.3^d	20.2 ± 2.4^a	16.8 ± 1.7^b
F. solani	3.5	13.3 ± 0.1^a	10.0 ± 0.2^f	8.6 ± 0.0^c		35.2 ± 0.7^a
	7	14.8 ± 2.2^c	11.8 ± 0.3^e	10.4 ± 0.7^d
	14	18.1 ± 0.0^b	13.6 ± 0.0^a	13.2 ± 0.6^c
	28	25.7 ± 0.3^f	22.6 ± 0.1^b	20.9 ± 0.1^c
R. solani	3.5	10.1 ± 1.1^a	9.9 ± 0.6^e	7.2 ± 0.4^b		29.4 ± 0.4^c
	7	13.2 ± 0.1^e	11.4 ± 1.1^d	9.6 ± 2.5^f
	14	15.6 ± 1.4^f	12.6 ± 0.2^d	10.2 ± 0.0^e
	28	22.5 ± 0.1^e	18.2 ± 1.6^f	15.6 ± 0.8^c

Data are represented as Mean ± SD of triplicate determinations. Means with different superscript letters in the same row under solvent extracts indicate significant difference (P < 0.05) in antimicrobial activity (mm) with regard to different solvent extracts tested. Means with different superscript letters in the same column under positive controls indicate significant difference (P < 0.05) in antimicrobial activity (mm) with regard to microbial strains tested.

Table 5. Minimum inhibitory concentration (MIC) values (mg/mL) of A. rutifolia leaf extracts and positive controls (µg/mL) against microbial strains.

Microbial strains	Solvent extracts
	Methanol extract	Chloroform extract	Hexane extract	Rifampicin	Terbinafine
	MIC (mg/mL)			MIC (µg/mL)
E. coli	7	14	14	40
P. multocida	7	14	14	40
B. subtilis	3.5	7	14	20
S. aureus	3.5	3.5	7	10
A. flavus	7	14	7		20
A. niger	3.5	7	14		10
F. solani	3.5	3.5	7		5
R. solani	7	3.5	14		10