Bioassay-guided isolation of antioxidant agents with analgesic properties from flowers of Tagetes patula

Figures & data

Figure 1.  Methyl protocatechuate (1) and diacetyl, methyl protocatechuate (1a).

Figure 2.  Patuletin (2), its derivatives (2a-2d) and patulitrin.

Scheme 1.  Bioassay guided isolation of antioxidant principles (1) and (2) from flowers of T. patula.

Scheme 2.  Isolation of antioxidant principle (1) from flowers of T. patula.

Figure 3.  α-Terthienyl (4).

Scheme 3.  Bioassay-guided isolation of antioxidant principles (2) and (3) from flowers of T. patula.

Table 1.  IC₅₀ values of antioxidant activity of different extracts and fractions from T. patula flowers following different extraction schemes using DPPH assay.

Samples	IC50 (µg/mL)
Scheme 1
Extracts
JFPE	>>200
JFM	39.67 ± 1.33
Fractions
JFMM	64.67 ± 2.9
JFMI	7.2 ± 0.3
Scheme-3
Extract
JF	28.0 ± 3.74
Phases
JF-P	>>100
JF-C	55.67 ± 3.38
JF-BuOH2	9.03 ± 0.08
JF-aq	>>100

Scheme 1, extracts, petroleum ether (JFPE) and methanol (JFM); fractions, methanol soluble fraction (JFMM) and methanol insoluble fraction (JFMI) of JFM. Scheme 3, extract, direct methanol extract of flowers (JF); phases of JF, petroleum ether (JF-P), chloroform (JF-C), butanol (JF-BuOH2), and aqueous (JF-aq). Values represents means ± SE (n = 3). IC₅₀= 50% inhibitory concentration of antioxidant activity.

Table 2.  Antioxidant activity of 1, 2, 3, quercetin and rutin using DPPH assay.

Doses (µg/mL)
Samples	2	3	4	5	10	20	25	IC50 (µg/mL)
Quercetin	36.87 ± 3.01	49.4 ± 1.71	62.42 ± 1.28	76.2 ± 0.48	77.67 ± 0.4	ND	ND	3.0 ± 0.15
Rutin	22.66 ± 0.84	30.26 ± 0.91	38.45 ± 1.66	46.11 ± 2.56	85.07 ± 1.15	86.0 ± 0.63	ND	5.56 ± 0.23
Methyl protocatechuate
Natural product (JFM7c) (1)	11.26 ± 1.71	ND	ND	23.32 ± 4.49	43.18 ± 10.06	67.26 ± 5.75	79.13 ± 3.78	13.17 ± 2.6^***###
Synthetic (1)	35.03 ± 2.24	54.55 ± 4.32	75.46 ± 5.13	90.08 ± 2.01	93.99 ± 2.55	ND	ND	2.8 ± 0.2
Patuletin (2)	28.64 ± 1.76	37.90 ± 1.92	47.50 ± 2.28	66.84 ± 8.00	85.47 ± 1.63	ND	ND	4.3 ± 0.25
Patulitrin (3)	11.50 ± 2.47	ND	ND	28.14 ± 1.28	51.92 ± 4.67	75.1 ± 1.84	86.7 ± 0.6	10.17 ± 1.16^*###

Values represent means ± SE (n = 3); IC₅₀ = 50% inhibitory concentration of antioxidant activity; ^*p < 0.05, ^**p < 0.01, ^***p < 0.005) as compared to rutin; ^#p < 0.05, ^##p < 0.01, ^###p < 0.005 as compared to quercetin; ND = not done.

Figure 4.  Flavonol (quercetin) and its glycoside (rutin).

Table 3.  Effect of patuletin and aspirin on acetic acid-induced writhing test in mice.

Treatment	Dose (mg/kg)	% Inhibition of writhes	IC50 (mg/kg)
Patuletin (2)	0.0033	47.14	0.143
	0.0332	39.73
	0.332	65.78
	3.22	60.0
	10	36.58
Aspirin	10	37.1	18.2
	20	53.0
	30	68.5
	50	72.0
	100	80.02

Number of writhes in control animals (0.9% acetic acid, 10 mL/kg) = 59.4 ± 4.98. Number of mice per dose = 3–5. Test substances were administered intraperitoneally before the administration of acetic acid.