Characterization of phenolic compounds from Phyllanthus emblica fruits using HPLC-ESI-TOF-MS as affected by an optimized microwave-assisted extraction

Figures & data

Table 1. Experimental design with the response surface methodology for total phenolic compounds (TPC) yield extracted from Phyllanthus emblica fruit using microwave-assisted extraction (MAE) method

Run number	Coded levels				Recovery of TPC (mg GAE/g)
	Microwave power (W), X1	Irradiation time (s), X2	Ethanol concentration (%), X3	Solvent-to-solid ratio (mL/g), X4
1	0(400)	0(30)	0(70)	0(20)	132.215
2	−1(320)	1(35)	1(75)	1(25)	104.524
3	0(400)	0(30)	0(70)	2(30)	114.090
4	0(400)	0(30)	−2(60)	0(20)	112.816
5	0(400)	−2(20)	0(70)	0(20)	115.239
6	−1(320)	1(35)	−1(65)	1(25)	119.452
7	0(400)	0(30)	0(70)	0(20)	121.239
8	0(400)	0(30)	0(70)	0(20)	126.063
9	0(400)	2(40)	0(70)	0(20)	117.483
10	0(400)	0(30)	0(70)	0(20)	132.174
11	1(480)	−1(25)	1(75)	−1(15)	115.417
12	1(480)	1(35)	1(75)	−1(15)	111.063
13	0(400)	0(30)	2(80)	0(20)	106.841
14	1(480)	−1(25)	−1(65)	−1(15)	108.905
15	0(400)	0(30)	0(70)	0(20)	132.435
16	−1(320)	−1(25)	1(75)	1(25)	106.764
17	1(480)	−1(25)	−1(65)	1(25)	133.021
18	1(480)	1(35)	−1(65)	−1(15)	105.734
19	−1(320)	−1(25)	1(75)	−1(15)	118.551
20	−1(320)	1(35)	1(75)	−1(15)	121.957
21	−1(320)	1(35)	−1(65)	−1(15)	112.532
22	−1(320)	−1(25)	−1(65)	1(25)	105.829
23	−1(320)	−1(25)	−1(65)	−1(15)	103.868
24	2(560)	0(30)	0(70)	0(20)	123.418
25	0(400)	0(30)	0(70)	0(20)	132.151
26	1(480)	1(35)	−1(65)	1(25)	127.112
27	1(480)	1(35)	1(75)	1(25)	112.667
28	−2(240)	0(30)	0(70)	0(20)	113.036
29	0(400)	0(30)	0(70)	−2(10)	105.676
30	1(480)	−1(25)	1(75)	1(25)	115.923

Figure 1. The effect of microwave power (a), extraction/irradiation time (b), ethanol concentration (c), and liquid-to-solid ratio (d) on the total phenolic yield extracted from Phyllanthus emblica fruits (n = 3)

Table 2. Estimated regression coefficients of the quadratic polynomial model and analysis of variance (ANOVA) for the experimental results of total phenolic contents extracted from Phyllanthus emblica fruit using microwave-assisted extraction (MAE) method

Source^a	Estimated coefficients	Standard error	Sum of squares	DF^b	Mean square	F-value	Prob > F
Model			2400.587	14	171.4705	15.46766	< 0.0001
Intercept	129.3795	1.359273		1
X1	2.380375	0.679637	135.9884	1	135.9884	12.26697	0.0032
X2	0.468792	0.679637	5.274375	1	5.274375	0.47578	0.5009
X3	−0.89738	0.679637	19.32677	1	19.32677	1.743389	0.2065
X4	1.837208	0.679637	81.00803	1	81.00803	7.307406	0.0164
X1X2	−2.50894	0.832382	100.7163	1	100.7163	9.085207	0.0087
X1X3	−1.86356	0.832382	55.56584	1	55.56584	5.012369	0.0408
X1X4	4.246438	0.832382	288.5157	1	288.5157	26.02583	0.0001
X2X3	−1.22819	0.832382	24.13511	1	24.13511	2.177131	0.1608
X2X4	−0.14544	0.832382	0.338433	1	0.338433	0.030529	0.8636
X3X4	−5.09281	0.832382	414.9878	1	414.9878	37.43437	< 0.0001
$X_1^2$	−2.72436	0.635742	203.5793	1	203.5793	18.36406	0.0007
$X_2^2$	−3.19086	0.635742	279.2672	1	279.2672	25.19156	0.0002
$X_3^2$	−4.82399	0.635742	638.2869	1	638.2869	57.57727	< 0.0001
$X_4^2$	−4.81036	0.635742	634.6864	1	634.6864	57.25248	< 0.0001
Residual			166.2862	15	11.08574
Lack of fit			56.15266	10	5.615266	0.25493	0.9686
Pure error			110.1335	5	22.0267
Cor total			2566.873	29
R2 = 0.9352 $R_{adj}^2$ = 0.8748 CV = 2.85

^a Coefficients refer to the general model;

^b Degree of freedom.

Figure 2. Response surface plots of the total phenolic yield extracted from Phyllanthus emblica fruits using microwave-assisted extraction as a function of significant interactions among factors: (a) microwave power and extraction/irradiation time; (b) microwave power and ethanol concentration; (c) microwave power and liquid-to-solid ratio; (d) extraction/irradiation time and ethanol concentration; (e) extraction/irradiation time and liquid-to-solid ratio; and (f) ethanol concentration and liquid-to-solid ratio

Table 3. The HPLC-ESI-TOF-MS identification and characterization of compounds isolated from Phyllanthus emblica fruit extracts by the optimized MAE method

Peak no.	TR [min]	Peak assignment	Molecular formula	m/z [M-H]¯	HPLC-ESI-MS/MS (negative) fragments, m/z	Reference
1	24.790	Mucic acid-1,4-lactone-3-O-gallate	C13H12O11	343.0336	MS^2, 191, 169, 147, 125, 85	Luo et al.^[Citation15] and Zhang et al.^[Citation16]
2	32.367	Hamamelitannin	C20H20O14	483.0841	MS^2, 313, 271, 211, 169, 125	Touriño et al.^[Citation29]
3	41.720	Isocorilagin	C27H22O18	633.0757	MS^2, 463, 301, 275, 169	Nampoothiri et al.^[Citation10]; Liu et al.^[Citation14]; Luo et al.^[Citation15]
4	45.055	Ethyl gallate	C9H10O5	197.0066	MS^2, 169, 124, 78	Zhang et al.^[Citation31]
5	54.944	Methyl gallate	C8H8O5	183.0310	MS^2, 124, 78, 59	Touriño et al.^[Citation29]
6	71.966	Ellagic acid	C14H6O8	301.0003	MS^2, 299, 257, 229	Nampoothiri et al.^[Citation10]
7	75.742	Quercetin-3-O-rhamnoside	C21H20O11	447.0948	MS^2, 301, 271, 151	Taamalli et al.^[Citation25]; Huang et al.^[Citation33]; Yamazaki et al.^[Citation34]
8	78.860	Undefined	C15H22O2	233.1567	MS^2, 217, 163, 75

Figure 3. Total ion current chromatogram of an emblic fruits extracts obtained using the optimized MAE method: (1) mucic acid-1,4-lactone-3-O-gallate, (2) hamamelitannin, (3) isocorilagin, (4) ethyl gallate, (5) methyl gallate, (6) ellagic acid, (7) quercetin-3-O-rhamnoside, and (8) an undefined compound

Table 4. The half maximal inhibitory concentration (IC_50, mg/mL) of sample, BHT, and ascorbic acid in antioxidant activities assays

Assays	IC50 (mg/mL)
	Sample	BHT	Ascorbic acid
DPPH	0.030 ± 0.002	0.045 ± 0.002	0.024 ± 0.001
ABTS^+	0.426 ± 0.06	0.215 ± 0.05	0.562 ± 0.08
Inhibition of peroxidation	0.578 ± 0.04	0.851 ± 0.11	0.617 ± 0.06
Reducing power	0.173 ± 0.02	0.065 ± 0.001	0.131 ± 0.003

IC₅₀: The concentration for a 50% inhibition and in the reducing power assay that means the concentration for a 0.5 absorbance at 700 nm.

Data are represented as means ± SE (n = 3).

Figure 4. Antioxidant activities of polyphenols of Phyllanthus emblica fruit extracts isolated using the optimized microwave-assisted extraction method. (a) DPPH radical scavenging activity. (b) ABTS⁺ scavenging activity. (c) Inhibition of lipid peroxidation. (d) Reducing power. Data are represented as means ± SE (n = 3)

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