Optimization of ultrasound-assisted hydroalcoholic extraction of phenolic compounds from walnut leaves using response surface methodology

Figures & data

Table 1. Independent variables and their coded and actual values used in the central composite rotatable design.

	Coded values
	−1.68	−1	0	+1	+1.68
Independent variables	Actual values
X1: ethanol concentration (% v/v)	12.17	29	54	79	95.83
X2: extraction time (min)	8.17	25	50	75	91.83
X3: liquid-to-solid ratio (v/w)	4.96	9	15	21	25.04

Table 2. Response values (mean ± SD, n = 3) of total phenolics content, total flavonoids content and antioxidant activity for given levels of variables (ethanol concentration, extraction time and liquid-to-solid ratio) in RSM.

Uncoded variables			Total phenolics content (mg GAE/L)	Total flavonoids content (mg QE/L)	Antioxidant activity (mmol Trolox/L)
Ethanol conc. (% v/v)	Liquid-to-solid ratio (v/w)	Extraction time (min)
54.0	15.0	50.0	3649.55 ± 113.77	960.52 ± 53.39	16.52 ± 0.55
29.0	9.0	25.0	5570.25 ± 165.04	1459.34 ± 69.63	18.67 ± 2.20
79.0	21.0	25.0	2405.63 ± 40.98	767.88 ± 31.37	11.02 ± 0.11
79.0	9.0	75.0	6491.06 ± 55.27	1875.72 ± 83.39	21.97 ± 0.26
29.0	21.0	75.0	2734.48 ± 18.35	686.89 ± 29.74	9.59 ± 0.13
29.0	21.0	25.0	2070.98 ± 66.93	623.22 ± 27.73	8.96 ± 0.52
29.0	9.0	75.0	5863.18 ± 316.25	1598.02 ± 86.86	17.86 ± 0.31
79.0	21.0	75.0	2435.81 ± 95.11	804.43 ± 29.92	11.54 ± 0.19
54.0	15.0	50.0	5674.17 ± 203.54	1692.24 ± 86.85	17.66 ± 0.22
79.0	9.0	25.0	6736.90 ± 27.08	2054.63 ± 95.34	22.27 ± 0.39
12.17	15.0	50.0	2318.60 ± 33.94	680.13 ± 35.95	8.56 ± 0.47
95.83	15.0	50.0	2402.05 ± 169.85	888.41 ± 30.31	9.55 ± 0.65
54.0	15.0	8.17	3278.65 ± 124.80	982.88 ± 47.74	17.51 ± 0.16
54.0	15.0	50.0	5248.12 ± 52.11	1393.69 ± 69.40	16.73 ± 2.72
54.0	15.0	91.83	4564.83 ± 88.92	1347.68 ± 53.42	15.85 ± 2.22
54.0	4.96	50.0	10408.45 ± 326.33	3028.20 ± 128.31	56.84 ± 1.88
54.0	25.04	50.0	2870.43 ± 21.25	850.19 ± 36.16	12.12 ± 0.35

Table 3. Regression coefficients and analysis of variance (ANOVA) of the predicted quadratic model to the responses total phenolics content, total flavonoids content and antioxidant activity.

Regression coefficients	Total phenolics content	Total flavonoids content	Antioxidant activity
β0	6331.23	1848.75	44.1676
β1 (ethanol concentration)	192.784	52.5929^a	0.767591
β2 (liquid-to-solid ratio)	−820.707^a	−237.924^a	−5.58349^a
β3 (extraction time)	65.7929^a	16.5622	0.1602
β12	−1.46542	−0.50908	−0.00300833
β13	−0.23443	−0.06894	0.000098
β23	0.538875	0.11704	0.00180833
β11	−1.41932^a	−0.34395^a	−0.00636786^a
β22	17.8106^a	5.48618^a	0.141704^a
β33	−0.52708^a	−0.12623	−0.00200929^a
Lack-of-fit	0.2808	0.1798	0.0000
R^2	95.84	93.167	81.3681

^aSignificant at p < 0.05.

Figure 1. Response surface plots of total phenolics content (mg GAE/L) at optimum ethanol concentration (% v/v) (a), extraction time (min) (b) and liquid-to-solid ratio (v/w) (c).

Figure 2. Response surface plots of total flavonoids content (mg QE/L) at optimum ethanol concentration (% v/v) (a), extraction time (min) (b) and liquid-to-solid ratio (v/w) (c).

Table 4. Parameters of calibration curves, LODs and LOQs for HPLC method validation and content of phenolic compounds in walnut leaves extracts (79% ethanol concentration, 9.0 v/w liquid to solid ratio, 25 min extraction time).

Analyte	λ (nm)	Concentrations (mg/L)					Retention time (min)	Calibration curve			LOD (mg/L)	LOQ (mg/L)	Content (mg/100 g DW)
		S1	S2	S3	S4	S5		Intercept	Slope	R^2
Vanillic acid	254	5	10	15	20	25	27.35	0.6042	1.16663 e − 005	0.9994	0.133	0.443	1.69 ± 0.23
Rutin	254	10	20	30	40	50	57.74	0.8447	4.74197 e − 006	0.9989	0.053	0.176	291.03 ± 19.63
Ellagic acid	254	10	20	30	40	50	60.81	−0.3113	2.21081 e − 005	0.9988	0.162	0.539	272.29 ± 15.83
Myricetin	254	10	20	30	40	50	61.40	−0.4929	1.38462 e − 005	0.9993	0.122	0.406	636.64 ± 26.04
Juglone	254	10	20	30	40	50	62.37	−0.0487	1.02449 e − 005	0.9991	0.117	0.390	545.61 ± 28.05
Quercetin	254	5	10	15	20	25	70.50	0.2500	1.01167 e − 005	0.9992	0.126	0.420	58.85 ± 4.12
Gallic acid	278	5	10	15	20	25	5.42	0.3109	1.26622 e − 005	0.9992	0.179	0.599	2.36 ± 0.16
Catechin hydrate	278	40	80	120	160	200	20.14	2.7311	5.68028 e − 005	0.9999	0.905	3.019	137.06 ± 4.60
Syringic acid	278	10	20	30	40	50	37.10	4.8145	7.80232 e − 006	0.9997	0.227	0.759	5.30 ± 0.25
Epicatechin	278	40	80	120	160	200	41.37	1.3485	5.73111 e − 005	0.9987	0.348	1.162	29.40 ± 0.88
trans-Cinnamic acid	278	5	10	15	20	25	68.32	−0.2451	4.99316 e − 006	0.9993	0.029	0.095	23.52 ± 2.80
Chlorogenic acid	300	10	20	30	40	50	29.07	1.4673	1.58729 e − 005	0.9995	0.243	0.812	14.52 ± 0.39
Caffeic acid	300	5	10	15	20	25	31.06	0.2171	8.52071 e − 006	0.9998	0.095	0.319	2.52 ± 0.13
p-Coumaric acid	300	5	10	15	20	25	46.13	0.1255	5.60511 e − 006	0.9989	0.033	0.109	14.65 ± 0.47
Ferulic acid	300	5	10	15	20	25	50.02	−0.0627	9.21424 e − 006	0.9997	0.045	0.130	12.38 ± 0.35
Sinapic acid	300	5	10	15	20	25	51.28	−0.2589	1.22805 e − 005	0.9994	0.050	0.167	66.07 ± 1.97
Salicylic acid	300	10	20	30	40	50	52.12	0.6164	2.71310 e − 005	0.9996	0.212	0.707	215.30 ± 10.47

Figure 3. Chromatogram at λ = 278 nm of an walnut leaves extract (79% ethanol concentration, 9.0 v/w liquid to solid ratio, 25 min extraction time) using 1% aqueous acetic acid solution in mobile phase A, methanol in mobile phase B, and the following gradient: 0–27 min 90% A, 27–55 min 90–60% A, 55–60 min 60% A, 60–62 min 60–56% A, 62–70 min 56% A, 70–71 min 56–90% A and 71–75 min 90% A. Peaks: 1, gallic acid; 2, catechin hydrate; 3, vanillic acid; 4, chlorogenic acid; 5, caffeic acid; 6, syringic acid; 7, epicatechin; 8, p-coumaric acid; 9, ferulic acid; 10, sinapic acid; 11, salicylic acid; 12, rutin; 13, ellagic acid; 14, myricetin; 15, juglone; 16, trans-cinnamic acid; 17, quercetin.