Impact of extraction parameters and their optimization on the nutraceuticals and antioxidant properties of aqueous extract mulberry leaf

Figures & data

Table 1. Performance parameters of the HPLC-DAD methods.

Nutraceutical compounds	λ (nm)	RT	LR	Linear equation	R^2	LOD	LOQ
Phenolic acids
Chlorogenic acid	280	26.09	5–1000	y = 1783.53x + 3612.98	0.999	0.530	1.607
Caffeic acid	280	34.56	5–1000	y = 365.67x + 7768.64	0.999	1.291	3.913
Flavonols
Kaempferol-7-O-glucoside	370	12.01	5–1000	y = 5177.72x + 407 313.48	0.999	1.824	5.526
Quercetin-3-rutinose	370	21.84	5–1000	y = 1920.13x + 340.65	0.999	0.492	1.490
Quercetin-3-O-glucoside	370	28.27	5–1000	y = 206.85x – 118.10	0.999	0.442	1.339
Kaempferol 3-(6-rhamnosylglucoside)	370	31.01	5–1000	y = 49402.79x + 15052.25	0.999	1.911	5.792
Quercetin 3-(6-malonylglucoside)	370	42.03	5–1000	y = 620.06x + 143.83	0.999	0.761	2.307
Kaempferol-3-glucoside	370	44.24	5–1000	y = 15.96x + 40.10	0.999	8.013	24.282
Kaempferol 3-(6-malonylglucoside)	370	52.40	5–1000	y = 4973.64x + 317.26	0.999	0.949	2.876
Other nutraceuticals
1-Deoxynojirimycin	254	10.70	6.25–100	y = 210.29x + 1508.17	0.999	0.426	1.291
Gamma-aminobutyric acid	262	19.86	5–8	y = 113913.85x + 131136.25	0.999	0.339	1.026

RT: retention time in minute; LR: linear range in µg/ml; R²: regression coefficient; LOD: limit of detection in µg/ml = 3.3 σ/s, and LOQ: limit of quantification in µg/ml (LR) = 10 σ/s, where (σ) is the standard deviation of intercept from calibration curve, (s) is the slope of calibration curve.

Figure 1. Representative HPLC chromatograms of nutraceuticals extract in aqueous mulberry leaf extract. This sample of aqueous mulberry leaf extract was obtained under the optimal extraction conditions: extraction temperature of 70°C, extraction time of 40 min, water to leaf powder ratio of 40:1 ml/g, particle size of 25 µm, and two extraction cycles. Peaks Identification, as ascertained with standards: chlorogenic acid (CHA), caffeic acid (CA), kaempferol-7-O-glucoside (K7G), quercetin-3-rutinose (Q3R), quercetin-3-O-glucoside (Q3G), kaempferol 3-(6-rhamnosylglucoside) (K3R), quercetin 3-(6-malonylglucoside) (Q3M), kaempferol-3-glucoside (K3G), kaempferol 3-(6-malonylglucoside) (K3M), 1-deoxynojirimycin (DNJ), and gamma aminobutyric acid (GABA).

Table 2. Effect of extraction temperature on the nutraceutical compounds and antioxidant properties of aqueous mulberry leaf extract.

Temp (°C)	Biocompounds content (mg/g db)											Antioxidants activities (mM/g db)
	CHA	CA	K7G	Q3R	Q3G	K3R	Q3M	K3G	K3M	DNJ	GABA	ABTS	CUPRAC	DPPH	FRAP
5	13.39^k	5.11^j	8.44^f	6.48^e	12.58^h	15.37^i	27.03^h	36.88^i	20.92^h	8.67^g	2.22^e	10.93^h	30.15^h	50.40^i	183.57^e
10	14.87^j	6.94^i	8.88^ef	6.94^de	13.05^g	17.68^h	27.22^h	42.19^h	21.06^h	8.81^g	2.35^e	11.08^h	30.60^gh	52.00^h	183.72^e
20	15.44^i	7.35^h	9.06^e	7.12^d	13.33^g	18.37^g	29.38^g	43.02^g	21.85^g	11.17^f	2.58^de	11.25^h	30.82^f	52.00^h	184.96^d
30	21.27^h	10.17^g	10.08^d	7.68^c	14.84^f	18.55^g	30.11^f	44.20^f	22.02^g	11.48^f	2.95^d	12.05^g	35.04^f	58.41^g	185.59^cd
40	23.69^g	13.42^e	10.97^c	9.55^b	16.31^e	21.76^f	35.86^e	45.59^e	25.54^f	12.10^e	3.58^c	15.58^f	40.43^e	61.61^f	186.05^c
50	32.21^d	17.68^c	11.02^c	10.32^a	18.72^d	24.86^e	42.76^d	54.65^d	28.89^e	12.71^d	3.86^bc	18.90^e	42.79^d	69.62^c	186.20^c
60	34.99^c	20.47^b	12.33^b	10.58^a	21.25^c	28.96^c	45.26^c	58.51^c	33.42^b	13.89^ab	4.12^b	23.43^b	47.27^c	71.75^b	187.10^b
70	38.24^a	22.12^a	14.82^a	10.45^a	23.17^ab	30.58^a	50.27^ab	60.06^ab	34.97^a	13.95^a	4.62^a	25.73^a	50.17^ab	74.96^a	187.92^a
80	36.09^b	20.73^b	15.06^a	10.17^a	23.53^a	29.85^b	50.38^ab	60.44^a	35.22^a	13.74^abc	4.68^a	25.49^a	50.31^a	73.89^a	187.76^ab
90	27.43^e	16.16^d	15.22^a	9.62^b	23.01^b	26.47^d	50.46^a	59.66^b	30.78^c	13.57^bc	4.71^a	21.96^c	50.49^a	64.81^d	187.60^ab
100	26.03^f	12.34^f	15.01^a	9.30^b	22.81^b	26.04^d	49.99^b	58.26^c	29.71^d	13.50^c	4.75^a	21.31^d	49.72^b	63.21^e	187.30^ab
PSD	0.14	0.13	0.17	0.16	0.15	0.17	0.16	0.16	0.16	0.12	0.16	0.17	0.17	0.39	0.25

Mean values with different letters in the same column are significantly different at p < 0.05 (Anova, Tukey). Temp: temperature; CHA: chlorogenic acid; CA: caffeic acid; K7G: kaempferol-7-O-glucoside; Q3R: quercetin-3-rutinose; Q3G: quercetin-3-O-glucoside; K3R: kaempferol-3-(6-rhamnosylglucoside); Q3M: quercetin-3-(6-malonylglucoside); K3G: kaempferol-3-glucoside; K3M: kaempferol-3-(6-malonylglucoside); DNJ: 1-deoxynojirimycin; GABA: gamma-aminobutyric acid; ABTS: 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid); CUPRAC: cupric ion reducing capacity; DPPH: 1,1-diphenyl-2-picrylhydrazyl; FRAP: ferric reducing antioxidant power capacity; and PSD: pooled standard deviation.

Figure 2. Effect of temperature (a), extraction time (b), water to leaf powder ratio (c), and particle size (d) on the total nutraceutical compounds (TNC) and the total antioxidant properties (TAP) of aqueous mulberry leaf extract.

Table 3. Effect of extraction time on the nutraceutical compounds and antioxidant properties of aqueous mulberry leaf extract.

Time (min)	Biocompounds content (mg/g db)											Antioxidants activities (mM/g db)
	CHA	CA	K7G	Q3R	Q3G	K3R	Q3M	K3G	K3M	DNJ	GABA	ABTS	CUPRAC	DPPH	FRAP
5	17.24^k	13.88^i	9.42^f	6.02^h	13.29^i	21.09^f	33.14^h	37.02^g	22.31^g	9.32^f	3.01^f	16.46^h	30.87^g	69.62^e	183.40^g
10	18.75^j	14.05^hi	11.06^e	6.28^h	14.54^h	22.12^e	35.66^g	42.98^f	22.50^g	9.69^e	3.48^ef	17.93^g	34.81^f	69.62^e	183.57^g
15	20.72^i	15.47^g	11.28^e	6.99^g	16.45^g	25.03^d	39.11^f	43.20^f	26.05^f	11.96^d	3.85^de	19.73^f	38.57^e	71.22^d	185.90^f
20	25.67^f	19.79^d	13.05^d	7.90^f	19.18^f	25.13^d	45.05^e	47.72^e	26.77^e	12.11^d	4.20^cd	22.87^e	42.02^d	72.82^c	186.05^ef
25	32.78^c	20.24^c	13.65^bc	8.78^e	21.71^e	29.24^c	48.72^d	48.95^d	28.23^d	12.76^c	4.41^c	24.49^d	44.37^c	74.42^b	186.70^de
30	38.24^b	22.12^b	14.82^a	10.45^bc	23.17^d	30.58^b	50.27^c	60.06^b	34.97^b	13.95^b	4.62^bc	25.73^c	50.17^b	74.96^b	187.92^b
40	40.38^a	26.77^a	14.78^a	11.60^a	23.96^abc	32.41^a	52.63^a	62.18^a	36.12^a	15.08^a	5.12^ab	27.90^a	53.21^a	77.62^a	190.72^a
50	31.16^d	19.34^e	14.06^b	10.83^b	24.05^ab	32.01^a	52.78^a	62.41^a	36.44^a	15.14^a	5.25^a	27.58^a	53.34^a	74.42^b	190.10^a
60	28.15^e	17.02^f	13.95^bc	10.30^c	24.38^a	29.04^c	52.87^a	60.31^b	33.60^c	13.86^b	5.28^a	26.61^b	53.44^a	72.82^c	187.45^bc
90	23.41^g	15.21^g	13.87^bc	9.61^d	23.84^bc	29.00^c	51.05^b	50.96^c	33.65^c	13.65^b	5.32^a	26.17^bc	53.39^a	71.22^d	186.99^cd
120	22.34^h	14.36^h	13.56^c	9.26^d	23.56 ^cd	28.96^c	50.80^b	50.41^c	33.51^c	12.81^c	5.35^a	26.26^b	53.16^a	70.15^de	186.99^cd
PSD	0.15	0.15	0.16	0.15	0.16	0.14	0.16	0.20	0.17	0.13	0.18	0.18	0.18	0.39	0.22

Mean values with different letters in the same column are significantly different at p < 0.05 (Anova, Tukey). CHA: chlorogenic acid; CA: caffeic acid; K7G: kaempferol-7-O-glucoside; Q3R: quercetin-3-rutinose; Q3G: quercetin-3-O-glucoside; K3R: kaempferol-3-(6-rhamnosylglucoside); Q3M: quercetin-3-(6-malonylglucoside); K3G: kaempferol-3-glucoside; K3M: kaempferol-3-(6-malonylglucoside); DNJ: 1-deoxynojirimycin; GABA: gamma-aminobutyric acid; ABTS: 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid); CUPRAC: cupric ion reducing capacity; DPPH: 1,1-diphenyl-2-picrylhydrazyl; FRAP: ferric reducing antioxidant power capacity; and PSD: pooled standard deviation.

Table 4. Effect of water to leaf powder ratio on the nutraceutical compounds and antioxidant properties of aqueous mulberry leaf extract.

WPR (ml/g)	Biocompounds content (mg/g db)											Antioxidants activities (mM/g db)
	CHA	CA	K7G	Q3R	Q3G	K3R	Q3M	K3G	K3M	DNJ	GABA	ABTS	CUPRAC	DPPH	FRAP
10	18.79^g	18.44^e	9.26^e	6.17^g	17.31^e	23.43^f	43.67^f	57.22^e	26.12^e	12.76^e	3.48^e	22.14^f	34.27^h	56.80^f	171.90^h
20	19.06^g	20.01^d	9.34^e	6.32^g	17.42^e	23.87^ef	44.09^ef	57.64^de	26.44^e	13.22^d	3.64^e	22.20^f	36.40^g	56.80^f	172.20^gh
30	30.14^f	20.47^d	10.75^d	8.47^f	18.34^d	24.05^e	44.27^e	57.82^d	26.13^e	13.50^cd	4.25^d	23.61^e	42.24^f	68.01^e	172.84^g
40	35.53^e	24.63^c	12.25^c	9.46^e	20.99^c	26.30^d	46.52^d	59.07^c	29.98^d	13.60^c	4.52^cd	25.11^d	47.05^e	72.82^d	177.50^f
50	36.11^d	24.99^bc	12.33^c	9.55^de	21.02^c	26.66^d	46.88^d	59.43^c	30.27^d	13.73^c	4.76^bc	25.20^d	48.77^d	73.35^cd	181.23^e
60	38.55^c	25.21^b	12.57^c	9.93^cd	21.16^c	27.27^c	47.49^c	60.04^b	30.44^cd	14.66^b	4.98^ab	27.02^c	49.04^d	74.96^bc	182.17^d
70	39.41^b	25.33^b	13.59^b	10.25^c	21.94^b	29.01^b	49.23^b	61.78^a	30.85^bc	14.91^ab	5.01^ab	28.02^ab	50.72^c	76.56^ab	185.90^c
80	40.15^a	26.58^a	14.93^a	10.77^b	22.22^b	29.32^b	49.54^b	61.89^a	31.09^b	15.01^a	5.06^ab	28.37^a	52.03^b	77.09^a	188.10^b
90	40.27^a	26.68^a	14.92^a	11.48^a	23.78^a	32.78^a	52.50^a	62.06^a	35.85^a	15.05^a	5.09^ab	28.34^ab	52.44^b	77.62^a	190.40^a
100	40.38^a	26.77^a	14.78^a	11.60^a	23.96^a	32.41^a	52.63^a	62.18^a	36.12^a	15.08^a	5.12^a	27.90^b	53.21^a	77.62^a	190.72^a
PSD	0.16	0.18	0.13	0.15	0.16	0.15	0.14	0.15	0.17	0.10	0.12	0.16	0.16	0.56	0.25

Mean values with different letters in the same column are significantly different at p < 0.05 (Anova, Tukey). WPR: water to leaf powder ratio; CHA: chlorogenic acid; CA: caffeic acid; K7G: kaempferol-7-O-glucoside; Q3R: quercetin-3-rutinose; Q3G: quercetin-3-O-glucoside; K3R: kaempferol-3-(6-rhamnosylglucoside); Q3M: quercetin-3-(6-malonylglucoside); K3G: kaempferol-3-glucoside; K3M: kaempferol-3-(6-malonylglucoside); DNJ: 1-deoxynojirimycin; GABA: gamma-aminobutyric acid; ABTS: 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid); CUPRAC: cupric ion reducing capacity; DPPH: 1,1-diphenyl-2-picrylhydrazyl; FRAP: ferric reducing antioxidant power capacity; and PSD: pooled standard deviation.

Table 5. Effect of particle size on the nutraceutical compounds and antioxidant properties of aqueous mulberry leaf extract.

PS (µm)	Biocompounds content (mg/g db)											Antioxidants activities (mM/g db)
	CHA	CA	K7G	Q3R	Q3G	K3R	Q3M	K3G	K3M	DNJ	GABA	ABTS	CUPRAC	DPPH	FRAP
270	35.53^e	24.63^d	12.25^d	9.46^d	20.99^e	26.30^c	46.52^e	59.07^d	29.98^c	13.60^c	4.52^b	25.11^d	47.05^c	72.82^d	177.50^d
150	38.11^c	25.77^c	13.17^c	10.64^c	24.63^d	26.71^c	50.16^d	59.85^c	30.17^c	13.73^c	4.61^b	26.84^c	47.18^bc	74.96^c	178.70^c
48	40.02^b	27.33^b	15.31^b	11.76^b	26.26^b	30.82^b	51.79^c	61.73^b	32.72^b	14.19^b	4.82^ab	31.20^b	47.32^abc	75.49^bc	179.37^b
25	43.96^a	28.04^a	18.88^a	12.34^a	26.52^ab	31.05^ab	53.05^ab	64.45^a	33.43^a	14.47^a	4.97^a	31.40^ab	47.59^ab	77.09^ab	182.00^a
18	37.67^d	24.10^e	18.99^a	12.59^a	26.74^a	31.25^ab	53.27^a	64.51^a	33.56^a	14.49^a	5.03^a	31.58^ab	47.68^ab	77.62^a	182.17^a
13	35.22^e	22.35^f	19.15^a	12.61^a	25.39^c	31.33^a	52.87^b	64.68^a	33.52^a	14.51^a	5.05^a	31.61^a	47.73^a	78.16^a	182.17^a
PSD	0.12	0.12	0.16	0.15	0.16	0.16	0.14	0.17	0.17	0.10	0.11	0.14	0.19	0.67	0.23

Mean values with different letters in the same column are significantly different at p < 0.05 (Anova, Tukey). PS: particle size; CHA: chlorogenic acid; CA: caffeic acid; K7G: kaempferol-7-O-glucoside; Q3R: quercetin-3-rutinose; Q3G: quercetin-3-O-glucoside; K3R: kaempferol-3-(6-rhamnosylglucoside); Q3M: quercetin-3-(6-malonylglucoside); K3G: kaempferol-3-glucoside; K3M: kaempferol-3-(6-malonylglucoside); DNJ: 1-deoxynojirimycin; GABA: gamma-aminobutyric acid; ABTS: 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid); CUPRAC: cupric ion reducing capacity; DPPH: 1,1-diphenyl-2-picrylhydrazyl; FRAP: ferric reducing antioxidant power capacity; and PSD: pooled standard deviation.

Figure 3. Effect of the extraction cycle on the total nutraceutical compounds (TNC) and the total antioxidant properties (TAP) of aqueous mulberry leaf extract.