Optimization of ultrasound-assisted phytomolecules extraction from moringa leaves (Moringa oleifera Lam) using response surface methodology

Figures & data

Table 1. Independent variables and the specified parameter ranges in the experimental design for ultrasound-assisted extraction.

Input Variables	Symbol	Levels
	Xi	−1	0	+1
Amplitude (%)	X1	60	70	80
Temperature (°C)	X2	25	35	45
Extraction Time (Min)	X3	20	30	40

Table 2. Dependent variables specified in the experimental design for ultrasound-assisted extraction.

Response	Unit	Acronym
Extraction Yield	Percentage (%)	EY
Total Phenolic Content	Milligrams of gallic acid equivalents per gram (mg GAE/g)	TPC
Total Flavonoid Content	Milligrams of quercetin equivalents per gram (mg QE/g)	TFC
Radical Scavenging Activity	Percentage (%)	RSA

Table 3. Parameters for the central composite design regarding the independent variables and experimental responses.

	Factors			EY (%)		TPC (mg GA/ g)		TFC (mg QE/g)		RSA (%)
Experimental Run	X1 (%)	X2 (°C)	X3 (Min)	Experimental	Predicted	Experimental	Predicted	Experimental	Predicted	Experimental	Predicted
1	60.00	25.00	40.00	14.72	15.37	120.74	119.86	15.4	15.89	69.32	69.94
2	60.00	45.00	20.00	12.78	13.88	119.24	116.54	14.33	13.91	64.51	64.37
3	80.00	25.00	40.00	17.51	17.17	125.27	127.92	17.11	17.36	76.96	77.91
4	70.00	18.18	30.00	16.68	16.83	123.81	122.86	16.59	16.70	74.66	74.14
5	70.00	35.00	30.00	22.01	22.31	144.63	145.71	20.87	20.20	83.01	83.56
6	60.00	45.00	40.00	13.56	14.01	121.74	120.14	14.89	15.00	67.02	68.44
7	70.00	35.00	30.00	22.12	22.31	145.12	145.71	19.73	20.20	81.71	83.56
8	70.00	35.00	46.82	19.96	19.27	142.01	140.87	19.44	18.48	81.50	79.98
9	70.00	35.00	30.00	22.01	22.31	144.52	145.71	20.03	20.20	85.00	83.56
10	86.82	35.00	30.00	16.74	16.44	114.56	110.53	16.05	16.01	72.09	72.05
11	80.00	25.00	20.00	19.26	19.57	122.62	124.17	17.82	17.54	80.14	79.52
12	70.00	51.82	30.00	15.01	13.78	115.19	116.21	14.18	14.31	68.32	67.71
13	70.00	35.00	13.18	21.56	21.17	133.47	134.68	16.51	17.71	77.53	77.91
14	70.00	35.00	30.00	22.16	22.31	145.35	145.71	19.57	20.20	83.17	83.56
15	80.00	45.00	40.00	15.12	16.15	118.11	119.82	15.43	16.05	69.44	69.65
16	80.00	45.00	20.00	17.18	17.30	115.14	115.97	16.26	15.59	73.18	73.37
17	70.00	35.00	30.00	23.28	22.31	149.22	145.71	20.92	20.20	85.08	83.56
18	53.18	35.00	30.00	12.82	12.04	100.12	104.22	13.08	13.36	58.87	57.78
19	60.00	25.00	20.00	16.75	16.49	118.12	116.36	16.24	15.44	63.17	63.76
20	70.00	35.00	30.00	22.09	22.31	145.42	145.71	20.12	20.20	83.19	83.56

Table 4. Analysis of Variance (ANOVA) findings for the extraction yield.

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	228.92	9	25.44	34.97	< 0.0001	Significant
A (AMPLITUDE)	23.34	1	23.34	32.09	0.0002
B (TEMPERATURE)	11.27	1	11.27	15.50	0.0028
C (TIME)	4.40	1	4.40	6.05	0.0337
AB	0.0545	1	0.0545	0.0749	0.7899
AC	0.8192	1	0.8192	1.13	0.3135
BC	0.7813	1	0.7813	1.07	0.3244
A²	117.28	1	117.28	161.26	< 0.0001
B²	88.37	1	88.37	121.50	< 0.0001
C²	7.86	1	7.86	10.81	0.0082
Residual	7.27	10	0.7273
Lack of Fit	6.05	5	1.21	4.95	0.0519	Non-significant
Pure Error	1.22	5	0.2444
Cor Total	236.19	19
R^2	0.9692	–	–	–	–	–
Adj. R^2	0.9415	–	–	–	–	–

Figure 1. 2D and 3D graphs depicting the relationship between extraction yield and both amplitude and temperature.

Table 5. Analysis of Variance (ANOVA) pertaining to the total phenol content.

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	3747.90	9	416.43	52.00	< 0.0001	Significant
A (AMPLITUDE)	47.93	1	47.93	5.99	0.0345
B (TEMPERATURE)	53.45	1	53.45	6.67	0.0273
C (TIME)	46.14	1	46.14	5.76	0.0373
AB	35.11	1	35.11	4.38	0.0627
AC	0.0313	1	0.0313	0.0039	0.9514
BC	0.0050	1	0.0050	0.0006	0.9806
A²	2646.96	1	2646.96	330.54	< 0.0001
B²	1233.97	1	1233.97	154.09	< 0.0001
C²	113.35	1	113.35	14.16	0.0037
Residual	80.08	10	8.01
Lack of Fit	64.62	5	12.92	4.18	0.0713	Non-significant
Pure Error	15.46	5	3.09
Cor Total	3827.98	19
R^2	0.9791
Adj. R^2	0.9603

Figure 2. 2D and 3D graphs depicting the relationship between amplitude and extraction time for total phenol content.

Table 6. Analysis of Variance (ANOVA) pertaining to the total flavonoid content.

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	64.86	9	7.21	52.02	< 0.0001	Significant
A (AMPLITUDE)	1.91	1	1.91	13.77	0.0040
B (TEMPERATURE)	5.16	1	5.16	37.28	0.0001
C (TIME)	0.2835	1	0.2835	2.05	0.1830
AB	0.9730	1	0.9730	7.02	0.0243
AC	1.30	1	1.30	9.41	0.0119
BC	0.0703	1	0.0703	0.5076	0.4925
A²	27.59	1	27.59	199.20	< 0.0001
B²	16.15	1	16.15	116.56	< 0.0001
C²	22.12	1	22.12	159.65	< 0.0001
Residual	1.39	10	0.1385
Lack of Fit	1.08	5	0.2169	3.60	0.0929	Non-Significant
Pure Error	0.3009	5	0.0602
Cor Total	66.24	19
R^2	0.9791
Adj. R^2	0.9603

Figure 3. 2D and 3D graphs depicting the relationship between Amplitude and temperature for total flavonoid content.

Table 7. Analysis of variance (ANOVA) pertaining to the radical scavenging activity (antioxidant activity).

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	1200.02	9	133.34	79.28	< 0.0001	significant
A-AMPLITUDE	245.76	1	245.76	146.13	< 0.0001
B-TEMPERATURE	49.89	1	49.89	29.66	0.0003
C-TIME	5.19	1	5.19	3.08	0.1096
AB	22.85	1	22.85	13.59	0.0042
AC	30.34	1	30.34	18.04	0.0017
BC	2.20	1	2.20	1.31	0.2789
A²	626.33	1	626.33	372.41	< 0.0001
B²	287.65	1	287.65	171.03	< 0.0001
C²	38.31	1	38.31	22.78	0.0008
Residual	16.82	10	1.68
Lack of Fit	8.43	5	1.69	1.00	0.4982	not significant
Pure Error	8.39	5	1.68
Cor Total	1216.84	19
R^2	0.9862
Adj. R^2	0.9737

Figure 4. 2D and 3D graphs depicting the relationship between Amplitude and temperature for radical scavenging activity.

Figure 5. Perturbation plot indicating (Left to Right), EY, TPC, TFC and RSA produced with reference to each variable.

Figure 6. Linear regression graphs depicting the predicted and experimental values for dependent variables.

Table 8. Qualitative data of the extracted moringa leaf extract with optimized parameters.

Response factors	Extraction Yield	Total Phenol Content	Total Flavonoid Content	Radical Scavenging Activity
Mean Value at 95% confidence level	23.41%	144.9 GA mg/g	24.23 QE mg/g	84.82%

Figure 7. GC-MS chromatogram of moringa extract by optimized UAE method.

Table 9. List of identified compounds using GC-MS with optimized extraction parameters.

S.No	Compound Name	Area	RT (Min)	Molecular formula	Molecular Weight (g/mol)	Biological Acitvity	Reference
1.	2-Amino-4,4,6,6-tetramethyl-4,6-dihydrothieno[2,3-c]furan-3-carbonitrile	148962.48	2.80	C6H4OS	222.31	Pain-relieving, Anti-anginal, Pain-relieving (non-opioid), Blood pressure-lowering, Anti-arthritic, Treatment for dementia, Neurotransmitter uptake inhibitor	(Brintha et al., 2017)
2.	1,2-Benzisothiazol-3-amine tbdms	242890.46	3.69	C7H6N2S	264.46	β-lactam antibiotic adjuvants	(Jin et al., 2020)
3.	2(3H)-Furanone, 5-acetyldihydro-	551328.56	5.30	C6H8O3	128.13	Flavouring compound	(Michel et al., 2021)
4.	Hydroquinone	501591.67	5.81	C42H63O7P	222.32	Anticancer Anti-fungal, Anti-oxidant Anti-bacterial,	(Ren et al., 2019)
5.	2-Acetylamino-2-cyano-acetamide	392374.83	6.83	C3H5N3O	141.13	Adulticidal Activity	(Rajalakshmi et al., 2022)
6.	2,5-Furandione, 3-methyl-	124596.35	8.24	C5H4O3	128.12	Antimicrobial activity	(Abed et al., 2023)
7.	10-Methoxycoryn-18-en-17-yl acetate	3163887.43	9.34	C22H28N2O3	368.50	Anticancer	(Divyalakshmi & Thoppil, 2022)
8.	Spiro[5.alpha.-androstane-3,2'-thiazolidine], 3'-acetyl	3163887.43	10.70	C23H37NOS	375.60	Not Reported	–
9.	1-(4-Bromo-phenyl)-3-isopropylimino-propan-1	998807.87	11.86	C11H14BrNO	256.14	Not Reported	–
10.	1,2,3-Propanetriol, 1-acetate	253011.88	12.27	C5H10O4	134.13	Antibacterial	(Van et al., 2023)
11.	1,2,4-Cyclopentanetriol	201734.09	12.56	C5H10O3	118.13	Antioxidant and Antimicrobial activity	(Yassin et al., 2022)
12.	5-Dimethyl (chloromethyl) silyloxypentadecane	557733.14	13.76	C16H34	143.80	Not Reported	–
13.	Hexasiloxane,1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl-	158334.78	14.76	C12H38O5Si6	430.94	Antibacterial and Antifungal	(Sbayou et al., 2014)
14.	Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl-	164796.10	16.73	C14H42O6Si7	503.07	Antioxidant and Antibacterial	(Ezez et al., 2023)
15.	1-Monolinoleoylglycerol trimethylsilyl ether	275926.03	19.08	C27H56O4Si2	500.9	Anti-infective, Antioxidative, Anti-arthritis, Anti-inflammatory, Anti-asthmatic, Diuretic, and Type II Diabetes treatment	(Senthil et al., 2016)
16.	Succinic acid, (4-methoxyphenyl) ester	19977.0	21.75	C11H12O5	224.21	GHB and GABAB neural receptors	(Kowalczyk & Kulig, 2014)
17.	n-Hexadecanoic acid	777701.00	25.11	C16H32O2	256.42	17-beta hydroxysteroid dehydrogenase inhibitor, Testosterone Hydroxylase inhibitor, Arachidonic acid inhibitor, increase aromatic amino acid decarboxylase acti	(Mohammad et al., 2019)
18.	Glutaric acid	905822.62	25.74	C18H30O4	300.4	Not reported	–
19.	Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl-	351836.97	28.04	C16H48O7Si8	577.2	Not reported	–
20.	Eicosanoic acid, methyl ester	6331138.88	28.94	C21H42O2	326.55	Not reported	–
21.	Distearylthiodipropionate	1774525.14	30.16	C42H82O4S	683.2	Antioxidant	–
22.	Palmitic Acid, TMS derivative	5569.14	31.24	C19H40O2Si	328.60	Food Emulsifier	(Baothman et al., 2023)
23.	.beta. Carotene	1415516.97	33.22	C40H56	536.90	Antioxidant	(Evans & Lawrenson, Cochrane Eyes & Vision Group 2017)
24.	5H-Cyclopropa[3,4]benz[1,2-e]azulen-5-one,9,9a-bis(acetyloxy)-1,1a,1b,2,4a,7a,7b,8,9,9a-decahydro-2,4a,7b-trihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-	150778167.50	36.21	C26H34O10	506.5	Anti-bacterial and anti-oxidant activity	(Majumder et al., 2019)
25.	1-Linolenoylglycerol, 2TMS derivative	292036.18	37.27	C27H52O4Si2	496.90	Antimicrobial, anti-inflammatory, anticancer, anti-oxidant and hypercholesterolemia activity	(Ibrahim & Abdul-Hafeez, 2023)
26.	2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-propanoic acid	796104.28	43.47	C27H42O4	430.6	Anti-microbial and Anti-tumour	(Sheik et al., 2023)
27.	17-(1,5-Dimethylhexyl)-10,13-dimethyl-3-styrylhexadecahyd rocyclopenta[a]phenanthren-2-one	11373950.94	44.18	C29H50O	414.7	Not Reported	–
28.	Glycerin 1-palmitate 2-myristate 3-stearate	6250342.34	45.20	C51H98O6	807.3	Not Reported	–

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Availability of data and material

The datasets generated and/or analyzed during the research are made available upon a reasonable request from the corresponding author.