Optimization of ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharide from radix Morindae officinalis by response surface methodology
Jie Shen, Zhenying Mei, Zhiqin Xu, Zhimin Zhao, Depo Yang, Xinjun Xu
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou; Guangdong Great Bay Area Institute of Southern Chinese Medicines, Huaiji, China
|Date of Submission||10-Oct-2019|
|Date of Decision||31-Oct-2019|
|Date of Acceptance||21-Apr-2020|
|Date of Web Publication||20-Oct-2020|
No. 132, East Waihuan Road, Guangzhou Higher Education Mega Centre, Guangzhou 510006
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: In this study, we aimed to establish an efficient method for the extraction of radix Morindae officinalispolysaccharide (MOP) by applying ultrasonic technology and by optimizing the parameters through response surface methodology (RSM) based on the central composite design. Materials and Methods: Ultrasound-assisted enzymatic extraction (UAEE) was performed to extract the MOP. We applied an orthogonal array design to optimize the concentration of enzymes (cellulose, pectinase, and papain). The extraction parameters were optimized based on the RSM technique. Furthermore, the effects of ultrasound-assisted enzymatic treatment on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity by the MOP was optimized using the RSM technique. Results: According to our results, 1.0% cellulase, 1.5% pectinase, and 1.0% papain were the optimum concentrations of the enzyme (calculated based on the percentage dry weight of radix Morindae officinalis powder). The optimum conditions of extraction were as follows: 21 mL/g solvent–solid ratio, 280 W, pH of 5.3, and temperature of 50°C. Under these conditions, the yield of MOPs and DPPH-scavenging activity of MOP were 23.68% ± 0.52% (n = 3) and 117.26 ± 2.73 mg Vitamin C/100 g dry weight (n = 3), respectively. The overall desirability was 1.03 ± 0.01 (n = 3). Conclusion: UAEE was effective in the extraction of MOP, and RSM technique was adequate to design and optimize the extraction parameters.
Keywords: 2,2-diphenyl-1-picrylhydrazyl, central composite design, radix Morindae officinalis, polysaccharides, ultrasound-assisted enzymatic extraction, yield
|How to cite this article:|
Shen J, Mei Z, Xu Z, Zhao Z, Yang D, Xu X. Optimization of ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharide from radix Morindae officinalis by response surface methodology. Phcog Mag 2020;16:662-9
|How to cite this URL:|
Shen J, Mei Z, Xu Z, Zhao Z, Yang D, Xu X. Optimization of ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharide from radix Morindae officinalis by response surface methodology. Phcog Mag [serial online] 2020 [cited 2021 Jul 26];16:662-9. Available from: http://www.phcog.com/text.asp?2020/16/71/662/298689
- Ultrasound-assisted enzymatic extraction method showed a great potential in enhancing the yield and 2,2-diphenyl-1-picrylhydrazyl scavenging activity of MOP. Moreover, response surface methodology with central composite design was indicated to be a reliable technique for optimizing the conditions of ultrasound-assisted enzymatic extraction, including solvent-solid ratio, power, pH, and extraction temperature.
Abbreviations used: DPPH: 2,2-Diphenyl-1-picrylhydrazyl; FT-IR: Fourier-transform infrared spectrophotometer; MOP: Radix Morindae officinalis polysaccharide; OD: Overall desirability; RMO: Radix Morindae officinalis; RSM: Response surface methodology; UAEE: Ultrasound-assisted enzymatic extraction; VC: Vitamin C.
| Introduction|| |
Radix Morindae officinalis (RMO) is the dried root of M. officinalis How, which belongs to the genus Morinda of the Rubiaceae family and has been cultivated widely in Guangdong, Guangxi, and Fujian provinces in China. Previous investigations have demonstrated the pharmacological activities of RMO, such as antioxidant, antiosteoporosis, antidepressant, and antirheumatoid activities. In addition, RMO contains many compounds, including polysaccharides, oligosaccharides, iridoid glycosides, anthraquinones, organic acids, and volatile oils., At present, researchers are focusing on functional factors of health foods, such as active polysaccharides. Radix Morindae officinalis polysaccharide (MOP) show antioxidant, anti-fatigue, and antiosteoporosis activity.
The extraction of MOP was initially attempted by using hot water extraction method, but it took a long time and also resulted in low yield. Recent development in the research has led to the extraction of compounds that would be difficult or impossible to extract, such as ultrasonic, microwave, and enzymatic extraction techniques.,, Ultrasonic extraction is a low energy-consuming method which provides high yield of polysaccharides as the materials can be physically mixed with high intensity through ultrasound cavitation. In addition, enzymatic method of extraction destroys the cell walls to release the intracellular substances. Ultrasound-assisted enzymatic extraction (UAEE) is advantageous as it applies both the aforementioned techniques. It is widely used in the extraction of polysaccharides., A previous study reported the yield of polysaccharides extracted from the dandelion leaves as 14.05% ±0.95% by using UAEE technique, 9.84% ± 0.20% using enzyme-assisted technique, and 8.84% ± 0.36% using ultrasonic-assisted technique. Another study conducted on the Atratylodes macrocephala polysaccharides extracted after UAEE showed higher yield and higher antioxidant activity than that of ultrasonic-assisted extraction and enzyme-assisted extraction under the same conditions. Hence, to obtain the crude extract-containing MOP with high yield and high antioxidant activity, we adopted UAEE technique in this study.
Response surface methodology (RSM) effectively explores the interactions of independent variables and helps to optimize the extraction process. The advantages of RSM include reduced time spent on optimization and decreased overall costs of extraction. Many studies have focused on the optimization of polysaccharide content and antioxidant activity from A. macrocephala, Trapa quadrispinosa, and Arthrocnemum indicum leaves using RSM technique. Moreover, the overall desirability (OD) has been developed for calculating the optimization of extract condition for independent variables on multiple responses. In this research, we developed an UAEE method of MOP using RSM with the aim on improving the yield of MOP and exploring their antioxidant activity.
| Materials and Methods|| |
RMO collected from Guangzhou of China was identified as the root of M. officinalis How. After the roots were dried and sieved (40 mesh), they were kept in closed desiccators until use.
Chemicals and apparatus
Cellulose (50 U/mg, LOT: H16A9S58744), pectinase (500 U/mg, LOT: L19S9 L70223), papain (800 U/mg, LOT: P19M9B56188), and D-glucose (purity: 99.0%, LOT: Q08A10N85042) were obtained from Yuanye Biotechnology Co., Ltd. Vitamin C (VC, purity: ≥99%, LOT: A103535) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Concentrated sulfuric acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), phenol, and absolute alcohol were of analytical grade.
Reflux extraction and enzyme inactivation was conducted on a water bath (HH, Jincheng Guosheng Laboratory Instrument Factory, Jintan, China) was applied to extraction. UAEE procedure was conducted on a Kunshan ultrasonic cleaner (KQ-400DE, Kunshan Ultrasonic Instruments Co., Ltd, China) with an ultrasound power of 80-400 W. A rotary evaporator (N-1200A, Eyela Instruments Co., Ltd., Shanghai China) was used to concentrate the obtained extracts. A freeze dryer (Martin Christ, Osterode, Germany) was used to dry the extracts. MOP was quantitative determined using phenol-sulfuric acid method on an ultraviolet-visible spectrophotometer (UV-2600, Shimadzu Corporation, Kyoto, Japan). DPPH radical-scavenging activity was measured on a microplate spectrophotometer (Flex Station 3, Molecular devices Co., Ltd., Sunnyvale, USA). Infrared spectra were obtained on a Fourier transform infrared spectrophotometer (FT-IR, Perkin Elmer, Waltham, USA).
The powder of RMO (5 g) was extracted thrice by 95% ethanol reflux for 2 h at 80°C to remove small molecular materials, monosaccharides, and oligosaccharides. After the residues were vacuum-dried, compound enzymes were added with the given concentration and ratio, and the pH was adjusted by phosphate buffer. The mixture was extracted using an ultrasonic instrument under the designed conditions. The obtained extract was placed in 90°C water bath for 10 min. After filtration and condensation, the extract was added 4 fold amounts of anhydrous ethanol and saved at 4°C for one night. After centrifuging at 4000 rpm for 20 min, the crude MOP was freeze-dried.
Determination of the yield of MOP
MOP was estimated based on the phenol-sulfuric acid method. The yield of MOP (%) was determined from the following formula:
Where, w is the content of MOP determined from the standard curve prepared from D-glucose (%); m is the weight of MOP (g); and m0 is the weight of RMO powder (g).
Determination of antioxidant activity of MOP
Due to higher stability, low cost, and high efficiency, DPPH is widely used to detect the ability of test compounds to eliminate the free radicals., It is an effective tool used in the evaluation of antioxidant activity of various polysaccharides, such as in Passiflora edulis Sims peel,Gentiana macrophylla, and A. macrocephala. In this study, antioxidant activity of the MOP was determined using DPPH method. After incubating in the dark for 30 min, the quenching of blue color formed was detected at 517 nm. Antioxidant activity of each extract was calculated by the percentage inhibition of DPPH radical formation:
Where A0 is the absorbance of ethanol plus DPPH solution without MOP, A1 is the absorbance of MOP plus DPPH solution, and A2 is the absorbance of MOP plus ethanol.
VC was selected as the control. The antioxidant activity of MOP was calculated using the standard curve of VC and expressed as mg VC per 100 g dry weight of RMO (mg VC/100 g).
Screening of the composition of compound enzymes
Orthogonal array design was employed to study the effects of different proportions of compound enzymes (cellulose, pectinase, and papain) on the yield of MOP and conducted with three factors and three levels [Table 1]. The constant extraction condition was solvent-solid ratio (10 mL/g), power (400 W), pH (5.5), temperature (50°C), and time (120 min).
Selection of extraction condition
Various conditions of MOP extraction were evaluated respectively by single-factor experiments, including solvent-solid ratio (5–25 mL/g), power (80-400 W), pH (3.5–7.5), extraction time (15–150 min), and temperature (30°C–70°C).
Central composite design
Based on single-factor experiments, four independent variables (solvent-solid ratio, X1; power, X2; pH, X3; and temperature, X4) were shown to have significant effects on the response variables (the yield of MOP, Y1; the DPPH radical-scavenging activity of MOP, Y2) and further optimized by central composite design. Each independent variable was set at 5 levels [Table 2], and 30 sets of experiments were implemented in random design [Table 3].
|Table 2: Levels of different process variables in central composite design|
Click here to view
Design Expert Software (Version 8.0.6, USA) was used in the statistical analysis. The behavior of the system was displayed by the following model:
Where Y was the response variable, X was the independent variable, and β was the coefficient.
Analysis of variance was implemented to detect the significant level. The yield (d1) and DPPH radical scavenging activity (d2) of MOP were transformed into OD:
Where d i is the desirability value, Y i is the response value, Ymin is the minimum response value, Ymax is the maximum response value, and n was the number of response variables (n = 2).
Characterization of MOP
The characterization of MOP was determined in the range of 4000–400 cm−1 by FT-IR.
| Results and Discussion|| |
Orthogonal array design of concentration of compound enzymes
Compared with 7.39% in the group without enzyme, the yield of MOP increased from 10.69% to 12.37% as cellulose increased from 0.5% to 1.5% [Figure 1]a, increased from 8.89% to 11.18% as pectinase increased from 0.5% to 1.5% [Figure 1]b, and increased from 9.2% to 10.62% as papain increased from 0.25% to 1.0% [Figure 1]c, and then, it became steady. Therefore, 1.0%–2.0% cellulose, 1.0%-2.0% pectinase, and 0.5%–1.5% papain were used in further experiments. The impacts of different compound enzyme proportions on MOP extraction were investigated by a L9(34) orthogonal test, and the results [Table 1] show varying degrees of influence as follows: papain (C) >pectinase (B) >cellulose (A) (R C >R B > R A). The optimal combination was A1B2C2 (1.0% cellulase, 1.5% pectinase, and 1.0% papain) and the corresponding yield of MOP was 17.50% ±0.24%.
|Figure 1: Effects of extraction variables on the yield of MOP: (a) Cellulase concentration; (b) pectinase concentration; (c) papain concentration; (d) solvent-solid ratio; (e) ultrasonic power; (f) pH; (g) extraction temperature; (h) extraction time|
Click here to view
Single-factor design for solvent-solid ratio
The yield of MOP significantly increased from 11.77% to 21.44%, with solvent-solid ratio increasing from 5 to 20 mL/g [Figure 1]d. However, the yield no longer changed above 20 mL/g. Therefore, 20 mL/g was selected as the optimal solvent-solid ratio.
Single-factor design for ultrasonic power
The yield of MOP increased from 16.54% to 19.85%, s with ultrasonic power increasing from 80 to 240 W [Figure 1]e, which might be because ultrasonic wave facilitates the rupture of cell walls and the dissolution of MOP. Then, the yield started to decrease at higher power, which might be due to the hydrolysis and aggregation of MOP under excessive power. Therefore, 240 W was selected as the optimal value.
Single-factor design for pH
The yield of MOP increased from 12.05% to 17.22%, with pH increasing from 3.5 to 5.5 [Figure 1]f. Then, a significant reduction in the yield was observed when pH was exceeded 5.5. This might be because pH had an important effect on the activity of enzymes and different enzymes had different suitable pH range to damage cell structures and release MOP. Therefore, pH 5.5 was selected as the optimum pH.
Single-factor design for extraction temperature
The yield of MOP increased with extraction temperature and reached 17.33% at 50°C [Figure 1]g. Then, the yield rapidly decreased when temperature exceeded 50°C. Compound enzymes had a suitable reaction temperature of 50°C, the enzyme activity might decrease or inactive at a lower or higher temperature.
Single-factor design for extraction time
The yield of MOP increased in the range of 15–60 min and then leveled off; the maximum value of 17.00% was observed at 60 min [Figure 1]h. Increasing extraction time could promote the dissolution of MOP, but excessive time could cause high-energy cost and low efficiency. Therefore, 60 min was chosen as the optimum extraction time.
According to the single-factor study, extraction time had a modest effect compared with other independent variables. Thus, extraction time was fixed as 60 min, and other conditions were further optimized by central composite design.
Results of central composite design for MOP extraction
The quadratic polynomial model that reflected the relationship between independent variables (X1, X2, X3, X4) with the yield of MOP (Y1) is shown as follows:
As shown from [Table 4], F-value of 107.23 (P < 0.001) confirmed the significance of model. R2 of 0.9901 showed goodness of model fitting. Pre-R2 of 0.9669 was in accord with Adj-R2 of 0.9809. Coefficient of variation (CV) of 2.37% (<5.00%) revealed that the model was repeatable. Adequate precision of 32.124 (>4) indicated an adequate signal. The lack of fit was not statistically significant (F = 0.40, P = 0.9006). X1, X2, X3, X4, X1X3, X2X3, X2X4, X3X4, X12, X22, X32, and X42 were highly significant (P < 0.01) and X1X4 was significant (P < 0.05).
|Table 4: Analysis of variance for the yield and the DPPH radical-scavenging activity of MOP|
Click here to view
[Figure 2] shows the interaction of independent variables. The yield increased with solvent-solid ratio increasing from 5 to 23 mL/g, with no significant increase exceeding this ratio. The yield reached a peak at 260 W and decreased above 260 W [Figure 2]a. This might be due to the cavitation formed by ultrasonic waves that can dissolve polysaccharides by increasing the penetration of solvent into the medicinal materials, but cavitation performance was decreased at high ultrasonic power. The yield increased with pH and reached a peak at pH 5.2 [Figure 2]b. In addition, the yield increased as the extraction temperature increased in the initial stage and then decreased [Figure 2]c. The other interactions showed similar tendencies [Figure 2]d, [Figure 2]e, [Figure 2]f]. This result agrees with previous studies; Wu et al. reported that pHof 5 and temperature of 51.5°C were optimal to extract polysaccharides from pumpkin by UAEE with the compound enzyme of cellulose, pectinase, and papain. Wu et al. reported that 55°C was suitable for polysaccharide extraction from cup plant with the compound enzyme of cellulose, pectinase, and papain.
|Figure 2: Response surface showing the effects of extraction variables on the yield of MOP: (a) Solvent-solid ratio and ultrasonic power; (b) solvent-solid ratio and pH; (c) solvent-solid ratio and extraction temperature; (d) ultrasonic power and pH; (e) ultrasonic power and extraction temperature; (f) pH and extraction temperature|
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Results of central composite design for DPPH radical scavenging activity of MOP
The quadratic polynomial model that reflected the relationship between independent variables (X2, X3, and X4) with antioxidant activity of MOP (Y1) is shown as follows:
As shown in [Table 4], F-value (F = 248.07, P < 0.01) of the model suggests that the model is significant. R2 (0.9957) showed a good fit of the model. Pre-R2 of 0.9788 was in accord with adj-R2 (0.9917), CV of 1.00% (<5.00%) implied that the model was reliable, and adequate precision of 49.016 (>4) indicated an adequate signal. The lack of fit was not significant (F = 2.08, P = 0.2161). X1, X2, X3, X4, X1X4, X2X4, X12, X22, X32, and X42 were highly significant (P < 0.01) and X1X2 and X2X3 were significant (P < 0.05).
The interaction of independent variables on DPPH radical scavenging activity of MOP can be seen from response surface plots [Figure 3]. Scavenging activity was positively correlated with ultrasonic power ranging from 80 to 230 W, whereas it correlated negatively above 230 Pu et al. reported that DPPH radical-scavenging percentage increased with ultrasonic power lower than 220 W and then decreased at higher power in the extraction of A. macrocephala polysaccharides by UAEE. Increasing solvent-solid ratio (5–18 mL/g) led to the increase in scavenging activity and further increase in the ratio made the activity decrease slightly [Figure 3]a. Higher pH contributed to the increase in the scavenging activity. The scavenging activity decreased when pH was over 5.3 [Figure 3]b, which may inhibit the compound enzyme at higher pH. The scavenging activity was maximum at 50°C, and then, it began to decrease [Figure 3]c, which may be because the activity of the compound enzymes (cellulose, pectinase, and papain) increased with the temperature until 52.7°C and decreased thereafter. Furthermore, the scavenging activity initially increased and then decreased as variables increased [Figure 3]d, [Figure 3]e, [Figure 3]f.
|Figure 3: Response surface showing the effects of extraction variables on the DPPH radical scavenging activity of MOP: (a) Solvent-solid ratio and ultrasonic power; (b) solvent-solid ratio and pH; (c) solvent-solid ratio and extraction temperature; (d) ultrasonic power and pH; (e) ultrasonic power and extraction temperature; (f) pH and extraction temperature|
Click here to view
Determination and experimental validation of the optimal conditions
By transforming the yield (d1) and the DPPH scavenging activity (d2) into the new response (OD), the optimum conditions of UAEE procedure obtained from the model were as follows: solvent-solid ratio of 21.36 mL/g, ultrasonic power of 278.13 W, pH of 5.29, and temperature of 50.29°C. For operational convenience, the optimal values were as follows: 21 mL/g, 280 W, 5.3 pH, and 50°C. As a result, experimental OD (1.03 ± 0.01, n = 3) were in accord with the predicted value of 1.015, which validated the adequacy of the model. The yield and DPPH scavenging activity of MOP were 23.68% ±0.52% (n = 3) and 117.26 ± 2.73 mg VC/100 g (n = 3), respectively.
Characterization of MOP
The total carbohydrate content of MOP was 77.42% ± 0.64%. [Figure 4] shows the FT-IR spectra of MOP. The broadly-stretched intense peak at 3257.20cm−1 was assigned to the O-H stretching vibration and the weak peak at 2930.08cm−1 represented the C-H asymmetric stretching vibration. The absorption peak at 1588.79 cm−1 was ascribed to C=O stretching vibrations. The absorption peaks at 1397.22 and 1273.31 cm−1 can be ascribed to C-H wagging vibrations, peaks at 1124.87 and 1021.43 cm−1 indicated the glucopyranoside unit. The peaks in 1000-800 cm−1 suggested a furan ring. Peaks at 931.83 cm−1, 871.56 cm−1, and 825.09 cm−1 indicated the existence of fructose with β-configuration glycosidic bonds. This was a typical polysaccharide structure. There is growing interest in antioxidant activities of polysaccharides. In general, the compounds have antioxidant activities containing the functional groups of-OH,-C=O,-SH,-COOH, and-O-. In addition, the hydroxyl groups in polysaccharides might provide electrons to reduce the radicals and react with the free radicals to end the radical chain reaction. MOP have been reported to possess antioxidant abilities. These previous reports have contributed to the research on antioxidant activities of polysaccharides.
|Figure 4: The Fourier transform infrared spectrophotometer spectra of Morindae officinalis Radix|
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| Conclusion|| |
UAEE process was optimized for the yield and DPPH scavenging activity of MOP using RSM with central composite design. The optimum proportion of cellulose, pectinase, and papain were 1.0%, 1.5%, and 1.0%, respectively. The optimum extraction conditions including time, solvent-solid ratio, ultrasonic power, pH, and extraction temperature were 60 min, 21 mL/g, 280 W, 5.3, and 50°C, respectively. The yield of MOP and DPPH scavenging activity of MOP was 23.68% and 117.26 mg VC/100 g, respectively. UAEE technology was applicable for MOP extraction and provided theoretical references for the extraction of active constituents from the natural products.
The authors greatly appreciate the financial support from the National Key Research and Development Program of China (2017YFC1701100), the Innovation Team of Modern Agricultural Industrial Technology System of Guangdong Province (2019KJ142).
Financial support and sponsorship
This work was financial supported by the National Key Research and Development Program of China (2017YFC1701100), the Innovation Team of Modern Agricultural Industrial Technology System of Guangdong Province (2019KJ142).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]