Optimize the extraction conditions of maca polysaccharides and study their antioxidant activity and lipid-lowering effects through in vitro experiments. Researchers including Feng Kang, Li Aimin, Wu Xiaolei, Gao Xiaodong, and Li Zijie from the Jiangnan University School of Bioengineering / Key Laboratory of Glycochemistry and Biotechnology of the Ministry of Education optimized the extraction conditions of maca polysaccharides using ultrasonic-assisted hot water extraction combined with response surface methodology. The antioxidant activity of maca polysaccharides was evaluated by the free radical scavenging rate, and the lipid-lowering effect was based on the binding ability of maca polysaccharides with sodium taurocholate. Under the conditions of extraction temperature of 50 °C, extraction time of 42 minutes, ultrasonic power of 220 W, and a material-to-liquid ratio (g/mL) of 1:32, the polysaccharide extraction rate reached a maximum of 11.0%. Three polysaccharide components were separated from crude maca polysaccharides using an ion exchange column, named maca polysaccharides 1, 2, and 3. Among them, crude maca polysaccharides, maca polysaccharides 1, and maca polysaccharides 2 exhibited strong antioxidant activity, with optimal scavenging rates for DPPH free radicals of 83.9%, 81.8%, and 63.7%, respectively, and optimal scavenging rates for hydroxyl radicals of 73.3%, 56.8%, and 76.7%, respectively. In addition, maca polysaccharides 3 showed binding rates of 49.1% and 32.3% for sodium taurocholate and glycocholic acid in vitro, respectively. The study indicates that crude maca polysaccharides, maca polysaccharides 1 and 2 possess good antioxidant activity, while maca polysaccharides 3 has certain lipid-lowering effects.
Maca (Lepidium meyenii Walp.) is a biennial herbaceous plant of the Brassicaceae family, native to the Andes Mountains at altitudes above 3,500 m in South America, with oval leaves and a root resembling a small round radish, also known as Inca radish. Maca is rich in nutrients and has a long cultivation history in Peru, earning it the titles of “Peruvian ginseng” and “South American ginseng.” China successfully introduced maca in 2003, and a certain scale of cultivation industry has been formed in Yunnan and Xinjiang. There are various extraction processes for maca polysaccharides, including hot water extraction, ultrasonic-assisted extraction, and microwave-assisted extraction. Zheng Pengpeng et al. optimized the extraction rate of maca polysaccharides using hot water extraction, achieving an extraction rate of 9.6% at an extraction temperature of 82 °C. Hao Limin et al. optimized hot water extraction of maca polysaccharides, achieving an extraction rate of 15.9%, but at a high extraction temperature of 100 °C. Studies have shown that extraction temperature can affect the structure and biological activity of polysaccharides, and high-temperature extraction can lead to partial degradation, reducing molecular weight and antioxidant activity. Pu Yaowu et al. compared ultrasonic, microwave, and hot water extraction methods, finding that ultrasonic extraction was the most effective, followed by microwave, with hot water extraction being the least effective. Ultrasonic-assisted hot water extraction can reduce extraction temperature and time.
Maca polysaccharides have various effects, including anti-fatigue[5-6], anti-tumor, immune regulation, and endocrine regulation, with very low toxicity, showing good potential for development as functional foods. Currently, research on the activity of maca polysaccharides has mostly focused on anti-fatigue and antioxidant activity, with little research on their lipid-lowering effects. This study optimized the extraction conditions of maca polysaccharides using ultrasonic-assisted hot water extraction, reducing extraction temperature and time; at the same time, this study utilized in vitro experiments to investigate their potential lipid-lowering effects, aiming to provide a basis for the development of maca polysaccharide products.
1 Materials and Methods
1.1 Materials and Reagents
The experimental material used maca roots harvested from the Shangri-La region of Yunnan in 2017, naturally air-dried until the experiment.
Amylase (384 U/mL), glucoamylase (300 U/mL), Novozymes (China) Biotechnology Co., Ltd.; chitosan, Shanghai Maiklin Biochemical Technology Co., Ltd.; anhydrous ethanol, chloroform, n-butanol were analytical grade, phenol was reagent grade, China National Pharmaceutical Group Chemical Reagents Co., Ltd.; 1,1-diphenyl-2-trinitrophenylhydrazine [2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl, DPPH], mass fraction of 97%, TCI (Shanghai) Chemical Industrial Development Co., Ltd.; o-phenylenediamine (mass fraction of 99%), sodium taurocholate (mass fraction of 97%), glycocholic acid (mass fraction of 98%), furfural solution (mass fraction 99%), trypsin (250 U/mg), Adamas Reagent Co., Ltd.
1.2 Instruments and Equipment
SB25-12DTDN ultrasonic cleaning machine, Ningbo Xinzhizheng Biotechnology Co., Ltd.; HL-2 constant flow peristaltic pump, Shanghai Huxi Analysis Instrument Factory Co., Ltd.; DEAE-650M ion exchange column, HW-65F dextran gel column, Tosoh Corporation, Japan; BIO-RAD iMark enzyme marker, Bio-Rad Laboratories, USA; FD-1000 freeze dryer, N-1100 rotary evaporator, Tokyo Rika Kikai Co., Ltd.
1.3 Experimental Methods
1.3.1 Extraction of Maca Polysaccharides
1.3.1.1 Extraction Method
Cut the maca roots into pieces, crush them into powder, and sieve through a 100-mesh screen to obtain maca powder. Mix 50 g of maca powder with distilled water in a certain ratio, and perform ultrasonic-assisted hot water extraction, filter to obtain maca water extract, and concentrate under reduced pressure to 250 mL; add 2 mL of amylase to the maca water extract, react at 60 °C for 3 hours, then add 2 mL of glucoamylase, react at 60 °C for 2 hours, heat at 100 °C for 10 minutes to inactivate the enzymes, and centrifuge to collect the supernatant; add three times the volume of anhydrous ethanol to the supernatant, and perform alcohol precipitation at a final concentration of 75% ethanol solution at 4 °C for 12 hours, then redissolve the precipitate with distilled water; use the Sevag method to repeatedly operate on the redissolved solution to remove proteins; freeze-dry the water extract solution that has had starch and protein removed to obtain crude maca polysaccharides (MCP).
1.3.1.2 Determination of Extraction Rate
First, determine the total sugar content (expressed as glucose) using the phenol-sulfuric acid method, and then calculate the extraction rate of maca polysaccharides using the conversion factor.
Standard curve preparation: Accurately weigh 20 mg of glucose into a 100 mL volumetric flask and dilute to volume with distilled H2O. Take 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mL of glucose stock solution into test tubes, add water to make up to 2 mL, then sequentially add 1 mL of 6% phenol aqueous solution and 5 mL of concentrated sulfuric acid, shake for 10 minutes, let stand for 10 minutes to develop color, and measure the absorbance at 490 nm. Plot the standard curve with glucose mass concentration ρglucose (mg/mL) on the x-axis and absorbance A on the y-axis.
The conversion factor F calculation can be seen in formula (1).
(1)
In formula (1), m1 is the mass of maca polysaccharides, mg; ρm is the total sugar mass concentration of maca polysaccharides (expressed as glucose), mg/mL; D is the dilution factor.
The calculation of the extraction rate of maca polysaccharides can be seen in formula (2).
(2)
In formula (2), w is the extraction rate of maca polysaccharides; m2 is the mass of maca dry powder, mg; ρm is the total sugar mass concentration of maca polysaccharides (expressed as glucose), mg/mL; D is the dilution factor; F is the conversion factor.
1.3.1.3 Response Surface Experimental Design
Based on the principles of Box-Behnken central composite experimental design, a three-factor three-level response surface analysis method was used to optimize the extraction process of maca root polysaccharides. Based on preliminary single-factor experiments, the experimental levels of independent variables were coded as -1, 0, and 1 (see Table 1), and a total of 29 experimental points were designed, with the central experiments repeated 5 times to estimate experimental error.
Table 1 Response Surface Experimental Design Factors and Levels for Maca Polysaccharides Extraction Conditions
1.3.2 Separation of Maca Polysaccharides Components
Take 200 mg of crude maca polysaccharides, dissolve in 2 mL of distilled H2O, and separate through an anion exchange column DEAE-650M at an elution rate of 1 mL/min. First, elute with distilled H2O to collect all neutral polysaccharides, and then elute with a sodium chloride aqueous solution gradient (0.1, 0.2, 0.4, 0.6 mol/L). The polysaccharide content in each tube was determined using the phenol-sulfuric acid method, and the tubes containing polysaccharides were collected, concentrated by rotary evaporation at 50 °C, and freeze-dried to obtain samples. The freeze-dried samples were further purified using a dextran gel column (HW-65F) to achieve desalting and purification, then the samples containing polysaccharides were concentrated by rotary evaporation and freeze-dried to obtain purified polysaccharide samples.
1.3.3 In Vitro Antioxidant Activity Experiment
Using vitamin C (Vc) as a positive control and glucose (Glc) as a negative control for comparison of antioxidant activity.
1.3.3.1 Determination of DPPH Free Radical Scavenging Activity
Mix 0.5 mL of samples with different mass concentrations (1, 2, 4, 6, 8, 10 mg/mL) with 0.5 mL of DPPH (0.04 mg/mL, dissolved in anhydrous methanol), shake thoroughly and let stand at room temperature for 40 minutes, and measure the absorbance at 517 nm.
DPPH Scavenging Rate
(3)
In formula (3), A1 represents the absorbance with anhydrous methanol replacing the sample, A2 represents the absorbance with anhydrous methanol replacing the DPPH solution, and A3 represents the absorbance after the reaction of the DPPH solution and the sample.
1.3.3.2 Determination of Hydroxyl Radical Scavenging Activity
Mix 0.5 mL of sample aqueous solution at different mass concentrations (1, 2, 4, 6, 8, 10 mg/mL) with 0.5 mL of ferrous sulfate solution (6 mmol/L) and 1 mL of H2O2 (6 mmol/L), let stand for 10 minutes, then add 0.5 mL of salicylic acid solution (6 mmol/L), mix, let stand for 30 minutes, and measure the absorbance at 510 nm.
Hydroxyl Radical Scavenging Rate
(4)
In formula (4), A4 represents the absorbance without adding the sample; A5 represents the absorbance after the reaction with the sample.
1.3.4 In Vitro Lipid-Lowering Activity Experiment
The in vitro lipid-lowering activity determination of maca polysaccharide components was modified based on reported methods[12-13]. Take 1 mL of standard solutions of sodium taurocholate and glycocholic acid at different concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mmol/L) into stoppered test tubes, add 1 mL of 0.25% freshly prepared furfural solution, mix, ice bath for 5 minutes, then add 5 mL of 70% H2SO4 aqueous solution and incubate at 70 °C for 10 minutes, take out and ice bath for 2 minutes, measure the absorbance at 510 nm. Plot the standard curve with bile salt concentration on the x-axis and absorbance on the y-axis, and calculate the bile salt concentration in the sample based on the standard curve. The experimental process for bile salt binding is shown in Figure 1. Add 20 mg of polysaccharide powder into a stoppered test tube, add 1 mL of 0.01 mol/L HCl solution, mix thoroughly, and incubate at 37 °C for 1 hour to simulate gastric digestion, then adjust the pH to 6.3 using 0.1 mol/L NaOH solution, and add 2 mL of 10 mg/mL trypsin, mix and incubate at 37 °C for 1 hour to simulate intestinal digestion. Mix with 2 mL of 0.5 mmol/L glycocholic acid and sodium taurocholate solutions, incubate at 37 °C for 1 hour, and centrifuge at 4,000 rpm for 30 minutes using a 3 kDa ultrafiltration tube. If the polysaccharides bind with bile salts, the permeate will contain unbound bile salts, and the bile salt concentration in the permeate can be measured to determine the binding rate of the polysaccharides with bile salts. Take 1 mL of permeate into a stoppered test tube, add 1 mL of 0.25% freshly prepared furfural solution, mix, ice bath for 5 minutes, then add 5 mL of 70% H2SO4 aqueous solution and incubate at 70 °C for 10 minutes, take out and ice bath for 2 minutes, measure the absorbance of the permeate. Use glucose as a negative control and chitosan as a positive control, with the binding rate of chitosan as 100%, to calculate the binding rates of various polysaccharides to sodium taurocholate.
Figure 1 Experimental Process for Maca Polysaccharide Bile Salt Binding
1.4 Statistical Analysis
Response surface experimental design and analysis were conducted using Design-Expert 10 software. The remaining experimental data were expressed as “mean ± standard deviation,” and data processing and significance analysis were performed using Prism 8.0 software.
2 Results and Analysis
2.1 Optimization of Extraction Conditions for Maca Polysaccharides
2.1.1 Response Surface Experimental Results
Zhang et al. found that when studying the effects of different temperatures on the structure and function of mulberry polysaccharides, the molecular weight of polysaccharides extracted at 30 °C was 536.45 kDa, while at 90 °C, the molecular weight was only 110.23 kDa, and polysaccharides extracted at low temperatures showed better antioxidant activity than those extracted at high temperatures. Xiang Haote et al. studied the effects of temperature and time on the extraction efficiency of maca polysaccharides and found that above 70 °C, the extraction rate continuously decreased with increasing temperature and time. Therefore, this study chose to extract polysaccharides using ultrasonic-assisted hot water extraction and optimized the extraction rate using response surface methodology. The experimental scheme was designed using Design-Expert 10 software, as shown in Table 2. According to this experimental scheme, maca polysaccharides were extracted following the method in section 1.3.1.1, and the polysaccharide content was determined using the phenol-sulfuric acid method, with the extraction rates shown in Table 2.
Table 2 Response Surface Experimental Results for Maca Polysaccharides Extraction Conditions
2.1.2 Response Surface Model Fitting Analysis
The model’s lack of fit value P=0.260 6>0.05, and the CV value is 2.93%, indicating that only 2.93% of the total variation cannot be explained by the model, demonstrating good model repeatability; the determination coefficient R2=0.981 9 and the adjusted determination coefficient are close, indicating a good degree of model fitting.
2.1.3 Analysis of Interaction Effects of Factors
The contour plots and response surfaces of the interaction effects of factors on the extraction rate of maca polysaccharides are shown in Figure 2. From the contour plots, the strength of the interaction between two factors can be observed; the more circular the contour, the weaker the interaction, while the more elliptical the contour, the stronger the interaction.[16] The response surface curves show the degree of influence of each factor on the response value (i.e., the extraction rate of maca polysaccharides); the steeper the surface, the greater the degree of influence, and the flatter the surface, the lesser the degree of influence.[17] Among the interactions of the four factors, three representative results were selected. Comparing Figures 2(a) and 2(b), it can be found that the interaction between temperature and time is stronger than that between temperature and ultrasonic power. From Figure 2(c), it can be observed that the effect of the material-to-liquid ratio on the extraction rate of maca polysaccharides is greater than that of time.
Figure 2 Effects of Interaction of Factors on Extraction Rate of Maca Polysaccharides
Through the analysis using Design-Expert 10 software, the optimal extraction conditions were determined to be an extraction temperature of 50.23 °C, ultrasonic power of 217.74 W, extraction time of 42.35 minutes, and a material-to-liquid ratio of 1:32.34 g/mL, with a theoretical extraction rate of 11.86%. Considering the convenience of actual extraction, the values of each factor were adjusted to an extraction temperature of 50 °C, ultrasonic power of 220 W, extraction time of 42 minutes, and a material-to-liquid ratio of 1:32 g/mL, resulting in an actual extraction rate of 11.0%, which is close to the theoretical predicted value.
2.2 Results of Separation of Maca Polysaccharides Components
The DEAE-650M anion exchange column was used to separate the components of maca polysaccharides. The concentration of polysaccharides in each tube was measured using the phenol-sulfuric acid method, as shown in Figure 3. The fixed elution rate was 1 mL/min, first eluting with distilled H2O to obtain the highest content of neutral maca polysaccharides 1 (MPS1), and then eluting with a sodium chloride concentration gradient to separate two components with lower content, named maca polysaccharides 2 (MPS2) and maca polysaccharides 3 (MPS3). Afterward, MPS1, MPS2, and MPS3 were further purified to remove NaCl. For example, the tubes containing MPS1 were combined and freeze-dried, re-dissolved in 1 mL of distilled H2O, and further purified using the dextran gel column HW-65F, collecting and freeze-drying the purified MPS1 again to obtain the final MPS1 polysaccharide sample.
Figure 3 Separation of Crude Maca Polysaccharides Components
2.3 Analysis of In Vitro Antioxidant Activity of Maca Polysaccharides
2.3.1 DPPH Free Radical Scavenging Effect Analysis
The DPPH free radical scavenging rates of maca polysaccharides are shown in Figure 4. It can be seen from Figure 4 that as the concentration of polysaccharides increases, all three polysaccharides showed an upward trend in DPPH free radical scavenging effect. Among them, MPS1 exhibited the best DPPH free radical scavenging effect, reaching an optimal scavenging rate of 81.8% at a concentration of 5 mg/mL. MPS2 showed a significant increase in scavenging effect after exceeding 5 mg/mL, reaching a scavenging effect of 63.7% at a concentration of 10 mg/mL. In addition, crude polysaccharides MCP showed a scavenging rate similar to MPS1 at concentrations above 3 mg/mL, reaching an optimal scavenging rate of 83.9% at 5 mg/mL. The half-inhibitory concentrations (IC50) of MCP and MPS1 were 2.411 mg/mL and 2.339 mg/mL, respectively. MPS1 accounts for the majority of MCP, indicating that MPS1 is the main component responsible for DPPH free radical scavenging. Vitamin C exhibited a rapid increase in DPPH free radical scavenging effect with concentrations ranging from 0.001 to 0.005 mg/mL, increasing from 35.8% to 81.6%, and reaching an optimal scavenging rate of 99% at a concentration of 1 mg/mL. The scavenging effects of MCP, MPS1, and MPS2 were all lower than that of vitamin C.
Figure 4 DPPH Free Radical Scavenging Effect of Maca Polysaccharides
2.3.2 Analysis of Hydroxyl Radical Scavenging Effect
The hydroxyl radical scavenging rates of maca polysaccharides are shown in Figure 5. It can be seen from Figure 5 that MCP, MPS1, and MPS2 all exhibited good scavenging effects on hydroxyl radicals, and the scavenging rates increased with the increase in polysaccharide concentration. Among them, MCP reached a scavenging rate of 73.3% at a concentration of 10 mg/mL, with a half-inhibitory concentration (IC50) of 2.388 mg/mL. MPS2 reached a scavenging rate of 76.7% at 10 mg/mL, while the scavenging effect of MPS1 changed little with increasing concentration, reaching 39.8% at 1 mg/mL, which is higher than that of MCP and MPS2, and 56.8% at 10 mg/mL. MPS3 showed no scavenging effect on hydroxyl radicals, with scavenging rates similar to the negative control group at all concentrations.
Figure 5 Hydroxyl Radical Scavenging Effect of Maca Polysaccharides
2.4 Analysis of In Vitro Lipid-Lowering Activity of Maca Polysaccharides
Cholesterol in the liver is degraded into bile acids and secreted into the intestines, of which 95% of bile acids are reabsorbed and returned to the liver through the portal vein, forming bound bile acids that are secreted back into the intestines. This process is known as enterohepatic circulation.[18] Bile acids play a crucial role in maintaining the balance of cholesterol in the human body. Once the enterohepatic circulation of bile acids is obstructed, the liver compensates for the deficiency of bile acids by degrading more cholesterol, leading to a decrease in cholesterol in the blood.[19] Both glycocholic acid and sodium taurocholate are primary bile acids that are typically combined with sodium or potassium ions to form bound primary bile salts, which is the main form of bile acids. The primary mechanism of polysaccharides in lipid-lowering is to form a network structure in solution, which can adsorb bile acids in the small intestine and prevent their reabsorption.
The binding rates of maca polysaccharides to two bile salts are shown in Figure 6. Compared to the negative group, all four polysaccharides showed significant binding to sodium taurocholate, but only MCP and MPS3 showed significant differences in binding to glycocholic acid (P<0.05). Using chitosan as a positive control, MPS3 exhibited the best binding ability to both bile salts in vitro, with a binding rate of 49.1% for sodium taurocholate and 32.3% for glycocholic acid. The inconsistent binding abilities of the two bile salts may be related to their molecular structures; sodium taurocholate has a larger molecular weight than glycocholic acid, making it easier for the network structure formed by MPS to encapsulate and bind. Additionally, comparing the four polysaccharides, it can be inferred that the network structure formed by MPS3 may be denser.
Figure 6 Binding Ability of Maca Polysaccharides to Bile Salts
3 Conclusion
This study employed ultrasonic-assisted hot water extraction and, after response surface optimization, obtained the optimal extraction conditions of an extraction temperature of 50 °C, ultrasonic power of 220 W, extraction time of 42 minutes, and a material-to-liquid ratio of 1:32 g/mL, resulting in an actual extraction rate of 11.0%. MCP, MPS1, and MPS2 exhibited strong antioxidant activity, with optimal scavenging rates for DPPH free radicals of 83.9%, 81.8%, and 63.7%, respectively, and optimal scavenging rates for hydroxyl radicals of 73.3%, 56.8%, and 76.7%; although MPS3 had no antioxidant activity, it demonstrated binding rates of 49.1% and 32.3% for sodium taurocholate and glycocholic acid in vitro, suggesting that maca polysaccharides may reduce lipid levels by adsorbing and binding bile salts, thereby decreasing enterohepatic circulation.
References: (omitted)
Citation format: Feng Kang, Li Aimin, Wu Xiaolei, et al. Study on optimization of maca polysaccharides extraction and in vitro antioxidant and hypolipidemic activities[J]. Journal of Food Science and Technology, 2021, 39(2): 48-55.
Funding project: National Natural Science Foundation of China (21778023).
Editor: Zhang Yiqun, Li Ning
Review: Ye Hongbo