Extraction of Total Saponins from Sixiangteng of Yao Medicine Based on Response Surface Methodology and Its Application

Abstract

The application discloses a kind of extraction method of Yao medicine total saponins of Hexangteng based on response surface method optimization and application thereof, the application utilizes response surface method to optimize the extraction process of total saponins of Hexangteng, obtains a kind of method of Yao medicine total saponins of Hexangteng, the extraction method is as follows: the medicinal material of Hexangteng is pulverized, and Hexangteng powder is obtained, then ethanol is added to ultrasonic extraction, and the extraction process conditions are as follows: extraction temperature is 65-70 DEG C, the volume concentration of ethanol is 70-80%, extraction time is 120-150 min, and the ratio of solid to liquid is 1g:25-30 mL.The extraction method process is stable, simple to operate, good repeatability, feasible, higher extraction efficiency, low cost, high yield of total saponins of Hexangteng.The application also determines the antioxidant activity of the obtained total saponins of Hexangteng, and the results show that total saponins of Hexangteng has certain inhibition and clearance effect on DPPH free radical, and has certain reducing power, which provides a theoretical basis for further development and utilization of Hexangteng.

Description

Technical Field

[0001] This invention belongs to the field of total saponin extraction technology, specifically relating to a method for extracting total saponins from the Yao herb *Liufangteng* based on response surface methodology optimization and its application. Background Technology

[0002] Hexagonal Vine ( Cissus hexangularis Thorel ex Planch. is a vine belonging to the genus Thorel in the Vitaceae family. Also known as square-stemmed broad-stemmed vine, winged-stemmed white powder vine, and hexagonal vine, it is distributed in Guangxi, Fujian, Guangdong, and Hainan provinces. The vine is used medicinally, either dried or fresh. It has a pungent and slightly bitter taste, is cool in nature, and enters the liver and kidney meridians. It has the functions of dispelling wind, activating collaterals, and promoting blood circulation. It is mainly used to treat rheumatic joint pain, lumbar muscle strain, and traumatic injuries. It is a commonly used medicinal material among the Yao ethnic group. The hexagonal vine is the hexagonal vine in the Yao medicine "Tiger and Ox Drill Wind." It has been included in the 2014 edition of the "Quality Standards for Yao Medicinal Materials of Guangxi Zhuang Autonomous Region (Volume 1)" under the Yao medicine name "Luobangzhun." Yao medicine often uses it for uterine obstruction (rheumatic joint pain), lower back pain (lumbar muscle strain), traumatic injuries, and boils and carbuncles.

[0003] The chemical composition of *Hexagrammus* is complex, containing flavonoids, saponins, sugars, phenols, anthraquinones, coumarins, cardiac glycosides, sterols, alkaloids, and volatile oils. Modern pharmacological studies have shown that *Hexagrammus* species possess various potential biological activities, including antibacterial, anti-inflammatory, antioxidant, anti-allergic, antitumor, endothelin and snake venom antagonism, and diabetes treatment. However, recent research on *Hexagrammus* has mainly focused on its chemical components, while research on its extraction processes and antioxidant activity is almost nonexistent. Saponins are an important component of the natural product chemical library, possessing various biological activities such as antitumor, anti-inflammatory, immunomodulatory, antiviral, antifungal, and hepatoprotective activities. Many traditional Chinese medicines, such as ginseng, polygala, platycodon, licorice, anemarrhena, and bupleurum, contain saponins as their main active ingredients. Numerous studies have shown that saponins have good efficacy in expectoration, antitussive, sedative, antipyretic, antibacterial, anticancer, metabolic and immune regulation, and treatment of cardiovascular diseases and diabetes. However, there are currently almost no research reports on the extraction process of total saponins from the Yao medicine Liufangteng, and there are also no reports on the optimization of the extraction process and antioxidant activity of total saponins from Yao medicine Liufangteng using response surface methodology. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting total saponins from the Yao medicinal herb *Ligusticum striatum* based on response surface methodology (RSM) optimization, and its application. This invention utilizes RSM to optimize the extraction process of total saponins from *Ligusticum striatum*, resulting in a stable, simple, reproducible, and feasible extraction method with a high yield of total saponins. Furthermore, this invention uses in vitro antioxidant experiments to measure the DPPH free radical scavenging rate of total saponins from *Ligusticum striatum*, demonstrating that the extracted total saponins possess good antioxidant activity, providing a theoretical basis for the further development and utilization of *Ligusticum striatum*.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] An extraction method for total saponins from the Yao herb *Ligusticum striatum* (also known as *Ligusticum striatum*) optimized using response surface methodology is disclosed. Based on the Box-Behnken experimental design principle, Design-Expert software is used to design and optimize the extraction temperature, ethanol concentration, and extraction time. The extraction method involves pulverizing *Ligusticum striatum* to obtain powder, then adding ethanol for ultrasonic extraction. The extraction conditions are: extraction temperature of 65℃~70℃, ethanol volume concentration of 70%~80%, extraction time of 120min~150min, and a solid-liquid ratio of 1g:25-30mL.

[0007] Preferably, the extraction method of total saponins from *Ligusticum striatum* based on response surface methodology of the present invention, according to the Box-Behnken experimental design principle, uses Design-Expert software to design and optimize the extraction temperature, ethanol concentration and extraction time of total saponins from *Ligusticum striatum*. The optimal extraction conditions for extracting total saponins from *Ligusticum striatum* are: extraction temperature 70℃, ethanol volume concentration 74%, and extraction time 150.00 min.

[0008] Furthermore, the specific steps of the response surface methodology include:

[0009] ① Single-factor experiment: The medicinal material of *Hylocereus undatus* was pulverized to obtain *Hylocereus undatus* powder. 1.00g of *Hylocereus undatus* powder was accurately weighed into a 250ml conical flask, and 25ml of 70% ethanol was added. The extraction was carried out using three methods: ultrasonic extraction, reflux extraction, and static extraction, for 1h respectively. After centrifugation, the effects of different extraction methods on the extraction rate of total saponins in *Hylocereus undatus* were studied, and the optimal extraction method was determined to be ultrasonic extraction. Several groups of single-factor experiments were designed with extraction temperature, ethanol concentration, and extraction time as the three single factors.

[0010] ② Response surface methodology optimization:

[0011] Using the extraction rate of total saponins from *Symplocos rubra* as the response factor, a response surface methodology was developed based on the single-factor experiment in step ① using the Box-Behnken principle in Design-Expert software. A quadratic polynomial equation model was established between the extraction rate response value Y and the three factors: extraction temperature A, ethanol concentration B, and extraction time C. The model is: Y = +24.24 - 0.0232 A - 0.87 B + 0.85 C + 0.68 AB + 0.39 AC - 0.12 BC - 1.11 A^2 -1.58 B^2 - 0.26 C^2;

[0012] ③ By solving the regression equation using Design-Expert software, the optimal extraction conditions for extracting total saponins from the Yao herb Liufangteng were obtained: extraction temperature 70℃, ethanol volume concentration 74%, and extraction time 150.00 min.

[0013] Preferably, the extraction method for total saponins of *Ligusticum striatum* based on response surface methodology optimizes the extraction process conditions, including extraction temperature, ethanol concentration, and extraction time, using Design-Expert software based on the Box-Behnken experimental design principle. The extraction method is as follows: *Ligusticum striatum* is pulverized to obtain *Ligusticum striatum* powder, and then ethanol is added for ultrasonic extraction. The extraction process conditions are: extraction temperature of 70℃, ethanol volume concentration of 80%, extraction time of 120 min, and material-to-liquid ratio of 1 g: 25 mL.

[0014] Preferably, in the above-mentioned method for extracting total saponins from Yao medicine *Liufangteng* based on response surface methodology optimization, the ultrasonic extraction frequency is 50 kHz.

[0015] Furthermore, another objective of this invention is to provide the application of the total saponins from *Ligusticum striatum* extracted using the response surface methodology-optimized extraction method in the preparation of antioxidant drugs and food antioxidants. These antioxidant drugs and food antioxidants exhibit a certain inhibitory and scavenging effect on DPPH free radicals and also possess a certain reducing ability.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention optimizes the extraction process of total saponins from *Symplocos rubra* using response surface methodology. Using the total saponin extraction rate as the indicator, a combination of single-factor experiments and Box-Benhken response surface methodology was employed to investigate three factors: extraction temperature, ethanol concentration, and extraction time. This yielded a method for extracting total saponins from *Symplocos rubra*, overcoming the limitations of insufficient precision in orthogonal experiments and ensuring the scientific validity and stability of the extraction process. The extraction method for total saponins from *Symplocos rubra* optimized by this invention is stable, simple to operate, has good repeatability, is feasible, has high extraction efficiency, low cost, and a high yield of total saponins from *Symplocos rubra*.

[0018] This invention also conducted in vitro antioxidant activity (DPPH free radical scavenging and reducing ability) experiments on the extracted total saponins from *Symplocos rubra*. The experimental results showed that the total saponins from *Symplocos rubra* have a certain inhibitory and scavenging effect on DPPH free radicals and also possess a certain reducing ability. The extraction method for total saponins from *Symplocos rubra* optimized by response surface methodology provided by this invention, and its application, provide a theoretical basis for the industrial extraction of total saponins from *Symplocos rubra* and the further development and utilization of *Symplocos rubra*. Attached Figure Description

[0019] Figure 1 Standard curve of total saponins from *Symplocos rubrum*;

[0020] Figure 2 The effect of different extraction temperatures on extraction rate;

[0021] Figure 3 The effect of different ethanol concentrations on extraction rate;

[0022] Figure 4 The effect of different extraction times on extraction rate;

[0023] Figure 5 Response surface analysis and contour plots of saponin yield to temperature and ethanol concentration;

[0024] Figure 6 Response surface analysis and contour plots of temperature and time on saponin yield;

[0025] Figure 7 Response surface analysis and contour plot of saponin yield with respect to time and ethanol concentration;

[0026] Figure 8 DPPH free radical scavenging ability;

[0027] Figure 9 The reducing power of different solutions. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0029] Example 1

[0030] 1. Materials and Methods

[0031] 1.1 Materials and Instruments

[0032] The samples of *Hylocereus undatus* were collected from the wild in various parts of Guangxi and Guangdong, and identified by Professor Wei Songji of Guangxi University of Traditional Chinese Medicine as *Hylocereus undatus*, a plant belonging to the genus *Hylocereus* of the family Vitaceae. Cissus hexangularis The vine stems of *Thorel ex Planch*, with the origin detailed in Table 1; ginsenoside Rg1 (standard) (Shanghai Anpu Experimental Technology Co., Ltd., batch number: T7740050), vitamin C (China National Institutes for Food and Drug Control, batch number: 100425-201504), and 2,2-biphenyl-1-picrylhydrazyl ≥98% (DPPH) (Shanghai Maclean, batch number: C12169642); perchloric acid, glacial acetic acid, vanillin, anhydrous ethanol, methanol, ferric chloride hexahydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride, potassium hexacyanoferrate, trichloroacetic acid, and sodium dihydrogen phosphate used in the experiment were all analytical grade, and distilled water was used.

[0033] UV-2550 UV-Vis spectrophotometer (Shimadzu Corporation, Japan); KQ-500GDV thermostatic CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); KS260-CS25 controlled oscillator (IKA GmbH, Germany); CX-A560 high-speed multi-functional grinder (Shanghai Yuanwo Co., Ltd.); 541-10000-00-1 vortex oscillator (Heidolph Instruments GmbH & Cn. KG); XS-205DU 0.1mg~220g electronic balance (Mettler-Toledo GmbH, Germany); 17605291 500-5000μl pipette (Sartorius Biohit); Millipore Simplicity-UV ultrapure water system (Mettler Corporation, USA); AE-200 electronic analytical balance (Mettler-Toledo GmbH, Germany); Finnpipette F3 (NZ44644) 20-200μl pipette (Thermo Scientific); ROTANTA-460 centrifuge (Hettich, Germany); 17601410 100-1000μl pipette (Sartorius Biohit); HH-S6 digital display constant temperature water bath (Jintan Medical Instrument Factory).

[0034]

[0035] 1.2 Experimental Methods

[0036] 1.2.1 Extraction of total saponins from *Symplocos rubra*

[0037] Place 1.00g of Hexagonal Vine Powder in a 250ml Erlenmeyer flask, add 25ml of 70% ethanol, extract by sonication, combine the extracts, concentrate, and then make up to 100mL in a volumetric flask. Take 50ml and put it into a centrifuge tube, centrifuge for 5min, discard the residue, take 2ml of the supernatant, and make up to 5ml with methanol for later use.

[0038] 1.2.2 Determination of total saponins from *Symplocos rubra*

[0039] Accurately weigh 7.18 mg of ginsenoside Rg1 standard into a 50 ml volumetric flask, dissolve the weighed ginsenoside Rg1 in methanol, dilute to volume, and shake well to obtain the concentration (0.1436 mg / ml). Take 0.1 ml, 0.3 ml, 0.5 ml, 0.7 ml, 0.9 ml, 1.1 ml, and 1.3 ml of the reference solution into 15 ml centrifuge tubes, place them in an N-EVAP116 nitrogen blower and dry at 40 °C. Add 0.2 ml of 5% vanillin-glacial acetic acid solution and 0.8 ml of perchloric acid, shake at 70 °C for 15 min, incubate on ice for 3 min, add 5 ml of glacial acetic acid, shake for 30 s, let stand for 15 min, and use the corresponding reagent as a blank to measure the absorbance at 545 nm. Plot a standard curve with absorbance as the ordinate and concentration as the abscissa. Place the extract of *Hexanthus chinensis* in a 25 mL volumetric flask, measure the absorbance according to the above method, and calculate the extraction rate. Formula (1) is as follows:

[0040]

[0041] C represents the total saponin content in the diluted sample solution calculated based on the standard curve, in mg / ml; D represents the dilution factor of the test solution, dimensionless; V represents the volume of the test solution, in ml; and M represents the mass of the *Hexagramambusa* powder, in g.

[0042] 1.2.3 Single-factor experiment

[0043] 1.2.3.1 Examination of Extraction Methods

[0044] Accurately weigh 1.00 g of *Hylocereus undatus* powder into a 250 ml Erlenmeyer flask, add 25 ml of 70% ethanol, and extract for 1 h using three methods: ultrasonic extraction, reflux extraction, and static extraction. Centrifuge and measure the absorbance at 545 nm according to the method in section "1.2.3". Calculate the total saponin content in *Hylocereus undatus*. The extraction rates of saponins from ultrasonic extraction and reflux extraction are not significantly different, but ultrasonic extraction is simpler to operate; therefore, ultrasonic extraction is the optimal method.

[0045] 1.2.3.2 Single-factor experiment on extraction of total saponins from *Symplocos rubra*

[0046] Six levels were set up for three factors: extraction temperature, ethanol concentration, and extraction time, to investigate the effect of each single factor on the extraction rate of total saponins from *Symplocos rubrum*, in order to determine the range of process parameters for the response surface methodology. Specifically, the extraction temperatures were 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃; the ethanol concentrations were 0%, 20%, 40%, 60%, 80%, and 100%; and the extraction times were 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min. Each group of experiments was repeated three times, and the mean value was taken.

[0047] 1.2.4 Response Surface Design

[0048] Based on the results of single-factor experiments, a Box-Behnken experimental design was used to investigate the effects of three factors on the extraction rate of total saponins from *Symplocos rubra*: extraction temperature (A), ethanol concentration (B), and extraction time (C). Specific factor codes and levels are shown in Table 2.

[0049]

[0050] 1.2.5 Determination of the antioxidant activity of total saponins from *Symplocos rubra*

[0051] 1.2.5.1 Determination of DPPH free radical scavenging ability

[0052] Take 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, and 0.05 mg / mL of sample solution, mix 5.0 mL of sample solution with 5.0 mL of DPPH solution, shake well, and let stand for 30 min. Measure the absorbance at 517 nm (A1). Zero the sample with anhydrous ethanol. The control group is the same volume of anhydrous ethanol, and the absorbance at 517 nm is measured under the same conditions (A0). The blank group is the same volume of anhydrous ethanol DPPH solution, and the absorbance at 517 nm is measured using the same method (A2). Perform the above operation with the same concentration of VC solution as a positive control. Calculate the clearance rate using the following formula (2):

[0053]

[0054] 1.2.5.1 Determination of reducing power

[0055] Take 1 mL of each sample solution with different concentrations (0.01, 0.03, 0.05, 0.1, 0.2 mg / mL), add 2.5 mL of 0.2 mol / L phosphate buffer (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide solution, mix well, and react in a 50℃ water bath for 30 min. After removing from the water bath, add 2.5 mL of 10% trichloroacetic acid, mix well, centrifuge at 5000 r / min for 10 min, take 2.5 mL of the supernatant, add 1.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride aqueous solution, mix, and react at room temperature for 10 min. Measure the absorbance at 700 nm. Use a blank reagent as a negative control and VC solution as a positive control. Measure each sample three times and take the average value. The higher the absorbance value, the stronger the total reducing power of the sample.

[0056] 2 Results and Discussion

[0057] 2.1 Standard curve of total saponins

[0058] Plot the standard curve as described in 1.2.2: Y = 29.49265X - 0.06827, linear correlation R0 2 =0.99964. (For example...) Figure 1 As shown.

[0059] 2.2 Single-factor experiment

[0060] 2.2.1 Effect of extraction temperature

[0061] The effect of extraction temperature on extraction rate is shown in the figure. Figure 2 When the extraction temperature is between 30℃ and 60℃, the extraction rate of total saponins increases with increasing extraction temperature. This is because the increase in extraction temperature promotes the dissolution of total saponin molecules, thus increasing the extraction rate. However, the yield decreases when the extraction temperature exceeds 70℃. Considering that the solvent will evaporate and be lost at higher temperatures, affecting the measurement results, and that excessively high temperatures may cause oxidation and degradation of saponins, resulting in the loss of effective components, the optimal extraction temperature is 70℃.

[0062] 2.2.2 Effect of ethanol concentration

[0063] The effect of ethanol concentration on extraction rate is shown in the figure. Figure 3 .Depend on Figure 3 It can be seen that the yield of total saponins increases with increasing ethanol concentration, but decreases after exceeding 80%; therefore, 80% ethanol concentration is considered the optimal concentration. This is because when the polarity of ethanol is close to that of the total saponins in *Hylocereus undatus*, it is beneficial for the dissolution of total saponins, thus increasing the yield. However, when the ethanol concentration is too high, the polarity of the solvent is too low, which actually reduces the extraction rate of total saponins. Therefore, 80% ethanol concentration was selected as the optimal ethanol concentration, serving as the central point for the response surface optimization experiment.

[0064] 2.2.3 Effect of extraction time

[0065] The effect of extraction time on extraction rate is shown in the figure. Figure 4 The yield of total saponins increases with increasing extraction time. This is because when the extraction time is too long, some of the total saponins are degraded due to oxidation, resulting in a decrease in the total saponin extraction rate. The yield decreases after 120 min, and the yield increase at 180 min is less than the yield at 120 min; therefore, 120 min is the optimal extraction time.

[0066] 2.3 Box-Behnken experimental design and analysis of variance

[0067] 2.3.1 Response Surface Experiment

[0068] Based on the Box-Behnken experimental design, a total of 17 experimental sites were designed, consisting of 12 factorial experiments and 5 central experiments. The experimental results are shown in Table 3, and the results of the analysis of variance are shown in Table 4.

[0069]

[0070]

[0071] 2.3.2 Establishment and Analysis of Response Surface Model

[0072] Response surface methodology was performed on the Box-Behnken experiment results in Table 3, yielding a quadratic polynomial regression model for the extraction response value (Y) of total saponins from *Symplocos rubra* in relation to three factors: extraction temperature (A), ethanol concentration (B), and extraction time (C): Y = + 24.24 - 0.0232 A - 0.87 B + 0.85 C + 0.68 AB + 0.39 AC - 0.12 BC -1.11 A^2 - 1.58 B^2 - 0.26 C^2. In the design, A represents extraction temperature, B represents ethanol concentration, and C represents extraction time. The magnitude and sign of the coefficients in the equation indicate the degree and direction of influence of each factor on the index value (yield). The quadratic term of ethanol concentration (B^2) has the greatest impact on the yield of total saponins from *Symplocos rubra*. The quadratic term of extraction temperature (A^2), ethanol concentration (B), and extraction time (C) have a significant impact on the yield of total saponins from *Symplocos rubra*.

[0073] Analysis of variance was performed on the mathematical model for the extraction of total saponins from *Symplocos rubrum*. Table 4 shows that: P = 0.0001 < 0.001, indicating that the differences in the regression model are extremely significant and statistically significant. The lack-of-fit term P = 0.03121 > 0.05, indicating no significant difference in the lack-of-fit term, suggesting good fit and suitability for analyzing experimental results. The coefficient of the lack-of-fit term R0... Adj 2 =0.9693 indicates that 96.93% of the actual values ​​can be explained by this model; correlation coefficient R 2 = 0.9865 indicates a high correlation between the theoretical and actual values. The P-values ​​in the table show that the effects of the three factors on the yield of hexagonal glycosides extracted by ultrasound, from largest to smallest, are: ethanol concentration (B), extraction time (C), and extraction temperature (A). Significantly influential factors include B, C, AB, AC, A², and B², while A, BC, and C² are insignificant. The linear terms have an extremely significant overall effect (P < 0.001), and the quadratic terms have an extremely significant overall effect (P < 0.001).

[0074] 2.3.3 Response Surface Analysis

[0075] To visually represent the analysis results, response surface methodology and contour plots were generated using Design-Expert 10.0.4 software. The results are as follows: Figures 5 to 7 .according to Figure 5 It can be seen that the yield of hexagonal vine saponins has a clear quadratic parabolic relationship with extraction temperature and ethanol concentration. The curve is very steep, indicating that both have a significant impact on the total saponin yield. The contour plot shows that the saponin yield reaches its maximum when the extraction temperature is between 65℃ and 75℃ and the ethanol concentration is between 70% and 80%. According to... Figure 6 It can be seen that the total saponin extraction rate from *Symplocos rubra* exhibits a distinct quadratic parabolic relationship with extraction temperature, with a steep curve. Furthermore, the contour plot shows that the total saponin extraction rate increases with increasing extraction time, indicating a significant interaction between the two factors on the total saponin yield. The total saponin yield reaches its maximum at an extraction temperature between 70℃ and 80℃ and an extraction time of 150 min. Increasing the extraction time and appropriately raising the extraction temperature both contribute to improving the extraction rate of the target product. Figure 7 shows that the total saponin yield from *Symplocos rubra* significantly increases with increasing extraction time. The saponin yield from *Symplocos rubra* also exhibits a distinct quadratic parabolic relationship with ethanol concentration, with a very steep curve. The maximum extraction rate of total saponins from *Symplocos rubra* occurs at an ethanol concentration between 70% and 80% and an extraction time of 150 min.

[0076] 2.3.4 Optimization Experiment Verification

[0077] Response surface methodology analysis revealed the optimal extraction conditions for total saponins from *Hylocereus undatus* as follows: extraction temperature 70.70℃, ethanol concentration 74.02%, and extraction time 150 min, yielding a saponin yield of 24.99 mg / g. In practical operation, to facilitate parameter setting, the optimal extraction conditions were modified to an extraction temperature of 70.00℃, an ethanol concentration of 74%, and an extraction time of 150 min. After modification, three parallel experiments were conducted on the optimal process, yielding a total saponin extraction rate of 25.64 mg / ml (RSD = 1.2%). The RSD between the measured and predicted values ​​was 1.82%, showing good agreement with the model prediction. The experimental results indicate that the optimized saponin extraction process is stable, feasible, and reproducible, and the extraction process optimized by response surface methodology can be used in practice.

[0078] 2.3.5 Total saponin content of Hexagonal Vine samples from different origins

[0079] Two portions of each of *Symplocos rubrum* from different origins, each weighing 1.00g, were extracted using the optimal extraction scheme optimized by response surface methodology. After color development, the absorbance was measured at 545nm, and the concentration was obtained according to the standard curve. The saponin content was calculated as shown in Table 5 below. Origin 1 had the highest total saponin content, and the content was in the following order: Origin 1 > Origin 7 > Origin 4 > Origin 3 > Origin 5 > Origin 8 > Origin 2 > Origin 6. Origin 6 had the lowest total saponin content.

[0080]

[0081] 2.4 Antioxidant activity of total saponins from *Symplocos rubra*

[0082] 2.4.1 Determination of DPPH free radical scavenging ability:

[0083] The DPPH free radical scavenging ability is shown in Figure 8. Within the concentration range of 0.01-0.08 mg / ml, the DPPH scavenging ability increases with increasing concentration, exhibiting a clear dose-dependent linear relationship. Above 0.08 mg / ml, the DPPH free radical scavenging rate of total saponins from *Symplocos rubra* tends to reach equilibrium. Therefore, total saponins from *Symplocos rubra* have a certain DPPH free radical scavenging effect. At the same concentration, the overall DPPH free radical scavenging ability of VC solution is greater than that of total saponins from *Symplocos rubra*.

[0084] 2.4.2 Determination of reducing power:

[0085] The reducing power of different solutions is shown in the figure. Figure 9Within a certain concentration range, the reducing power of total saponin extracts at different concentrations is clearly directly proportional to the concentration; the higher the total saponin concentration, the stronger the reducing power. However, when the concentration reaches 0.03 mg / ml, the absorbance value is less than 1.0, indicating that the reducing power of the sample is far lower than that of vitamin C.

[0086] 3. Conclusion

[0087] Previous experiments showed that the material-to-liquid ratio had no significant effect on the extraction rate of total saponins from *Hylocereus undatus*. Only three factors (extraction temperature, ethanol concentration, and extraction time) were investigated. A superior mathematical model was established using Box-Behnken response surface methodology to accurately identify the optimal experimental point. Based on the single-factor experiments, the response surface methodology was applied to the extraction process of total saponins from *Hylocereus undatus*, yielding the optimal extraction process as follows: extraction temperature 70℃, ethanol concentration 74%, and extraction time 150.00 min. Under these conditions, the saponin content of *Hylocereus undatus* was 24.989 mg / g. Comparison of total saponin content from different origins revealed that *Hylocereus undatus* from Dawang Village, Liuma Town, Beiliu City, Guangxi Province (origin #1) had the highest total saponin content. This experiment also investigated the in vitro antioxidant activity (DPPH free radical scavenging and reducing ability) of total saponins from *Hylocereus undatus*. The results showed that total saponins from *Hylocereus undatus* have a certain inhibitory and scavenging effect on DPPH free radicals and also possess a certain reducing ability.

[0088] Example 2

[0089] An extraction method for total saponins from the Yao herb *Ligusticum striatum* (also known as *Ligusticum striatum*) optimized using response surface methodology is disclosed. Based on the Box-Behnken experimental design principle, Design-Expert software is used to design and optimize the extraction temperature, ethanol concentration, and extraction time. The extraction method involves pulverizing *Ligusticum striatum* to obtain powder, then adding ethanol for ultrasonic extraction at a frequency of 50 kHz. The extraction conditions are: extraction temperature 70℃, ethanol volume concentration 74%, extraction time 150.00 min, and a solid-liquid ratio of 1 g:25 mL.

[0090] Example 3

[0091] An extraction method of total saponins from the Yao medicine Cissus hexangularis based on response surface method optimization. According to the Box-Behnken experimental design principle, Design-Expert software is used to design and optimize the technological conditions of the extraction temperature, ethanol concentration, and extraction time for extracting total saponins from the Yao medicine Cissus hexangularis. The extraction method of the total saponins from the Yao medicine Cissus hexangularis is as follows: The Cissus hexangularis medicinal materials are pulverized to obtain Cissus hexangularis powder, and then ethanol is added for ultrasonic extraction. The ultrasonic frequency is 50 KHz, and the extraction technological conditions are: the extraction temperature is 65 °C, the ethanol volume concentration is 70%, the extraction time is 130 min, and the solid-liquid ratio is 1 g∶30 mL.

[0092] Example 4

[0093] An extraction method of total saponins from the Yao medicine Cissus hexangularis based on response surface method optimization. According to the Box-Behnken experimental design principle, Design-Expert software is used to design and optimize the technological conditions of the extraction temperature, ethanol concentration, and extraction time for extracting total saponins from the Yao medicine Cissus hexangularis. The extraction method of the total saponins from the Yao medicine Cissus hexangularis is as follows: The Cissus hexangularis medicinal materials are pulverized to obtain Cissus hexangularis powder, and then ethanol is added for ultrasonic extraction. The ultrasonic frequency is 50 KHz, and the extraction technological conditions are: the extraction temperature is 68 °C, the ethanol volume concentration is 75%, the extraction time is 140 min, and the solid-liquid ratio is 1 g∶28 mL.

[0094] Example 5

[0095] An extraction method of total saponins from the Yao medicine Cissus hexangularis based on response surface method optimization. According to the Box-Behnken experimental design principle, Design-Expert software is used to design and optimize the technological conditions of the extraction temperature, ethanol concentration, and extraction time for extracting total saponins from the Yao medicine Cissus hexangularis. The extraction method of the total saponins from the Yao medicine Cissus hexangularis is as follows: The Cissus hexangularis medicinal materials are pulverized to obtain Cissus hexangularis powder, and then ethanol is added for ultrasonic extraction. The extraction technological conditions are: the extraction temperature is 70 °C, the ethanol volume concentration is 80%, the extraction time is 120 min, and the solid-liquid ratio is 1 g∶25 mL.

Claims

1. A method for extracting total saponins from the Yao herb *Liufangteng* based on response surface methodology optimization, characterized in that, Based on the Box-Behnken experimental design principle, the extraction temperature, ethanol concentration, and extraction time of total saponins from the Yao herb *Liufangteng* were designed and optimized using Design-Expert software. The extraction method for total saponins from *Liufangteng* is as follows: *Liufangteng* medicinal material is pulverized to obtain *Liufangteng* powder, then ethanol is added for ultrasonic extraction. The extraction conditions are: extraction temperature of 65℃~70℃, ethanol volume concentration of 70%~80%, extraction time of 120min~150min, and a solid-liquid ratio of 1g:25-30mL. The specific steps of the response surface methodology include: ① Single-factor experiment: The medicinal material of *Hylocereus undatus* was pulverized to obtain *Hylocereus undatus* powder. 1.00g of *Hylocereus undatus* powder was accurately weighed into a 250ml conical flask, and 25ml of 70% ethanol was added. The extraction was carried out using three methods: ultrasonic extraction, reflux extraction, and static extraction, for 1h respectively. After centrifugation, the effects of different extraction methods on the extraction rate of total saponins in *Hylocereus undatus* were studied, and the optimal extraction method was determined to be ultrasonic extraction. Several groups of single-factor experiments were designed with extraction temperature, ethanol concentration, and extraction time as the three single factors. ② Response surface methodology optimization: Using the extraction rate of total saponins from *Symplocos rubra* as the response factor, a response surface methodology was conducted based on the single-factor experiment in step ① using the Box-Behnken principle in Design-Expert software. A quadratic polynomial equation model was established between the extraction rate response value Y and the three factors: extraction temperature A, ethanol concentration B, and extraction time C. The model is: Y = + 24.24 -0.0232 A - 0.87 B + 0.85 C + 0.68 AB + 0.39 AC - 0.12 BC - 1.11 A^2 - 1.58 B^2 - 0.26 C^2; ③ The optimal extraction process conditions for extracting total saponins from the Yao herb *Liufangteng* were obtained by solving the regression equation using Design-Expert software.

2. The extraction method for total saponins from *Liufangteng* (a type of herb) based on response surface methodology optimization according to claim 1, characterized in that... Based on the Box-Behnken experimental design principle, the extraction temperature, ethanol concentration, and extraction time of total saponins from the Yao medicine Liufangteng were designed and optimized using Design-Expert software. The optimal extraction conditions for total saponins from the Yao medicine Liufangteng were found to be: extraction temperature 70℃, ethanol volume concentration 74%, and extraction time 150.00 min.

3. The extraction method for total saponins from *Liufangteng* (a type of herb) based on response surface methodology optimization according to claim 1, characterized in that... Based on the Box-Behnken experimental design principle, the extraction temperature, ethanol concentration, and extraction time of total saponins from the Yao herb *Liufangteng* were designed and optimized using Design-Expert software. The extraction method for total saponins from *Liufangteng* was as follows: *Liufangteng* medicinal material was pulverized to obtain *Liufangteng* powder, and then ethanol was added for ultrasonic extraction. The extraction process conditions were: extraction temperature of 70℃, ethanol volume concentration of 80%, extraction time of 120 min, and material-to-liquid ratio of 1 g: 25 mL.

4. The extraction method for total saponins from *Liufangteng* (a type of herb) based on response surface methodology optimization according to claim 1, characterized in that... The ultrasonic frequency for ultrasonic extraction is 50 kHz.

5. The application of total saponins from *Liufangteng* obtained by the extraction method according to any one of claims 1-3 in the preparation of antioxidant drugs and food antioxidants.

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