Radix bupleuri extract as well as preparation method and application thereof

Abstract

According to the radix bupleuri extract and the preparation method thereof, a surfactant-assisted infrared radiation extraction mode is adopted, the amphiphilic effect of a surfactant and infrared radiation heating are utilized, the extraction rate of active ingredients is remarkably increased, and the method is easy to operate, high in extraction efficiency and suitable for industrial production. And a new thought is provided for extraction and application of radix bupleuri. The extraction process conditions are optimized by adopting a method of combining a single factor with a Box-Behnken response surface test, antioxidant performance research is carried out on extraction of active substances of radix bupleuri by virtue of surfactant-assisted infrared radiation, and a theoretical basis is provided for wide application of the traditional Chinese medicine radix bupleuri. The radix bupleuri extract can be directly applied to development of traditional Chinese medicine cosmeceutical products or pure natural green Chinese herbal medicine washing and caring products, secondary separation of a surfactant is not needed, the extraction process can be simplified, and the production cost can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicinal material extraction, and particularly relates to a bupleurum extract and a preparation method and application thereof. Background Art

[0002] Radix bupleuri is pungent and bitter in nature, and has antiviral, anti-tumor, anti-depressant, and immune-enhancing effects. It is a commonly used medicinal material in clinical Chinese medicine. The genus Bupleuri is widely distributed and has many species. Among them, northern Bupleurum is the most important type, mainly distributed in Inner Mongolia, Henan, North China, Northeast China, Shaanxi and Gansu. The medicinal ingredients of Bupleurum are very complex and diverse, mainly saikosaponins. Among them, saikosaponin a is the key to the efficacy of Bupleurum because of its high content and significant efficacy. The structure of saikosaponin a Figure 1 Therefore, studying new extraction technology of Bupleurum is of great significance for the mechanism of Bupleurum's pharmacological effects and the development of new drugs or new products of Bupleurum saponins.

[0003] At present, the extraction process of saikosaponin in Bupleurum includes solvent extraction, ultrasound-assisted extraction, etc. However, the effect of ultrasound can easily break the carbon-carbon bonds in the effective molecules, destroy the active ingredients, and reduce the stability of the extract. In addition, the traditional extraction method not only uses toxic low-boiling organic solvents, but also has the disadvantages of a long extraction process, high extraction temperature, high energy consumption, the effective molecular structure is easily affected and destroyed by factors such as high temperature, and requires a lot of labor. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a bupleurum extract and a preparation method thereof in view of the deficiencies of the above-mentioned prior art. The method adopts a surfactant-assisted infrared radiation extraction method, which is green and environmentally friendly and has high extraction efficiency, and provides a new idea for the extraction and application of bupleurum saponins in bupleurum.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for preparing a bupleurum extract, the method comprising the following steps:

[0007] S1. Weigh the Radix Bupleuri, crush and grind it, pass it through a 50-mesh sieve, and dry it to obtain Radix Bupleuri powder;

[0008] S2, prepare a surfactant aqueous solution with a volume fraction of 1% to 10% with deionized water, wherein the surfactant is one or more of fatty alcohol polyoxyethylene ether AEO-9, sodium cocoyl sarcosinate CS-30, potassium cocoyl glycinate YCK30K or sodium fatty acid methyl ester sulfonate MES;

[0009] S3. Place the bupleurum powder obtained in S1 into a reactor, add the surfactant aqueous solution obtained in S2, the amount ratio of the bupleurum powder to the surfactant aqueous solution is 1g:20-80mL; connect a condenser to the top of the reactor, place an infrared heating device on one side of the reactor and heat it to 50°C for extraction, the infrared power is 100-250W, stir and extract for 5-120min, cool to room temperature, and obtain the bupleurum extract.

[0010] Preferably, the surfactant in S2 is fatty alcohol polyoxyethylene ether AEO-9, and the volume fraction of the surfactant aqueous solution is 7.5%.

[0011] Preferably, the dosage ratio of the Bupleurum powder and the surfactant aqueous solution in S3 is 1g:40mL.

[0012] Preferably, the infrared power in S3 is 250W and the extraction time is 60min.

[0013] The present invention also provides a bupleurum extract prepared by the method, wherein the components of the bupleurum extract include: 0.2-0.8% of saikosaponin a, 0.1-0.5% of saikosaponin c, 0.15-0.6% of saikosaponin d, 0.5-1.2% of other saponin compounds, 1.0-3.5% of polysaccharide compounds, 1.2-2.6% of flavonoid compounds, 1.8-3.0% of volatile oil, 2.5-10.0% of surfactant, and 77.8-92.55% of deionized water.

[0014] The present invention also provides an application of the Bupleurum extract in traditional Chinese medicine cosmetic products and pure natural green Chinese herbal medicine cleaning products.

[0015] The present invention has the following significant technical effects:

[0016] 1. The present invention provides a new method for preparing bupleurum extract by using surfactant-assisted infrared radiation. It is a new green, environmentally friendly and efficient extraction method. The method is simple to operate and has high extraction efficiency. It effectively solves the problem that the traditional extraction method uses volatile low-boiling organic solvents to pollute the environment, and the heating method has high energy consumption and is difficult to industrialize. It provides a new idea for the extraction and application of saikosaponins in bupleurum. The bupleurum extract of the present invention can be directly applied to the development of traditional Chinese medicine cosmetic products or pure natural green Chinese herbal medicine washing and care products, without the need for secondary separation of surfactants, which can simplify the extraction process and reduce production costs.

[0017] 2. Surfactants are amphiphilic molecules. When combined with bioactive substances, they can enhance their hydrophilicity and weaken their hydrophobicity, significantly improve the extraction rate of bioactive substances, and be green and environmentally friendly. Bupleurum saponins contain a large number of hydroxyl groups (-OH), while AEO-9 is a non-ionic surfactant fatty alcohol polyoxyethylene ether, which contains a large number of oxygen atoms and ether bonds (-O-) in its molecular structure. The lone pair of electrons on the oxygen atom causes AEO-9 to form hydrogen bonds with the -OH in Bupleurum saponins, thereby allowing more Bupleurum saponins to be extracted into the AEO-9 aqueous solution, greatly improving the extraction efficiency. In addition, AEO-9 has good biodegradability and is environmentally friendly. It can be used in combination with other types of anionic, nonionic, and cationic surfactants. It has outstanding synergistic effects and can greatly reduce the use and consumption of additives. Compared with traditional heating, infrared radiation heating is a process that uses infrared rays as a heat source to heat the extractant, which can greatly shorten the extraction time and improve the extraction efficiency.

[0018] 3. The present invention adopts the method of single factor combined with Box-Behnken response surface experiment to systematically study the influence of key factors such as surfactant concentration, liquid-to-solid ratio, extraction time, infrared power, etc. on the extraction effect of the main component saikosaponin a, obtains the optimal extraction process conditions, and studies the antioxidant properties of surfactant-assisted infrared radiation extraction of Bupleurum active substances, providing a theoretical basis for the widespread application of the traditional Chinese medicine Bupleurum.

[0019] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the molecular structure of saikosaponin a of the present invention;

[0021] Figure 2 is the HPLC chromatogram of the saikosaponin a reference substance and the bupleurum extract of Example 2;

[0022] Figure 3 is the saikosaponin a standard curve of Example 2;

[0023] Figure 4 is a residual analysis diagram of the response surface regression model of Example 2;

[0024] Figure 5 1 is the response surface diagram and contour diagram of the interaction of various factors in Example 2 on the extraction rate of saikosaponin a;

[0025] Figure 6 This is the test result of the antioxidant capacity of the Bupleurum extract in Example 3. DETAILED DESCRIPTION

[0026] The instruments and reagents used in the present invention are as follows:

[0027] Instruments: DAD-3000 series diode array detector, Xtimate TM C18 liquid chromatography column;

[0028] Reagents: Bupleurum saponin a reference substance, Nanjing Yuanzhi Biotechnology Co., Ltd.; surfactant fatty alcohol polyoxyethylene ether AEO-9, Shandong Junda Chemical Co., Ltd., analytical grade; phosphoric acid, methanol and acetonitrile, Tianjin Kangkede Technology Co., Ltd., chromatographic grade; Northern Bupleurum, batch number: 221007, purchased from Gansu Shengyuan Chinese Medicinal Materials Co., Ltd., and identified by Li Xixiang, a Chinese medicine pharmacist at Gansu Provincial Hospital of Traditional Chinese Medicine, as the dried root of Bupleurum Chinense DC. of the Apiaceae family.

[0029] Example 1

[0030] This embodiment is a method for preparing a Bupleurum extract, which comprises the following steps:

[0031] S1. Weigh the Radix Bupleuri, crush and grind it, pass it through a 50-mesh sieve, and dry it to obtain Radix Bupleuri powder;

[0032] S2. Prepare surfactant aqueous solution: accurately measure 1.0, 2.5, 5.0, 7.5, and 10.0 mL of surfactant respectively and place them in a volumetric flask, dilute to 100 mL with deionized water, and obtain surfactant aqueous solutions with volume fractions of 1%, 2.5%, 5%, 7.5%, and 10%, respectively; the surfactants include fatty alcohol polyoxyethylene ether AEO-9, sodium cocoyl sarcosinate CS-30, potassium cocoyl glycinate YCK30K, and sodium fatty acid methyl ester sulfonate MES;

[0033] S3. Take the Bupleurum powder obtained in S1 and place it in a 50mL round-bottom flask, add the surfactant aqueous solution obtained in S2, the dosage ratio of Bupleurum powder to surfactant aqueous solution is 1g:20-80mL, place an infrared bulb on one side of the round-bottom flask as a heat source and heat it to 50°C for extraction, the infrared power is 100-250W, and a condenser tube with condensed water is connected above the round-bottom flask to prevent the loss of extraction solvent and analyte. Stir and extract for 5-120min, cool to room temperature, and obtain Bupleurum extract.

[0034] The main components and relative contents of the Bupleurum extract prepared by the method of the present invention are shown in Table 1:

[0035] Table 1 Main components and relative contents of Bupleurum extract

[0036] Element Relative content Saikosaponin A 0.2～0.8％ Saikosaponin C 0.1～0.5％ Saikosaponin d 0.15～0.6％ Other saponin compounds 0.5～1.2％ Polysaccharide compounds 1.0～3.5％ Flavonoids 1.2～2.6％ Volatile Oil 1.8～3.0％ Surfactants 2.5～10.0％ Deionized water 77.8～92.55％

[0037] Example 2

[0038] Bupleurum saponin a is the most abundant chemical component in Bupleurum, and is also the main physiologically active component. In pharmacology, the content of Bupleurum saponin a is usually used as a quality evaluation index of Bupleurum. This example uses a single factor experiment to study the effects of extractant, extractant concentration, liquid-solid ratio, extraction time, and infrared power on the extraction rate of Bupleurum saponin a, and then optimizes the extraction process based on the Box-Behnken response surface method.

[0039] Among them, the calculation method of saikosaponin a extraction rate is:

[0040] The Bupleurum extract was centrifuged at a speed of 4000 rpm for 10 minutes, and the supernatant was taken and filtered with a 0.45 μm filter membrane for HPLC analysis. The chromatographic conditions were: Xtimate TM C18 chromatographic column, 250 mm×4.6 mm, 5 μm, column temperature: 35°C, flow rate: 1 mL / min, injection volume: 10 μL, mobile phase: acetonitrile-1.5% phosphoric acid aqueous solution, gradient elution: 0-25 min, 25%-90% acetonitrile, 25-30 min, 90% acetonitrile; detection wavelength: 210 nm.

[0041] The blank sample, reference substance and Bupleurum extract were analyzed by HPLC. Figure 2 As shown, Figure 2 a is AEO-9 aqueous solution (blank), Figure 2 b is saikosaponin a methanol (reference substance), Figure 2 c is the extract of Bupleurum chinense extracted with 10% AEO-9, showing that the retention time of saikosaponin a is (13±0.1) min. The peak shape is good at this retention time, indicating that saikosaponin a is completely separated from other components in the extract.

[0042] Standard solutions of saikosaponin a with concentrations of 0.0110, 0.0219, 0.0438, 0.175, 0.35 and 0.70 mg / mL were prepared, and the linear regression equation between the peak area and the mass concentration of saikosaponin a was obtained by HPLC analysis: y = 59.501x + 0.2016 (R 2 =0.9998). Standard curve see Figure 3 , indicating that saikosaponin a has a good linear relationship in the range of 0.011-0.7 mg / mL. The peak area of ​​saikosaponin a was obtained by HPLC, and the mass concentration of saikosaponin a in the bupleurum extract was obtained by substituting it into the linear curve. Then, the mass of saikosaponin a in the bupleurum extract was calculated according to the volume of the extractant.

[0043] The calculation method of the extraction rate of saikosaponin a (mg / g) is: the mass of saikosaponin a in Bupleurum extract / the mass of Bupleurum powder.

[0044] 1. Effect of extractants on the extraction rate of saikosaponin a

[0045] Deionized water, 10% CS-30 aqueous solution, 10% YCK30K aqueous solution, 10% AEO-9 aqueous solution, and 10% MES aqueous solution were used as extractants, with deionized water as a control, to investigate the effect on the extraction rate of saikosaponin a. The extraction conditions were: 0.5 g of bupleurum powder, 20 mL of extractant, and 50°C for 30 min. The results are shown in Table 2. The surfactant AEO-9 has a better extraction effect.

[0046] Table 2 Effect of different extractants on the extraction rate of saikosaponin a

[0047]

[0048] 2. Effect of extractant concentration on the extraction rate of saikosaponin a

[0049] Five different concentrations of AEO-9 aqueous solution with volume fractions of 1.0%, 2.5%, 5.0%, 7.5% and 10.0% were selected as extraction solvents for research. The extraction conditions were: 0.5g of Bupleurum powder, 20ml of extractant, and 50℃ extraction for 30min. Three parallel experiments were conducted in each group, and the average extraction rate was calculated. The results are shown in Table 3. 7.5% was selected as the optimal concentration.

[0050] Table 3 Effect of different concentrations of AEO-9 on the extraction rate of saikosaponin a

[0051]

[0052]

[0053] 3. Effect of liquid-solid ratio on the extraction rate of saikosaponin a

[0054] The extraction conditions are: 0.5 g of bupleurum powder, 20 mL of 10% AEO-9 aqueous solution extractant, and extraction at 50°C for 30 min. The results are shown in Table 4, and 40 mL: 1 g is selected as the optimal liquid-solid ratio.

[0055] Table 4 Effect of different liquid-solid ratios on the extraction rate of saikosaponin a

[0056]

[0057] 4. Effect of extraction time on the extraction rate of saikosaponin a

[0058] The extraction conditions are: 0.5 g of bupleurum powder, 20 mL of 10% AEO-9 aqueous solution extractant, and extraction at 50° C. The results are shown in Table 5, and 60 min is the most suitable extraction time.

[0059] Table 5 Effect of different extraction time on the extraction rate of saikosaponin a

[0060]

[0061]

[0062] 5. Effect of infrared power on the extraction rate of saikosaponin a

[0063] The extraction conditions are: 0.5g of bupleurum powder, 20mL of 10% AEO-9 aqueous solution extractant, and 30min of extraction time. The results are shown in Table 6. The results show that the increase of infrared power makes the system temperature rise faster, the permeability of the cell membrane is enhanced and the molecular movement is accelerated, and bupleurosaponin a is easier to be extracted, so 250W is selected as the optimal infrared power.

[0064] Table 6 Effect of infrared power on the extraction rate of saikosaponin a

[0065]

[0066] The extraction process was optimized based on the Box-Behnken response surface methodology, and the results were as follows:

[0067] On the basis of single factor experiment, surfactant concentration (A), liquid-solid ratio (B), extraction time (C) and infrared power (D) were selected as investigation factors, with three levels of each factor (Table 7). The extraction rate of saikosaponin a (R) was used as the evaluation index. Three repetitions were set for each group, and a total of 29 experimental points were designed. Design Expert 8.0.6 software was used for response surface analysis.

[0068] Table 7 Box-Behnken Experiment Factor Levels

[0069]

[0070]

[0071] Table 8 Response surface methodology test results

[0072]

[0073]

[0074] By using Design Expert 8.0.6 software for linear regression analysis, we can obtain the regression equation of the relationship between the extraction rate of saikosaponin a (R) and the four factors A, B, C, and D:

[0075] R=7.75+0.30A-0.027B+0.39C+0.82D-0.25AB+0.40AC-0.20AD+0.063B C-0.13BD-0.24CD-0.42A 2 -0.011B 2 -0.38C 2 -0.20D 2

[0076] Through model analysis, the optimal extraction conditions are surfactant concentration 7.89%, liquid-solid ratio 33.28:1 (mL: mg), extraction time 70.13min, infrared power 229.35W, and the predicted extraction rate under these conditions is 8.4288mg / g. In order to facilitate practical operation, the above conditions were revised to 7.9% AEO-9 concentration, liquid-solid ratio 33:1 (mL: mg), extraction time 70min, infrared power 230W, and three parallel experiments were carried out under these conditions. The average extraction rate of saikosaponin a was 8.4221mg / g, and the relative error between the experimental value and the predicted value was 0.08%, indicating that the above regression equation has a good fit with the experimental results and can be used for regression analysis.

[0077] The regression model was subjected to variance and residual analysis. The variance analysis is shown in Table 9. The significance of the extraction rate of saikosaponin a is determined by the size of the F value and the P value. The model value F is 36.6 and P < 0.001, indicating that the model terms are extremely significantly different. R 2 =0.9734, indicating that the model fitting results are relatively ideal.

[0078] Table 9 Variance analysis table of regression model

[0079]

[0080]

[0081] Note: P < 0.001, extremely significant difference; 0.001 ≤ P < 0.01, highly significant difference; 0.01 ≤ P < 0.05, significant difference.

[0082] The residual analysis results are as follows Figure 4 As shown in the figure, all the scattered points are close to the same straight line, indicating that the extraction rate of saikosaponin a in the model has a good fit with the four factors.

[0083] Design Expert 8.0.6 software was used to draw the response surface and contour map ( Figure 5). As can be seen from the figure, the response surface of AB, AC, AD, and CD combined in pairs has a large curvature, and the contour lines are elliptical, indicating that the above four groups have obvious interactions, the extraction rate is high, and the influence is significant. When BC and BD are combined in pairs, the response surface has a small curvature, and the contour lines are close to a circle, indicating that the interaction between liquid-solid ratio and extraction time, liquid-solid ratio and infrared power is not obvious, the extraction rate is small, and there is no significant effect. This theoretical simulation result is consistent with the results of the previous single-factor optimization infrared power test. The infrared-assisted extraction method has a higher extraction effect, which is completely consistent with the significance test results.

[0084] Example 3

[0085] This example is a determination of the antioxidant capacity of the Bupleurum extract, including the ABTS and DPPH free radical scavenging capacity.

[0086] Prepare ABTS and DPPH reaction solutions. Dilute the Bupleurum extract into six different concentration gradient sample solutions with volume fractions of 0.2, 0.5, 1.0, 1.5, 3.0, and 5.0%, take 5 mL of each concentration gradient sample solution, add it to 5 mL of ABTS and DPPH reaction solution, react at room temperature in the dark for 30 minutes, measure the absorbance at ultraviolet wavelengths of 734 nm (ABTS) and 517 nm (DPPH), and calculate the scavenging rate of ABTS and DPPH free radicals according to the following formula.

[0087]

[0088] Wherein, A0 is the absorbance of 5 mL DPPH or ABTS solution + 5 mL anhydrous ethanol; A1 is the absorbance of 5 mL ABTS or DPPH solution + 5 mL sample solution; A2 is the absorbance of 5 mL anhydrous ethanol + 5 mL sample solution.

[0089] The scavenging rate of ABTS and DPPH free radicals extracted from Bupleurum chinense by infrared radiation surfactant is as follows Figure 6 As shown in the figure, the scavenging rate of the two free radicals by the Bupleurum extract showed an obvious dose-dependent relationship. With the increase of the concentration of the Bupleurum extract, the scavenging rate of the two free radicals also gradually increased, and the scavenging rate of the extracts at different concentrations for ABTS was higher than that for DPPH. The ABTS scavenging rate was as high as 86.03%, and the DPPH scavenging rate was as high as 72.96%. The half-scavenging concentration (IC50) of the Bupleurum extract for ABTS and DPPH free radicals obtained by this method was 50 ) were 1.02% and 1.93% respectively, indicating that the active ingredients extracted by the surfactant-assisted infrared radiation extraction method have strong antioxidant activity and have good application prospects in the field of natural green Chinese herbal antioxidant daily chemicals.

[0090] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a Bupleurum extract, characterized in that: The method comprises the following steps: S1. Weigh the Radix Bupleuri, crush and grind it, pass it through a 50-mesh sieve, and dry it to obtain Radix Bupleuri powder; S2, prepare a surfactant aqueous solution with a volume fraction of 1% to 10% with deionized water, wherein the surfactant is one or more of fatty alcohol polyoxyethylene ether AEO-9, sodium cocoyl sarcosinate CS-30, potassium cocoyl glycinate YCK30K or sodium fatty acid methyl ester sulfonate MES; S3. Place the bupleurum powder obtained in S1 into a reactor, add the surfactant aqueous solution obtained in S2, the amount ratio of the bupleurum powder to the surfactant aqueous solution is 1g:20-80mL; connect a condenser to the top of the reactor, place an infrared heating device on one side of the reactor and heat it to 50°C for extraction, the infrared power is 100-250W, stir and extract for 5-120min, cool to room temperature, and obtain the bupleurum extract.

2. The method according to claim 1, characterized in that The surfactant S2 is fatty alcohol polyoxyethylene ether AEO-9, and the volume fraction of the surfactant aqueous solution is 7.5%.

3. The method according to claim 1, characterized in that: The dosage ratio of the Bupleurum powder and the surfactant aqueous solution in S3 is 1g:40mL.

4. The method according to claim 1, characterized in that The infrared power of S3 is 250W and the extraction time is 60min.

5. A Bupleurum extract prepared by the method according to any one of claims 1 to 4.

6. The Bupleurum extract according to claim 5, characterized in that The components of the bupleurum extract include: 0.2-0.8% of saikosaponin a, 0.1-0.5% of saikosaponin c, 0.15-0.6% of saikosaponin d, 0.5-1.2% of other saponin compounds, 1.0-3.5% of polysaccharide compounds, 1.2-2.6% of flavonoid compounds, 1.8-3.0% of volatile oil, 2.5-10.0% of surfactant, and 77.8-92.55% of deionized water.

7. Use of the Bupleurum extract according to claim 5 or 6 in traditional Chinese medicine cosmetics and pure natural green Chinese herbal medicine cleaning products.

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