Weeping forsythia extract and preparation method thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a fructus forsythiae extract and a preparation method thereof.The method comprises the following steps that 1, pretreatment is conducted, specifically, fructus forsythiae is added into water to be braised, then dried and ground, and fructus forsythiae powder is obtained; (2) adding the fructus forsythiae powder into a deep-eutectic solvent, then adding an enzyme preparation, carrying out enzymolysis extraction under an ultrasonic condition, and after the enzymolysis extraction is finished, filtering out precipitates to obtain a fructus forsythiae extract; wherein the enzyme preparation comprises cellulase, pectinase and saccharifying enzyme; the eutectic solvent is prepared from choline chloride, glycerol and polyethylene glycol in a molar ratio of 1: 1: (2-3). The fructus forsythiae extract obtained by the method has a very high elimination rate on DPPH free radicals, superoxide anion free radicals and ABTS free radicals, and can be applied to antioxidant products as a raw material.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a Forsythia suspensa extract and a preparation method thereof. Background Art

[0002] Forsythia suspensa is a plant of the genus Forsythia in the family Oleaceae. Its fruit is a famous Chinese medicinal material with the effects of clearing away heat and detoxifying, relieving heat and wind, and reducing swelling and dispersing nodules. Forsythia suspensa was first recorded in Shennong's Herbal Classic and is regarded as a "holy medicine for sores". It is widely used in Chinese patent medicine preparations such as Shuanghuanglian Injection, Yinqiao Jiedu Tablets, and Lianhua Qingwen Capsules. Forsythia suspensa is native to central and northern China and is widely distributed in Shanxi, Henan, Shaanxi and other places. It is one of the "Top Ten Jin Medicines" in Shanxi Province. The fruit is harvested in autumn when it is just beginning to ripen and is still green. Impurities are removed, steamed, and dried in the sun. It is commonly known as Qingqiao. When the fruit is harvested when it is fully ripe, dried in the sun, and impurities are removed, it is called Laoqiao.

[0003] The fruit of Forsythia suspensa contains chemical components such as phenylethanol glycosides, lignans, flavonoids, cyclohexylethanol derivatives and alkaloids. Among them, phenylethanol glycosides and lignans are the two major representative characteristic components of Forsythia suspensa. So far, several phenylethanol glycoside compounds such as forsythiaside AE, eugenol glycoside, anal hydroxy eugenol glycoside, akebia phenylethanol glycoside B, and echinacoside have been isolated from the fruit of Forsythia suspensa; lignan compounds such as forsythiaside, forsythiaside, arctigenin, arctiin, podocarpone, podocarpone glycoside, and larchresinol have been isolated. Modern pharmacological studies have confirmed that Forsythia suspensa has anti-inflammatory, antioxidant, antibacterial, anticancer, antiviral, antiallergic, and neuroprotective effects. Among them, antioxidant capacity is the most important indicator for evaluating the efficacy of Forsythia suspensa.

[0004] Deep eutectic solvents (DESs) refer to two-component or three-component eutectic mixtures composed of a certain stoichiometric ratio of hydrogen bond acceptors (such as quaternary ammonium salts) and hydrogen bond donors (such as compounds such as amides, carboxylic acids and polyols). Due to the hydrogen bonding between the components, the melting point of DESs is significantly lower than that of its individual components, making it appear as a dense and uniform liquid at room temperature. DESs are recognized as a new type of green extraction solvent due to its many advantages such as simple preparation process, low cost, environmental friendliness, low volatility, high solubility, structural designability and easy biodegradability. These characteristics make DESs show excellent application prospects in the field of traditional Chinese medicine extraction. Summary of the invention

[0005] To this end, the present invention provides a Forsythia suspensa extract prepared by extraction with a low eutectic solvent, wherein the extract has high antioxidant activity.

[0006] The technical contents of the present invention are:

[0007] A Forsythia suspensa extract, the preparation method of which comprises the following steps:

[0008] (1) Pretreatment: adding Forsythia suspensa to water and stewing, then drying and grinding to obtain Forsythia suspensa powder;

[0009] (2) adding the Forsythia powder to a low eutectic solvent, then adding an enzyme preparation, performing enzymatic extraction under ultrasonic conditions, and filtering out the precipitate to obtain a Forsythia extract;

[0010] The enzyme preparations are cellulase, pectinase and saccharifying enzyme; the low eutectic solvent is choline chloride, glycerol and polyethylene glycol in a molar ratio of 1:1:(2-3).

[0011] Preferably, the water content of the deep eutectic solvent is 20-30%.

[0012] Further preferably, the low eutectic solvent is choline chloride, glycerol and polyethylene glycol in a molar ratio of 1:1:3, and the water content is 20%.

[0013] Preferably, in step (2), the liquid-to-solid ratio of the low eutectic solvent to the Forsythia suspensa powder is 25-35 mL / g.

[0014] Preferably, the weight ratio of the enzyme preparation to the Forsythia suspensa powder is 1:(10-20); specifically, it can be 1:10, 10:20, 1:15, 1:14, etc., preferably 1:15.

[0015] More preferably, the weight ratio of cellulase, pectinase and saccharifying enzyme in the enzyme preparation is 0.25:(0.17-1):(0.25-0.4).

[0016] Further preferably, the stewing temperature in step (1) is 90-100° C. and the stewing time is 5-10 min.

[0017] Preferably, in step (2), the ultrasonic power of enzymatic hydrolysis is 200-500 W, the temperature is 40-60° C., and the ultrasonic time is 5-10 min.

[0018] The Forsythia suspensa extract obtained by the above preparation method.

[0019] An antioxidant product comprises the forsythia extract described above.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The present invention provides a method for preparing a Forsythia suspensa extract, wherein the Forsythia suspensa that has been stewed and dried is used as a raw material, the powder is ground into powder, and then added into a low eutectic solvent, and an enzyme preparation is added for ultrasonic extraction, wherein the active enzyme contained in the Forsythia suspensa can degrade forsythoside A, stewing the Forsythia suspensa first can kill the enzyme, and then the enzyme preparation is added into the low eutectic solvent for enzymatic extraction, and ultrasound, enzyme and low eutectic solvent are combined and applied to Forsythia suspensa extraction for the first time, and the Forsythia suspensa extract obtained by the method has a high elimination rate for DPPH free radicals, superoxide anion free radicals and ABTS free radicals, and can be used as a raw material for antioxidant products. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The DPPH radical elimination rates of the Forsythia suspensa extracts obtained in the examples of the present invention and the comparative examples are shown; the significant differences are indicated as follows: the same letters indicate no significant difference between the two groups; different letters indicate significant difference between the two groups.

[0023] Figure 2 The superoxide anion radical elimination rates of the Forsythia suspensa extracts obtained in the examples of the present invention and the comparative examples are shown; the significant differences are indicated as follows: the same letters indicate no significant difference between the two groups; different letters indicate significant difference between the two groups.

[0024] Figure 3 The ABTS free radical elimination rates of the Forsythia suspensa extracts obtained in the examples of the present invention and the comparative examples; the significant differences are indicated as follows: the same letters indicate no significant difference between the two groups; different letters indicate significant difference between the two groups. DETAILED DESCRIPTION

[0025] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0026] The present invention is further described below by way of specific examples. If no specific experimental steps or conditions are specified in the examples, the conventional steps or conditions described in the literature in the art can be used. If no manufacturer is specified for all reagents, they can be obtained through conventional commercial channels.

[0027] In the following examples, the source information of some products is as follows:

[0028] Forsythia suspensa was purchased from Hongwang Seedling Planting Professional Cooperative in Jiangxian County; cellulase and pectinase were purchased from Henan Anrui Biotechnology Co., Ltd., where the cellulase activity was 100,000 U / g and the pectinase activity was 50,000 U / g; saccharifying enzyme was purchased from Hebei Chuangzhiyuan Biotechnology Co., Ltd., where the enzyme activity was 50,000 U / g.

[0029] Preparation Example Preparation of deep eutectic solvent

[0030] A certain molar ratio of choline chloride (Chlinechlo-ride, Chcl) and a hydrogen bond donor (glycerol, polyethylene glycol) were placed in a round-bottom flask, heated in a 90°C water bath and stirred for 6-12 hours until a uniform, transparent, colorless liquid was formed, and dried with P2O5 at room temperature for at least 2 weeks. A certain volume of DESs was dissolved in a certain volume of citric acid buffer (0.1M, pH = 4.5) to prepare a DESs aqueous solution with a water content of 30%. The composition and ratio of the low eutectic solvent are shown in Table 1.

[0031] Table 1 Composition and ratio of DESs

[0032]

[0033]

[0034] Examples 1-3

[0035] The present embodiment provides a method for preparing a Forsythia suspensa extract, comprising the following steps: (1) adding Forsythia suspensa to water, simmering in the water at 100° C. for 5 minutes, then drying the Forsythia suspensa in an oven, grinding the Forsythia suspensa into powder, and filtering through an 80-mesh sieve to obtain Forsythia suspensa powder; (2) adding the Forsythia suspensa powder to a low eutectic solvent (Preparation Example 2), then adding cellulase, pectinase and saccharifying enzyme, performing enzymatic hydrolysis and ultrasonic extraction, and filtering out the precipitate to obtain a Forsythia suspensa extract. The specific parameter ratios are shown in Table 2.

[0036] Table 2 Specific raw materials and process parameters in Examples 1-3

[0037]

[0038] Example 4

[0039] The present embodiment provides a method for preparing a Forsythia suspensa extract, comprising the following steps: (1) adding Forsythia suspensa to water, simmering in the water at 90° C. for 10 minutes, then drying the Forsythia suspensa in an oven, grinding the Forsythia suspensa into powder, and filtering through an 80-mesh sieve to obtain Forsythia suspensa powder; (2) taking 15 g of Forsythia suspensa powder and adding it to a low eutectic solvent (Preparation Example 1), then adding 0.25 g of cellulase, 0.375 g of pectinase, and 0.375 g of saccharifying enzyme, performing enzymatic hydrolysis and ultrasonic extraction at a temperature of 50° C. for 8 minutes, and filtering out the precipitate after the extraction to obtain a Forsythia suspensa extract.

[0040] Comparative Example 1

[0041] This comparative example provides a method for preparing a Forsythia suspensa extract, which differs from Example 3 only in that a different low eutectic solvent is used. This comparative example uses the low eutectic solvent obtained in Preparation Example 3, and the rest is the same as Example 3.

[0042] Comparative Example 2

[0043] This comparative example provides a method for preparing a Forsythia suspensa extract, which differs from Example 3 only in that a different low eutectic solvent is used. This comparative example uses the low eutectic solvent obtained in Preparation Example 4, and the rest is the same as Example 3.

[0044] Comparative Example 3

[0045] This comparative example provides a method for preparing a Forsythia suspensa extract, which is different from Example 3 only in that the enzyme preparation does not include saccharifying enzyme, and the enzyme preparation in this comparative example includes 0.4 g of cellulase and 0.6 g of pectinase, and the rest is the same as Example 3.

[0046] Comparative Example 4

[0047] This comparative example provides a method for preparing a Forsythia suspensa extract, which is different from Example 3 only in that the enzyme preparation does not include pectinase, and the enzyme preparation in this comparative example includes 0.4 g of cellulase and 0.6 g of saccharifying enzyme, and the rest is the same as Example 3.

[0048] Comparative Example 5

[0049] This comparative example provides a method for preparing a Forsythia suspensa extract, which is different from Example 3 only in that the enzyme preparation only includes cellulase. The enzyme preparation in this comparative example includes 1 g of cellulase, and the rest is the same as Example 3.

[0050] Comparative Example 6

[0051] This comparative example provides a method for preparing a Forsythia suspensa extract, which differs from Example 3 in that the low eutectic solvent is replaced with water, namely, (1) adding Forsythia suspensa to water, simmering in the water at 90°C for 10 min, then drying the Forsythia suspensa in an oven, grinding it into powder, and passing it through an 80-mesh sieve to obtain Forsythia suspensa powder; (2) taking 15 g of Forsythia suspensa powder and adding it to 450 mL of water, then adding 0.25 g of cellulase, 0.375 g of pectinase, and 0.375 g of saccharifying enzyme, performing enzymatic hydrolysis and ultrasonic extraction at 50°C for 8 min, and filtering out the precipitate after the extraction to obtain the Forsythia suspensa extract.

[0052] Comparative Example 7

[0053] This comparative example provides a method for preparing a Forsythia suspensa extract, which differs from Example 3 in that no enzyme preparation is added, namely, (1) adding Forsythia suspensa to water, simmering in the water at 90°C for 10 min, then drying the Forsythia suspensa in an oven, grinding it into powder, and passing it through an 80-mesh sieve to obtain Forsythia suspensa powder; (2) taking 15 g of Forsythia suspensa powder and adding it to 450 mL of water, performing enzymatic hydrolysis and ultrasonic extraction at 50°C for 8 min, and filtering out the precipitate after the extraction to obtain the Forsythia suspensa extract.

[0054] Experimental Example 1

[0055] 1 Test sample: Forsythia suspensa extracts prepared in Examples 1-4 and Comparative Examples 1-7

[0056] 2. Superoxide anion free radical elimination rate detection method:

[0057] Pipette 1mL of the sample to be tested and 4.5mL of 0.05M Tris-HCL (pH=8.2) solution, mix well, and react in a water bath at 25℃ for 20min. Add 0.3mL of 0.05M pyrogallol, and react in a water bath at 25℃ for 4min. Add 1mL of 8mmol / L HCL to terminate the reaction, measure the absorbance at a wavelength of 325nm, and calculate the clearance rate according to the following formula:

[0058] Wherein, R: superoxide anion free radical scavenging rate; A: absorbance of the experimental group; B: absorbance of the blank group (pure water replaces the sample).

[0059] 3DPPH free radical elimination rate detection method

[0060] Pipette 1ml of the sample solution + 1ml of 0.2mmol / L DPPH, react for 30min in the dark, measure the absorbance at 517nm with a UV-visible spectrophotometer, and calculate the elimination rate according to the following formula:

[0061] DPPH clearance rate (%) = [1-(Ai-Aj) / A0] × 100%

[0062] The absorbance of the experimental group was recorded as Ai; the control group was measured by adding 1ml sample solution + 1ml anhydrous ethanol, recorded as Aj. The blank group was measured by adding 1ml anhydrous ethanol + 1ml 0.2mmol / L DPPH, recorded as A0.

[0063] 4ABTS(C 18 H 24 N6O6S4) free radical elimination ability detection method

[0064] Take 0.2 ml of 7.4 mmol / L ABTS solution and 0.2 ml of 2.6 mmol / L K2S2O8, mix them, and let them stand at room temperature in the dark for 12 hours to prepare ABTS stock solution; dilute the mixture 50 times with anhydrous ethanol to make its absorbance at 734 nm 0.7±0.02 as ABTS working solution (anhydrous ethanol can be replaced by pH 7.4 phosphate buffer or 95% ethanol, which can be diluted 40-50 times).

[0065] Prepare a 2ml EP tube, add 0.8ml ABTS working solution, and then add 0.2ml of the sample solution to be tested. Shake for 10s, let stand for 6min, and record the absorbance value as A. For the control blank, replace 0.2ml of the sample solution with 0.2ml of 95% ethanol, and record the absorbance value as A0. Pipette 300ul of solution and process the result. The clearance rate = (A0-A) / A0*100%.

[0066] The antioxidant capacity of the samples was tested according to the above method, and the results were shown in Table 3 and Table 4. Figure 1-3 .

[0067] Table 3 Antioxidant capacity of Forsythia suspensa extracts of Examples 1-4 and Comparative Examples 1-7 (mean ± standard deviation)

[0068]

[0069]

[0070] Note: Different letters in the same column represent significant differences, p < 0.05

[0071] From Table 3 and Figure 1-3 It can be seen that the Forsythia extract provided by Examples 1-4 of the present invention has a good DPPH radical elimination rate, superoxide anion radical elimination rate and ABTS radical elimination rate. The difference between Comparative Examples 1 and 2 and Example 3 is that the composition and proportion of the low eutectic solvent are different, and the results in Table 3 show that the DPPH radical, ABTS radical and superoxide anion radical elimination rates are significantly different from those in Example 3, indicating that the selection and proportion of HBDs in the low eutectic solvent affect the radical elimination ability of the Forsythia extract. The difference between Comparative Examples 3-5 and Comparative Example 7 and Example 3 is that the selection of enzyme preparations is different, and the difference between Comparative Example 6 and Example 3 is that the low eutectic solvent is replaced with water as a solvent. It can be seen from Table 3 that the elimination rates of the three free radicals are all significant, indicating that the selection of the low eutectic solvent and the enzyme preparation will affect the radical elimination ability of the Forsythia extract, and the Forsythia extract obtained by the preparation method selected by the present invention has a better ability to eliminate free radicals and can be applied to antioxidant products.

[0072] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a Forsythia suspensa extract, characterized in that: The method comprises the following steps: (1) pretreatment: adding Forsythia suspensa to water and stewing, then drying and grinding to obtain Forsythia suspensa powder; (2) adding Forsythia suspensa powder to a low eutectic solvent, then adding an enzyme preparation, performing enzymatic extraction under ultrasonic conditions, and filtering out the precipitate after the extraction to obtain Forsythia suspensa extract; The enzyme preparation comprises cellulase, pectinase and saccharifying enzyme; the low eutectic solvent comprises choline chloride, glycerol and polyethylene glycol in a molar ratio of 1:1:(2-3).

2. The preparation method according to claim 1, characterized in that: The water content of the deep eutectic solvent is 20-30%.

3. The preparation method according to claim 2, characterized in that: In the step (2), the liquid-to-solid ratio of the low eutectic solvent to the Forsythia suspensa powder is 25-35 mL / g.

4. The preparation method according to claim 1, characterized in that: The weight ratio of the enzyme preparation to the Forsythia suspensa powder is 1:(10-20).

5. The preparation method according to claim 1, characterized in that: The weight ratio of the cellulase, pectinase and saccharifying enzyme is (1-3): (2-4): (1-4).

6. The preparation method according to claim 1, characterized in that: In the step (2), the ultrasonic power is 200-500W, the ultrasonic time is 5-10min, and the ultrasonic temperature is 40-60°C.

7. The preparation method according to claim 1, characterized in that: The stewing temperature in step (1) is 90-100° C. and the stewing time is 5-10 minutes.

8. A Forsythia suspensa extract, characterized in that Obtained according to any one of the preparation methods described in claims 1-7.

9. An antioxidant product, characterized in that: The invention comprises the Forsythia suspensa extract according to claim 8.

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