Method for integrally extracting active ingredients in vitis davidii and application

Abstract

The invention discloses a method for integrally extracting active ingredients in vitis davidii and application of the active ingredients. According to the method, the contents of total polyphenols, total flavonoids and total proanthocyanidins in vitis davidii juice, seeds and residues are measured, and the availability of vitis davidii resources is discussed. Results show that part of the extract has certain DPPH (1, 1-diphenyl-2-picrylhydrazyl) removing capacity and tyrosinase inhibiting capacity and can relieve H2O2-induced LO2 cell damage. The invention also accidentally discovers that the 95% ethanol elution part of the vitis davidii juice has obvious toxicity to Hepg2 cells in a range of 150-600mu g / ml, namely, the vitis davidii juice has an obvious anti-liver cancer effect. The discovery of the invention can break through the bottleneck of existing anticancer activity, and provides a new strategy for targeted therapy of liver cancer. Overall processing and utilization of the vitis davidii can be achieved, and reference is provided for quality evaluation and comprehensive development and utilization of the vitis davidii.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine extraction and preparation technology and application, and particularly relates to a method for integrally extracting active ingredients from thorny grapes and applications thereof. Background Art

[0002] Vitis davidii is a wild grape germplasm resource unique to China. It is the only plant in the grape family with thorns and is widely distributed in Hunan, Jiangxi, Fujian and other places.

[0003] The thick skin and numerous seeds of thorny grapes make them less palatable when eaten directly. However, they can be transformed into highly nutritious juice, specialty wine, or high-value-added extracts to fully tap their economic value. The processing of thorny grapes produces byproducts such as skin residue and seeds, which account for up to 30%-40%. Traditional processing methods can lead to resource waste and environmental pollution.

[0004] In response to the above problems, it is urgent to develop an efficient and environmentally friendly technology for extracting active ingredients from Vitis thornyii and expand its innovative application models in food, medicine, high-end cosmetics and other fields to enhance resource utilization value and market competitiveness. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for overall extraction of active ingredients from Vitis thunbergii.

[0006] Another object of the present invention is to provide the use of the above method.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for extracting active ingredients from thorny grapes, comprising the following steps:

[0009] S1. Weighing Vitis serrata fruits, removing the seeds, squeezing and extracting, centrifuging, filtering, and freeze-drying the filtrate to obtain freeze-dried Vitis serrata juice powder; drying the filter residue to obtain dry Vitis serrata pomace; and drying the Vitis serrata seeds to obtain dry Vitis serrata seeds.

[0010] S2. The lyophilized powder of the thorny grape juice obtained in step S1 is freeze-dried, and then subjected to adsorption separation using a macroporous resin to obtain a 30% ethanol elution fraction of the thorny grape juice and a 95% ethanol elution fraction of the thorny grape juice;

[0011] S3, taking the dried Vitis serrata pomace obtained in step S1, performing ultrasonic-enhanced extraction with 70% ethanol, recovering the solvent, and obtaining a 70% ethanol ultrasonic extract of Vitis serrata pomace;

[0012] S4. Taking the dried Vitis thorni seeds obtained in step S1, performing ultrasonic-enhanced extraction with 70% ethanol, and recovering the solvent to obtain a 70% ethanol ultrasonic extract of Vitis thorni seeds.

[0013] Furthermore, the adsorption separation is as follows: first eluting with 3 to 5 times the volume of AB-8 macroporous resin with water, then eluting with 5 to 9 times the volume of AB-8 macroporous resin with 30% ethanol, and finally eluting with 6 to 9 times the volume of AB-8 macroporous resin with 95% ethanol.

[0014] Furthermore, the elution rate of the adsorption separation is 2±0.5 BV / h.

[0015] Furthermore, the intensity of the ultrasonic enhanced extraction is 20±5W / cm 2 .

[0016] Furthermore, the ultrasonic enhanced extraction time is 30±5 min.

[0017] An extract of active ingredients from Vitis thorni, comprising at least one of Vitis thorni juice, Vitis thorni pomace, Vitis thorni seeds, a 30% ethanol elution fraction of Vitis thorni juice, a 95% ethanol elution fraction of Vitis thorni juice, a 70% ethanol ultrasonic extract of Vitis thorni pomace, and a 70% ethanol ultrasonic extract of Vitis thorni seeds obtained by the above method.

[0018] Applications of the above-mentioned method for extracting the active ingredients from Vitis thunbergii as a whole include at least one of the following applications:

[0019] (1) Application in the preparation of products for treating liver damage;

[0020] (2) Application in the preparation of products with antioxidant effects;

[0021] (3) Application in the preparation of products with whitening and freckle-removing effects;

[0022] (4) Application in the preparation of products with therapeutic effects on liver cancer.

[0023] Furthermore, the product described in application (1) is a drug having the effect of treating liver damage or a food having an auxiliary protective function against chemical liver damage.

[0024] Furthermore, the product described in application (2) is a medicine or cosmetic with antioxidant effect.

[0025] Furthermore, the product described in application (3) is a cosmetic with whitening and freckle-removing effects.

[0026] Furthermore, the product described in application (4) is a drug having a therapeutic effect on liver cancer.

[0027] Furthermore, the application is the use of the 70% ethanol extract of Vitis thorni pomace or the 70% ethanol extract of Vitis thorni seeds obtained by the method in the preparation of a drug for treating liver damage or a food having an auxiliary protective function against chemical liver damage.

[0028] Furthermore, the application is the use of the 95% ethanol elution fraction of the spiny grape juice obtained by the method in the preparation of a medicine having a therapeutic effect on liver cancer.

[0029] The present invention has the following advantages and effects compared to the prior art:

[0030] The invention measures the contents of total polyphenols, total flavonoids and total proanthocyanidins in the juice, seeds and residues of Vitis thunbergii and explores the availability of Vitis thunbergii resources.

[0031] The experiment found that the 70% ethanol extract of Vitis thorny seeds, the 30% ethanol elution fraction of Vitis thorny juice, and the 95% ethanol elution fraction of Vitis thorny juice all have a certain DPPH scavenging ability; the tyrosinase inhibition rate of Vitis thorny juice was very low and its IC50 value could not be calculated. The 70% ethanol ultrasonic extract of Vitis thorny pomace and the 70% ethanol ultrasonic extract of Vitis thorny seeds both have a certain tyrosinase inhibition ability.

[0032] Further experiments revealed that Vitis pilosa juice and Vitis pilosa pomace 70% ethanol extracts showed no significant toxicity to L02 cells, while Vitis pilosa seed 70% ethanol extract showed significant toxicity to L02 cells at concentrations ranging from 200 to 800 μg / mL. Freeze-dried Vitis pilosa juice, Vitis pilosa juice-30% ethanol elution fractions, and Vitis pilosa juice-95% ethanol elution fractions showed no significant repair effect on H2O2-induced L02 cell damage. However, Vitis pilosa pomace 70% ethanol extract at concentrations ranging from 25 to 400 μg / mL and Vitis pilosa seed 70% ethanol extract at concentrations ranging from 6.25 to 100 μg / mL all showed a protective effect against H2O2-induced L02 cell damage.

[0033] The present invention unexpectedly discovered that freeze-dried Vitis thorni juice powder and the 30% ethanol-eluted fraction of Vitis thorni juice showed no significant toxicity to HepG2 cells. However, the 95% ethanol-eluted fraction of Vitis thorni juice showed significant toxicity to HepG2 cells at concentrations ranging from 150 to 600 μg / ml, indicating a significant anti-liver cancer effect. This suggests that innovative extraction methods can overcome the current bottleneck in anti-cancer activity and provide a new strategy for targeted liver cancer treatment.

[0034] The invention can realize the overall processing and utilization of the thorn grapes, and provide a reference for the quality evaluation and comprehensive development and utilization of the thorn grapes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a chart showing the percentage of dry products from various parts of thorny grapes;

[0036] Figure 2 This is a flow chart of the process for preparing samples from various parts of Vitis thunbergii;

[0037] Figure 3This is the result of DPPH inhibition rate determination;

[0038] Figure 4 This is the result of tyrosinase inhibition rate determination;

[0039] Figure 5 This is a graph showing the results of measuring the effects of various Vitis thornyii samples on L02 cell proliferation;

[0040] Figure 6 This is the result of the determination of the effect of each sample of Vitis thunbergii on the growth of L02 damaged cells induced by H2O2, compared with the normal group ### P < 0.001; compared with the model group *P < 0.05, **P < 0.01, ***P < 0.001;

[0041] Figure 7 This is the result of the determination of the effect of various samples of Vitis thornysis on the proliferation of Hepg2 cells, compared with the normal group # P<0.05, ## P<0.01; compared with the model group *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0042] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0043] The spiny grape fruits involved in the following embodiments were provided by Guizhou Debao Agricultural Science and Technology Co., Ltd.

[0044] Example 1:

[0045] 1. Sample preparation

[0046] Take 120g of Vitis serrata fruit, remove the seeds and squeeze the juice, centrifuge and filter to obtain Vitis serrata pomace, dry the heavy residue to obtain 5.30g of dry product, pre-freeze the filtered filtrate at -80℃, freeze-dry it in a freeze dryer to obtain 7.20g of Vitis serrata juice freeze-dried powder, dry the Vitis serrata seeds to obtain 10.40g of dry Vitis serrata seeds, calculate the percentage of dry Vitis serrata pomace, dry Vitis serrata juice and dry Vitis serrata seeds in the total weight of the dry product respectively. The results are as follows: Figure 1 shown.

[0047] 6.8051 g of the above-mentioned lyophilized powder of thorny grape juice was subjected to adsorption separation by AB-8 macroporous resin. The mixture was first eluted with 3 to 5 times the volume of AB-8 macroporous resin with water, then with 5 to 9 times the volume of AB-8 macroporous resin with 30% ethanol, and finally with 6 to 9 times the volume of AB-8 macroporous resin with 95% ethanol at an elution rate of 2 BV / h. 155.9 mg of the 30% ethanol elution fraction and 43.3 mg of the 95% ethanol elution fraction were obtained.

[0048] The obtained dried grape seeds were crushed in a mortar and subjected to ultrasonic enhanced extraction with 70% ethanol at an extraction intensity of 20W / cm 2 , time 30min, recover the solvent, and obtain 0.8037g of its extract; the obtained dry product of the prickly grape pomace was extracted with 70% ethanol by ultrasonic extraction at an extraction intensity of 20W / cm 2 , time 30min, recover the solvent and obtain 1.1145g of its extract.

[0049] The process flow chart is shown in Figure 2 .

[0050] 2. Activity Study

[0051] 2.1 Determination of total polyphenols, total flavonoids, and total proanthocyanidins (Table 1)

[0052] Table 1 Determination of total polyphenols, total flavonoids and total proanthocyanidins

[0053]

[0054] 2.2 Determination of DPPH inhibition rate

[0055] Accurately weigh an appropriate amount of DPPH and use anhydrous ethanol to prepare a 0.2 mmol / L DPPH working solution. Add 190 μL of DPPH working solution to each test well of a 96-well plate. Add 10 μL of anhydrous ethanol or the sample to be tested to the well. Set up a sample control group, mix gently, react in the dark at room temperature for 20 minutes, and then use a microplate reader to measure the absorbance of each test well at 517 nm. Calculate the DPPH inhibition rate according to the formula.

[0056] DPPH inhibition rate (%) = [1-(A1-A2) / (A3-A4)] x 100%.

[0057] Wherein: A1 is the sample group, A2 is the sample control group, A3 is the control group, and A4 is the blank control group.

[0058] Sample group A1: 190 mL DPPH working solution + 10 mL sample;

[0059] Sample control group A2: 190 mL anhydrous ethanol + 10 mL sample;

[0060] Control group A3: 190 mL DPPH working solution + 10 mL anhydrous ethanol;

[0061] Blank control group A4: 190 mL anhydrous ethanol + 10 mL anhydrous ethanol.

[0062] The DPPH clearance rates of the samples were determined as shown in Table 2 below.

[0063] Table 2 DPPH clearance rate

[0064]

[0065]

[0066] like Figure 3 As shown, 70% ethanol extract of Vitis thorn seed, 30% ethanol elution fraction of Vitis thorn juice, 95% ethanol elution fraction of Vitis thorn juice

[0067] The ethanol-eluted fractions all had certain DPPH scavenging abilities, with IC50 values of 13.19 mg / mL, 2.622 mg / mL, and 2.583 mg / mL, respectively.

[0068] 2.3. Determination of tyrosinase inhibition rate

[0069] Accurately weigh 0.025g of L-tyrosine and dissolve it in 50ml of PBS buffer using ultrasound to obtain an L-tyrosine solution. Prepare the solution immediately. Add mushroom tyrosinase to a concentration of 500U / ml in PBS buffer to obtain the tyrosinase solution. Set up a 96-well microtiter plate with solvent background wells (Ta), solvent reaction wells (Tb), sample background wells (Tc), and sample reaction wells (Td). The Ta group is the solvent background group, without the addition of the L-tyrosine substrate solution or the sample solution; the Tb group is the solvent reaction group, with the addition of the L-tyrosine substrate solution but no sample solution; the Tc group is the sample background group, without the addition of the L-tyrosine substrate solution but with the sample solution; and the Td group is the sample reaction group, with both the L-tyrosine substrate solution and the sample solution. Prepare three replicates for each group.

[0070] Refer to Table 3 for reagent addition amounts. Add L-tyrosine solution, sample solution / solvent, and PBS buffer to each well in sequence, mix thoroughly, and incubate at 37°C for 10 minutes. Then add 20 μL of tyrosinase solution to each well in sequence. Mix and react at 37°C for 5 minutes ± 5 seconds, then immediately place in a microplate reader for measurement at a wavelength of 475 nm.

[0071] Note: The time from adding tyrosinase solution to measuring absorbance in each well should be kept consistent (5min±5s). Calculation formula:

[0072] Tyrosinase inhibition rate (%) = [1-(Td-Tc) / (Tb-Ta)] × 100%.

[0073] Table 3 Sample loading table for tyrosinase activity inhibition experiment

[0074]

[0075] The results are as follows Figure 4As shown in the results, the tyrosinase inhibition rate of Vitis thornyii juice was very low and its IC50 value could not be calculated. The 70% ethanol ultrasonic extract of Vitis thornyii pomace and the 70% ethanol ultrasonic extract of Vitis thornyii seeds both had certain tyrosinase inhibition ability, and their IC50 were 6.638mg / mL and 5.318mg / mL respectively.

[0076] 2.4 Effects of Vitis thornyii extracts on H2O2-induced liver damage

[0077] 2.4.1 Effects of samples on L02 cell proliferation

[0078] In a 96-well plate, LO2 cells in the logarithmic phase were plated at 8 × 10 3 Each well was inoculated with complete medium and incubated at 37°C, 5% CO2 for 24 hours before replacing the medium with fresh culture medium. The cells were divided into two groups: a normal group and groups treated with different concentrations of the Vitis thornyii sample. The working solution for each treatment group was prepared with complete medium. The normal group received 100 μL of complete medium as a control, while the treated groups received the medium containing the extract at the appropriate concentration. Three replicate wells were set up for each experimental group, and culture was continued for 24 hours. Cell viability in each group was assessed using the CCK-8 assay to evaluate the effect of the extract on LO2 cell proliferation. Cell viability = OD value of the test well / average OD value of the normal group.

[0079] The results are as follows Figure 5 As shown, the 70% ethanol extracts of Vitis thorni juice and Vitis thorni pomace had no obvious toxicity to L02 cells, while the 70% ethanol extract of Vitis thorni seeds had obvious toxicity to L02 cells in the concentration range of 200-800 μg / ml.

[0080] 2.4.2 Effects of samples on the growth of H2O2-induced L02 damaged cells

[0081] In a 96-well plate, LO2 cells in the logarithmic phase were plated at 8 × 10 3 Cells were seeded per well, complete medium was added to each well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The original medium was discarded. The cells were then divided into different groups: normal, model, and drug-treated. The normal group received serum-free DMEM, while both the model and treatment groups received 600 μM H2O2 solution. Six replicate wells were incubated for 1 hour, and the medium was discarded. The treatment groups were then replaced with solutions of different concentrations of Vitis thornyii samples, while the control group remained unchanged for an additional 24 hours. Following incubation, the cells were washed twice with PBS buffer, and CCK-8 reagent was added and incubated for an additional 1.5 hours. Cell viability was determined by absorbance. Cell viability = OD value of the test well / average OD value of the normal group.

[0082] The results are as follows Figure 6As shown in the results, the freeze-dried powder of Vitis pilosa juice, the fraction eluted with 30% ethanol and the fraction eluted with 95% ethanol had no obvious repair effect on H2O2-induced L02 damaged cells. The 70% ethanol extract of Vitis pilosa pomace had the effect of alleviating H2O2-induced LO2 cell damage in the concentration range of 25-400 μg / mL, and the 70% ethanol extract of Vitis pilosa seeds had the effect of alleviating H2O2-induced LO2 cell damage in the concentration range of 6.25-100 μg / mL.

[0083] 2.5 Effects of Vitis thornyii extracts on HepG2 cell proliferation (anti-liver cancer)

[0084] 2.5.1 Effects of samples on Hepg2 cell proliferation

[0085] Hepg2 cells in the logarithmic phase were plated at 8 × 10 3 Each well was inoculated with complete culture medium, and cultured at 37°C and 5% CO2 for 24 hours before replacing with new culture medium. The cells were divided into two groups: a normal group and a group treated with different concentrations of thorny grape samples. The working solution of each concentration treatment group was prepared with complete culture medium. The normal group added 100 μL of complete culture medium as a control, and the treatment group added culture medium containing the extract according to the concentration. Each experimental group had 3 replicate wells and continued to culture for 24 hours. The CCK-8 method was used to detect the viability of each group of cells and evaluate the effect of the extract on the proliferation of Hepg2 cells. Cell viability = OD value of the test well / average OD value of the normal group.

[0086] The results are as follows Figure 7 As shown, the freeze-dried powder of Vitis serrata juice and the 30% ethanol-eluted fraction of Vitis serrata juice showed no significant toxicity to HepG2 cells, indicating a weak anti-hepatocarcinogenic effect. However, the 95% ethanol-eluted fraction of Vitis serrata juice showed significant toxicity to HepG2 cells at concentrations ranging from 150 to 600 μg / ml, indicating a strong anti-hepatocarcinogenic effect. The 70% ethanol ultrasonically-extracted Vitis serrata seed and 70% ethanol ultrasonically-extracted Vitis serrata pomace showed no significant toxicity to HepG2 cells, indicating a weak anti-hepatocarcinogenic effect.

[0087] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for extracting the active ingredients from Vitis vinifera as a whole, characterized by: The steps include: S1. Weighing Vitis serrata fruits, removing the seeds, squeezing and extracting, centrifuging, filtering, and freeze-drying the filtrate to obtain freeze-dried Vitis serrata juice powder; drying the filter residue to obtain dry Vitis serrata pomace; and drying the Vitis serrata seeds to obtain dry Vitis serrata seeds. S2. The lyophilized powder of the thorny grape juice obtained in step S1 is lyophilized and subjected to adsorption separation using a macroporous resin to obtain a 30% ethanol elution fraction of the thorny grape juice and a 90% ethanol elution fraction of the thorny grape juice; S3, taking the dried Vitis serrata pomace obtained in step S1, performing ultrasonic-enhanced extraction with 70% ethanol, recovering the solvent, and obtaining a 70% ethanol ultrasonic extract of Vitis serrata pomace; S4. Taking the dried Vitis thorni seeds obtained in step S1, performing ultrasonic-enhanced extraction with 70% ethanol, and recovering the solvent to obtain a 70% ethanol ultrasonic extract of Vitis thorni seeds.

2. The method for extracting the active ingredients from Vitis vinifera as a whole according to claim 1, characterized in that: The adsorption separation is as follows: first eluting with water 3 to 5 times the volume of the AB-8 macroporous resin, then eluting with 30% ethanol 5 to 9 times the volume of the AB-8 macroporous resin, and finally eluting with 95% ethanol 6 to 9 times the volume of the AB-8 macroporous resin.

3. The method for extracting the active ingredients from Vitis vinifera as a whole according to claim 2, characterized in that: The elution rate of the adsorption separation is 2±0.5 BV / h.

4. The method for extracting the active ingredients from Vitis vinifera as a whole according to claim 1, characterized in that: The intensity of the ultrasonic enhanced extraction is 20±5W / cm 2 .

5. The method for extracting the active ingredients from Vitis vinifera as a whole according to claim 4, characterized in that: The ultrasonic enhanced extraction time is 30±5min.

6. An extract of active ingredients from Vitis thunbergii, characterized by: The invention comprises at least one of the following: Vitis thorni juice, Vitis thorni pomace, Vitis thorni seeds, 30% ethanol elution fraction of Vitis thorni juice, 95% ethanol elution fraction of Vitis thorni juice, 70% ethanol ultrasonic extract of Vitis thorni pomace, and 70% ethanol ultrasonic extract of Vitis thorni seeds, obtained by the method described in any one of claims 1 to 5.

7. Use of the method according to any one of claims 1 to 5, characterized in that: Include at least one of the following applications: (1) Application in the preparation of products for treating liver damage; (2) Application in the preparation of products with antioxidant effects; (3) Application in the preparation of products with whitening and freckle-removing effects; (4) Application in the preparation of products with therapeutic effects on liver cancer.

8. The use according to claim 7, characterized in that: The product described in application (1) is a drug having the effect of treating liver damage or a food having an auxiliary protective function against chemical liver damage; The product described in application (2) is a drug or cosmetic having an antioxidant effect; The product described in application (3) is a cosmetic with whitening and freckle-removing effects; The product described in application (4) is a drug having a therapeutic effect on liver cancer.

9. The use according to claim 7, characterized in that: The application is to use the 70% ethanol extract of Vitis thorne pomace or the 70% ethanol extract of Vitis thorne seeds obtained by the method in preparing medicine for treating liver damage or food with auxiliary protective function for chemical liver damage.

10. The use according to claim 7, characterized in that: The application is the use of the 95% ethanol elution component of the thorn grape juice obtained by the method in preparing medicine with liver cancer therapeutic effect.

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