Extraction method of flavone in folium turpiniae and application of flavone in preparation of medicine for treating prostate cancer

Abstract

The invention provides an extraction method of flavone in folium turpiniae and application of the flavone in preparation of prostate cancer drugs. Through the methods of grinding, degreasing, enzymolysis, fermentation and the like, the extraction rate of flavone in turpinia arguta leaves is increased, and meanwhile, the anti-cancer activity of the flavone-containing extract is improved; the flavone-containing extract obtained by the method provided by the invention has a relatively strong inhibition effect on proliferation of prostatic cancer PC-3 cells, a theoretical basis can be provided for an anti-tumor effect of turpinia formosana leaf flavone, and development and application of turpinia formosana leaves are promoted.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a method for extracting flavonoids from leaves of Scutellaria baicalensis and an application thereof in preparing a prostate cancer drug. Background Art

[0002] Prostate cancer (PC) is one of the main cancer-related deaths in middle-aged and elderly men. It is a common male malignancy, second only to lung cancer, and its incidence rate shows a clear upward trend with age. In the early stage of prostate cancer, it is localized and is often treated with radical prostatectomy and other combined treatments. 99% of patients survive for more than 10 years. However, there are no obvious symptoms in the early stage and it is not easy to detect. Hormone deprivation therapy is generally used for middle and late stage prostate cancer, but after a period of remission, drug resistance will occur and relapse will occur, and it will progress to hormone-independent prostate cancer, which is difficult to treat and has a poor prognosis. Since the method is prone to drug resistance, finding and studying treatment methods with long-lasting efficacy and few side effects has become a research hotspot; currently, human prostate cancer cells (PC-3 cells) are a human prostate cancer cell line that is widely used in prostate cancer research.

[0003] Turpinia arguta Seem is a plant of the family Turpinaceae, mainly distributed in southern my country, such as Jiangxi, Hunan, Guizhou, and Sichuan. It is also known as the two-finger sword and the thousand-beat hammer. Its leaves are bitter and cold in nature, and have the effect of clearing away heat and detoxifying. It is one of the traditional Chinese herbal medicines in my country and can be used to treat sore throat, upper respiratory tract inflammation, etc. The main active ingredients of Turpinia arguta leaves are triterpenoids, phenolic acid compounds, macrocolumn compounds, flavonoids, and alkaloids; among them, flavonoids have multiple pharmacological effects such as enhancing immunity and anti-tumor.

[0004] Therefore, it is necessary to develop a new solution to improve the above problems. Summary of the invention

[0005] The present invention provides a method for extracting flavonoids from leaves of Scutellaria baicalensis, comprising the following steps:

[0006] S1. After degreasing the leaves of Scutellaria serrata, an enzymatic hydrolysis solution is added for enzymatic hydrolysis to obtain a Scutellaria serrata leaf hydrolysate; the enzymatic hydrolysis solution comprises a final concentration of 1% cellulase and 0.1% pectinase; the degreasing treatment comprises the following steps: suspending the ground Scutellaria serrata leaf powder in water, adding petroleum ether for extraction, and obtaining a reaction solution 1; allowing the reaction solution 1 to stand until stratification, and discarding the upper layer of petroleum ether after stratification;

[0007] S2. After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is added to the yeast solution, and the colony count is 1×10 7CFU / mL, cultured in a constant temperature box at 37°C for 12h to obtain a solid-liquid mixture; centrifuged, collected the mixed liquid in the solid-liquid mixture, and distilled and extracted at 75°C-80°C for 1h-2h to obtain the first ethanol; the mass volume ratio of the enzymatic hydrolysate to the yeast liquid was 5:1; the ratio of the distillation extraction time to the volume of the mixed liquid was 1min:5mL;

[0008] S3, mixing the first ethanol and ethyl acetate in a volume ratio of 2:1 to prepare an extract;

[0009] S4, using the extracting solution to extract the remaining amount of the enzymatic hydrolysate, and after the extraction is completed, centrifugation is performed to obtain the supernatant to obtain a flavonoid extract.

[0010] Optionally, the extraction in step S4 is performed with ultrasound-assisted extraction, and the ultrasound time is 20 minutes.

[0011] In a second aspect, the present invention provides a method for extracting flavonoids from leaves of Scutellaria baicalensis and its use in preparing a prostate cancer drug.

[0012] The beneficial effects of the present invention include:

[0013] (1) The extraction method provided by the present invention has a high extraction rate for flavonoids in the leaves of Scutellaria baicalensis, and the process operation is simple, which is conducive to large-scale production;

[0014] (2) The extraction method provided by the present invention combines enzymatic hydrolysis and fermentation in the extraction process; using self-produced ethanol for extraction can retain the active substances in the leaves of Scutellaria baicalensis to a greater extent;

[0015] (3) The flavonoid extract obtained by the extraction method provided by the present invention can inhibit the proliferation of PC-3 cells to a large extent, and has great application prospects in the treatment of prostate cancer. DETAILED DESCRIPTION

[0016] The present invention will be further illustrated by the following examples.

[0017] The embodiment of the present invention provides a method for extracting flavonoids from the leaves of Scutellaria baicalensis, comprising the following steps:

[0018] S1. After degreasing the leaves of Scutellaria baicalensis, an enzymatic hydrolysis solution is added to perform enzymatic hydrolysis to obtain an enzymatic hydrolyzate of the leaves of Scutellaria baicalensis; the enzymatic hydrolysis solution comprises a final concentration of 1% cellulase and 0.1% pectinase;

[0019] S2. After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is added to the yeast solution, and the colony count is 1×10 7CFU / mL, incubate in a constant temperature box at 37°C for 12 hours to obtain a solid-liquid mixture; centrifuge to collect the mixed liquid in the solid-liquid mixture, and distill and extract at 75°C-80°C to obtain the first ethanol;

[0020] S3, mixing the first ethanol and ethyl acetate in a volume ratio of 2:1 to prepare an extract;

[0021] S4, using the extracting solution to extract the remaining amount of the enzymatic hydrolysate, and after the extraction is completed, centrifugation is performed to obtain the supernatant to obtain a flavonoid extract.

[0022] Specifically, the defatting treatment in step S1 includes the following steps: suspending the ground Scutellaria baicalensis leaf powder in water, adding petroleum ether for extraction to obtain a reaction solution 1; allowing the reaction solution 1 to stand until stratification, and discarding the upper layer of petroleum ether after stratification.

[0023] Specifically, in step S2, the mass volume ratio of the enzymatic hydrolysate to the yeast liquid is 5:1, and the ratio of the distillation extraction time to the volume of the mixed liquid is 1 min:5 mL.

[0024] Specifically, the extraction in step S4 is assisted by ultrasound, and the ultrasound time is 20 minutes.

[0025] Specifically, the yeast is from China Center for Type Culture Collection, named Saccharomyces sp., and the collection number is CCTCC CY 20081258.

[0026] In some embodiments, the yeast liquid needs to be activated before use, comprising the following steps:

[0027] S31, inoculate the yeast into potato dextrose agar (PDA) medium, culture at 28°C for 96h, and adjust the bacterial solution concentration to 10 7 -10 8 CFU / mL;

[0028] S32, the concentration is 10 7 -10 8 The yeast with a CFU / mL was transferred into the seed culture medium and cultured in a shaking incubator at 28°C and 120 rpm for 96 h to obtain a yeast culture liquid.

[0029] In some embodiments, the composition of the PDA culture medium includes: 200 g of potato, 20 g of glucose, 20 g of agar, and 1000 mL of distilled water mixed.

[0030] In some embodiments, the composition of the seed culture medium includes sucrose 20.0 g, NaNO 3 1.5g, K 2 HPO 4 1.5g, KH2 PO 4 1.0g, NaCl 0.5g, MgSO 4 7H 2 O 0.1g, CuSO 4 ·H 2 O 0.01g, FeSO 4 7H 2 O 0.01g, 1000mL distilled water.

[0031] In a second aspect, the present invention provides a method for extracting flavonoids from leaves of Scutellaria baicalensis and its use in preparing a prostate cancer drug.

[0032] Example 1

[0033] Embodiment 1 of the present invention provides a method for extracting flavonoid extract from the leaves of Scutellaria baicalensis, comprising the following steps:

[0034] S0, taking the collected round leaves of Scutellaria baicalensis and evenly spreading them on a porcelain plate, drying them at 60°C for 4 hours, and then drying them; grinding the leaves obtained by drying, and passing them through an 80-100 mesh sieve to obtain round leaves of Scutellaria baicalensis powder;

[0035] S1. suspending the powder of the leaves of the Rhizoma Coptidis in water, adding petroleum ether for extraction, allowing to stand for stratification, and then aspirating the upper layer of petroleum ether to obtain a defatted material of the leaves of the Rhizoma Coptidis;

[0036] 20 g of the defatted leaves of the safflower were added to 100 mL of an enzymatic hydrolysis solution for enzymatic hydrolysis for 4 h at a temperature of 37° C. The enzymatic hydrolysis was completed to obtain an enzymatic hydrolyzate; the enzymatic hydrolysis solution included a final concentration of 1% cellulase and 0.1% pectinase;

[0037] S3, take 50g of the enzymatic hydrolysate, add 10mL of yeast solution, and the colony count is 1×10 7 CFU / mL, cultured in a constant temperature box at 37°C for 12 hours to obtain a solid-liquid mixture; centrifuged, collected the mixed liquid in the solid-liquid mixture, and distilled and extracted at a constant temperature of 78°C in a distillation kettle to obtain the first ethanol; wherein the ratio of the distillation extraction time to the mixed liquid volume is 1min:5mL;

[0038] S4, take 1g of the enzymatic hydrolysate, add 35mL of the leaching compound solution, that is, the liquid-to-solid ratio is 1:35, the compound solution includes 20mL of the first ethanol, 10mL of ethyl acetate (the volume ratio of the first ethanol and ethyl acetate is 2:1), 5mL of water, and adjust the pH to 4.5 at the same time, perform ultrasonic extraction for 25min, and obtain a flavonoid crude extract; ultrasonic extraction is performed twice in total;

[0039] S5. After the ultrasonic extraction is completed, the crude flavonoid extract is centrifuged, the supernatant is taken, and blown to near dryness using nitrogen at 40°C;

[0040] S6. After nitrogen blowing is completed, the volume is fixed to 2 mL using 60% first ethanol to obtain a flavonoid extract.

[0041] Example 2

[0042] Example 2 of the present invention provides a method for extracting flavonoid extracts from the leaves of Scutellaria baicalensis, which is different from Example 1 in that the liquid-to-solid ratio in step S4 is 25:1, and other conditions and steps remain consistent.

[0043] Example 3

[0044] Example 2 of the present invention provides a method for extracting flavonoid extracts from the leaves of Scutellaria baicalensis, which is different from Example 1 in that the liquid-to-solid ratio in step S4 is 45:1, and other conditions and steps remain the same.

[0045] Comparative Example 1

[0046] Comparative Example 1 of the present invention provides a method for extracting flavonoid extracts from the leaves of Scutellaria baicalensis, which is different from Example 1 in that fermentation is performed directly without enzymatic hydrolysis, and other conditions and steps remain the same.

[0047] Comparative Example 2

[0048] Comparative Example 2 of the present invention provides a method for extracting flavonoid extracts from the leaves of Scutellaria baicalensis, which is different from Example 1 in that no fermentation is performed after enzymatic hydrolysis, and ordinary ethanol and ethyl acetate are used in combination for extraction, while other conditions and steps remain consistent.

[0049] Comparative Example 3

[0050] Comparative Example 3 of the present invention provides a method for extracting flavonoid extracts from the leaves of Scutellaria baicalensis, which is different from Example 1 in that only the first ethanol is used for extraction in step S4, and other conditions and steps remain consistent.

[0051] Comparative Example 4

[0052] Comparative Example 3 of the present invention provides a method for extracting flavonoid extracts from the leaves of Scutellaria baicalensis, which is different from Example 1 in that ultrasound assistance is not used in the extraction process in step S4, and other conditions and steps remain consistent.

[0053] Performance Verification

[0054] 1. Extraction and calculation of flavonoids from the leaves of Scutellaria baicalensis

[0055] F1. Accurately weigh 10 mg of rutin standard and dissolve and dilute it with 70% ethanol to obtain 0.2, 0.4, 0.6, 0.8, and 1 (mg / mL) rutin standard solutions;

[0056] F2. Take 500 μL of rutin standard solution of each concentration and add 30% ethanol to 1.0 mL. Add 0.5 mL of 0.05 g / mL NaNO 2 Solution, let stand for 6 minutes;

[0057] F3, after standing, add 0.3mL of 0.1g / ml Al(NO 3 ) 3 The solution was shaken and allowed to stand for 6 minutes. After the standing was completed, 3 mL of 0.04 g / mL NaOH solution was added, the volume was made up to the mark with water, the solution was shaken and allowed to stand for 15 minutes. The absorbance was measured at 510 nm, and the regression equation of the standard curve was y = 0.7159x - 0.0104, R 2 =0.999;

[0058] F4. Accurately pipette 1 mL of the flavonoid extracts from Example 1 and Comparative Examples 1-4 into a volumetric flask, and add 0.5 mL of 0.05 g / mL NaNO 2 The absorbance of each solution is measured in turn, and the concentration of rutin in the sample solution is obtained from the standard working curve, which is used as the concentration of total flavonoids, and then the content of total flavonoids is calculated.

[0059] Total flavonoid yield / %=(nCV / 1000m)×100%.

[0060] Wherein: C is the total flavonoids mass concentration (mg / mL); n is the dilution multiple of the extract; V is the volume of the extract (mL); m is the mass of the leaves of Scutellaria baicalensis (g); the flavonoids extraction rates of each embodiment are shown in Table 1;

[0061] 2. Effects of the flavonoid extracts obtained in each example on PC-3 cell proliferation

[0062] D1, PC-3 cells were cultured in DMEM / F12 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum, 500 μL GlutaMAX, 100 μg / mL streptomycin and 100 U / ml penicillin at 37°C and 5% CO 2 Culture in an incubator;

[0063] D2, take the PC-3 cells cultured to the logarithmic growth phase in step D1 and adjust the cell concentration to 2×10 4 / mL, inoculated in a 96-well plate, 100 μL / well; the extracts prepared in the embodiment and the comparative example were diluted by the same multiple, 10 μL / well, and a group without extract was set as a negative control group;

[0064] D3: Incubate the 96-well plate at 37°C and 5% CO 2After culturing in the incubator for 24 hours, the cells were taken out and CCK-8 reagent was added, and the cells were reacted in the dark for 1 hour. After the reaction was completed, the absorbance value of each group was measured at 490 nm using an enzyme reader.

[0065] D4. Calculate the relative cell viability using the formula: (OD value of the experimental group - OD value of the blank) / (OD value of the negative control - OD value of the blank control) × 100%; The viability of the negative control group and the extracts in each embodiment on PC-3 cells evaluated by the CCK-8 method is shown in Table 1;

[0066] Table 1 Flavonoid extraction rate in Examples and Comparative Examples

[0067] Average flavonoid extraction rate Mean relative cell viability Negative control group / 78.5％ Example 1 3.81％ 11.3％ Example 2 3.54％ 13.44％ Example 3 3.79％ 12.69％ Comparative Example 1 1.88％ 28.77％ Comparative Example 2 3.79％ 35.69％ Comparative Example 3 1.56％ 27.54％ Comparative Example 4 1.65％ 26.65％

[0068] Results Analysis

[0069] From Examples 1-3, combined with Table 1, it can be seen that the extraction method provided by the present invention has a higher extraction rate for flavonoids, and in the inhibition experiment of PC-3 cells, it can effectively inhibit the average relative activity of PC-3 cells.

[0070] It can be seen from Example 1 and Comparative Example 1 that when the Schizonepeta tenuifolia leaves in Comparative Example 1 are directly fermented without enzymatic hydrolysis, the flavonoid extraction rate will be reduced and the content of active substances will be reduced. As can be seen from Table 1, the average flavonoid extraction rate of Comparative Example 1 is reduced, and the average relative cell viability is increased.

[0071] It can be seen from Example 1 and Comparative Example 2 that when the leaves of the Scutellaria baicalensis in Comparative Example 2 were enzymatically hydrolyzed and then fermented with ordinary ethanol, the flavonoid extraction rate did not change significantly, and the content of active substances decreased. Combined with the results in Table 1, the use of the extraction method in Comparative Example 2 resulted in a decrease in the content of active substances in the extract, and an increase in the average relative cell viability, which was higher than that of Comparative Example 1 and Comparative Examples 2-3, indicating that the extraction method provided in Example 1 of the present invention can retain the active substances in the leaves of the Scutellaria baicalensis to a large extent, thereby inhibiting the average relative cell viability of PC-3 cells.

[0072] It can be seen from Example 1 and Comparative Example 3 that when the Scutellaria baicalensis leaves in Comparative Example 3 are extracted only with the first ethanol without adding ethyl acetate to the composite extract, the flavonoid extraction rate of the extraction method in Comparative Example 3 is significantly reduced, thereby affecting the average relative cell viability of PC-3 cells.

[0073] It can be seen from the examples and comparative example 4 that when ultrasound assistance is not used during the extraction in the extraction method of comparative example 4, the flavonoid extraction rate is significantly lower than that of example 1, thereby increasing the average cell viability.

[0074] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A method for extracting flavonoids from the leaves of Rhizoma Cyperi, characterized in that: The following steps are involved: S1. After degreasing the leaves of Scutellaria serrata, an enzymatic hydrolysis solution is added for enzymatic hydrolysis to obtain a Scutellaria serrata leaf hydrolysate; the enzymatic hydrolysis solution comprises a final concentration of 1% cellulase and 0.1% pectinase; the degreasing treatment comprises the following steps: suspending the ground Scutellaria serrata leaf powder in water, adding petroleum ether for extraction, and obtaining a reaction solution 1; allowing the reaction solution 1 to stand until stratification, and discarding the upper layer of petroleum ether after stratification; S2. After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is added to the yeast solution, and the colony count is 1×10 7 CFU / mL, culturing in a constant temperature box at 37°C for 12 hours to obtain a solid-liquid mixture; centrifuging, collecting the mixed liquid in the solid-liquid mixture, and distilling and extracting at 75°C-80°C to obtain the first ethanol; the mass volume ratio of the enzymatic hydrolysate to the yeast liquid is 5:1; the ratio of the distillation extraction time to the volume of the mixed liquid is 1min:5mL; S3, mixing the first ethanol and ethyl acetate in a volume ratio of 2:1 to prepare an extract; S4, using the extracting solution to extract the remaining amount of the enzymatic hydrolysate, and after the extraction is completed, centrifugation is performed to obtain the supernatant to obtain a flavonoid extract.

2. The method according to claim 1, characterized in that The extraction in step S4 is assisted by ultrasound, and the ultrasound time is 20 minutes.

3. Use of the flavonoid extract obtained by the method according to claim 1 in the preparation of prostate cancer drugs.