Extract of overground part of liquorice for preventing and treating benign prostatic hyperplasia and preparation of extract

Abstract

The preparation method comprises the following steps: extracting the overground part of a glycyrrhiza plant by using ethanol to obtain an alcohol extract; mixing the alcohol extract with water, and carrying out water precipitation separation to obtain a precipitate; and further enriching and purifying the precipitate to obtain the licorice extract. According to the invention, the alcohol extract is subjected to water precipitation separation treatment to obtain the licorice extract especially rich in isopentenyl flavanone compounds, and the licorice extract is especially suitable for effectively preventing and treating benign prostatic hyperplasia.

Description

Technical Field

[0001] The present invention relates to a purified liquorice extract, and more particularly to the production or preparation of a purified liquorice extract that can be used for preventing and treating prostatic hyperplasia. Background Art

[0002] Liquorice root has a long history of medicinal use and a large market demand, but its above-ground parts (including stems and leaves) have not been given sufficient attention and effectively utilized for a long time. Although there have been relevant studies on the medicinal value of the above-ground parts of liquorice, especially its medicinal value in preventing and treating prostate diseases, there is still a lack of sufficient understanding of the effective parts of the extract and its specific applications. Summary of the Invention

[0003] The object of the present invention is to provide a purified liquorice extract that is particularly suitable for effectively preventing and treating prostatic hyperplasia.

[0004] According to a first aspect of the present invention, there is provided a method for preparing a liquorice extract particularly for preventing and treating prostatic hyperplasia, comprising:

[0005] providing the above-ground parts of a Glycyrrhiza plant, wherein the Glycyrrhiza plant is selected from at least one of the group consisting of Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L., and Glycyrrhiza inflata Bat.;

[0006] extracting the above-ground parts of the Glycyrrhiza plant with ethanol to obtain an ethanol extract;

[0007] mixing the obtained ethanol extract with water for water precipitation separation to obtain a precipitate; and

[0008] further enriching and purifying the obtained precipitate to obtain a liquorice extract.

[0009] According to the preparation method of the present invention, the ethanol extract can be concentrated and dried and then added with water for water precipitation separation, or the ethanol can be recovered and directly added with water for water precipitation separation without drying to improve the separation degree.

[0010] According to the preparation method of the present invention, the enrichment and purification treatment may include: subjecting the obtained precipitate to resin adsorption treatment, then performing ethanol elution treatment on the adsorbed resin, and then collecting the eluate and drying it to obtain a liquorice extract. The resin used may be a macroporous adsorption resin or a polyamide resin. The volume fraction of the eluting ethanol is preferably 60%-95%.

[0011] According to the preparation method of the present invention, the enrichment and purification treatment may also include: subjecting the obtained precipitate to organic solvent extraction treatment, then collecting the extract and drying it to obtain a liquorice extract. The organic solvent may be ethyl acetate.

[0012] According to the preparation method of the present invention, the enrichment and purification treatment may further include: performing alkali dissolution and acid precipitation treatment on the obtained precipitate, and then collecting the precipitate and drying it to obtain the licorice extract. When performing alkali dissolution, the pH value of the solution may be 8 or above, and when performing acid precipitation, the pH value of the solution may be 4-5.

[0013] According to the preparation method of the present invention, the volume fraction of the extraction ethanol may be 40%-95%, more preferably 60%-80%. In addition, the ethanol extraction temperature is preferably not lower than 70°C (but lower than the boiling point temperature of ethanol).

[0014] According to the preparation method of the present invention, the above-ground part of the glycyrrhiza plant is preferably the crushed stems and leaves, which is more conducive to subsequent extraction.

[0015] According to another aspect of the present invention, there is also provided a licorice extract for preventing and treating prostate hyperplasia, which is prepared according to the above method.

[0016] According to still another aspect of the present invention, there is also provided the use of the above licorice extract in the preparation of a drug for preventing and treating prostate hyperplasia.

[0017] According to still another aspect of the present invention, there is also provided a licorice extract for preventing and treating prostate hyperplasia, which contains or is rich in flavonoids and stilbenoids, especially isopentenyl dihydroflavonoids.

[0018] The present invention first obtains an ethanol extract rich in flavonoid compounds by extracting the above-ground part of licorice with ethanol, then obtains a water precipitation precipitate rich in water-insoluble flavonoids by performing water precipitation separation treatment on the ethanol extract, and then further enriches and purifies the precipitate to obtain a licorice extract mainly rich in isopentenyl dihydroflavonoids. The licorice extract prepared according to the present invention is particularly suitable for effectively preventing and treating prostate hyperplasia. This method is simple, efficient and low-cost, and is very suitable for industrial-scale production.

[0019] The licorice extract produced according to the present invention may contain up to 58% of flavonoid compounds. By verifying the effectiveness of the obtained licorice extract in preventing and treating prostate hyperplasia, the active substances therein are further pointed to isopentenyl dihydroflavonoids. Description of the drawings:

[0020] Figure 1 is the HPLC chromatogram of ethanol extracts with different concentrations;

[0021] Figure 2 is the HPLC chromatogram of the preliminary separation and enrichment product of the water precipitation of the ethanol extract;

[0022] Figure 3It is the HPLC chromatogram of the purified product enriched by solvent extraction, or alkali solution-acid precipitation, or resin adsorption-ethanol elution of the precipitate obtained by ethanol extraction and water precipitation;

[0023] Figure 4a , Figure 4b , Figure 4c , Figure 4d , Figure 4e and Figure 4f They are respectively the quantitative detection results of prostate index and related factors T, DHT, EGF, BAX, and BCL-2 related to hyperplasia in serum in the efficacy verification experiment of different purified products;

[0024] Figure 5 They are the HE staining photos of prostate tissue sections in the efficacy verification experiment of different purified products;

[0025] Figure 6 They are the Masson staining photos of prostate tissue sections in the efficacy verification experiment of different purified products; and

[0026] Figure 7 It is the total ion current chromatogram detected by UPLC-MS of the purified product obtained by ethyl acetate extraction and macroporous resin purification of the precipitate of the aerial part of Glycyrrhiza uralensis after ethanol extraction and water precipitation. Specific Embodiments

[0027] The present invention will be further described below in conjunction with specific examples and drawings. Those skilled in the art should understand that these examples and drawings are only for better understanding of the present invention and are not used to make any limitations.

[0028] Preparation of ethanol extracts by selecting different concentrations of ethanol

[0029] 5 g of the powder of the stems and leaves of Glycyrrhiza uralensis was placed in a 100 mL conical flask, and the ethanol concentrations were 45%, 60%, 70%, 80% and 95% respectively. The mass ratio of material to liquid was 1:10. The reflux extraction was carried out for 60 min at a temperature of 75 °C, and the extraction was carried out 3 times. The filtrates were combined and dried.

[0030] The above process was repeated 3 times to obtain 3 samples of the ethanol extract.

[0031] Next, the determination of the total flavonoid content and the detection of the HPLC fingerprint were carried out.

[0032] The total flavonoid content was determined by ultraviolet spectrophotometry. Using quercetin as the reference substance, methanol as the solvent, and 10% KOH as the color-developing agent, it was determined with an ultraviolet spectrophotometer at 400 nm.

[0033] HPLC fingerprint detection: The sample was dissolved in 70% ethanol, and Agilent SB-C was used. 18Chromatographic column, using acetonitrile as mobile phase A and 0.5% formic acid aqueous solution as mobile phase B, adjusting the proportion of mobile phases A and B in different time periods to set gradient elution conditions, detection wavelength 280 nm, column temperature 30 °C, flow rate 0.8 mL·min -1 , and the injection volume is 10 μL.

[0034] Figure 1 are HPLC chromatograms of ethanol extracts with different concentrations.

[0035] Table 1 shows the active ingredient contents and fingerprint similarities of ethanol extracts with different concentrations.

[0036] Table 1

[0037]

[0038] Note: For multiple comparisons, different letters indicate significant differences (P < 0.05), and the same letters indicate no significant differences (P > 0.05).

[0039] As Figure 1 shown in and Table 1, the chemical compositions of extracts at various concentrations are very close, and the similarities between the fingerprint chromatograms and the reference chromatogram are all above 0.90.

[0040] Obtaining an ethanol extract with 70% ethanol and then performing water precipitation separation

[0041] Take 30 g of the above-ground powder of Glycyrrhiza uralensis Fisch., reflux extract with 70% ethanol, recover the ethanol to obtain a non-alcoholic extract solution, and perform water precipitation in two groups. For group A, perform water precipitation without drying, directly add water until the extract accounts for about 1 / 10 of the total solution weight, let it stand for 24 h and then centrifuge, collect the supernatant and precipitate, and concentrate and dry them; for group B, perform water precipitation after drying, concentrate and dry the extract solution to obtain the extract, dissolve it with water at a solid-liquid ratio of 1:10, let it stand for 24 h and then centrifuge, collect the supernatant and precipitate, and concentrate and dry them. Repeat 3 times.

[0042] Figure 2 is the HPLC chromatogram of the preliminary separation and enrichment product of the ethanol extract by water precipitation; Table 2 shows the determination results of the flavonoid contents and HPLC fingerprint similarities of the separation and enrichment products of the ethanol extract after drying and without drying directly by water precipitation.

[0043] Table 2

[0044]

[0045] Figure 2 And Table 2 show that after water precipitation of the ethanol extract, components with larger water solubility and polarity are separated into the supernatant part, and components with smaller water-insolubility and polarity are separated into the precipitate part. The flavonoid content in the precipitate is more than doubled compared with the ethanol extract. During the extraction process, directly adding water for precipitation without drying the extract can not only reduce one drying step, improve production efficiency, reduce energy consumption and costs, but also increase the flavonoid content in the precipitate.

[0046] The precipitate obtained by ethanol extraction and water precipitation is adsorbed by macroporous resin and then eluted with ethanol to obtain purified licorice extract

[0047] According to the polarity of macroporous resins, 6 types of resins in 4 categories of non-polar, weakly polar, medium polar and polar were selected. 2 g of the water-precipitated product of the concentrated and dried ethanol extract (70% ethanol) was adsorbed by the corresponding macroporous resins and eluted with 70% ethanol.

[0048] Table 3 shows the separation and enrichment effects of macroporous resins with different polarities on the precipitation of ethanol extracts.

[0049] Table 3

[0050]

[0051] Table 3 shows that there are slight differences in the extract yield, flavonoid content and flavonoid transfer rate among resin types with different polarities. The flavonoid transfer rates of the 6 resins are all above 75%. It shows that macroporous resins with various polarities are all suitable for further enrichment and purification of the water-precipitated product of ethanol extracts. Among them, the flavonoid content of the AB-8 purified product increased by 36.2%.

[0052] The precipitate obtained by ethanol extraction and water precipitation is adsorbed by polyamide resin and then eluted with ethanol to obtain purified licorice extract

[0053] Dissolve 2 g of the water-precipitated product of the above ethanol extract (70% ethanol) in methanol, add an equal amount of polyamide resin, mix well, evaporate to dryness and load into a column, and elute successively with water, 20%, 40%, 60%, 80%, 95% ethanol, and collect the eluates and dry them respectively.

[0054] Table 4 shows the separation and enrichment effects of polyamide resin on the precipitation of ethanol extracts.

[0055] Table 4

[0056]

[0057] Table 4 shows that polyamide resin can effectively separate and purify flavonoids in the water-precipitated product of ethanol extracts: the flavonoid transfer rates are the highest (about 20%) and the flavonoid contents (concentrations) are also relatively high when eluting with 60% and 80% ethanol, and the flavonoid content is the highest (exceeding 92%) when eluting with 95% ethanol. Considering the flavonoid content and transfer rate comprehensively, the enrichment and purification effect is good with elution of 60% - 95%.

[0058] The precipitate obtained by ethanol extraction and water precipitation is prepared into purified licorice extract after solvent extraction, alkali dissolution and acid precipitation, or resin adsorption and elution

[0059] (1) Take 1 g of the water precipitation of the above-mentioned ethanol extract (70% ethanol), dissolve it in 10 mL of methanol, adjust the pH to 8 with 5% sodium carbonate, let it stand for 12 h and filter. Combine the filtrates, adjust the pH to 4.5 with dilute hydrochloric acid, let it stand for 12 h and filter to obtain the precipitate. Repeat this twice, collect the precipitate and dry it. (2) Take 1 g of the above precipitate, extract it twice with ethyl acetate at a solid-liquid ratio of 1:10 for 12 h each time. Collect the extraction solutions, recover the solvent and dry. (3) Take 1 g of the above precipitate, dissolve it in a small amount of methanol, add 1 g of polyamide and mix well. After drying, load it onto a polyamide resin column, rinse it with 45% ethanol to remove impurities, elute it with 90% ethanol, collect the eluate, recover ethanol and dry. (4) Take 1 g of the above precipitate, dissolve it in a small amount of methanol, add 1 g of polyamide and mix well. After drying, load it onto an AB-8 macroporous resin column, rinse it with 45% ethanol to remove impurities, elute it with 80% ethanol, collect the eluate, recover ethanol and dry. (5) Take 1 g of the above precipitate, first purify it by extraction with ethyl acetate using the above method, and then purify it with AB-8 macroporous resin to obtain the combined purified product of ethyl acetate and macroporous resin.

[0060] Figure 3 Figure 4 is the HPLC chromatogram of the purified product obtained by purifying the ethanol extract water precipitation by 5 methods; Table 5 shows the effects of enrichment and purification by the above methods.

[0061] Table 5

[0062]

[0063] Table 5 and Figure 3 It shows that: alkali dissolution-acid precipitation, organic solvent extraction and resin adsorption-ethanol elution can all significantly enrich and purify the flavonoids in the precipitate, and significantly increase the flavonoid content. Among them, the combined use of ethyl acetate extraction and macroporous resin adsorption-ethanol elution can reach a flavonoid content of 58%.

[0064] Efficacy experiment of licorice extract in the treatment of benign prostatic hyperplasia

[0065] Preparation of test substances: The aerial parts of Glycyrrhiza uralensis Fisch. were refluxed and extracted with 75% ethanol at a solid-liquid ratio of 1:10 at 75 °C. The ethanol extract was divided into supernatant and precipitate by water precipitation at a ratio of 1:15. Three groups of compounds with different polarities were prepared as test substances by using organic solvent extraction or macroporous resin adsorption-ethanol elution enrichment and purification methods, namely, the ethyl acetate extract of the supernatant of ethanol extract water precipitation (CTSQY), the ethyl acetate extract of the precipitate of ethanol extract water precipitation (CTCDY), and the product eluted with 95% ethanol after removing impurities with 55% ethanol by AB-8 macroporous resin adsorption of the precipitate of ethanol extract water precipitation (CTCDDK95).

[0066] Experimental treatment: Seven groups of treatments were set up, namely blank (control), model, CTSQY, CTCDDK95, CTCDY, Qianliekang (positive drug), and Baoliezhi (positive drug). Each group had 8 male SD rats, and they were adaptively fed for 1 week before modeling. For the model and drug administration groups, each rat was subcutaneously injected with testosterone propionate with soybean oil as the solvent at a dose of 50 mg / kg body weight, while the blank group was injected with 1 mL / kg body weight of soybean oil. The injection was given every other day for 4 consecutive weeks. During the modeling period, the rats in the drug administration groups were given intragastric administration of the enriched product according to the dose converted from the flavonoid content. The doses of the three test substances were 149 mg / kg, 167 mg / kg, and 170 mg / kg. During the test period, the rats were routinely fed, their body weights were measured weekly, and their living conditions were observed. After fasting for 12 h in the 5th week, blood samples were collected from the abdominal aorta, and organs such as the prostate, kidney, testis, liver, spleen, and thymus were harvested.

[0067] Result analysis: Prostatic lesions are the most direct indicators reflecting prostatic hyperplasia. The detection results of the prostate index are shown in Figure 4a , and the HE staining photos of the prostate tissue sections are shown in Figure 5 (HE staining of prostate tissue sections at 100× magnification: A: blank control; B: model; C: CTSQY; D: CTCDDK95; E: CTCDY; F: Qianliekang; G: Baoliezhi), and the Masson staining photos are shown in Figure 6 (Masson staining of prostate tissue sections at 100× magnification: A: blank control; B: model; C: CTSQY; D: CTCDDK95; E: CTCDY; F: Qianliekang; G: Baoliezhi). Prostatic hyperplasia may be related to hormonal disorders such as testosterone (T) and dihydrotestosterone (DHT) in the body. Genes related to cell proliferation and apoptosis, such as epidermal growth factor (EGF), pro-apoptotic gene (BAX), and B-cell lymphoma-2 gene (BCL-2), may lead to prostatic hyperplasia. The detection results of the above 5 indicators are shown in Figure 4b 、 Figure 4c 、 Figure 4d 、 Figure 4e 、 4f . Experiments have shown that although both the supernatant and precipitate enriched products of the alcohol extraction and water precipitation of the above-ground part of licorice have the effect of reducing the hyperplasia induced by modeling, the purified products prepared by extracting with ethyl acetate (CTCDY) or adsorbing with AB-8 macroporous resin and eluting with ethanol (CTCDDK95) from the precipitate of alcohol extraction and water precipitation have significantly higher efficacy than the ethyl acetate extract of the supernatant of alcohol extraction and water precipitation (CTSQY), and the measurement results of some of their indicators are also significantly better than those of the 2 positive drugs, returning to the blank level. This indicates that the precipitate separated by water precipitation from the alcohol extract contains compounds that can effectively prevent and treat prostatic hyperplasia.

[0068] Compound analysis experiment on the efficacy site for preventing and treating prostatitis hyperplasia

[0069] Preparation of the analyte: The precipitate obtained by water precipitation of the ethanol extract was then extracted with ethyl acetate, and further adsorbed onto AB-8 macroporous resin, and impurities were removed with 45% ethanol and eluted with 80% ethanol.

[0070] UPLC-MS detection method: Acquity UPLC HSS T3 chromatographic column was used, mobile phase A was acetonitrile, and mobile phase B was 0.1% formic acid aqueous solution, with gradient elution. Detection was carried out in both positive and negative ion modes of mass spectrometry, and the total ion chromatogram (TIC) is shown in Figure 7 : where the upper is the negative ion mode; the lower is the positive ion mode.

[0071] Results of identification of main chemical components: Based on relative retention time, relative molecular mass, molecular formula, primary mass spectrometry data and secondary mass spectrometry fragment ion data, 20 compounds were identified and analyzed: 13 flavonoid compounds and 7 stilbene compounds. The flavonoid compounds are mainly dihydroflavones, and 12 of them carry isopentenyl groups. The list of compounds is arranged in order of retention time as follows: 5,7,3',5'-tetrahydroxy-dihydroflavone, pinocembrin, α,α'-dihydro-3,5,3',4'-tetrahydroxy-5'-isopentenyl stilbene, 8-isopentenyl kaempferol, 8-isopentenyl naringenin, glycyrrhisoflavone, α,α'-dihydro-3,3',4'-trihydroxy-5-O-isopentenyl-6-isopentenyl stilbene, 6-isopentenyl eriodictyol, uralensol or glycyrinin P, 6-isopentenyl quercetin-3-methyl ether, 5,7,3'-trihydroxy-4'-isopentenyl dihydroflavone, 5,7,4'-trihydroxy-6-isopentenyl isoflavone, α,α'-dihydro-3,5-dihydroxy-4'-acetoxy-5'-isopentenyl stilbene, α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diisopentenyl stilbene, lupiwighteone, α,α'-dihydro-3,5,3',4'-tetrahydroxy-4,5'-diisopentenyl stilbene, α,α'-dihydro-3,5,4'-trihydroxy-4,5'-diisopentenyl stilbene, α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diisopentenyl stilbene, glabridin, neo-uralensol. Considering that isopentenyl flavonoid compounds have hormonal activity and can also regulate cell proliferation and apoptosis, it is inferred that the smaller polar components in the aerial parts of licorice that effectively prevent and treat benign prostatic hyperplasia are mainly flavonoids and stilbenes, especially dihydroflavonoid compounds with isopentenyl groups.

Claims

1. A preparation method of a licorice extract especially for preventing and treating benign prostatic hyperplasia, comprising: providing the aerial parts of a Glycyrrhiza plant, wherein the Glycyrrhiza plant is selected from at least one of the group consisting of Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L., and Glycyrrhiza inflata Bat.; using ethanol to extract the aerial parts of the Glycyrrhiza plant to obtain an ethanol extract; mixing the obtained ethanol extract with water for water precipitation separation to obtain a precipitate; and further enriching and purifying the obtained precipitate to obtain a licorice extract.

2. The preparation method according to claim 1, wherein the enrichment and purification treatment comprises: subjecting the obtained precipitate to resin adsorption treatment, then subjecting the adsorbed resin to ethanol elution treatment, and then collecting the eluate and drying it to obtain a licorice extract.

3. The preparation method according to claim 2, wherein the volume fraction of the eluting ethanol is 60%-95%.

4. The preparation method according to claim 1, wherein the enrichment and purification treatment comprises: subjecting the obtained precipitate to organic solvent extraction treatment, then collecting the extract and drying it to obtain a licorice extract.

5. The preparation method according to claim 1, wherein the enrichment and purification treatment comprises: subjecting the obtained precipitate to alkali dissolution and acid precipitation treatment, then collecting the precipitate and drying it to obtain a licorice extract.

6. The preparation method according to claim 1, wherein the volume fraction of the extracting ethanol is 40%-95%.

7. The preparation method according to claim 6, wherein the ethanol extraction temperature is at least 70°C.

8. A licorice extract for preventing and treating benign prostatic hyperplasia, prepared by the method according to any one of claims 1-7.

9. Use of the licorice extract according to claim 8 in the preparation of a drug for preventing and treating benign prostatic hyperplasia.

10. A licorice extract for preventing and treating prostatitis, comprising isopentenyl dihydroflavonoids.

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