Extract of overground part of liquorice for preventing and treating prostatitis 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 supernate; and further enriching and purifying the supernate to obtain the liquorice extract. According to the invention, the alcohol extract is subjected to water precipitation separation treatment to obtain the licorice extract containing or enriching flavonoid glycoside compounds, and the licorice extract is especially suitable for effectively preventing and treating prostatitis.

Description

Technical Field

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

[0002] Licorice 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 licorice, 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, etc. Summary of the Invention

[0003] The object of the present invention is to provide a licorice extract that is particularly suitable for effectively preventing and treating prostatitis.

[0004] According to one aspect of the present invention, there is provided a preparation method of a licorice extract particularly for preventing and treating prostatitis, 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] Using ethanol to extract the above-ground parts of the Glycyrrhiza plant to obtain an ethanol extract;

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

[0008] Further enriching and purifying the obtained supernatant to obtain a licorice extract.

[0009] According to the preparation method of the present invention, the enrichment and purification treatment preferably includes: performing resin adsorption treatment on the obtained supernatant, then performing ethanol elution treatment on the adsorbed resin, and then collecting the eluate and drying it to obtain a licorice extract. The resin used in this case can be macroporous adsorption resin or polyamide resin.

[0010] Other enrichment and purification treatments can also be adopted. In particular, the ethyl acetate-n-butanol extract is further purified by combining with AB-8 macroporous resin, and the total flavonoid content of the obtained licorice extract can reach more than 59%.

[0011] According to the preparation method of the present invention, the volume fraction of the eluting ethanol can be 20% - 60%, preferably 30% - 40%.

[0012] According to the preparation method of the present invention, the volume fraction of the extracted ethanol can be 30% to 90%, 60% to 80%, and more preferably 70% to 75%. Additionally, the ethanol extraction temperature is preferably not lower than 70°C (but lower than the boiling point temperature of ethanol).

[0013] According to the preparation method of the present invention, the above-ground part of the Glycyrrhiza plant provided is preferably the crushed stems and leaves to be more conducive to subsequent extraction.

[0014] According to another aspect of the present invention, there is also provided a Glycyrrhiza extract for preventing and treating prostatitis, prepared according to the above method.

[0015] According to yet another aspect of the present invention, there is also provided the use of the above Glycyrrhiza extract in the preparation of a drug for preventing and treating prostatitis.

[0016] According to yet another aspect of the present invention, there is also provided a Glycyrrhiza extract for preventing and treating prostatitis, which contains or is rich in flavonoid glycosides.

[0017] The present invention first obtains an ethanol extract rich in flavonoid compounds by extracting the above-ground part of Glycyrrhiza with ethanol, then obtains a water-sedimentation supernatant rich in water-soluble flavonoid compounds by subjecting the ethanol extract to water-sedimentation separation treatment, and then further enriches and purifies the water-sedimentation supernatant to obtain a Glycyrrhiza extract mainly enriched in flavonoid glycosides. The Glycyrrhiza extract prepared according to the present invention is particularly suitable for effectively preventing and treating prostatitis. This method is simple, efficient, and low-cost, and is very suitable for industrial-scale production.

[0018] The Glycyrrhiza extract produced according to the present invention can contain up to 59% of flavonoid compounds. By verifying the effectiveness of the obtained Glycyrrhiza extract in preventing and treating prostatitis, the active substances therein are further pointed to flavonoid glycosides. Description of the Drawings

[0019] Figure 1 is the HPLC chromatogram of ethanol extracts with different concentrations;

[0020] Figure 2 is the HPLC chromatogram of the water-sedimentation separation product of the ethanol extract;

[0021] Figure 3 is the HPLC chromatogram of the Glycyrrhiza extract purified from the water-sedimentation separation product by different enrichment and purification methods;

[0022] Figure 4a 、 Figure 4b and Figure 4c are respectively the prostate index and the number of lecithin corpuscles and white blood cells in the prostatic fluid in the efficacy verification experiments of different purified products;

[0023] Figure 5a 、Figure 5b and Figure 5c are the expression levels of related inflammatory factors TNF-α, IL-6, and IFN-γ in the sera of the efficacy verification experiments of different purified substances, respectively;

[0024] Figure 6a 、 Figure 6b and Figure 6c are the expression levels of IgG, DHT, and PSA in the sera of the efficacy verification experiments of different purified substances, respectively;

[0025] Figure 7 is the HE staining result (×100) of the prostate tissue sections in the efficacy verification experiment of the macroporous resin purified product; and

[0026] Figure 8 is the total ion current chromatogram of the liquid mass detection of the licorice extract purified by macroporous resin after the supernatant of the ethanol extraction and water precipitation of the aerial parts of licorice is extracted with ethyl acetate and n-butanol. 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] Prepare ethanol extracts with different concentrations of ethanol

[0029] 5 g of the powder of the stems and leaves of Glycyrrhiza uralensis Fisch. is placed in a 100 mL conical flask. The ethanol concentrations (hereinafter, the ethanol concentration refers to its volume fraction unless otherwise specified) are 90%, 75%, 70%, 60%, 45%, and 30% respectively, the mass ratio of material to liquid is 1:10, ultrasonic extraction is carried out for 30 min at a temperature of 75 °C, and the extraction is carried out 3 times. The filtrates are combined and dried.

[0030] The above process is 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 are carried out.

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

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

[0034] Figure 1 They are HPLC chromatograms of ethanol extracts at different concentrations. Table 1 shows the contents of active ingredients and the similarity of fingerprint spectra of ethanol extracts at different concentrations.

[0035] Table 1

[0036]

[0037] Note: For multiple comparisons, significant differences exist when the letters are different (*P<0.05), and no significant differences exist when they are the same (P>0.05).

[0038] 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 spectra and the control spectrum are all above 0.98. It should be particularly noted that when the ethanol concentration is 70% and 75%, the extraction yields of (total) flavonoids are significantly higher than those at other concentration values, reaching 2.82% and 2.98% respectively. This indicates that ethanol, especially ethanol with a concentration of 70% - 75%, can effectively extract the flavonoids contained in the above-ground part of licorice. As further discussed below, the flavonoids contained in this ethanol extract can be further selectively separated into flavonoid glycosides that can effectively prevent and treat prostatitis.

[0039] After obtaining the ethanol extract with 70% ethanol, perform water precipitation separation

[0040] Take 30 g of the above-ground powder of licorice, reflux extract with 70% ethanol, recover the ethanol to obtain a non-alcoholic extract solution, and divide it into two groups for water precipitation. Group A: Water precipitation without drying: directly add water until the extract accounts for about 1 / 10 of the total solution weight, let it stand for natural sedimentation for 24 h, and then collect the supernatant and precipitate for concentration and drying respectively; Group B: Water precipitation with drying: Concentrate and dry the extract solution to obtain the extract, add water and stir to dissolve it according to a solid-liquid ratio of 1:10, let it stand for natural sedimentation for 24 h, and then collect the supernatant and precipitate for concentration and drying respectively. Repeat 3 times. Detect the total flavonoid content and fingerprint spectra of the supernatant and precipitate respectively.

[0041] Figure 2 They are HPLC chromatograms of the enriched substances separated by water precipitation of the ethanol extract. Table 2 shows the corresponding experimental results.

[0042] Table 2

[0043]

[0044] Figure 2Table 2 shows that the water precipitation treatment can separate the flavonoid compounds contained in the ethanol extract into two parts: the water-soluble components (mainly flavonoid compounds with relatively large polarity) separated and enriched in the supernatant, and the water-insoluble components (mainly flavonoid compounds with relatively small polarity) separated and enriched in the precipitate. The chemical components of the supernatant are similar whether the water precipitation is carried out after drying or not after extraction, and the similarity of the fingerprint is 0.949. The flavonoid content in the supernatant is slightly higher when the water precipitation is carried out directly without drying. Directly adding water for precipitation without drying the extract during the extraction process can not only reduce one drying step, improve production efficiency, reduce energy consumption and costs, but also increase the flavonoid content in the supernatant.

[0045] The supernatant of the ethanol extract and water precipitation is adsorbed by macroporous resin and eluted with different concentrations of ethanol to obtain purified licorice extract

[0046] Take 2 ml of the concentrated supernatant of the ethanol extract (70% ethanol) after water precipitation (natural sedimentation), add it to a macroporous resin column of HPD600 with an inner diameter of 2 cm, and elute it successively with ethanol gradients of 10%, 20%, 30%, 40%, 50%, 60%, and 70% at a speed of 2 BV / h. When 3 BV is eluted, change to the next concentration for elution. Collect the eluates separately, dry them, and determine the flavonoid content and the similarity of the fingerprint.

[0047] Table 3 shows the enrichment and purification effects of macroporous resin on the supernatant of the ethanol extract after water precipitation.

[0048] Table 3

[0049]

[0050] Table 3 shows that the flavonoids in the supernatant of the ethanol extract after water precipitation can be effectively separated and purified by macroporous resin adsorption and ethanol elution: the flavonoid content in the ethanol eluates of 20%-60% is relatively high, and the elution efficiency is the highest at concentrations of 30% and 40%.

[0051] The supernatant of water precipitation is prepared into licorice extract by different purification methods

[0052] Take the supernatant of the ethanol extract (70% ethanol) after water precipitation respectively, and enrich and purify it by 5 methods: (1) extract it successively with ethyl acetate and n-butanol, and collect the n-butanol extract; (2) adsorb it with HPD600 macroporous resin, wash away impurities with water and elute it with 45% ethanol, and collect the eluate; (3) adsorb it with AB-8 macroporous resin, wash away impurities with water and elute it with 45% ethanol, and collect the eluate; (4) first extract it with ethyl acetate and n-butanol, then adsorb the n-butanol extract with AB-8 macroporous resin, wash away impurities with water and elute it with 45% ethanol, and collect the eluate; (5) first extract it with ethyl acetate and n-butanol, then adsorb the n-butanol extract with polyamide resin, wash away impurities with water and elute it with 45% ethanol, and collect the eluate. Determine the total flavonoid content and the HPLC fingerprint of the purified products respectively.

[0053] Figure 3 The HPLC fingerprints of 5 purified products are shown. Table 4 shows the corresponding experimental results.

[0054] Table 4

[0055]

[0056] As Figure 3 As shown in and Table 4, all five methods can efficiently purify flavonoids. For both macroporous resins with ethanol elution, the flavonoid content can be increased by more than three times. The flavonoid content in the supernatant is 14.15%, and after purification, it is increased to 47.46% and 46.87% respectively. The ethyl acetate-n-butanol extract is further purified by combining with AB-8 macroporous resin, and the total flavonoid content can reach more than 59%.

[0057] Efficacy experiment of licorice extract in the treatment of prostatitis

[0058] Preparation of test substances: The raw materials of the aerial parts of Glycyrrhiza uralensis Fisch. were the same as before. 75% ethanol was used as the extraction solvent, and the material-liquid ratio was 1:10. The ethanol extract was obtained by reflux extraction at 75°C. The ethanol extract was precipitated with water at a ratio of 1:10, divided into supernatant and precipitate. A total of three test substances were prepared: ① The supernatant of ethanol extraction and water precipitation was successively extracted with ethyl acetate and n-butanol to obtain the test substance (CTSQYZ); ② The supernatant of ethanol extraction and water precipitation was adsorbed with HPD600 macroporous resin, decontaminated with water, and eluted with 60% ethanol to obtain the test substance (CTSQDK60); ③ The precipitate obtained by water precipitation of the ethanol extract (CTCD).

[0059] Experimental treatment: Sham operation, model, three test substances, and two positive control drugs (Prostate Plus and Proscar) were set up, with a total of seven experimental treatments. Eight male SD rats in each group were housed separately in cages and adaptively raised for 1 week for modeling. After anesthesia, an incision about 1 cm long was made longitudinally in the lower abdomen of the model group and the drug administration groups to expose the prostate and seminal vesicles on the dorsal side of the bladder. A 10 g / L carrageenan saline solution was injected into both seminal vesicles at a dose of 0.05 mL per side per rat. The sham operation group had the same modeling surgery, but carrageenan was not injected. One week after the modeling surgery, the corresponding test drugs were administered by gavage according to the equivalent conversion of the purified product dose based on the total flavonoid content. The doses of CTSQYZ, CTSQDK60, and CTCD were 268 mg / kg, 217 mg / kg, and 252 mg / kg per day respectively, and the positive drugs were administered according to the dose conversion in the instructions. The drugs were continuously administered for 4 weeks. The body weight was measured weekly and the living conditions were observed. Then, after fasting for 12 h, blood samples were collected from the abdominal aorta, and organs such as the prostate, kidney, testis, liver, spleen, and thymus were harvested.

[0060] Result analysis: The prostate index, the number of lecithin corpuscles and white blood cells in prostatic fluid can directly reflect the inflammatory condition. HE sections of prostate tissue can show the damage and inflammation of prostate tissue. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) are important indicators reflecting inflammation. Immunoglobulin G (IgG), dihydrotestosterone (DHT) and prostate-specific antigen (PSA) can reflect the occurrence of inflammation from the perspectives of secretion and immunology. Figure 4a 、 Figure 4b and Figure 4c , which are the prostate index, the number of lecithin corpuscles and white blood cells in prostatic fluid in the efficacy verification experiments of different purified products respectively; Figure 5a 、 Figure 5b and Figure 5c , which are the expression levels of related inflammatory factors TNF-α, IL-6, IFN-γ in serum in the efficacy verification experiments of different purified products respectively; Figure 6a 、 Figure 6b and Figure 6c , which are the expression levels of IgG, DHT and PSA in serum in the efficacy verification experiments of different purified products respectively. Figure 7 is the HE staining photo of prostate tissue section (×100), where: A: Sham operation; B: Model; C: CTSQYZ; D: CTSQDK60; E: CTCD; F: Qianliekang; G: Baolizhi. The index detection results in Figures 4 - 7 show that although the resin purified products of the precipitate and supernatant of the ethanol extraction and water precipitation of the aerial part of Glycyrrhiza uralensis Fisch. both have the effect of reducing the inflammation induced by modeling, the ethyl acetate-n-butanol sequential extraction part (CTSQYZ) of the supernatant of ethanol extraction and water precipitation with larger polarity and the 60% ethanol elution part adsorbed by macroporous resin (CTSQDK60) have significantly higher efficacy than the ethanol extraction and water precipitation precipitate (CTCD) part, and the efficacy shown by some of their detection indexes is stronger than that of the positive drug and has recovered to the level of the sham operation group. This indicates that the supernatant separated by water precipitation treatment of the ethanol extract contains compounds that can effectively prevent and treat prostatitis.

[0061] Analysis experiment of flavonoids in the efficacy site for preventing and treating prostatitis

[0062] Using the UPLC-MS method, the components of the purified product obtained by extracting the supernatant of ethanol extraction (70%) and water precipitation with ethyl acetate-n-butanol and then adsorbing with AB-8 macroporous resin and eluting with 45% ethanol were detected:

[0063] Acquity UPLC HSS T3 chromatographic column, mobile phase A is acetonitrile, B is 0.1% formic acid aqueous solution, gradient elution. Detection was carried out in positive and negative ion modes of mass spectrometry, and the total ion chromatogram (TIC) is shown in Figure 8 , where the upper figure is the negative ion mode and the lower figure is the positive ion mode.

[0064] Identification results of main chemical components: According to the relative retention time, relative molecular mass, molecular formula, primary mass spectrometry data and secondary mass spectrometry fragment ion data, 45 compounds were analyzed and identified: 31 flavonoid glycosides, 7 organic acids, 3 flavonoid aglycones, 2 chromones, 1 coumarin and 1 phenyl ethanol glycoside. The compound names are arranged in the order of retention time as follows: gallic acid, catechin or epicatechin, gentisic acid, p-hydroxycinnamic acid, urolsin, rutin isomer, cryptochlorogenic acid, puerarin, vicenin-2, apigenin-6-C-glucoside-8-C-xyloside, quercetin-3,7-di-O-glucoside, isoschaftoside,schaftoside, genistein-8-C-apiosylglucoside, rutin, forsythoside A, phloretin-C-diglucoside, liquiritin, isoquercitrin, orientin, kaempferol-3-O-rutinoside, luteoloside, naringin, isorhamnetin-3-O-rutinoside, astragalin, hesperidin, puerarin isomer, luteolin-4'-O-glucoside, pinostrobin-7-O-glucoside, azelaic acid, hyperoside, isoliquiritin, quercetin-7-O-glucoside, p-hydroxybenzoic acid, quercitrin, puerarin apioside, daidzin, myricetin, scopoletin, isoliquiritigenin, vitexin, isovitexin, isorhamnetin-3-O-neohesperidoside, prim-O-glucosylcimifugin, 5-O-methylvisamminol. Flavonoid compounds account for 76%, among which flavonoid glycosides account for 69%. Since most flavonoid glycoside compounds have anti-inflammatory activity, it is inferred that the larger polar components in the aerial part of Glycyrrhiza uralensis Fisch. effective in preventing and treating prostatitis should mainly be flavonoid glycoside compounds.

Claims

1. A method for preparing a licorice extract especially for preventing and treating prostatitis, 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.; obtaining an ethanol extract by using ethanol to extract the aerial parts of the Glycyrrhiza plant; mixing the obtained ethanol extract with water for water precipitation separation to obtain a supernatant; and further enriching and purifying the obtained supernatant to obtain a licorice extract.

2. The preparation method according to claim 1, wherein the enrichment and purification treatment comprises: subjecting the obtained supernatant 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 resin used for the resin adsorption treatment is a macroporous adsorption resin or a polyamide resin.

4. The preparation method according to claim 2, wherein the volume fraction of the eluting ethanol is 20% - 60%.

5. The preparation method according to claim 1, wherein the volume fraction of the extracting ethanol is 30% - 90%.

6. The preparation method according to claim 5, wherein the ethanol extraction temperature is at least 70 °C.

7. The preparation method according to claim 1, wherein the provided aerial parts of the Glycyrrhiza plant are mainly crushed stems and leaves.

8. A licorice extract for preventing and treating prostatitis, 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 prostatitis.

10. A licorice extract for preventing and treating prostatitis, comprising flavonoid glycosides.

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