WO2023065860A1 - 独一味酚苷的提取方法及防治肝纤维化药物或保健品的应用 - Google Patents

独一味酚苷的提取方法及防治肝纤维化药物或保健品的应用 Download PDF

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WO2023065860A1
WO2023065860A1 PCT/CN2022/117143 CN2022117143W WO2023065860A1 WO 2023065860 A1 WO2023065860 A1 WO 2023065860A1 CN 2022117143 W CN2022117143 W CN 2022117143W WO 2023065860 A1 WO2023065860 A1 WO 2023065860A1
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total phenolic
extract
phenolic glycosides
duyiwei
glycosides
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潘正
邓杰
谢亚均
万果果
陈志伟
曹文富
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重庆医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

Definitions

  • the invention relates to the technical field of traditional Chinese medicines and health care products, in particular to a method for extracting unique phenol glycosides and the application of drugs or health care products for preventing and treating liver fibrosis.
  • hepatitis B virus invasion, non-alcoholic fat, or hepatic cholestasis damages the liver, causing liver tissue repair ability disorder, resulting in fibronectin (FN), ⁇ -smooth muscle actin ( ⁇ -SMA) and collagen- ⁇ 1 (Coll- ⁇ 1) and other extracellular matrix are excessively deposited, eventually forming liver fibrosis.
  • FN fibronectin
  • ⁇ -SMA ⁇ -smooth muscle actin
  • Coll- ⁇ 1 collagen- ⁇ 1
  • TGF- ⁇ transforming growth factor- ⁇
  • TGF- ⁇ signaling pathway also regulates many biological responses, including cell proliferation, apoptosis, differentiation, autophagy, and immune responses. Therefore, inhibiting the activation of TGF- ⁇ pathway has become one of the important targets for anti-fibrosis.
  • Lamiophlomis rotata (Benth.) Kudo
  • Lamiophlomis rotata (Benth.) Kudo
  • It is also a commonly used drug in the "Chinese Pharmacopoeia” for treating various surgical wounds, bleeding and other diseases, and its related oral preparations have curative effects Accurate and safe.
  • Duyiwei granule is composed of the water extract of Duyiwei medicinal material, which is concentrated and dried, and then added with appropriate excipients. The dosage is large, and the effective substances are not clear. So far, 127 chemical components such as iridoids, flavonoids and phenylethanol glycosides have been identified from the unique flavor (Zhan-HuCui, et al. Traditional uses, phytochemistry, pharmacology and toxicology of Lamiophlomis rotata (Benth.) Kudo: areview.RSC Advances.2020, 10:11463–11474), which type or components have anti-hepatic fibrosis effect, so far, there is no report.
  • one of the objects of the present invention is to provide the application of phenolic glycoside mixture in the preparation of health products or medicines for preventing or treating liver fibrosis, said phenolic glycoside mixture containing verbascoside, forsythiaside B, 4-hydroxybenzoic acid Any two or more of the eight phenolic glycosides, icariin H1, Decaffeoylverbascoside, cosmoside, luteolin and apigenin.
  • the phenolic glycoside mixture may be any two or more of the above eight phenolic glycosides.
  • the mixture of phenolic glycosides is the total phenolic glycoside extract of unique herb; the total phenolic glycoside extract of unique herb contains verbascoside, forsythiaside B, 4-hydroxybenzoic acid, icariin H1, Decaffeoylverbascoside, Cosmoside, luteolin and apigenin are 8 kinds of phenolic glycosides.
  • the mixture of phenolic glycosides is extracted from Duyiwei, and contains any two or more of the above-mentioned 8 kinds of phenolic glycosides.
  • the total phenolic glycosides extract of the unique flavor contains 8 species of verbascoside, forsythiaside B, 4-hydroxybenzoic acid, icariin H1, Decaffeoylverbascoside, cosmoside, luteolin and apigenin phenolic glycosides, and the sum of the ion peak areas of the liquid chromatography-mass spectrum of the above eight phenolic glycosides accounts for ⁇ 60% of the total peak areas of the unique total phenolic glycosides extract.
  • the extraction method of the total phenolic glycosides extract of Duyiwei includes the following steps: weighing the aerial part of the unique medicinal material, pulverizing it, adding water 10 times the volume of the medicinal material, decocting twice for 2 hours each time, filtering and combining The filtrate was left to stand for 12 hours, and the supernatant was absorbed by the treated polyamide adsorption resin bed. After the adsorption, it was first eluted with 5 times the amount of water of the polyamide adsorption resin bed, and then eluted with 75% ethanol for analysis, and the analysis solution was collected. , recovering the solvent, concentrating at 90° C. and then drying to obtain the total phenolic glycosides extract.
  • the extraction method of the total phenolic glycosides extract of Duyiwei includes the following steps: weighing the aboveground part of the unique medicinal material, pulverizing it, adding water 10 times the volume of the medicinal material, decocting twice for 2 hours each time, filtering and combining The filtrate was left to stand for 12 hours, and the supernatant was absorbed by the Bio-Gel P-2 polyacrylamide gel resin bed. After adsorption, it was first eluted with 5 times the amount of water of the Bio-Gel P-2 polyacrylamide gel resin bed.
  • the particle size of the gum resin is 45-90 ⁇ m.
  • the phenolic glycoside mixture consists of 6-10 parts by weight of verbascoside, 3-5 parts by weight of forsythiaside B, 2-3 parts by weight of 4-hydroxybenzoic acid, and 1.5-2 parts by weight of epimedium Huoside H1, 1-1.5 parts by weight of Decaffeoylverbascoside, 1-1.5 parts by weight of cosmoside, 1-1.5 parts by weight of luteolin and 1-1.5 parts by weight of apigenin.
  • the health product or medicine is a composition composed of a mixture of phenolic glycosides and acceptable excipients in health products or medicines.
  • the formulation of the health care product or medicine is decoction, granule, powder, capsule, tablet or oral liquid.
  • the unique total phenolic glycoside extract can be made into oral and non-oral dosage forms.
  • the second object of the present invention is to provide a method for extracting the total phenolic glycosides of Duyiwei, which includes the following steps: weighing the aboveground part of the unique medicinal material, pulverizing it, adding water 10 times the volume of the medicinal material, and decocting twice, each time After 2 hours, filter and combine the filtrate, let it stand for 12 hours, take the supernatant and absorb it through the treated polyamide adsorption resin bed. Deanalysis, collecting the analysis solution, recovering the solvent, concentrating at 90°C and then drying to obtain the total phenolic glycosides extract.
  • the second object of the present invention is to provide a method for extracting the total phenolic glycosides of Duyiwei, which includes the following steps: weighing the aboveground part of the unique medicinal material, pulverizing it, adding water 10 times the volume of the medicinal material, and decocting twice, each time After 2 hours, filter and combine the filtrates, let stand for 12 hours, take the supernatant and absorb it through the Bio-Gel P-2 polyacrylamide gel resin bed.
  • the particle size of the polyacrylamide gel resin is 45-90 ⁇ m.
  • the total phenolic glycosides extract obtained in the above extraction method contains verbascoside, forsythiaside B, 4-hydroxybenzoic acid, icariin H1, Decaffeoylverbascoside, cosmoside, mignonette
  • icariin H1 Decaffeoylverbascoside
  • cosmoside mumbleette
  • 8 phenolic glycosides components of apigenin and apigenin there are 8 phenolic glycosides components of apigenin and apigenin, and the sum of the ion peak areas of the liquid chromatography-mass spectrum of the 8 phenolic glycoside components accounts for ⁇ 60% of the total peak area of the total phenolic glycosides extract of the unique herb.
  • the total phenolic glycoside extract of Duyiwei can inhibit the proliferation of hepatic stellate cells (LX-2) cells, and promote Apoptosis of LX-2 cells after TGF- ⁇ activation.
  • the total phenolic glycoside extract of Duyiwei can also significantly reduce the inflammatory response and liver fibrosis degree of CCl4 mice liver tissue, H&E staining, Sirius red and Masson staining, the fibrosis degree of the liver tissue of model animals is weakened, and the degree of liver fibrosis of model animals is reduced.
  • ALT and AST in plasma can inhibit the proliferation of hepatic stellate cells (LX-2) cells, and promote Apoptosis of LX-2 cells after TGF- ⁇ activation.
  • the total phenolic glycoside extract of Duyiwei can also significantly reduce the inflammatory response and liver fibrosis degree of CCl4 mice liver tissue, H&E staining, Sirius red and Masson staining, the fibro
  • the unique total phenolic glycoside extract can significantly reduce the expression of Smad2, 3, 4 mRNA and protein levels in liver tissue, promote the expression of Smad7 mRNA and protein levels (Smad family inhibitors), and then inhibit the TGF- ⁇ /Smad signaling pathway Excessively activate and reduce the secretion of extracellular matrix such as fibronectin (FN), ⁇ -smooth muscle actin ( ⁇ -SMA) and collagen- ⁇ 1 (Coll- ⁇ 1), and ultimately prevent the occurrence and development of liver fibrosis.
  • Smad2 Smad7 mRNA and protein levels
  • TGF- ⁇ /Smad signaling pathway Excessively activate and reduce the secretion of extracellular matrix such as fibronectin (FN), ⁇ -smooth muscle actin ( ⁇ -SMA) and collagen- ⁇ 1 (Coll- ⁇ 1)
  • the present invention clarifies that the total phenolic glycoside extract of Duyiwei has anti-hepatic fibrosis effect, and clarifies 8 kinds of phenolic glycoside components (actascoside, forsythiaside B, 4-hydroxybenzoic acid, icariin H1, Decaffeoylverbascoside, Cosmoside, luteolin and apigenin) have anti-hepatic fibrosis effects, and they can be used to prepare health care products or medicines for preventing or treating liver fibrosis.
  • phenolic glycoside components actascoside, forsythiaside B, 4-hydroxybenzoic acid, icariin H1, Decaffeoylverbascoside, Cosmoside, luteolin and apigenin
  • the extraction method of the unique total phenolic glycosides extract of the present invention adopts Bio-Gel P-2 polyacrylamide gel resin, and the interior of Bio-Gel P-2 polyacrylamide gel resin is a network structure with a particle diameter of 45 ⁇ 90 ⁇ m, with the same column bed volume, its adsorption capacity is larger than that of ordinary irregular polyamide particles (solid structure inside), so it can more fully adsorb the classified components in the solution.
  • the total phenolic glycosides extract The yield is higher; and because the Bio-Gel P-2 polyacrylamide gel resin is a spherical particle, the liquid medicine is easier to pass through the resin bed during the separation and purification process, and it is not suitable for clogging, which can ensure the smooth progress of the purification process.
  • Figure 1 UPLC-TOF/MS chromatogram of the total phenolic glycosides extract of the unique ESI positive ion mode
  • Figure 2 The structure of the compound identified by the UPLC-TOF/MS spectrum of the total phenolic glycosides extract of Duyiwei;
  • Fig. 3 The effect of the total phenolic glycosides extract and eight kinds of phenolic glycosides composition on the proliferation of hepatic stellate cells;
  • Figure 5 The effect of the total phenolic glycosides extract of Duyiwei on the CCl4 liver fibrosis model
  • Fig. 6 the effect of the total phenolic glycosides extract of Duyiwei on alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of CCl4 model mice;
  • Figure 7 The effect of the total phenolic glycosides extract of Duyiwei on the TGF- ⁇ /Smad signaling pathway in the liver of model mice.
  • Example 1 Extraction preparation and UPLC-TOF/MS analysis of the unique total phenolic glycoside extract of the present invention
  • Method 1 Weigh 5 kg of the aboveground part of Duyiwei, pulverize it, add water 10 times the volume of the medicinal material, decoct twice for 2 hours each time, filter and combine the filtrate, let it stand for 12 hours, take the supernatant and pass it through the treated polyamide ( 10 to 30 mesh) adsorption resin bed adsorption, after adsorption, first eluted with 5 times the amount of water of the adsorption resin bed, and then eluted with 75% ethanol for analysis, collected the analysis solution, recovered the solvent, concentrated at 90°C, Dried to obtain 203.6 g of the total phenolic glycosides extract of Duyiwei.
  • Method 2 Weigh 5kg of the aboveground part of Duyiwei, crush it, add water 10 times the volume of the medicinal material, decoct twice, 2 hours each time, filter and combine the filtrate, let it stand for 12 hours, take the supernatant and pass it through Bio-Gel P- 2 polyacrylamide gel resin bed (particle size 45 ⁇ 90 ⁇ m) adsorption, first eluted with 5 times the amount of water of Bio-GelP-2 polyacrylamide resin bed after adsorption, then eluted with 75% ethanol for analysis, collected Analyze the solution, recover the solvent, concentrate at 90°C, and dry to obtain 248.9 g of the total phenolic glycosides extract of Dumoi.
  • the test solution was detected by ESI positive ion mode, and a total of 16 components were detected and identified, as shown in Figure 1, peaks 1 to 16, the components were 1: Decaffeoylverbascoside, 2: 4-hydroxybenzoic acid, 3: Epimedium Glycoside H1, 4: Campneoside II, 5: Forsythiaside C, 6: Forsythiaside B, 7: leolin-7-O-[ ⁇ -D-apiose(6 ⁇ 1)]- ⁇ -glucoside, 8 : Isovacoside, 9: Verbacoside, 10: Luteolin, 11: Cosmoside, 12: Dehydroacteoside, 13: LeucosceptosideA, 14: Luteolin, 15: Apigenin, 16: Apigenin7-O-(6 '-(E)-p-coumaroyl- ⁇ -D-galactopyranoside, the structure of its corresponding compound is shown in Figure 2, of which there are 6 kinds of flavonoids, mainly
  • the total phenolic glycosides extract obtained by method 1 was also detected by liquid chromatography-mass spectrometry, which also contained the above-mentioned 8 iconic components, and accounted for more than 60% of the total peak area of all ion peaks in liquid chromatography-mass spectrometry.
  • the anti-hepatic fibrosis experiment will be carried out on the total phenolic glycoside extract of Duyiwei and the composition composed of the finished products of these iconic components.
  • Example 2 Effects of the total phenolic glycoside extract and phenolic glycoside composition of Duyiwei on the proliferation and apoptosis of hepatic stellate cells
  • phenolic glycoside composition solution preparation solution Dilute the phenolic glycoside composition solution preparation solution into different concentrations of phenolic glycoside composition solutions: 8 ⁇ g/mL, 16 ⁇ g/mL, 31 ⁇ g/mL, 62 ⁇ g/mL, 124 ⁇ g/mL.
  • LX-2 cells human semi-activated hepatic stellate cells
  • LX-2 cells were normally subcultured and subplated. After 12 hours of adherent growth, different concentrations of total phenolic glycoside extracts of Duyiwei and phenolic glycoside composition solutions of different concentrations and compositions were added, and after 48 hours of intervention , CCK-8 kit detection and analysis of the effect on the proliferation of LX-2 cells.
  • the total phenolic glycoside extract and the phenolic glycoside composition of Duyiwei can effectively inhibit the proliferation of LX-2 cells, and the 200 ⁇ g/mL total phenolic glycoside extract of Duyiwei has the most obvious inhibitory effect (P ⁇ 0.001, Compared with the blank group), their half-inhibitory concentration (IC 50 ) is slightly different, wherein, the IC 50 value of the total phenolic glycosides extract of Duyiwei is 78.1 ⁇ g/mL, and the IC 50 value of the phenolic glycoside composition is 80.8 ⁇ g/mL mL.
  • HSCs activated hepatic stellate cells
  • LX-2 cells were normally subcultured and divided into plates. After 12 hours of adherent growth, 10ng/mL TGF- ⁇ was added for 12 hours of stimulation, and different concentrations of total phenolic glycoside extracts of Duyiwei and different concentrations of phenolic glycoside composition solutions were added, AnnexinV/PI Cell Apoptosis Detection kit detects the apoptosis of LX-2 cells after TGF- ⁇ activation.
  • the phenolic glycoside composition formed also has the effect of inhibiting proliferation and promoting apoptosis of hepatic stellate cells; the experiment found that the phenolic glycoside composition group of the unique total phenolic glycoside extract and eight kinds of phenolic glycosides can effectively promote TGF - Apoptosis of LX-2 cells after ⁇ activation, but the total phenolic glycosides extract of unique herb has a better effect; both can inhibit the proliferation of hepatic stellate cells (LX-2), among which, the total phenolic glycosides extract of unique herb has an IC 50 The value is 78.1 ⁇ g/mL, which is slightly lower than that of the iconic component mixture group (IC
  • Example 3 Effect of the total phenolic glycosides extract of Duyiwei on the CCl4 liver fibrosis model
  • C57 male mice (body weight 22-25g) were randomly divided into control group, CCl 4 induction group, high, medium and low (50mg/kg, 100mg/kg, 200mg/kg) dosage groups of Duyiwei total phenolic glycosides extract , 10 in each group.
  • CCl 4 was prepared with corn oil to a concentration of 10%, and 0.5 mL/kg of CCl 4 corn oil solution was injected intraperitoneally, and the model was established every Monday and Thursday for 6 weeks.
  • the administration group of total phenolic glycosides extract of Duyiwei was administered after the first CCl 4 injection followed by CCl 4 injection.
  • liver tissues were harvested for HE staining of pathological sections.
  • the results showed that the hepatic lobule structure in the normal group was clear, the hepatic plates were arranged neatly in a cord shape, the portal area did not expand, and there was no inflammatory cell infiltration; in the model group, a large number of inflammatory cell infiltration and hepatic cell swelling and necrosis were seen; All of them were alleviated to varying degrees, and the improvement was more significant in the Duyiwei high-dose total phenolic glycosides group (A in Figure 5).
  • Masson staining is a specific staining of collagen used to display fibers and inflammatory factors in tissues.
  • a large area of blue positive areas can be seen in the liver tissue of mice in the model group; each drug group was compared with the model group.
  • the area of the blue positive area decreased to varying degrees, and the improvement was more significant in the high-dose group of total phenolic glycosides alone (Fig . Collagen deposition in the liver tissue and reduces the inflammatory response that accompanies this process.
  • the effect of the total phenolic glycosides extract of Duyiwei on the liver morphology of CCl 4 model mice the liver of the mice in the blank group was dark red, no obvious nodules were found on the surface, the edges were smooth and sharp, and the texture was soft. The livers of mice in the model group were dark brown, shrinking in size, with granules, rough surface, dull edges, darkened color and hard texture.
  • Each administration group of total phenolic glycosides of Duyiwei the color of the liver was dark red, the surface nodules were less than those of the model group, the edges were basically normal, and there were granules. D in 5).
  • the color, volume, hardness and nodular changes of the liver tissue of the mice were improved to varying degrees.
  • the effect of the total phenolic glycosides extract of Duyiwei on the liver function of CCl 4 model mice Before and after the intervention of the total phenolic glycosides of Duyiwei, the kit detected the activity values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse plasma. Compared with the normal control group, the serum ALT and AST activities of the model group were significantly increased (P ⁇ 0.001); compared with the model group, the total phenolic glycosides extract group of Duyiwei could significantly reduce the serum ALT and AST activities ( Figure 6) . Among them, the high-dose group of total phenolic glycosides of Duyiwei had a more significant improvement.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • mice Liver tissues of mice were harvested, and immunohistochemical assay (IHC) technique was used to detect changes in the deposition of extracellular matrix such as TGF- ⁇ , FN, ⁇ -SMA and Col1- ⁇ 1 in mouse liver.
  • IHC immunohistochemical assay
  • the mouse liver tissue was collected, and the method and primers in the reference (JinfengLiu et al.Praziquantel ameliorates CCl 4 -induced liver fibrosis in mice by inhibiting TGF- ⁇ /Smad signaling via up-regulating Smad7 in hepatic stellatecells.Br J Pharmacol.2019,176:4666–4680), detecting the gene expression level changes of the Smad protein family downstream of TGF- ⁇ in liver tissue.

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Abstract

本发明属于医药及保健品领域，具体涉及独一味酚苷的提取方法及防治肝纤维化药物或保健品的应用。本发明独一味总酚苷提取物中标志性成分为毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoylverbascoside、大波斯菊苷、木犀草素和芹菜素8种成分，上述8种成分具有抗肝纤维化的作用，可用来制备预防和治疗肝纤维化的药物，或辅助治疗CCl4肝损伤的保健品。本发明独一味总酚苷提取物的提取方法采用Bio-Gel P-2聚丙烯酰胺凝胶树脂，使独一味总酚苷提取物的分离纯化更顺利、得率更高。

Description

独一味酚苷的提取方法及防治肝纤维化药物或保健品的应用
本申请要求于2021年10月21日提交中国专利局、申请号为CN202111228588.2、发明名称为“独一味酚苷的提取方法及防治肝纤维化药物或保健品的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及中药及保健品技术领域，特别涉及独一味酚苷的提取方法及防治肝纤维化药物或保健品的应用。
背景技术
长期的乙肝病毒侵害、非/酒精性脂肪、或肝胆汁淤积对肝脏的损伤，引起肝组织修复能力失调，导致粘连蛋白(FN)、α-平滑肌肌动蛋白(α-SMA)和胶原-α1(Coll-α1)等细胞外基质过度沉积，最终形成肝纤维化。
研究表明转化生长因子-β(transforming growth factor-β，TGF-β)的过度活化是肝纤维化发生发展的主要因素之一(Meng X M,et al.TGF-β:the master regulator of fibrosis[J]Nature Reviews Nephrology,2016,48:)；TGF-β信号通路的过度激活，促进细胞外基质(ECM)的过度沉积，进一步导致肝硬化或肝细胞癌的发生。此外，TGF-β信号通路还调节许多生物反应，包括细胞增殖、凋亡、分化、自噬和免疫反应等。因此，抑制TGF-β通路激活成为抗纤维化重要的靶点之一。
独一味为唇形科植物独一味Lamiophlomis rotata(Benth.)Kudo的干燥地上部分，也是《中国药典》收载治疗多种外科手术后的刀口创伤、出血等疾病的常用药，其相关口服制剂疗效确切，且安全性好。
2010～2013年，张怡课题组研究发现独一味颗粒具有较好的抗肝纤维化作用(杨晗,张怡,谭万初,等.独一味颗粒抗大鼠肝纤维化的实验研究[J].上海中医药杂志,2010,044:64-67；张怡,代夏欢,解秀翠,等.独一味颗粒对实验性肝纤维化大鼠TGF-β1/Smads信号通路的影响[J].中国中医基础医学杂志,2013,019(012):1401-1403,1407.)，但未见相关后继报 道。
独一味颗粒以独一味药材的水提取物，浓缩干燥后，加适当的赋形剂组成，服用量大，药效物质不明确。迄今为止从独一味中已鉴定环烯醚萜、黄酮和苯乙醇苷等127个化学成分(Zhan-HuCui,et al.Traditional uses,phytochemistry,pharmacology andtoxicology of Lamiophlomis rotata(Benth.)Kudo:areview.RSC Advances.2020,10:11463–11474)，究竟哪一类或哪些成分具有抗肝纤维化的作用，迄今为止，未见报道。
发明内容
鉴于此，本发明的目的之一是提供酚苷混合物在制备预防或治疗肝纤维化保健品或药物中的应用，所述酚苷混合物含有毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoylverbascoside、大波斯菊苷、木犀草素和芹菜素8种酚苷成分中的任意2种或2种以上。所述酚苷混合物可以只是上述8种酚苷成分中的任意2种或2种以上。
优选地，所述酚苷混合物为独一味总酚苷提取物；所述独一味总酚苷提取物含有毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoylverbascoside、大波斯菊苷、木犀草素和芹菜素8种酚苷成分。，即酚苷混合物从独一味中提取获得，并含有上述8种酚苷成分中的任意两种或两种以上。
优选地，所述独一味总酚苷提取物中含有毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoylverbascoside、大波斯菊苷、木犀草素和芹菜素8种酚苷成分，且上述8种酚苷成分的液相色谱-质谱的离子峰面积之和占所述独一味总酚苷提取物全部峰面积总和的比例为≥60％。
优选地，所述独一味总酚苷提取物的提取方法包括以下步骤：称取独一味地上部分药材，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过处理的聚酰胺吸附树脂床吸附，吸附后先用聚酰胺吸附树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后干燥，得到所述独一味总酚苷提取物。
优选地，所述独一味总酚苷提取物的提取方法包括以下步骤，称取独 一味地上部分药材，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过Bio-Gel P-2聚丙烯酰胺凝胶树脂床吸附，吸附后先用Bio-Gel P-2聚丙烯酰胺凝胶树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后干燥，得到所述独一味总酚苷提取物；所述Bio-Gel P-2聚丙烯酰胺凝胶树脂的粒径为45～90μm。
优选地，所述酚苷混合物由6～10重量份的毛蕊花糖苷、3～5重量份的连翘酯苷B、2～3重量份的4-羟基苯甲酸、1.5～2重量份的淫羊藿苷H1、1～1.5重量份的Decaffeoylverbascoside、1～1.5重量份的大波斯菊苷、1～1.5重量份的木犀草素和1～1.5重量份的芹菜素组成。
优选地，所述保健品或药物为酚苷混合物与保健品中或药物中可接受的辅料组成的组合物。
优选地，所述保健品或药物的制剂为汤剂、颗粒剂、散剂、胶囊剂、片剂或口服液。
优选地，所述独一味总酚苷提取物可以制成口服剂型和非口服剂型。
本发明的目的之二是提供一种独一味总酚苷提取物的提取方法，包括以下步骤：称取独一味地上部分药材，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过处理的聚酰胺吸附树脂床吸附，吸附后先用聚酰胺吸附树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后干燥，得到所述独一味总酚苷提取物。
本发明的目的之二是提供一种独一味总酚苷提取物的提取方法，包括以下步骤：称取独一味地上部分药材，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过Bio-Gel P-2聚丙烯酰胺凝胶树脂床吸附，吸附后先用Bio-Gel P-2聚丙烯酰胺凝胶树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后干燥，得到所述独一味总酚苷提取物；所述Bio-Gel P-2聚丙烯酰胺凝胶树脂的粒径为45～90μm。
优选地，上述提取方法中获得的所述独一味总酚苷提取物中含有毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoylverbascoside、 大波斯菊苷、木犀草素和芹菜素8种酚苷成分，且8种酚苷成分的液相色谱-质谱的离子峰面积之和占所述独一味总酚苷提取物全部峰面积总和的比例为≥60％。
所述独一味总酚苷提取物，以及由上述8种酚苷成分中任意2种或2种以上组成的酚苷组合物均可抑制肝星状细胞(LX-2)细胞的增殖，并促进TGF-β活化后LX-2细胞的凋亡。所述独一味总酚苷提取物还可显著减轻CCl 4小鼠肝组织炎症反应、肝纤维化程度，H&E染色、天狼星红及马松染色，模型动物肝组织的纤维化程度减弱，降低模型动物血浆中ALT和AST。所述独一味总酚苷提取物通过可显著降低肝组织中Smad2、3、4mRNA和蛋白水平的表达，促进Smad7mRNA和蛋白水平(Smad家族抑制剂)的表达，进而抑制TGF-β/Smad信号通路过度激活，并减少纤连蛋白(FN)、α-平滑肌肌动蛋白(α-SMA)和胶原-α1(Coll-α1)等细胞外基质的分泌，最终防治肝纤维化的发生发展。
本发明明确独一味总酚苷提取物具有抗肝纤维化的作用，并明确8种酚苷成分(毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoylverbascoside、大波斯菊苷、木犀草素和芹菜素)的组合物具有抗肝纤维化作用，它们可用于制备预防或治疗肝纤维化保健品或药物。
本发明所述独一味总酚苷提取物的提取方法采用Bio-Gel P-2聚丙烯酰胺凝胶树脂，Bio-Gel P-2聚丙烯酰胺凝胶树脂内部为网状结构，粒径为45～90μm，相同柱床体积，其吸附载量较普通不规则聚酰胺颗粒状(内部为实心结构)大，因此对溶液中分类成分吸附更充分，相应的，所述独一味总酚苷提取物的得率更高；又由于Bio-Gel P-2聚丙烯酰胺凝胶树脂为球状颗粒，分离纯化过程中，药液更易通过树脂床，不宜堵塞，可保障纯化过程的顺利进行。
说明书附图
图1 ESI正离子模式独一味总酚苷提取物UPLC-TOF/MS色谱图；
图2独一味总酚苷提取物UPLC-TOF/MS图谱鉴定的化合物结构；
图3独一味总酚苷提取物及八种酚苷的酚苷组合物对肝星状细胞增殖的影响；
图4独一味总酚苷提取物及八种酚苷的酚苷组合物对TGF-β活化肝 星状细胞的影响；
图5独一味总酚苷提取物对CCl 4肝纤维化模型的影响；
图6独一味总酚苷提取物对CCl 4模型小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)影响；
图7独一味总酚苷提取物对模型小鼠肝脏中TGF-β/Smad信号通路的影响。
具体实施方式
以下结合实施例对本发明进一步说明，但本发明并不局限于这些实施例。在不脱离本发明原理的前提下，还可以做出若干变型和改进，这些也应视为属于本发明的保护范围。
实施例一：本发明独一味总酚苷提取物的提取制备与UPLC-TOF/MS分析
1、独一味总酚苷提取物的提取制备
方法一：称取独一味地上部分5kg，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过处理的聚酰胺(10～30目)吸附树脂床吸附，吸附后先用吸附树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后，干燥得到独一味总酚苷提取物203.6g。
方法二：称取独一味地上部分5kg，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过Bio-Gel P-2聚丙烯酰胺凝胶树脂床(粒径45～90μm)吸附，吸附后先用Bio-GelP-2聚丙烯酰胺树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后，干燥得到独一味总酚苷提取物248.9g。
2、独一味总酚苷提取物的UPLC-TOF/MS分析
(1)色谱及质谱条件
①色谱条件:Waters Acquity UPLC BEH Cl8 column柱(100mm×2.1mm,1.8μm)，柱温25℃，流速为0.2mL/min，进样量2μL，流动相为甲醇(A)和0.01甲酸％水(B)，梯度洗脱程序：0～30min，20～70％A。
②质谱条件:TOF MS-MS/MS模式，电喷雾离子源(ESI)，在正离子检 测模式下采集，正离子:喷雾电压(IS)～5500eV，去簇电压(DP)～100eV，碰撞电压(CE)～10eV；质量数扫描范围m/z 100～1500Da。
(2)供试品的制备
称取方法二提取制备的独一味总酚苷提取物12.14mg置于10mL容量瓶中，加55％的甲醇/水稀释至刻度并摇匀，即得供试品溶液。
(3)结果分析
通过ESI正离子模式检测供试品溶液，共检测并鉴定出16个组成成分，如图1中1～16峰，成分分别为1：Decaffeoylverbascoside，2：4-羟基苯甲酸，3：淫羊藿苷H1，4：Campneoside II，5：连翘酯苷C，6：连翘酯苷B，7：Leuteolin-7-O-[β-D-apiose(6→1)]-β-glucoside，8：异毛蕊花糖苷，9：毛蕊花糖苷，10：木犀草苷，11：大波斯菊苷，12：Dehydroacteoside，13：LeucosceptosideA，14：木犀草素，15：芹菜素，16：Apigenin7-O-(6'-(E)-p-coumaroyl-β-D-galactopyranoside，其对应的化合物的结构如图2所示，其中黄酮类化合物6种，主要包括：木犀草苷、大波斯菊苷、木犀草素和芹菜素等；苯乙醇苷类化合物7种，主要包括：连翘酯苷B、连翘酯苷C、毛蕊花糖苷、异毛蕊花糖苷和Dehydroacteoside等；有机酚酸及其苷类3个，为4-羟基苯甲酸、淫羊藿苷H1和CampneosideII。
依据各化合物在独一味总酚苷提取物液相色谱-质谱的色谱离子峰的面积比例，最终确定其标志性成分8种，为：毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoylverbascoside、大波斯菊苷、木犀草素和芹菜素，8种标志性成分占液相色谱-质谱的离子峰全部峰面积总和的60％以上。对方法一获得的独一味总酚苷提取物也进行液相色谱-质谱检测，同样含有上述8种标志性成分，且占液相色谱-质谱的离子峰全部峰面积总和的60％以上。
下面将对独一味总酚苷提取物以及由这些标志性成分的成品所组成的组合物进行抗肝纤维化实验。
实施例二：独一味总酚苷提取物和酚苷组合物对肝星状细胞增殖和凋亡的影响
1、溶液配置
(1)独一味总酚苷提取物溶液制备
称取实施例1中方法二提取制备的20.0mg干燥的独一味总酚苷提取物置于10mL容量瓶中，加DMSO 10mL至刻度并摇匀，得到独一味总酚苷提取物溶液准备液，浓度为2.0mg/mL。
将独一味总酚苷提取物溶液准备液稀释为不同浓度的独一味总酚苷提取物溶液：10μg/mL、25μg/mL、50μg/mL、100μg/mL、200μg/mL。
(2)酚苷组合物溶液的配制
实验药品：毛蕊花糖苷、连翘酯苷B、淫羊藿苷H1和Decaffeoylverbascoside为本实验室分离纯化所得，经紫外、红外、核磁和质谱检测，确定其结构，经HPLC检测纯度≥98％。木犀草素、芹菜素和4-羟基苯甲酸均购于上海麦克林生化试剂有限公司，纯度均≥98％；大波斯菊苷购于成都曼斯特生物科技有限公司，纯度≥98％。
参考实施例1提取的独一味总酚苷提取物中的八种酚苷之间的离子峰面积之比，分别精密称取毛蕊花糖苷25.6mg、连翘酯苷B12.2mg、4-羟基苯甲酸6.7mg、淫羊藿苷H14.7mg、Decaffeoylverbascoside 3.8mg、大波斯菊苷3.3mg、木犀草素2.9mg和芹菜素2.7mg，置于10mL容量瓶中，加DMSO 10mL至刻度并摇匀，得到酚苷组合物溶液准备液，浓度为6.19mg/mL。
将酚苷组合物溶液准备液稀释为不同浓度的酚苷组合物溶液：8μg/mL、16μg/mL、31μg/mL、62μg/mL、124μg/mL。
2、独一味总酚苷提取物和酚苷组合物对肝星状细胞增殖的影响
实验分组及药物干预情况如表1：
表1实验分组及药物干预情况
编号 组别 给药 浓度
N 空白组 DMSO 0.1％
TPLR 独一味提取物组 独一味总酚干预48h 10～200μg/mL
MIX 酚苷组合物组 酚苷组合物干预48h 8～124μg/mL
LX-2细胞(人半活化肝星状细胞)正常传代分板，贴壁生长12h后，加入不同浓度的独一味总酚苷提取物、不同浓度和组成的酚苷组合物溶 液，干预48h后，CCK-8试剂盒检测分析对LX-2细胞增殖情况的影响。
结果如图3所示，独一味总酚苷提取物和酚苷组合物可有效抑制LX-2细胞的增殖，其中，独一味总酚苷提取物200μg/mL抑制效果最明显(P<0.001，与空白组比较)，它们的半抑制浓度(IC 50)略有区别，其中，独一味总酚苷提取物的IC 50值为78.1μg/mL，酚苷组合物的IC 50值为80.8μg/mL。
3、独一味总酚苷提取物及酚苷组合物对TGF-β活化肝星状细胞凋亡的影响
活化的肝星状细胞(HSC)发生凋亡被认为是肝纤维化逆转、ECM降解过程中重要的形成机制，现应用流式技术检测药物干预后的细胞凋亡情况。
实验分组及药物干预情况如下表：
LX-2细胞正常传代分板，贴壁生长12h后，加入10ng/mL的TGF-β激动12h，加入不同浓度的独一味总酚苷提取物和不同浓度的酚苷组合物溶液，AnnexinV/PI CellApoptosis Detection试剂盒检测TGF-β活化后LX-2细胞的凋亡情况。
实验结果显示，不同浓度的独一味总酚苷提取物和酚苷组合物均可有效促进TGF-β活化后LX-2细胞的凋亡。其中，100μg/mL的独一味总酚苷提取物，可诱导18.8％的活化LX-2细胞凋亡，62.0μg/mL酚苷组合物干预后，可诱导18.2％的活化LX-2细胞凋亡，如图4所示。
CCK-8和流式细胞仪检测实验结果，均明确独一味总酚苷提取物体外可抑制肝星状细胞(LX-2)增殖，促进肝星状细胞凋亡。同时，依据独一味总酚苷提取物的UPLC-TOF/MS分析实验结果，按照八个标志性成分所占液相色谱-质谱的离子峰全部峰面积的比例，选取对应的化合物单体，配制成的酚苷组合物，同样对肝星状细胞具有抑制增殖和促进凋亡的作用；实验发现，独一味总酚苷提取物和八种酚苷的酚苷组合物组，均可有效促进TGF-β活化后LX-2细胞的凋亡，但独一味总酚苷提取物效果更好；均可抑制肝星状细胞(LX-2)增殖，其中，独一味总酚苷提取物的IC 50值为78.1μg/mL，略低于标志性成分混合物组(IC 50＝80.8μg/mL)，表明独一味总酚苷提取物的药理活性略优于八种酚苷的酚苷组合物组。依据上述试验 结果，最终选取独一味总酚苷提取物进行整体动物实验，深入挖掘独一味总酚苷提取物抗肝纤维化的药理作用和作用机制。
实施例三：独一味总酚苷提取物对CCl 4肝纤维化模型的影响
将C57雄性小鼠(体重在22～25g)随机分为对照组、CCl 4诱导组、独一味总酚苷提取物高、中、低(50mg/kg、100mg/kg、200mg/kg)剂量组，每组10只。除正常对照组外，CCl 4用玉米油配制成浓度为10％，CCl 4玉米油溶液腹腔注射0.5mL/kg，每周一、四造模，持续6周。独一味总酚苷提取物给药组在第一次CCl 4注射后伴随CCl 4注射后给药。
CCl 4诱导后2天，收取肝组织，进行病理切片HE染色。结果显示正常组肝小叶结构清晰，肝板排列整齐呈条索状，汇管区无扩大，无炎症细胞浸润；模型组可见大量炎症细胞浸润，肝细胞肿胀坏死；各用药组与模型组相比，均有不同程度减轻，其中，独一味总酚苷高剂量组与改善更为显著(图5中的A)。
独一味总酚苷提取物对CCl 4模型小鼠肝脏胶原纤维化和沉积的影响：天狼星红(Sirius red)染色是胶原纤维特异性染色，将石蜡切片用天狼星红染色，观察小鼠肝脏组织纤维化情况。结果显示，正常组除血管壁外，肝窦有少量表达；模型组可见汇管区大量胶原沉积，纤维间隔形成、增粗并包绕成假小叶；各用药组与模型组相比，均有不同程度减轻，其中，独一味总酚苷高剂量组与改善更为显著(图5中的B)。
Masson染色是胶原用来显示组织中纤维以及炎性因子的特异性染色，独一味总酚苷干预前后，模型组小鼠肝脏组织中，可见大面积蓝色阳性区域；各用药组与模型组相比，蓝色阳性区域面积均有不同程度减小，其中，独一味总酚苷高剂量组与改善更为显著(图5中的C)，表明独一味总酚苷提取物组可降低CCl 4肝脏组织的胶原沉积，并降低此过程中伴随的炎症反应。
独一味总酚苷提取物对CCl 4模型小鼠肝脏形态的影响：空白组小鼠的肝脏暗红色，表面未见有明显结节，边缘光滑较锐巧，质地柔软。模型组小鼠的肝脏暗褐色，体积缩小，有颗粒状物，表面粗糙，边缘变钝，色泽变暗，质地硬。独一味总酚苷各给药组：肝脏颜色呈暗红色，表面结节较模型组减少，边缘基本正常，有颗粒状物，但与模型组比较，颗粒状物 体积缩小、数量明显减少(图5中的D)。总之独一味总酚苷干预后，小鼠肝脏组织色泽、体积、硬度及结节样改变等情况均有不同程度的改善。
独一味总酚苷提取物对CCl 4模型小鼠肝脏功能的影响：独一味总酚苷干预前后，试剂盒检测小鼠血浆中谷丙转氨酶(ALT)、谷草转氨酶(AST)的活力值。与正常对照组相比，模型组血清ALT、AST活性显著升高(P<0.001)；与模型组相比，独一味总酚苷提取物组均能显著降低血清ALT、AST活性(图6)。其中，独一味总酚苷高剂量组与改善更为显著。
独一味总酚苷提取物对模型小鼠肝脏中TGF-β/Smad信号通路的影响：
1)独一味总酚苷提取物对模型小鼠肝脏ECM沉积的影响。
收取小鼠肝组织，采用免疫组织化学染色(Immunohistochemistry assay,IHC)技术，检测小鼠肝脏TGF-β、FN、α-SMA和Col1-α1等细胞外基质沉积的变化。如图7中的A染色结果显示，CCl 4肝纤维化模型组TGF-β、FN、α-SMA和Col1-α1染色明显高于正常对照组。独一味总酚苷各剂量组干预后，肝脏组织中染色区域比模型组减少明显，说明独一味总酚苷能抑制小鼠肝脏组织中TGF-β、FN、α-SMA和Col1-α1表达，抑制肝纤维化后病变部位的细胞外基质沉积。
2)Western blotting方法检测小鼠肝脏中Smad家族蛋白表达水平变化。
独一味总酚苷干预前后，收取小鼠肝组织，采用Western blotting方法检测肝脏组织中TGF-β下游Smad蛋白家族的表达。如图7中的B，与正常对照组相比，模型组肝脏组织中TGF-β信号通路下游Smad 2、3、4蛋白水平表达显著升高(P<0.001)，Smad家族主要抑制转导分子Smad 7的蛋白水平表达显著降低；与模型组相比，独一味总酚苷提取物组均能显著降低肝脏组织中Smad 2、3、4蛋白水平的表达，升高Smad 7蛋白水平的表达。其中，独一味总酚苷高剂量组的抑制作用更为显著，结果表明，独一味总酚苷干预后，可抑制模型动物肝脏中TGF-β/Smad信号通路，延缓肝纤维化发生。
3)实时定量PCR方法检测小鼠肝脏中Smad家族基因表达水平变化。
独一味总酚苷干预前后，收取小鼠肝组织，参考文献中的方法和引物 (JinfengLiu et al.Praziquantel ameliorates CCl 4-induced liver fibrosis in mice byinhibiting TGF-β/Smad signalling via up-regulating Smad7 in hepatic stellatecells.Br J Pharmacol.2019,176:4666–4680)，检测肝脏组织中TGF-β下游Smad蛋白家族的基因表达水平变化。如图7中的C～G的qRT-PCR结果，与正常对照组相比，模型组肝脏组织中TGF-β信号通路下游Smad 2、3、4和FN的mRNA表达显著升高水平显著升高(P<0.001)，Smad7mRNA的表达水平显著降低；与模型组相比，独一味总酚苷提取物组均能显著降低肝脏组织中Smad 2、3、4和FN的mRNA表达水平表达，升高Smad 7mRNA表达水平，量效关系良好。其中，独一味总酚苷高剂量组的抑制作用更为显著，实验结果从基因水平证实了，独一味总酚苷可抑制模型动物肝脏中TGF-β/Smad信号通路，延缓肝纤维化发生。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (13)

  1. 酚苷混合物在制备预防或治疗肝纤维化保健品或药物中的应用，所述酚苷混合物含有毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoyl verbascoside、大波斯菊苷、木犀草素和芹菜素8种酚苷成分中的任意2种或2种以上。
  2. 如权利要求1所述的应用，其特征在于，所述酚苷混合物为独一味总酚苷提取物。
  3. 如权利要求2所述的应用，其特征在于，所述独一味总酚苷提取物含有毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoyl verbascoside、大波斯菊苷、木犀草素和芹菜素8种酚苷成分；所述毛蕊花糖苷、所述连翘酯苷B、所述4-羟基苯甲酸、所述淫羊藿苷H1、所述Decaffeoyl verbascoside、所述大波斯菊苷、所述木犀草素和所述芹菜素8种成分的液相色谱-质谱的离子峰面积之和占所述独一味总酚苷提取物的全部峰面积总和的比例为≥60％。
  4. 如权利要求3所述的应用，其特征在于，所述独一味总酚苷提取物的提取方法包括以下步骤：称取独一味地上部分药材，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过处理的聚酰胺吸附树脂床吸附，吸附后先用聚酰胺吸附树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后干燥，得到所述独一味总酚苷提取物。
  5. 如权利要求3所述的应用，其特征在于，所述独一味总酚苷提取物的提取方法包括以下步骤：称取独一味地上部分药材，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过Bio-Gel P-2聚丙烯酰胺凝胶树脂床吸附，吸附后先用Bio-Gel P-2聚丙烯酰胺凝胶树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后干燥，得到所述独一味总酚苷提取物；所述Bio-Gel P-2聚丙烯酰胺凝胶树脂的粒径为45～90μm。
  6. 如权利要求2所述的应用，其特征在于，所述独一味总酚苷提取物的质量浓度为10～200μg/mL。
  7. 如权利要求1所述的应用，其特征在于，所述酚苷混合物由6～10重量份的毛蕊花糖苷、3～5重量份的连翘酯苷B、2～3重量份的4-羟基苯甲酸、1.5～2重量份的淫羊藿苷H1、1～1.5重量份的Decaffeoyl verbascoside、1～1.5重量份的大波斯菊苷、1～1.5重量份的木犀草素和1～1.5重量份的芹菜素组成。
  8. 根据权利要求7所述的应用，其特征在于，所述酚苷混合物的质量浓度为8～124μg/mL。
  9. 如权利要求1所述的应用，其特征在于，所述保健品或药物为酚苷混合物与保健品中或药学上可接受的辅料组成的组合物。
  10. 如权利要求1所述的应用，其特征在于，所述保健品或药物的制剂为汤剂、颗粒剂、散剂、胶囊剂、片剂或口服液。
  11. 一种独一味总酚苷提取物的提取方法，包括以下步骤：称取独一味地上部分药材，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过处理的聚酰胺吸附树脂床吸附，吸附后先用聚酰胺吸附树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后干燥，得到所述独一味总酚苷提取物。
  12. 一种独一味总酚苷提取物的提取方法，包括以下步骤：称取独一味地上部分药材，粉碎，加入药材重量10倍体积的水，煎煮2次，每次2h，过滤后合并滤液，静置12h，取上清液通过Bio-Gel P-2聚丙烯酰胺凝胶树脂床吸附，吸附后先用Bio-Gel P-2聚丙烯酰胺凝胶树脂床5倍量的水洗脱，再用75％的乙醇洗脱解析，收集解析液，回收溶剂，在90℃条件下浓缩后干燥，得到所述独一味总酚苷提取物；所述Bio-Gel P-2聚丙烯酰胺凝胶树脂的粒径为45～90μm。
  13. 如权利要求12所述的方法，其特征在于，所述独一味总酚苷提取物中毛蕊花糖苷、连翘酯苷B、4-羟基苯甲酸、淫羊藿苷H1、Decaffeoyl verbascoside、大波斯菊苷、木犀草素和芹菜素8种酚苷成分的液相色谱-质谱的离子峰面积之和占所述独一味总酚苷提取物全部峰面积总和的比例为≥60％。
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