WO2019241901A1 - 一种基于肝-肠轴的护肝型肠道微生态调节剂及其制备方法 - Google Patents

一种基于肝-肠轴的护肝型肠道微生态调节剂及其制备方法 Download PDF

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WO2019241901A1
WO2019241901A1 PCT/CN2018/000264 CN2018000264W WO2019241901A1 WO 2019241901 A1 WO2019241901 A1 WO 2019241901A1 CN 2018000264 W CN2018000264 W CN 2018000264W WO 2019241901 A1 WO2019241901 A1 WO 2019241901A1
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extract
liver
parts
extraction
intestinal
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PCT/CN2018/000264
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English (en)
French (fr)
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贾福怀
晏永球
涂宏建
王俊
陶刚
熊菲菲
王彩霞
袁媛
许璐云
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宁波御坊堂生物科技有限公司
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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)
    • A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • 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)
    • 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)
    • A61K36/64—Orobanchaceae (Broom-rape family)
    • 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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • 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)
    • A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8969—Polygonatum (Solomon's seal)
    • 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
    • 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
    • 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
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • 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
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the invention relates to the field of biotechnology, in particular to a liver-gut axis-based hepatoprotective intestinal micro-ecological regulator and a preparation method thereof.
  • the liver is an organ whose main function is material metabolism and detoxification
  • the intestine is an organ whose main function is digestion and absorption of nutrients. It is generally believed that the two are independent and do not interfere with each other. With the development of science and technology and the continuous deepening of research, more and more evidence shows that the biological function of the liver and intestine are closely related and affect each other, and the theory of "liver-gut axis" was born. At the embryonic stage, the liver and intestines originate from the foregut. After maturation, the two organs are mainly related to each other through the portal vein: about 70% of the blood supply in the liver comes from the portal vein, and intestinal venous return is the main source of portal blood.
  • the liver can not only remove various toxins and intestinal-derived microorganisms from the intestine, but also regulate the structural function of the intestinal flora through bile acid secretion and enterohepatic circulation, and maintain the intestinal microecological balance, while the intestinal flora
  • Primary bile acids that can be excreted by the liver into the intestinal tract are converted into secondary bile acids, which are then reabsorbed by the intestinal wall.
  • Reusable bile acids can also stimulate bile secretion, clear the bile duct, and eliminate harmful mycotoxins and bacterial endotoxins. Substances play a role in protecting the liver and gall.
  • the intestinal microecology plays a vital role in the pathophysiological process of the liver, and when suffering from liver disease, it will in turn affect the structure and function of the intestinal microecology.
  • intestinal microorganisms can pass through the intestinal mucosal barrier through the portal vein to reach the liver, but can activate the liver reticular endothelial system and keep it in an activated state.
  • the intestinal mucosal barrier may be damaged, a large number of intestinal microorganisms and toxins will be transferred to the liver, destroy the liver's immune tolerance, and will over-activate the liver's inherent immune system to produce an inflammatory cascade.
  • the reaction causes bacteremia and endotoxemia, and induces liver damage.
  • liver When the liver is damaged and pathological changes occur, it will lead to a decline in its synthetic function, a sharp decrease in albumin in the plasma, and easy to cause fluid accumulation in the abdominal cavity.
  • the liver does not secrete bile, which results in a lack of bile salts in the intestine, which in turn causes portal hypertension.
  • Gastrointestinal congestion and hypoxia lead to portal hypertension gastrointestinal disease; reduced liver synthetic coagulation factors reduce the severity of gastrointestinal bleeding and further worsen the intestinal microecological balance.
  • the intestinal microecology is closely related to the liver, and the intestinal microecology disorders and liver diseases are causal to each other, and then cause a series of pathological changes in the body.
  • the close relationship between the two also suggests that when the liver disease occurs, attention should be paid to the intestinal microecological conditioning, and when the intestinal microecology is imbalanced, the protection of the liver should also be paid attention to. This provides a new idea for the research and prevention of intestinal microecological disorders and the pathogenesis of liver diseases.
  • liver-protecting intestinal microecological regulator based on this, which is safe and efficient, has small toxic and side effects, and is of stable and controllable quality, is an important issue that needs to be solved urgently.
  • the technical problem to be solved by the present invention is to address the current state of the art, and provide a safe and efficient, small toxic and side effects, stable and controllable quality, does not produce resistance and resistance, and does not accumulate in the body to protect the liver.
  • liver-gut axis-based intestinal micro-ecological regulator that regulates the micro-ecological function of the intestine.
  • Another technical problem to be solved by the present invention is to provide a method for preparing the liver-gut axis-based intestinal micro-ecological regulator based on the current state of the art in view of the current state of the art.
  • the main active ingredient can improve the yield of the extract, and can also effectively reduce the extraction time and energy consumption.
  • a liver-gut axis-based hepatoprotective intestinal micro-ecological regulator characterized in that the raw materials for preparing the regulator include the following components by weight
  • the conditioner is made of the following raw materials in parts by weight: 210 parts of groundweed grass, 165 parts of dentate snake grape leaf, 240 parts of raspberry, 210 parts of purslane, 140 parts of huangjing, 100 parts of cistanche, 16 parts of yeast ⁇ -glucan, 24 parts of fructooligosaccharide, and 30 parts of soybean oligosaccharide.
  • the conditioner includes Dioscorea chinensis extract, Dendrobium variabilis leaf extract, raspberry extract, purslane extract, Polygonatum extract, Cistanche extract, yeast ⁇ -glucan, oligomeric Fructose and soy oligosaccharides.
  • the regulator of the present invention uses the above-mentioned components as the main active substance. At the same time, it can also be preferably added with suitable auxiliary materials and prepared into any form suitable for oral administration, such as tablets, capsules, granules, and the like using conventional techniques in formulation. Agent, powder, pill or oral solution.
  • the extracts of Radix Caryophylla, grape leaf extract, and raspberry are all obtained by flash extraction, co-vacuum decompression, and post-processing.
  • the purslane extract , Polygonatum extract, Cistanche extract are obtained through post-treatment by mechanochemical cooperative ultrasonic assisted method.
  • flash extraction, combined vacuum decompression method and mechanochemical coordinated ultrasonic-assisted method to extract different types of components not only can retain the main active ingredients to the maximum extent, improve the yield of the extract, but also effectively reduce the extraction time and energy consumption. .
  • a method for preparing the above-mentioned liver-gut axis-based liver-protecting intestinal micro-ecological regulator includes the following steps;
  • the Dioscorea chinensis extract, the grape leaf extract of S. dentata, and the raspberry extract are all obtained through the following steps:
  • (B) Vacuum decompression extraction Open the communication valve between the flash extractor and the vacuum decompression extractor, and the material liquid enters the vacuum decompression extractor for extraction under the pressure difference until the extraction liquid is obtained;
  • the extraction temperature of the vacuum reduced pressure extraction is 40 to 65 ° C
  • the extraction pressure is 0.05 to 0.1MPa
  • the extraction time is 20 to 40min
  • the concentration concentration of the reduced pressure concentration is 50 ° C
  • the vacuum degree is controlled at -0.05.
  • ⁇ -0.08Mpa the relative density is controlled between 1.05 and 1.15.
  • the spray drying process parameters include a nozzle temperature of 160 to 180 ° C, an outlet temperature of 70 to 85 ° C, and a feeding speed of 10 to 20 mL / min.
  • the purslane extract, Huangjing extract, and Cistanche extract are obtained by the following steps:
  • the extraction temperature of the ultrasonic-assisted extraction is 40 to 80 ° C.
  • the extraction time is 20 to 80 min
  • the ultrasonic power is 100 to 500 W.
  • the regulator of the present invention a variety of natural products are scientifically and reasonably compatible, synergistically synergistically, while protecting the liver, promote intestinal micro-ecological regulation.
  • Diospyros chinensis and purslane are used in the same liver and large intestine meridian. They are used together to cool the blood and detoxify and treat diarrhea, especially for various intestinal tracts such as E. coli, dysentery, salmonella, shigella, staphylococcus
  • the pathogenic bacteria have a strong inhibitory effect, and when combined with the grape leaf of S. dentata, it further strengthens the inhibitory effect on pathogenic bacteria such as E.
  • coli and Staphylococcus aureus and yeast ⁇ -glucan, fructooligosaccharide, soybean Intestinal probiotics activated proliferation factors such as oligosaccharides can effectively promote the proliferation of probiotics, and can effectively inhibit the generation of intestinal spoilage substances and promote their excretion.
  • the organic combination of the above raw materials can effectively maintain the intestinal microecological balance; meanwhile, The combined use of Diospyros chinensis, S.
  • Cistanche deserticola has a strong anti-oxidation function, which reduces the generation of free radicals, thereby reducing the damage of free radicals on liver tissue and mitochondria, and thereby improving the antioxidant capacity of the liver.
  • Reduce lipid peroxidation, synergistic effect between the two and Huang Jing, can significantly enhance the body's ability to scavenge free radicals and lipid peroxides in cells, and cooperate with yeast ⁇ -glucan to promote immune cell development Specific binding can stimulate the immune activity of lymphocytes and macrophages in the body, effectively improve the body's immune capacity, promote the recovery of damaged tissues and organs such as the liver, and then improve the intestinal microecological balance via the liver-gut axis.
  • the present invention uses a variety of natural products in an organic combination to synergize from the microbial level, the cell level and the metabolic level to promote the excretion of liver toxins, repair damaged liver tissue, protect the liver, and at the same time can effectively regulate the intestinal microecological balance of the body And improve intestinal health.
  • composition of the present invention has significantly improved curative effect.
  • This group of contents is proposed under the guidance of the traditional Chinese medicine health care theory and modern medical research "liver-gut axis" theory. Yes, the components cooperate with each other for synergies, not just the superposition of individual substances;
  • the liver-protecting intestinal micro-ecological regulator of the present invention is mainly composed of traditional Chinese medicine and prebiotics of natural origin, and is basically a medicine-food homologue or a new food raw material. It is safe and has no toxic or side effects, and does not produce tolerance or resistance. Does not accumulate in the body, protects the liver and regulates the intestinal microecological function;
  • the flash extraction, the vacuum decompression method, and the mechanochemical coordinated ultrasonic-assisted method are used to extract different types of components, which can not only retain the main active components to the maximum extent, improve the yield of the extract, but also effectively Reduce extraction time and energy consumption.
  • the liver-gut axis-based intestinal micro-ecological regulator based on liver-gut axis in this embodiment is prepared from the following raw materials: 150 parts of gynospermum, 130 parts of grape leaf of dentate snake, 200 parts of raspberry, and purslane 150 parts, 120 parts of Huangjing, 60 parts of Cistanche, 8 parts of yeast ⁇ -glucan, 15 parts of fructooligosaccharides, and 20 parts of soybean oligosaccharides.
  • liver-gut axis-based liver-protecting intestinal micro-ecological regulator is as follows:
  • the liver-gut axis-based intestinal microecological regulator based on liver-gut axis in this embodiment is prepared using the following raw materials: 180 parts of gynospermum chinensis, 150 parts of vine leaf, 220 parts of raspberry, and purslane 180 parts, 140 parts of Huang Jing, 80 parts of Cistanche, 13 parts of yeast ⁇ -glucan, 20 parts of fructooligosaccharides, and 40 parts of soy oligosaccharides.
  • liver-gut axis-based liver-protecting intestinal micro-ecological regulator is as follows:
  • the extract was spray-dried, and the nozzle temperature was 165 ° C and the outlet temperature was 75. °C, feeding speed of 14mL / min, to obtain Dioscorea chinensis extract or Dendrobium variegata grape leaf extract or raspberry extract;
  • the liver-gut shaft-based intestinal micro-ecological regulator based on liver-gut axis in this embodiment is prepared using the following raw materials: 210 parts of Dianthus chinensis, 165 parts of Vitis variabilis leaves, 240 parts of raspberries, and purslane 210 parts, 140 parts of Huangjing, 100 parts of Cistanche, 16 parts of yeast ⁇ -glucan, 24 parts of fructooligosaccharides, and 30 parts of soybean oligosaccharides.
  • liver-gut axis-based liver-protecting intestinal micro-ecological regulator is as follows:
  • the concentration temperature was 60 °C, vacuum degree is controlled at -0.05 ⁇ -0.08Mpa, relative density is controlled at 1.05 ⁇ 1.15, extract is obtained; the extract is spray-dried, nozzle temperature is 160C, outlet temperature is 60 °C, feeding speed is 10mL / min, horse is obtained Fructus Jacaranda extract or Polygonatum extract or Cistanche extract;
  • the liver-gut axis-based intestinal microecological regulator based on the liver-gut axis in this embodiment is prepared using the following raw materials: 280 parts of Dianthus chinensis, 210 parts of grape leaves of Vigna dentata, 260 parts of raspberries, and Portulaca 280 parts, 170 parts of Huang Jing, 140 parts of Cistanche, 20 parts of yeast ⁇ -glucan, 45 parts of fructooligosaccharides, and 50 parts of soy oligosaccharides.
  • liver-gut axis-based liver-protecting intestinal micro-ecological regulator is as follows:
  • the extract was obtained; the extract was spray-dried, the nozzle temperature was 175 ° C, and the outlet temperature was 80 °C, feeding speed of 18mL / min, to obtain Dioscorea chinensis extract or Dendrobium grape leaf extract or raspberry extract;
  • the liver-gut axis-based intestinal micro-ecological regulator based on the liver-gut axis in this embodiment is prepared using the following raw materials: 210 parts of Diospyros chinensis, 165 parts of the grape leaf of the snake tooth, 240 parts of the raspberry, and purslane 210 parts, 140 parts of Huangjing, 100 parts of Cistanche, 16 parts of yeast ⁇ -glucan, 24 parts of fructooligosaccharides, and 30 parts of soybean oligosaccharides.
  • liver-gut axis-based liver-protecting intestinal micro-ecological regulator is as follows:
  • step (2) Add the yeast ⁇ -glucan, fructooligosaccharide and soybean oligosaccharides with corresponding weight components to the mixed extract prepared in step (1), and mix evenly to obtain liver protection based on the liver-gut axis. Intestinal microecological regulator.
  • the present invention performs a taking effect test on the regulators prepared in the embodiments, and is specifically an evaluation test of the efficacy of a liver-protecting intestinal microecological regulator based on a mouse model of intestinal microecological disorders with acute liver injury.
  • Test method 130 Kunming mice, weighing 20 ⁇ 2g, both male and female. After 1 week of adaptive feeding, they were randomly divided into 13 groups, 10 in each group, which were the normal control group, model control group, and Lizhu Changle group.
  • mice in each group were administrated with lincomycin, 0.3 mL / time, twice a day for three consecutive days, and mice in each group were injected intraperitoneally with 10% D-amino acid.
  • Galactose was administered at a dose of 0.8 g / kg to prepare a mouse model of acute liver injury accompanied by intestinal microecological disorders.
  • the mice in the normal control group and the model control group were intragastrically administered with an equal volume of normal saline.
  • the Lizhu Changle group was intragastrically administered with a Lizhu Changle solution of 50 million live bacteria / mL, and the dosage was 0.3.
  • the Baogan tablet group, the groups of Examples 1, 2, 4, and 5 and the dose group, extract group, and prebiotic group of Example 3 were all dissolved in physiological saline at a dose of 10.0 g / kg and then administered.
  • Stomach, the low-dose group of Example 3 and the high-dose group of Example 3 were dissolved with physiological saline at a dosage of 5.0 g / kg and 20.0 g / kg, respectively, and administered to the stomach once a day for seven consecutive days.
  • Mice were sacrificed by cervical dislocation method. All the mice were removed from the eyeballs to take blood, and the serum was separated.
  • ALT serum alanine aminotransferase
  • AST glutathraninase
  • the suspension was shaken on a shaker for 15 minutes to prepare a suspension.
  • the supernatant was collected after centrifugation to obtain intestinal bacteria.
  • the intestinal bacteria solution was serially diluted with sterile physiological saline, and selective culture of bifidobacteria, lactobacillus, enterobacter, and enterococcus was performed respectively. After the culture, the colony count was performed. The result was based on the number of colonies per gram of feces. Numeric representation.
  • mice in the control group were in good mental state during the test, their hair was shiny, their food, water and feces were normal, and they were sensitive to external stimuli. After gavage with lincomycin, the mice became mentally sick and acted. Slow down, food intake, water consumption, and wet stool. After intraperitoneal injection of D-galactosamine, the mice became debilitated, tired, lazy, rough, dull and dull, with hair loss, and the condition of wet stool became worse. . After seven days of treatment, the positive control group and each administration group improved to varying degrees, and their mobility, coat status, food intake, water intake, and dryness of the feces all improved to varying degrees.
  • Example 3 In the prebiotic group, although endotoxin, ALT, AST, and MDA all showed a decreasing trend, the difference was not significant (P> 0.05); SOD showed an upward trend, but the difference was not significant (P ⁇ 0.01), suggesting that the intestine was adjusted separately The micro-ecology helps to improve liver damage to a certain extent; the groups of Example 1, Group 2, Example 3, different dose groups of Example 3, extracts of Example 3, groups of Example 4 and groups of Example 5 are small Rat plasma endotoxin, ALT, AST, and MDA were all significantly reduced (P ⁇ 0.05, P ⁇ 0.01), and SOD was significantly increased (P ⁇ 0.01), suggesting that each group can effectively improve the status of liver injury.
  • mice (3) Observe the liver tissue of each group of mice after HE staining.
  • the normal control mice had normal hepatic lobules, no swelling, steatosis, and tissue necrosis, and no inflammatory cell infiltration in the hepatic lobules and the manifold area.
  • No fibrous connective tissue hyperplasia compared with the normal group, the model control group had poorly defined hepatic lobules with focal necrosis areas, marked hepatocyte edema, diffuse balloon-like changes, and hepatocyte slices in the lobular area.
  • Hepatocytes were still seen in the extract group of Example 3 and the group of Example 5.
  • Example 3 The number of bifidobacteria and lactobacillus in the intestine of mice in the extract group and Baogan tablet group showed an upward trend, and the number of enterobacteria and enterococci showed a decreasing trend, but the difference was not significant (P> 0.05), suggesting the use of liver protection drugs To a certain extent, it helps to regulate the intestinal microecology, but the effect is not very obvious in a short time;
  • Example 4 The number of Bifidobacterium and Lactobacillus in the intestinal tract of mice in the group and the Example 5 group was significantly increased (P ⁇ 0.05, P ⁇ 0.01), and the

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Abstract

一种基于肝-肠轴的护肝型肠道微生态调节剂及其制备方法，所述调节剂包括下列重量份的原料：地锦草100-300、显齿蛇葡萄叶60-210、覆盆子100-300、马齿苋100-300、黄精60-180、肉苁蓉50-150、酵母β-葡聚糖5-20、低聚果糖10-50、和大豆低聚糖10-60。

Description

一种基于肝-肠轴的护肝型肠道微生态调节剂及其制备方法 技术领域
本发明涉及生物技术领域，具体指一种基于肝-肠轴的护肝型肠道微生态调节剂及其制备方法。
背景技术
肝脏是以物质代谢和解毒为主要功能的器官，而肠道是以消化和吸收营养为主要功能的器官，人们普遍认为二者是相互独立、互不干扰的。随着科技的发展和研究的不断深入，越来越多的证据表明肝脏和肠道在生物学功能上是密切相关、相互影响的，“肝-肠轴”的理论也由此诞生。在胚胎阶段，肝脏和肠道共同起源于前肠，成熟后两器官主要通过门静脉相互关联：肝脏中约70％的血液供养源自门静脉，而肠道静脉血回流则是门静脉血的主要来源，很多经肠道吸收的营养物质及毒素都需要依赖肝脏进行代谢。人类肠道中栖息着大量的微生物，它们与人类共同生活、进化至今，逐步形成了较为稳定的肠道微生态系统，具有维持肠上皮细胞和肠黏膜屏障完整性、促进人体营养吸收和肠道蠕动、合成多种维生素、抑制致病菌群生长、免疫调节等生理功能，是人体健康不可或缺的要素。近年来，肠道微生态与健康和疾病的关系，日益成为全球生命科学研究领域的重点和热点。
肝脏不但可以清除来自肠道的各类毒素及肠源性的微生物，还可以通过胆汁酸分泌和肠肝循环来调节肠道菌群的结构功能，维护肠道微生态平衡，而肠道菌群能将肝脏排入肠道的初级胆汁酸转变为次级胆汁酸，进而被肠壁重吸收，得到重复利用的胆汁酸还能刺激胆汁分泌，能畅通胆道，消除霉菌毒素、细菌内毒素等有害物质，起到保肝护胆的作用。大量的临床研究显示，肠道微生态对肝脏病理生理过程起着至关重要的作用，而当罹患肝脏疾病时又会反过来影响肠道微生态的结构和功能。正常情况下，只有痕量的肠道微生物能穿过肠道黏膜屏障经门静脉而到达肝脏，反而可以激活肝脏网状内皮系统，使其持续处于活化状态。当肠道微生态平衡失调，肠道粘膜屏障就可能受损，大量肠道微生物及毒素将移位至肝脏，破坏肝脏的免疫耐受，并将过度激活肝脏固有的免疫系统而产生炎症级联反应，导致菌血症和内毒素血症，诱发肝脏损伤。而当肝脏受损发生病变时，会导致其合成功能下降，血浆中的白蛋白急剧减少，易造成腹腔积水；同时，肝脏分泌胆汁不足，使得肠道内缺乏胆盐，继而形成门静脉高压，造成胃肠道淤血、缺氧而出现门静脉高压性胃肠病；受损后的肝脏合成凝血因子减少，会加重胃肠道出血症状，进一步恶化肠道微生态平衡。由此可见，肠道微生态与肝脏之间密切相关，而肠道微生态失调与肝脏疾病间又互为因果，继而引发机体一系列病理变化。二者的密切关系也提示，当发生肝脏疾病时应注意肠道微生态的调理，而当肠道微生态失调时，也应注意对肝脏的保护。这为肠道微生态失调和肝脏疾病的发病机制研究与防治提供了全新的 思路。
随着社会的飞速发展，生活节奏也越来越快，人们的饮食结构和生活习惯都发生了很大的变化，长期地熬夜、饮食不规律、吸烟、大量饮酒以及不合理用药等等，都给我们的肝脏和肠道微生态带来沉重的负担。然而，市场现有的众多产品中，几乎都是对二者区别对待：常使用护肝片、联苯双酯、复方益肝灵等治疗各种肝病，以达到养肝、护肝的目的，但存在药物起效慢、服用周期长、生物利用度低的问题，甚至会引起恶心、呕吐、皮炎、皮疹等不良反应；常使用益生菌、益生元或者合生元等调理肠道微生态，这类肠道调节剂，特别是含有益生菌类的调节剂，多存在菌株单一且不稳定、难保存和菌株存活率低、定殖率低等问题，且对于许多存在肝脏疾病的人群，常出现治愈率低、易反复的现象。因此，正确认识肝脏与肠道微生态的关系，并基于此而开发出安全高效、毒副作用小、质量稳定可控的护肝型肠道微生态调节剂，是当前亟待解决的一项重要课题。
发明内容
本发明所要解决的技术问题是针对现有技术的现状，提供一种安全高效、毒副作用小、质量稳定可控、不会产生依耐性及抗药性、也不会在体内蓄积，起到护肝及调节肠道微生态功能的基于肝-肠轴的护肝型肠道微生态调节剂。
本发明所要解决的另一个技术问题是针对现有技术的现状，提供一种上述基于肝-肠轴的护肝型肠道微生态调节剂的制备方法，该制备方法不但能最大限度地保留各主要活性成分，提高提取物得率，还能有效减少提取时间，降低能耗。
本发明解决上述技术问题所采用的技术方案为；一种基于肝-肠轴的护肝型肠道微生态调节剂，其特征在于：按重量计，所述调节剂的制备原料包括以下组分
地锦草100～300份、显齿蛇葡萄叶60～210份、覆盆子100～300份、马齿苋100～300份、黄精60～180份、肉苁蓉50～150份、酵母β-葡聚糖5～20份、低聚果糖10～50份、大豆低聚糖10～60份。
优选地，所述调节剂由下列重量份数的原料制成；地锦草210份、显齿蛇葡萄叶165份、覆盆子240份、马齿苋210份、黄精140份、肉苁蓉100份、酵母β-葡聚糖16份、低聚果糖24份、大豆低聚糖30份。
优选地，所述调节剂包括地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物、马齿苋提取物、黄精提取物、肉苁蓉提取物、酵母β-葡聚糖、低聚果糖及大豆低聚糖。本发明的调节剂以上述组分为主要活性物质，同时，还可以优选地加入合适的辅料，采用制剂学的常规技术制备成任何一种适合口服的制剂形式，如片剂、胶囊剂、颗粒剂、粉剂、丸剂或口服液等。
作为改进，所述的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物均是通过闪式提取协同真空减压法再经后处理而获得，所述的马齿苋提取物、黄精提取物、肉苁蓉提取物则均是通过机械化学协同超声波辅助法再经后处理而获得。采用闪式提取协同真 空减压法和机械化学协同超声波辅助法对不同类别成分进行提取，不但能最大限度地保留各主要活性成分，提高提取物得率，还能有效减少提取时间，降低能耗。
一种上述基于肝-肠轴的护肝型肠道微生态调节剂的制备方法，其特征在于包括以下步骤；
(1)分别制备地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物；
(2)分别制备马齿苋提取物、黄精提取物、肉苁蓉提取物；
(3)将步骤(1)、(2)中制备的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物、马齿苋提取物、黄精提取物、肉苁蓉提取物进行混合，并加入相应重量组分的酵母β-葡聚糖、低聚果糖及大豆低聚糖，混合均匀，即得到所述的基于肝-肠轴的护肝型肠道微生态调节剂。
优选地，所述的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物均通过以下步骤获得：
(A)闪式提取：将粉碎后过10～80目筛的地锦草/显齿蛇葡萄叶/覆盆子加入提取罐中，加入8～30倍量的20～75％7醇作为提取溶剂，于室温下提取5～120s；
(B)真空减压提取：开启闪式提取器与真空减压提取器间的连通阀门，料液在压差作用下进入真空减压提取器进行提取，直至获得提取液；
(C)离心分离：将所得提取液进行离心分离，得澄清液；
(D)减压浓缩：将澄清液进行减压浓缩，得浸膏；
(E)喷雾干燥：将浸膏进行喷雾干燥，得到地锦草提取物/显齿蛇葡萄叶提取物/覆盆子提取物。
优选地，所述真空减压提取的提取温度为40～65℃，提取压力为0.05～0.1MPa，提取时间为20～40min所述减压浓缩的浓缩温度为50℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15。
优选地，所述喷雾干燥的工艺参数为喷口温度160～180℃，出口温度70～85℃，上料速度10～20mL/min。
优选地，所述的马齿苋提取物、黄精提取物、肉苁蓉提取物通过以下步骤获得：
(A)机械化学提取：将粗粉后的马齿苋/黄精/肉苁蓉加入球磨罐中，加入体积浓度为5％～30％的Na2CO3作为研磨助剂，提取时间为3min～15min，全程开启冷凝水；
(B)超声波辅助提取：将上述经机械化学提取得到的物料转入超声提取器中，加入10～30倍量水作为提取溶剂，得到提取液；
(C)离心分离；将所得提取液进行离心分离，得澄清液；
(D)减压浓缩；将澄清液进行减压浓缩，浓缩温度为60℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；
(E)喷雾干燥：将浸膏喷雾干燥，工艺参数为喷口温度130～160℃，出口温度60～80℃，上料速度10～20mL/min，得到马齿苋提取物/黄精提取物/肉苁蓉提取物。
优选地，所述超声波辅助提取的提取温度为40～80℃，提取时间为20～80min，超声功率为100-500W。
与现有技术相比，本发明的优点在于：
本发明调节剂的配方中，采用多种天然产物科学合理配伍，协同增效，在保护肝脏的同时，促进肠道微生态调节。地锦草与马齿苋同归肝、大肠经，配合使用，凉血解毒，同治泻痢，尤其是对大肠杆菌、痢疾杆菌、沙门氏菌、志贺氏菌、金黄色葡萄球菌等多种肠道致病菌具有较强的抑制作用，再配以显齿蛇葡萄叶，进一步加强对大肠杆菌、金黄色葡萄球菌等致病菌的抑制作用，而酵母β-葡聚糖、低聚果糖、大豆低聚糖等肠内益生菌活化增殖因子能有效促进益生菌增殖，并能有效抑制肠内腐败物质的生成和促进其排泄，上述原料有机结合，能有效维持肠道微生态的平衡；同时，地锦草、显齿蛇葡萄叶和马齿苋三者合用，能通过增加肝脏的排毒能力而起到保护肝脏的作用，而覆盆子能增强肝组织抗氧化酶类活性，抑制炎性因子生成，同时，肉苁蓉具有较强的抗氧化功能，使自由基生成减少，从而减轻自由基对肝组织及线粒体的损伤作用，进而提高肝脏抗氧化能力，减少脂质过氧化，二者与黄精配合作用协同增效，能显著增强机体清除细胞内自由基和脂质过氧化物的能力，并与酵母β-葡聚糖配合作用，能促使免疫细胞发生特异性结合，刺激体内淋巴细胞、巨噬细胞等产生免疫活性，有效提高机体免疫能力，促进肝脏等受损组织器官恢复，进而经肝-肠轴改善肠道微生态平衡。可见，本发明将多种天然产物有机组合使用，从微生物水平、细胞水平和代谢水平上协同作用，促进肝脏毒素排出、修复受损肝组织、保护肝脏，同时能有效调节机体肠道微生态平衡，增进肠道健康。
与单独使用药物或单独使用益生元相比，本发明组合物疗效显著性提高，该组含物是以传统中医药的养生保健理论及现代医学研究的“肝-肠轴”理论为指导下提出的，各组分间相互配合，协同增效，而不仅仅是单个物质的叠加；
本发明的护肝型肠道微生态调节剂以天然来源的中药和益生元为主，且基本为药食同源或者新食品原料，安全无毒副作用，不会产生依耐性、抗药性，也不会在体内蓄积，护肝及调节肠道微生态功能确切；
本发明的制备方法中，采用闪式提取协同真空减压法和机械化学协同超声波辅助法对不同类别成分进行提取，不但能最大限度地保留各主要活性成分，提高提取物得率，还能有效减少提取时间，降低能耗。
具体实施方式
以下结合实施例对本发明作进一步详细描述。
实施例1：
按重量计，本实施例的基于肝-肠轴的护肝型肠道微生态调节剂采用以下原料制备：地锦草150份、显齿蛇葡萄叶130份、覆盆子200份、马齿苋150份、黄精120份、肉苁蓉60份、酵母β-葡聚糖8份、低聚果糖15份、大豆低聚糖20份。
上述基于肝-肠轴的护肝型肠道微生态调节剂的制备方法为：
(1)分别制备地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物；
将粉碎后过10目筛的地锦革/显齿蛇葡萄叶/覆盆子加入提取罐中，加入10倍量的30％乙醇作为提取溶剂，于室温下提取10s；开启闪式提取器与真空减压提取器间的连通阀门，料液在压差作用下进入真空减压提取器，提取温度为40℃，提取压力为0.05MPa，提取时间为20min，得提取液：将所得提取液进行离心分离后进行减压浓缩，浓缩温度为50℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度160℃，出口温度70℃，上料速度10mL/min，得到地锦草提取物或显齿蛇葡萄叶提取物或覆盆子提取物：
(2)分别制备马齿苋提取物、黄精提取物、肉苁蓉提取物；
将粗粉后的马齿苋/黄精/肉苁蓉加入球磨罐中，加入5％的Na2CO3作为研磨助剂，提取时间为3min，全程开启冷凝水：再将上述经机械化学提取得到的物料转入超声提取器中，加入10倍量水作为提取溶剂，提取温度为40℃，提取时间为20min，超声功率为150W，得提取液；将所得提取液进行离心分离，后进行减压浓缩，浓缩温度为60℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度130℃，出口温度60℃，上料速度10mL/min，得到马齿苋提取物或黄精提取物或肉苁蓉提取物；
(3)将步骤(1)、(2)中制备的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物、马齿苋提取物、黄精提取物、肉苁蓉提取物进行混合，并加入相应重量组分的酵母β-葡聚糖、低聚果糖及大豆低聚糖，混合均匀，即得到基于肝-肠轴的护肝型肠道微生态调节剂。
实施例2：
按重量计，本实施例的基于肝-肠轴的护肝型肠道微生态调节剂采用以下原料制备：地锦草180份、显齿蛇葡萄叶150份、覆盆子220份、马齿苋180份、黄精140份、肉苁蓉80份、酵母β-葡聚糖13份、低聚果糖20份、大豆低聚糖40份。
上述基于肝-肠轴的护肝型肠道微生态调节剂的制备方法为：
(1)分别制备地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物；
将粉碎后过24目筛的地锦草或显齿蛇葡萄叶或覆盆子加入提取罐中，加入15倍量的40％乙醇作为提取溶剂，于室温下提取40s；开启闪式提取器与真空减压提取器间的连通阀门，料液在压差作用下进入真空减压提取器，提取温度为50℃，提取压力为0.07MPa，提取时间为25min，得提取液；将所得提取液进行离心分离后进行减压浓缩，浓缩温度为50℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度165℃，出口温度75℃，上料速度14mL/min，得到地锦草提取物或显齿蛇葡萄叶提取物或覆盆子提取物；
(2)分别制备马齿苋提取物、黄精提取物、肉苁蓉提取物；
将粗粉后的马齿苋或黄精或肉苁蓉加入球磨罐中，加入10％的Na2CO3作为研磨助剂，提取时间为10min，全程开启冷凝水；再将上述经机械化学提取得到的物料转入超声提取器中，加入20倍量水作为提取溶剂，提取温度为55℃，提取时间为40min，超 声功率为250W，得提取液；将所得提取液进行离心分离，后进行减压浓缩，浓缩温度为60℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度140℃，出口温度65℃，上料速度14mL/min，得到马齿苋提取物或黄精提取物或肉苁蓉提取物；
(3)将步骤(1)、(2)中制备的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物、马齿苋提取物、黄精提取物、肉苁蓉提取物进行混合，并加入相应重量组分的酵母β-葡聚糖、低聚果糖及大豆低聚糖，混合均匀，即得到基于肝-肠轴的护肝型肠道微生态调节剂。
实施例3：
按重量计，本实施例的基于肝-肠轴的护肝型肠道微生态调节剂采用以下原料制备：地锦草210份、显齿蛇葡萄叶165份、覆盆子240份、马齿苋210份、黄精140份、肉苁蓉100份、酵母β-葡聚糖16份、低聚果糖24份、大豆低聚糖30份。
上述基于肝-肠轴的护肝型肠道微生态调节剂的制备方法为：
(1)分别制备地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物；
将粉碎后过24目筛的地锦草或显齿蛇葡萄叶或覆盆子加入提取罐中，加入20倍量的55％乙醇作为提取溶剂，于室温下提取50s；开启闪式提取器与真空减压提取器间的连通阀门，料液在压差作用下进入真空减压提取器，提取温度为50℃，提取压力为0.08MPa，提取时间为25min，得提取液；将所得提取液进行离心分离后进行减压浓缩，浓缩温度为50℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度165℃，出口温度75℃，上料速度15mL/min，得到地锦草提取物或显齿蛇葡萄叶提取物或覆盆子提取物；
(2)分别制备马齿苋提取物、黄精提取物、肉苁蓉提取物；
将粗粉后的马齿苋或黄精或肉苁蓉加入球磨罐中，加入10％的Na2CO3作为研磨助剂，提取时间为4min，全程开启冷凝水；再将上述经机械化学提取得到的物料转入超声提取器中，加入15倍量水作为提取溶剂，提取温度为65℃，提取时间为30min，超声功率为350W，得提取液：将所得提取液进行离心分离，后进行减压浓缩，浓缩温度为60℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度160C，出口温度60℃，上料速度10mL/min，得到马齿苋提取物或黄精提取物或肉苁蓉提取物；
(3)将步骤(1)、(2)中制备的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物、马齿苋提取物、黄精提取物、肉苁蓉提取物进行混合，并加入相应重量组分的酵母β-葡聚糖、低聚果糖及大豆低聚糖，混合均匀，即得到基于肝-肠轴的护肝型肠道微生态调节剂。
实施例4；
按重量计，本实施例的基于肝-肠轴的护肝型肠道微生态调节剂采用以下原料制备： 地锦草280份、显齿蛇葡萄叶210份、覆盆子260份、马齿苋280份、黄精170份、肉苁蓉140份、酵母β-葡聚糖20份、低聚果糖45份、大豆低聚糖50份。
上述基于肝-肠轴的护肝型肠道微生态调节剂的制备方法为：
(1)分别制备地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物：
将粉碎后过50目筛的地锦草或显齿蛇葡萄叶或覆盆子加入提取罐中，加入30倍量的60％乙醇作为提取溶剂，于室温下提取100s；开启闪式提取器与真空减压提取器间的连通阀门，料液在压差作用下进入真空减压提取器，提取温度为60℃，提取压力为0.1MPa，提取时间为40min，得提取液；将所得提取液进行离心分离后进行减压浓缩，浓缩温度为50℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度175℃，出口温度80℃，上料速度18mL/min，得到地锦草提取物或显齿蛇葡萄叶提取物或覆盆子提取物；
(2)分别制备马齿苋提取物、黄精提取物、肉苁蓉提取物；
将粗粉后的马齿苋或黄精或肉苁蓉加入球磨罐中，加入25％的Na2CO3作为研磨助剂，提取时间为10min，全程开启冷凝水；再将上述经机械化学提取得到的物料转入超声提取器中，加入25倍量水作为提取溶剂，提取温度为75℃，提取时间为60min，超声功率为450W，得提取液；将所得提取液进行离心分离，后进行减压浓缩，浓缩温度为60℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度160℃，出口温度75℃，上料速度18mL/min，得到马齿苋提取物或黄精提取物或肉苁蓉提取物；
(3)将步骤(1)、(2)中制备的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物、马齿苋提取物、黄精提取物、肉苁蓉提取物进行混合，并加入相应重量组分的酵母β-葡聚糖、低聚果糖及大豆低聚糖，混合均匀，即得到基于肝-肠轴的护肝型肠道微生态调节剂。
对比例1：
按重量计，本实施例的基于肝-肠轴的护肝型肠道微生态调节剂采用以下原料制备；地锦草210份、显齿蛇葡萄叶165份、覆盆子240份、马齿苋210份、黄精140份、肉苁蓉100份、酵母β-葡聚糖16份、低聚果糖24份、大豆低聚糖30份。
上述基于肝-肠轴的护肝型肠道微生态调节剂的制备方法为：
(1)将相应重量份数的地锦草、显齿蛇葡萄叶、覆盆子、马齿苋、黄精及肉苁蓉加入提取罐中，加入20倍量水，浸泡30min后采用传统提取法进行提取，自煮沸后开始计时，提取60min后得提取液；将所得提取液进行离心分离后进行减压浓缩，浓缩温度为50℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；将浸膏喷雾干燥，喷口温度165℃，出口温度75℃，上料速度15mL/min，得到上述五种原料的混合提取物；
(2)向步骤(1)中制备的混合提取物中加入相应重量组分的酵母β-葡聚糖、低聚果糖及大豆低聚糖，混合均匀，即得到基于肝-肠轴的护肝型肠道微生态调节剂。
本发明对各实施例制备的调节剂进行了服用效果试验，具体为基于急性肝损伤伴发肠道微生态失调小鼠模型的护肝型肠道微生态调节剂功效评价试验。
试验方法：昆明种小鼠130只，体重20±2g，雌雄兼用，适应性喂养1周后，随机分成13组，每组10只，分别为正常对照组、模型对照组、丽珠肠乐组(阳性对照组1)、保肝片组(阳性对照组2)、实施例1组、实施例2组、实施例3低剂量组(5.0g/kg)、实施例3中剂量组(10.0g/kg)、实施例3高剂量组(20.0g/kg)、实施例3提取物组(将各中药提取物混合)、实施例3益生元组(将各益生元混合)、实施例4组、实施例5组。除正常对照组外，各组小鼠均用林可霉素灌胃，0.3mL/次，每天给药两次，连续给药三天，并将各组小鼠腹腔注射10％的D-氨基半乳糖，给药剂量为0.8g/kg，制备急性肝损伤伴发肠道微生态失调的小鼠模型。模型制备成功后，正常对照组和模型对照组小鼠均以等容量的生理盐水灌胃，丽珠肠乐组以0.5亿活菌/mL的丽珠肠乐溶液灌胃，给药剂量为0.3mL/次，保肝片组、实施例1、2、4、5组以及实施例3中剂量组、提取物组、益生元组均以10.0g/kg的给药剂量用生理盐水溶解后灌胃，实施例3低剂量组、实施例3高剂量组分别以5.0g/kg、20.0g/kg的给药剂量用生理盐水溶解后灌胃，每天给药一次，连续给药七天后，采用颈椎脱臼法处死小鼠，全部小鼠摘眼球取血，分离血清，测定内毒素、血清谷丙转氨酶(ALT)和谷革转氨酶(AST)水平；取各组小鼠肝左叶固定，按冰冻切片法制作切片，HE染色，光镜下观察肝组织病理学变化；肝各组小鼠肝右叶距边缘0.5cm处的一小块肝组织，用10％甲醛溶液固定，再用冷生理盐水制备10％肝匀浆，测定肝组织匀浆中丙二醛(MDA)和超氧化物歧化酶(SOD)含量；无菌采集盲肠内容物5.0g，按1.0g内容物加入4mL生理盐水的比例加入生理盐水，置于振荡器上振荡15min制成混悬液，离心后收集上清液，得到肠道菌液，将肠道菌液用无菌生理盐水连续稀释，分别进行双歧杆菌、乳酸杆菌、肠杆菌和肠球菌的选择性培养，培养后进行菌落计数，结果以每克粪便中菌落数的对数值表示。
试验结果：
(1)正常对照组小鼠在试验过程中精神状态良好，毛色有光泽，摄食、饮水及粪便正常，且对外界刺激反应灵敏；用林可霉素灌胃后，小鼠出现精神恍惚，行动减缓，摄食、饮水量减少，粪便稀湿等状况，腹腔注射D-氨基半乳糖后，小鼠精神萎靡，倦怠懒动，毛色粗糙暗淡无光泽，并伴有脱毛现象，粪便稀湿情况进一步加重。经过七天的治疗后，阳性对照组与各给药组均有不同程度好转，行动能力、被毛状况、摄食量、饮水量及粪便干燥程度等均有不同程度改善。
(2)与正常对照组比较，模型对照组小鼠血浆内毒素、ALT、AST、MDA均显著升高(p＜0.01)，SOD显著降低(P＜0.01)，提示肝损伤模型建立成功。与模型对照组比较，保肝片组小鼠血浆内毒素、ALT、AST、MDA均明显降低(P＜0.05，P＜0.01)，SOD显著升高(P＜0.01)；丽珠肠乐组与实施例3益生元组，虽内毒素、ALT、AST、MDA均呈降低趋势，但差异不显著(P＞0.05)；SOD呈上升趋势，但差异不显著(P＜0.01)，提示单独调节肠道微生态，在一定程度上有助于改善肝脏损伤状况；实施例1 组、实施例2组、实施例3不同剂量组、实施例3提取物组、实施例4组以及实施例5组小鼠血浆内毒素、ALT、AST、MDA均明显降低(P＜0.05，P＜0.01)，SOD显著升高(P＜0.01)，提示各组均能有效改普肝脏损伤状况，进一步组间对比结果表明，将提取物与益生元配伍使用，具有良好的协同增效作用，且采用闪式提取协同真空减压法和机械化学协同超声波辅助法所制各提取物的功效优于传统提取法。具体试验结果如表1所示。
表1：各试验组小鼠血浆内毒素、ALT、AST、MDA、SOD的含量比较(
Figure PCTCN2018000264-appb-000001
n＝10)
Figure PCTCN2018000264-appb-000002
与正常对照组比较： **P＜0.01；与模型对照组比较： #P＜0.05， ##P＜0.01
(3)观察各组小鼠肝组织经HE染色后切片可见，正常对照组小鼠肝小叶结构正常，无肿胀、脂肪变性、组织坏死等现象发生，肝小叶内及汇管区无炎性细胞浸润，无纤维结缔组织增生；与正常组相比，模型对照组小鼠肝小叶界限不清，内有局灶性坏死区，肝细胞明显水肿，呈弥漫性气球样变，小叶区出现肝细胞片状坏死伴炎性细胞浸润，汇管区亦见炎性细胞浸润，进一步证明造模成功；保肝片组小鼠肝小叶、汇管区结构趋于正常，病变范围缩小，炎性细胞浸润减少；丽珠肠乐组与实施例3益生元组稍有改善，但仍见肿胀，大片肝细胞水肿，气球样变，局灶性坏死及炎性细胞浸润仍明显；实施例1组、实施例2组、实施例3不同剂量组以及实施例4组，肝组织均无明显坏死，肝细胞有轻度水肿，大部分肝细胞结构正常；实施例3提取物组、实施例5组肝细胞仍见少量肿胀，部分肝细胞轻度水肿，偶见气球样变，局灶性坏死及炎性细胞浸润。由此也进 一步证明，本发明的调节剂可修复受损肝组织，对肝损伤有保护作用。
(4)与正常对照组比较，模型对照组小鼠肠道内双歧杆菌和乳酸杆菌数量显著减少(P＜0.01)，而肠杆菌和肠球菌数量显著增加(P＜0.01)，表明肠道微生态失调模型建立成功。与模型对照组比较，丽珠肠乐组小鼠肠道内双歧杆菌和乳酸杆菌数量显著增加(P＜0.01)，肠杆菌和肠球菌数量明显减少(P＜0.05，P＜0.01)；实施例3提取物组和保肝片组小鼠肠道内双歧杆菌和乳酸杆菌数量呈上升趋势，肠杆菌和肠球菌数量呈减少趋势，但差异不显著(P＞0.05)，提示使用护肝类药物，在一定程度上有助于调节肠道微生态，但短时间内效果不是十分明显；实施例1组、实施例2组、实施例3不同剂量组、实施例3益生元组、实施例4组以及实施例5组小鼠肠道内双歧杆菌和乳酸杆菌数量明显增加(P＜0.05，P＜0.01)，肠杆菌和肠球菌数量明显减少(P＜0.05，P＜0.01)提示各组均能有效促进肠道有益菌的生长与繁殖、抑制有害菌，调节肠道微生态，进一步组间对比结果表明，将提取物与益生元配伍使用，具有良好的协同增效作用，且验证了采用闪式提取协同真空减压法和机械化学协同超声波辅助法所制备提取物的功效优于传统提取法。具体试验结果如表2所示。
表2：各实验组小鼠肠道中的4种细菌定量检测结果比较(logN/g，
Figure PCTCN2018000264-appb-000003
n＝10)
Figure PCTCN2018000264-appb-000004
与正常对照组比较： **P＜0.01；与模型对照组比较： #P＜0.05， ##P＜0.01

Claims (10)

  1. 一种基于肝-肠轴的护肝型肠道微生态调节剂，其特征在于：按重量计，所述调节剂的制备原料包括以下组分
    地锦草100～300份、显齿蛇葡萄叶60～210份、覆盆子100～300份、马齿苋100～300份、黄精60～180份、肉苁蓉50～150份、酵母β-葡聚糖5～20份、低聚果糖10～50份、大豆低聚糖10～60份。
  2. 根据权利要求1所述的基于肝-肠轴的护肝型肠道微生态调节剂，其特征在于：所述调节剂由下列重量份数的原料制成
    地锦草210份、显齿蛇葡萄叶165份、覆盆子240份、马齿苋210份、黄精140份、肉苁蓉100份、酵母β-葡聚糖16份、低聚果糖24份、大豆低聚糖30份。
  3. 根据权利要求1所述的基于肝-肠轴的护肝型肠道微生态调节剂，其特征在于：所述调节剂包括地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物、马齿苋提取物、黄精提取物、肉苁蓉提取物、酵母β-葡聚糖、低聚果糖及大豆低聚糖。
  4. 根据权利要求3所述的基于肝-肠轴的护肝型肠道微生态调节剂，其特征在于：所述的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物均是通过闪式提取协同真空减压法再经后处理而获得，所述的马齿苋提取物、黄精提取物、肉苁蓉提取物则均是通过机械化学协同超声波辅助法再经后处理而获得。
  5. 一种权利要求1～4中任一权利要求所述基于肝-肠轴的护肝型肠道微生态调节剂的制备方法，其特征在于包括以下步骤：
    (1)分别制备地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物；
    (2)分别制备马齿苋提取物、黄精提取物、肉苁蓉提取物；
    (3)将步骤(1)、(2)中制备的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物、马齿苋提取物、黄精提取物、肉苁蓉提取物进行混合，并加入相应重量组分的酵母β-葡聚糖、低聚果糖及大豆低聚糖，混合均匀，即得到所述的基于肝-肠轴的护肝型肠道微生态调节剂。
  6. 根据权利要求5所述的基于肝-肠轴的护肝型肠道微生态调节剂的制备方法，其特征在于：所述的地锦草提取物、显齿蛇葡萄叶提取物、覆盆子提取物均通过以下步骤获得
    (A)闪式提取：将粉碎后过10～80目筛的地锦草/显齿蛇葡萄叶/覆盆子加入提取罐中，加入8～30倍量的20～75％乙醇作为提取溶剂，于室温下提取5～120s；
    (B)真空减压提取：开启闪式提取器与真空减压提取器间的连通阀门，料液在压差作用下进入真空减压提取器进行提取，直至获得提取液；
    (C)离心分离：将所得提取液进行离心分离，得澄清液；
    (D)减压浓缩：将澄清液进行减压浓缩，得浸膏；
    (E)喷雾干燥：将浸膏进行喷雾干燥，得到地锦草提取物/显齿蛇葡萄叶提取物/覆盆子提取物。
  7. 根据权利要求6所述的基于肝-肠轴的护肝型肠道微生态调节剂的制备方法，其特征在于：所述真空减压提取的提取温度为40～65℃，提取压力为0.05～0.1MPa，提取时间为20～40min；所述减压浓缩的浓缩温度为50℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15。
  8. 根据权利要求6所述的基于肝-肠轴的护肝型肠道微生态调节剂的制备方法，其特征在于：所述喷雾干燥的工艺参数为喷口温度160～180℃，出口温度70～85℃，上料速度10～20mL/min。
  9. 根据权利要求5所述的基于肝-肠轴的护肝型肠道微生态调节剂的制备方法，其特征在于：所述的马齿苋提取物、黄精提取物、肉苁蓉提取物通过以下步骤获得
    (A)机械化学提取：将粗粉后的马齿苋/黄精/肉苁蓉加入球磨罐中，加入体积浓度为5％～30％的Na2CO3作为研磨助剂，提取时间为3min～15min，全程开启冷凝水；
    (B)超声波辅助提取：将上述经机械化学提取得到的物料转入超声提取器中，加入10～30倍量水作为提取溶剂，得到提取液；
    (C)离心分离：将所得提取液进行离心分离，得澄清液；
    (D)减压浓缩：将澄清液进行减压浓缩，浓缩温度为60℃，真空度控制在-0.05～-0.08Mpa，相对密度控制在1.05～1.15，得浸膏；
    (E)喷雾干燥：将浸膏喷雾干燥，工艺参数为喷口温度130～160℃，出口温度60～80℃，上料速度10～20mL/min，得到马齿苋提取物/黄精提取物/肉苁蓉提取物。
  10. 根据权利要求9所述的基于肝-肠轴的护肝型肠道微生态调节剂的制备方法，其特征在于：所述超声波辅助提取的提取温度为40～80℃，提取时间为20～80min，超声功率为100～500W。
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