CN110742915A - Application of broadleaf holly leaf aqueous extract and pharmaceutical composition for regulating intestinal micro-ecology - Google Patents

Application of broadleaf holly leaf aqueous extract and pharmaceutical composition for regulating intestinal micro-ecology Download PDF

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CN110742915A
CN110742915A CN201911075450.6A CN201911075450A CN110742915A CN 110742915 A CN110742915 A CN 110742915A CN 201911075450 A CN201911075450 A CN 201911075450A CN 110742915 A CN110742915 A CN 110742915A
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intestinal
broadleaf holly
aqueous extract
holly leaf
group
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刘洪涛
陈嫚
杨庆雄
郑军平
邹小娟
胡白飞
殷明珠
张志刚
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Hubei College of Chinese Medicine
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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)
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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

Abstract

The invention relates to the technical field of functional food and medical health care, and discloses application of an aqueous extract of broadleaf holly leaf and a pharmaceutical composition for regulating intestinal microecology. The invention provides application of an aqueous extract of broadleaf holly leaf in preparing a medicament for regulating intestinal microecology and a pharmaceutical composition for regulating intestinal microecology. The invention researches the application of the broadleaf holly leaf aqueous extract in adjusting intestinal microecology through animal experiments, and finds that the broadleaf holly leaf aqueous extract effectively adjusts intestinal flora disorder and improves the barrier function of intestinal tracts by improving the diversity of mouse intestinal flora, reduces the expression of inflammatory factors of intestinal tissues and promotes the secretion of intestinal mucoproteins, thereby achieving the effects of reducing fat and weight, reducing blood sugar and reducing fat accumulation. The broadleaf holly leaf aqueous extract can regulate diseases such as obesity, type II diabetes mellitus, non-alcoholic fatty liver disease and the like caused by intestinal flora disorder.

Description

Application of broadleaf holly leaf aqueous extract and pharmaceutical composition for regulating intestinal micro-ecology
Technical Field
The invention relates to the technical field of functional food and medicine health care, in particular to application of an aqueous extract of broadleaf holly leaf and a pharmaceutical composition for regulating intestinal microecology.
Background
Intestinal micro-ecology refers to a large ecosystem of the host's intestinal tract and its neighboring microorganisms. Beneficial bacteria and harmful bacteria in the intestinal micro-ecosystem are mutually restricted and interdependent, and the intestinal micro-ecosystem balance is formed on the quality and quantity; once the internal and external environments of the organism change, beneficial bacteria are inhibited, and harmful bacteria propagate in the organism, so that the intestinal flora is disordered, pathological manifestations are generated, and a series of clinical symptoms are called intestinal flora disorder. Modern research results show that the coprophilous fungi of normal people are transplanted into the intestinal tracts of obese people or type II diabetics, so that the obesity phenotype of obese individuals can be obviously improved, the glucose metabolism level of human bodies can be improved, and the effects of losing weight and reducing blood sugar are achieved. It follows that modulation of gut flora disturbance is an effective way to improve the health status of a host.
Along with the improvement of living standard and the acceleration of life rhythm of people, people tend to obtain food in a convenient mode more and more, so that high-calorie fast food becomes the first choice; the excessive high calorie diet has led to an increase in the number of people suffering from diseases such as obesity, type ii diabetes, non-alcoholic fatty liver disease and inflammatory bowel disease. Disturbance of the intestinal flora may be the cause of the disease, or the result of the disease, or the outcome of the conversion of the cause and the result. Existing evidence indicates that obesity, type ii diabetes, non-alcoholic fatty liver disease, inflammatory bowel disease, and the like can cause intestinal dysbacteriosis and breakdown of intestinal barrier function; the breakdown of the intestinal barrier function will further lead to a disturbance of the intestinal flora, which may lead to an increase in symptoms of obesity, type ii diabetes, non-alcoholic fatty liver disease, and inflammatory bowel disease. The intestinal flora and the host have interdependence and mutual restriction relationship.
Current treatment for gut flora disorders include: (1) diet adjustment, enhancing the local defense barrier function of intestinal mucosa, and increasing fiber food; (2) antibacterial drug therapy, selecting proper antibiotics to inhibit bacteria which excessively reproduce, thereby indirectly supporting the bacteria which are insufficiently reproduced in intestinal tracts; (3) probiotics and prebiotics preparations to achieve the purpose of supporting normal flora. Although the traditional Chinese medicine composition has a treatment effect on diseases caused by imbalance of intestinal flora, the traditional Chinese medicine composition can cause side effects of drug dependence, drug resistance, liver and kidney injury and the like to different degrees.
The traditional Chinese medicine considers that: the root of diarrhea is not due to the spleen and stomach. Acute diarrhea is mostly predominant and is responsible for the spleen and stomach; chronic diarrhea is usually due to deficiency, and usually involves the spleen and kidney. The former should clear heat and resolve dampness, while the latter should tonify spleen and kidney. The broadleaf holly leaf is a traditional Chinese medicine, belongs to the Aquifoliaceae family, is a common plant substitutional tea in Chinese folks, is bitter and cold in property, mainly enters liver, lung and stomach channels, and has the effects of dispelling wind and clearing heat, improving eyesight and intelligence, and promoting the production of body fluid to quench thirst. The existing research suggests that the broadleaf holly leaves can regulate the intestinal flora disorder and improve the intestinal barrier function so as to improve the diseases such as obesity, type II diabetes mellitus, nonalcoholic fatty liver and the like caused by the intestinal flora disorder, but the regulation effect of the broadleaf holly leaves on the intestinal flora disorder in animals is not reported in documents and discovered in patents. Therefore, the research on the regulation and control of the intestinal microecology by the broadleaf holly leaves is of great significance, and the application provides the application of the broadleaf holly leaf aqueous extract and the composition thereof in the regulation of the intestinal microecology.
Disclosure of Invention
The invention aims to solve the problem that the regulation effect of broadleaf holly leaf on intestinal flora disturbance in the prior art is not yet applied, and provides application of a broadleaf holly leaf aqueous extract in preparing a medicine for regulating intestinal microecology and a medicine composition for regulating intestinal microecology, wherein the application can regulate diseases such as obesity, type II diabetes, non-alcoholic fatty liver disease and the like caused by the intestinal flora disturbance, and provides a new thought for research and development of broadleaf holly leaf.
In order to achieve the above objects, the present invention provides, in one aspect, an application of an aqueous extract of broadleaf holly leaf in the preparation of a medicament for regulating intestinal micro-ecology.
Preferably, said modulating gut microbiota comprises modulating gut flora disturbance and improving impaired gut barrier function.
Preferably, the dosage of the broadleaf holly leaf aqueous extract is 1-500mg/kg body weight.
In another aspect, the invention provides a pharmaceutical composition for regulating intestinal micro-ecology, which comprises an aqueous extract of broadleaf holly leaf, prebiotics and pharmaceutically acceptable excipients.
Preferably, the prebiotic is at least one of xylooligosaccharide, fructooligosaccharide, chitooligosaccharide, mannooligosaccharide and glucooligosaccharide.
Preferably, the pharmaceutical composition is in the form of capsules, tablets, pills, granules, syrup, powder, injection, solid beverage, beverage or baked food.
The invention researches the application of the broadleaf holly leaf aqueous extract in adjusting intestinal microecology through animal experiments, and finds that the broadleaf holly leaf aqueous extract effectively adjusts intestinal flora disorder and improves the barrier function of intestinal tracts by improving the diversity of mouse intestinal flora, reduces the expression of inflammatory factors of intestinal tissues and promotes the secretion of intestinal mucoproteins, thereby achieving the effects of reducing fat and weight, reducing blood sugar and reducing fat accumulation. The broadleaf holly leaf aqueous extract can regulate diseases such as obesity, type II diabetes mellitus, non-alcoholic fatty liver disease and the like caused by intestinal flora disorder.
Drawings
FIG. 1 is a graph of the effect of aqueous extract of Folum Ilicis on the regulation of body weight in mice with high-fat diet-induced intestinal bacteria disorders;
FIG. 2 is a graph showing the inhibitory effect of aqueous extract of Folum Ilicis on intestinal inflammation caused by high fat diet-induced intestinal flora disturbance;
FIG. 3 is a graph showing the effect of aqueous extract of Folum Ilicis on the promotion of intestinal mucin secretion in mice with high-fat diet-induced intestinal flora disturbance;
FIG. 4 is a graph of the effect of aqueous extract of Folum Ilicis on the regulation of blood glucose in mice with high-fat, high-sugar diet-induced intestinal flora imbalance;
FIG. 5 is a graph of the modulating effects of aqueous extract of Folum Ilicis on glucose intolerance and insulin resistance in high-fat, high-sugar mice;
fig. 6 and 7 are graphs showing the effect of aqueous extract of broadleaf holly leaf on the regulation of lipid metabolism in mice with high-fat high-cholesterol diet-induced intestinal flora disorder.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides an application of an aqueous extract of broadleaf holly leaf in preparing a medicament for regulating intestinal microecology.
In the use according to the invention, in particular, the regulation of intestinal micro-ecology comprises regulation of intestinal flora disturbances and improvement of impaired intestinal barrier function.
The intestinal flora disorder is a state that the diversity of the intestinal flora is reduced compared with the normal condition, and is specifically represented by imbalance of the diversity of the intestinal flora and imbalance of the proportion of beneficial bacteria and harmful bacteria.
The beneficial bacteria comprise lactobacillus and bifidobacterium.
The harmful bacteria include desulfurization vibrio and clostridium.
The flora diversity is a value obtained by computer algorithms Chao1, underlying species, PD white tree and Shannon.
The impaired intestinal barrier function is decreased intestinal mucin, decreased intestinal tight junction protein content or impaired intestinal mucus above the intestinal tract.
In the present invention, the aqueous extract of broadleaf holly leaf can be prepared by a conventional method. In one embodiment, the preparation method of the aqueous extract of broadleaf holly leaf comprises the following steps:
s1, grinding the dried broadleaf holly leaves into fine powder, soaking the fine powder in water for 30min, adding water according to the volume ratio of the broadleaf holly leaves to the water of 1:10, and decocting the mixture for 1h at 90-100 ℃;
s2, collecting the water decoction obtained in the step S1, filtering, adding water into the residue obtained in the step S1 again according to the volume ratio of the residue to the water of 1:5, decocting for 1 hour at 90-100 ℃, combining the two filtrates, and filtering again;
s3, concentrating the filtrate obtained in the step S2 under reduced pressure at 40-60 ℃ and 1.0KPa to form thick paste;
s4, freeze-drying the product obtained in the step S3 into powder to obtain the broadleaf holly leaf aqueous extract.
In the application of the invention, the dosage of the broadleaf holly leaf aqueous extract is 1-500mg/kg body weight. In particular embodiments, the aqueous extract of Folum Ilicis can be administered in an amount of 1mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight, 15mg/kg body weight, 25mg/kg body weight, 40mg/kg body weight, 50mg/kg body weight, 80mg/kg body weight, 100mg/kg body weight, 120mg/kg body weight, 150mg/kg body weight, 200mg/kg body weight, 250mg/kg body weight, 300mg/kg body weight, 400mg/kg body weight, or 500mg/kg body weight. In a preferred embodiment, the aqueous extract of Folum Ilicis is administered in an amount of 60-450mg/kg body weight.
In another aspect, the invention provides a pharmaceutical composition for regulating intestinal micro-ecology, which comprises an aqueous extract of broadleaf holly leaf, prebiotics and pharmaceutically acceptable excipients.
In the pharmaceutical composition of the present invention, specifically, the prebiotic is at least one of xylooligosaccharide, fructooligosaccharide, chitosan oligosaccharide, mannooligosaccharide, and glucooligosaccharide, and preferably, the prebiotic is xylooligosaccharide, chitooligosaccharide, or glucooligosaccharide.
In the pharmaceutical composition, prebiotics and pharmaceutically acceptable auxiliary materials are added into the aqueous extract of broadleaf holly leaf to prepare a pharmaceutical preparation or health-care food. In a specific embodiment, the pharmaceutical composition may be in the form of a capsule, a tablet, a pill, a granule, a syrup, a powder, an injection, a solid beverage, a beverage, or a baked food. Preferably, the pharmaceutical composition is in the form of capsules, granules or syrup.
After the broadleaf holly leaf aqueous extract is prepared into a pharmaceutical composition, the pharmaceutical composition can regulate diseases such as obesity, type II diabetes mellitus, non-alcoholic fatty liver disease and the like caused by intestinal flora disorder, and provides a new idea for research and development of broadleaf holly leaf.
The present invention will be described in detail by way of examples, but the scope of the present invention is not limited thereto.
In the examples of the present invention, "#" or "#" represents a statistical difference between the model group and the control group, and "#" or "# #" represents a significant difference between the treatment group and the model group.
Preparation example 1
The preparation example is used for explaining the preparation process of the aqueous extract of broadleaf holly leaf, and the specific preparation process is as follows:
s1, crushing the dried broadleaf holly leaves into fine powder, soaking the fine powder in water for 30min, and adding water according to the volume ratio of the broadleaf holly leaves to the water of 1:10 and decocting the mixture for 1h at 100 ℃;
s2, collecting the water decoction obtained in the step S1, filtering, adding water into the residue obtained in the step S1 again according to the volume ratio of the residue to the water of 1:5, decocting for 1 hour at 100 ℃, combining the two filtrates, and filtering again;
s3, concentrating the filtrate obtained in the step S2 under reduced pressure at 40 ℃ and 1.0KPa to form thick paste;
s4, freeze-drying the product obtained in the step S3 into powder to obtain the broadleaf holly leaf aqueous extract.
Example 1
This example illustrates the body weight regulation and intestinal inflammation inhibition of mice with high fat diet-induced intestinal bacteria disorders by aqueous extract of Folum Ilicis.
In order to research the regulation effect of the broadleaf holly leaf aqueous extract on the body weight of a mouse with intestinal flora disorder induced by high-fat diet and the inhibition effect on intestinal inflammation, a mouse model with the intestinal flora disorder induced by the high-fat diet is established and administration treatment is carried out. The method specifically comprises the following steps:
(1) male C57BL/6 mice 4-6 weeks old and 20 + -2 g in body weight were randomly assigned to normal diet group (NCD), high fat diet group (HFD), normal diet plus Kuding tea group (NCD-KD) and high fat diet plus Kuding tea group (HFD-KD).
(2) The breeding conditions are kept at proper temperature and humidity, and the normal standard feed and sterile drinking water are fed. After one-week environmental adaptation period, the NCD group and the NCD-KD group were given normal standard diet, the HFD group and the HFD-KD group were given high fat diet, the NCD group and the HFD group were given normal sterile drinking water, and the NCD-KD group and the HFD-KD group were given 1mg/mL drinking water of Folum Ilicis;
(3) the mice were monitored for body weight every 15 days for 4 months of feeding, and changes in body weight were recorded to prepare mouse growth curves (as shown in fig. 1).
(4) After the mice are fed for 4 months, fresh excrement of the mice is collected, genes in the excrement are extracted, second-generation high-throughput nucleic acid sequencing is carried out on the basis of amplified fragments of V3-V4 regions of 16S rRNA, and diversity values obtained on the basis of different algorithms of Chao1, underlying species, PD hole tree and Shannon through database information comparison and combined with bioinformatics analysis and statistical analysis are higher, and higher values represent better diversity. Table 1 shows the diversity values obtained by Chao1, underlying species, PD white tree and Shannon. The species of enteric bacteria in each experimental group were then identified and the relative amounts of the different bacteria were calculated. Table 2 shows the percentage of bifidobacteria, lactobacilli and desulfurization vibrio bacteria in the total intestinal bacteria. It can be found that in a mouse model group of intestinal flora disorder induced by high-fat diet, the contents of beneficial bacteria bifidobacteria and lactobacilli are obviously reduced, the content of proinflammatory bacteria desulfurization vibrio is obviously increased, the contents of the bifidobacteria and the lactobacilli in a broadleaf holly leaf treatment group are obviously increased, the specific gravity of the desulfurization vibrio is obviously reduced, and the intestinal flora disorder is obviously improved.
(5) Feeding mice for 4 months, anesthetizing and killing the mice, weighing 40mg of large intestine tissues of the mice, adding 800 mu L of RNA lysate to extract RNA, performing reverse transcription by using a cDNA rapid synthesis kit, detecting the expression of inflammation related factors by using RT-qPCR (reverse transcription-quantitative polymerase chain reaction), and using 2-ΔΔCTThe relative gene expression levels were calculated (as shown in FIG. 2).
The weight change results show that compared with the normal diet group, the weight of the mice in the high-fat diet group is obviously higher than that in the normal diet group and has a significant difference (P is less than 0.05), and the weight of the mice in the high-fat diet group plus broadleaf holly leaves is obviously reduced and has a significant difference (P is less than 0.05). The broadleaf holly leaf can regulate the weight of a mouse by regulating intestinal flora disorder induced by high-fat diet and has good weight-losing effect.
RT-qPCR results show that the expression levels of interleukin-1 beta (IL-1 β), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic factor-1 (MCP-1), tumor necrosis factor-alpha (TNF- α) and NLRP3 inflammasome (NLRP3) related to inflammatory factors of mice with high-fat diet are obviously increased, the higher the expression levels of the inflammatory factors such as IL-1 β, IL-6, IL-8, MCP-1, TNF- α and NLRP3 are, the more obvious the inflammation is represented, and the tendency that the intestinal inflammation expression of the mice fed with high-fat diet is obviously reduced after the ilex latifolia is treated is shown, so that the ilex latifolia can improve the intestinal inflammation caused by the disorder of the intestinal flora induced by the high-fat diet by regulating the intestinal flora and inhibiting the expression of the inflammatory factors.
TABLE 1 variation of the intestinal flora diversity of the groups of mice
(*P<0.05，**P<The 0.01vs normal diet group was,#P<0.05，##P<0.01vs high fat diet group)
Experimental grouping/diversity Chao1 Observedspecies PDwholetree Shannon
Group of normal diet 605.67±52.17 495.29±32.96 38.93±4.18 6.87±0.36
High fat diet group 434.72±55.88** 356.67±29.78** 31.59±3.44** 5.92±0.34**
Tea group of normally added broadleaf holly leaves 561.69±45.16 459.33±38.24 35.64±4.93 6.47±0.28
High fat broadleaf holly leaf group 599.32±61.32## 499.76±30.05## 38.84±5.02## 6.85±0.31##
TABLE 2 Change in the amount of bacteria in the intestinal tract of mice in each group
(*P<0.05，**P<The 0.01vs normal diet group was,#P<0.05，##P<0.01vs high fat diet group)
Experimental groups/contents (%) Bifidobacterium Lactobacillus strain Desulfurization vibrio
Group of normal diet 10.86±2.13 22.46±5.35 4.97±2.85
High fat diet group 0.14±0.02** 0.25±0.21** 27.52±4.67**
Tea group of normally added broadleaf holly leaves 11.22±1.18 18.78±5.53 5.62±1.35
High fat broadleaf holly leaf group 3.87±0.57## 1.76±0.63# 8.32±2.63##
Example 2
This example illustrates the effect of aqueous extract of Kuding tea on the promotion of intestinal mucin secretion in mice with high-fat diet-induced intestinal flora disturbance.
In order to research the promotion effect of the broadleaf holly leaf aqueous extract on the intestinal mucoprotein secretion of a mouse with high-fat diet-induced intestinal flora disorder, a mouse model with high-fat diet-induced intestinal flora disorder is established and the administration treatment is carried out, the method specifically comprises the following steps:
(1) male C57BL/6 mice 4-6 weeks old and 20 + -2 g in body weight were randomly assigned to normal diet group (NCD), high fat diet group (HFD), normal folium Ilicis (NCD-KD) and high fat folium Ilicis (HFD-KD).
(2) The breeding conditions are kept at proper temperature and humidity, and the normal standard feed and sterile drinking water are fed. After one-week environmental adaptation period, the NCD group and the NCD-KD group were given normal standard diet, the HFD group and the HFD-KD group were given high fat diet, the NCD group and the HFD group were given normal sterile drinking water, and the NCD-KD group and the HFD-KD group were given 0.5mg/mL drinking water of Folum Ilicis;
(3) after the mice are fed for 4 months, collecting fresh excrement of the mice, extracting genes in the excrement, carrying out second-generation high-throughput nucleic acid sequencing on the amplified fragment of the V3-V4 region of 16S rRNA, identifying the types of intestinal bacteria in each experimental group by database information comparison and combining bioinformatics analysis and statistical analysis, and calculating the relative content of different bacteria. Table 3 shows the percentage of bifidobacteria, lactobacilli and desulfurization vibrio bacteria in the total intestinal bacteria. It can be found that in a mouse model group of intestinal flora disorder induced by high-fat diet, the contents of beneficial bacteria bifidobacteria and lactobacilli are obviously reduced, the content of proinflammatory bacteria desulfurization vibrio is obviously increased, the contents of the bifidobacteria and the lactobacilli in a broadleaf holly leaf treatment group are obviously increased, the specific gravity of the desulfurization vibrio is obviously reduced, and the intestinal flora disorder is obviously improved.
(4) After feeding the mice for 4 months, the mice were sacrificed by anesthesia, and the mouse large intestine tissue was fixed in a 4% paraformaldehyde solution, paraffin sections were prepared, stained with alcian blue, and mucin secretion by the large intestine tissue goblet cells was observed under an optical microscope (see fig. 3).
As can be seen from fig. 3, compared with the normal diet group, the high-fat diet group has less intestinal mucin, incomplete intestinal tissue structure and damaged intestinal barrier, and after the high-fat diet is added with the broadleaf holly leaf, the intestinal mucin is obviously increased and the intestinal barrier structure is complete, which indicates that the broadleaf holly leaf has promotion and protection effects on the secretion of the intestinal mucin of the mice with intestinal flora disorder induced by the high-fat diet.
TABLE 3 Change in the amount of bacteria in the intestinal tract of mice in each group
(**P<The 0.01vs normal diet group was,##P<0.01vs high fat diet group)
Experimental groups/contents (%) Bifidobacterium Lactobacillus strain Desulfurization vibrio
Group of normal diet 27.73±5.37 16.24±2.73 1.87±0.25
High fat diet group 1.97±0.34** 0.14±0.07** 25.32±1.67**
Tea group of normally added broadleaf holly leaves 35.87±5.86 20.88±2.79 0.67±0.02
High fat broadleaf holly leaf group 19.13±2.23## 9.81±2.17## 2.87±0.25##
Example 3
This example illustrates the effect of aqueous extract of Folum Ilicis on the regulation of blood glucose in mice with high-fat, high-sugar diet-induced intestinal flora imbalance.
In order to research the regulation effect of the broadleaf holly leaf aqueous extract on blood sugar, insulin resistance and glucose tolerance of a mouse with high-fat and high-sugar diet, a mouse model with intestinal flora disorder induced by high-fat and high-sugar diet is established and administration treatment is carried out, and the method specifically comprises the following steps:
(1) male C57BL/6 mice 4-6 weeks old and 20 + -2 g in body weight were randomly assigned to normal control group (NCD), high-fat high-sugar group (HFHS), normal folium Ilicis group (NCD-KD) and high-fat high-sugar folium Ilicis group (HFHS-KD).
(2) The breeding conditions are kept at proper temperature and humidity, and the normal standard feed and sterile drinking water are fed. After one week of environmental adaptation period, the NCD group and the NCD-KD group were given normal standard diet, the HFHS group and the HFHS-KD group were given high-fat high-sugar diet, the NCD group and the HFHS group were given normal sterile drinking water, and the NCD-KD group and the HFHS-KD group were given 0.25mg/mL drinking water of Folum Ilicis.
(3) After the mice are fed for 4 months, collecting fresh excrement of the mice, extracting genes in the excrement, carrying out second-generation high-throughput nucleic acid sequencing on the amplified fragment of the V3-V4 region of 16S rRNA, identifying the types of intestinal bacteria in each experimental group by database information comparison and combining bioinformatics analysis and statistical analysis, and calculating the relative content of different bacteria. Table 4 shows the percentage of bifidobacteria, lactobacilli and desulfurization vibrio bacteria in the total intestinal bacteria. It can be found that in a mouse model group of intestinal flora disorder induced by high-fat diet, the contents of beneficial bacteria bifidobacteria and lactobacilli are obviously reduced, the content of proinflammatory bacteria desulfurization vibrio is obviously increased, the contents of the bifidobacteria and the lactobacilli in a broadleaf holly leaf treatment group are obviously increased, the specific gravity of the desulfurization vibrio is obviously reduced, and the intestinal flora disorder is obviously improved.
(4) After the mice were fed for 4 months, the mice were starved for 12 hours, and the fasting blood glucose levels of the mice were measured and plotted (as shown in FIG. 4).
(5) After starvation treatment of the mice for 12 hours, the weight of each mouse is weighed, the glucose dosage for intraperitoneal injection of each mouse is calculated according to 1g/kg of glucose, the blood glucose value of each mouse is recorded at 0min, 15min, 30min, 45min, 60min and 120min respectively, and a glucose tolerance map is drawn (as shown in figure 5).
(6) After the mice were fed with diets again for 2 days, the mice were starved for 6 hours, the weight of each mouse was weighed, the amount of insulin injected into the abdominal cavity of each mouse was calculated according to 0.75U/kg of insulin, and the blood glucose level of each mouse was measured and recorded at 0min, 15min, 30min, 45min, 60min and 120min, respectively, to draw an insulin resistance graph (as shown in fig. 5).
Fasting blood glucose results show: compared with the normal diet group, the fasting blood sugar of the high-fat high-sugar diet group is obviously increased and has significant difference (P is less than 0.05), and the fasting blood sugar value of the high-fat high-sugar and broadleaf holly leaf group is reduced to the normal level and also has significant difference (P is less than 0.05), so that the broadleaf holly leaf has good regulating effect on the hyperglycemia of the mice with intestinal flora disorder induced by high-fat high-sugar, and can be used for preventing and treating the hyperglycemia. The glucose tolerance results show that: the broadleaf holly leaf has good regulation effect on the glucose tolerance of mice and has significant difference (P is less than 0.05); the insulin resistance results show that: the broadleaf holly leaf has an enhancement effect on the insulin sensitivity of mice and also has a significant difference (P is less than 0.05). The broadleaf holly leaf has the advantages of reducing blood sugar, enhancing glucose tolerance and insulin sensitivity, and reducing the risks of diseases such as obesity, type II diabetes mellitus, non-alcoholic fatty liver disease and the like caused by intestinal flora disorder.
TABLE 4 Change in the amount of bacteria in the intestinal tract of mice in each group
(**P<The 0.01vs normal diet group was,#P<0.05，##P<0.01vs high fat and high sugar diet group)
Figure BDA0002262285610000111
Figure BDA0002262285610000121
Example 4
This example illustrates the effect of aqueous extract of Kuding tea on the regulation of lipid metabolism in mice with high-lipid and high-cholesterol diet-induced intestinal flora disorders.
In order to research the regulation effect of the broadleaf holly leaf aqueous extract on lipid metabolism of mice with intestinal flora disorder induced by high-fat and high-cholesterol diet, a mouse model with intestinal flora disorder induced by high-fat and high-cholesterol diet is established and administration treatment is carried out, the method specifically comprises the following steps:
(1) male C57BL/6 mice 4-6 weeks old and 20 + -2 g in body weight were randomly assigned to normal diet group (NCD), high-fat high-cholesterol diet group (HFHC), normal Folum Ilicis group (NCD-KD) and high-fat high-cholesterol Folum Ilicis group (HFHC-KD).
(2) The breeding conditions are kept at proper temperature and humidity, and the normal standard feed and sterile drinking water are fed. After one week of environmental adaptation period, the NCD group and the NCD-KD group were given normal standard diet, the HFHC group and the HFHC-KD group were given high-fat high-cholesterol diet, the NCD group and the HFHC group were given normal sterile drinking water, and the NCD-KD group and the HFHC-KD group were given 1.4mg/mL drinking water of Folum Ilicis.
(3) After the mice are fed for 4 months, collecting fresh excrement of the mice, extracting genes in the excrement, carrying out second-generation high-throughput nucleic acid sequencing on the amplified fragment of the V3-V4 region of 16S rRNA, identifying the types of intestinal bacteria in each experimental group by database information comparison and combining bioinformatics analysis and statistical analysis, and calculating the relative content of different bacteria. Table 5 shows the percentage of bifidobacteria, lactobacilli and desulfurization vibrio bacteria in the total intestinal bacteria. It can be found that in a mouse model group of intestinal flora disorder induced by high-fat diet, the contents of beneficial bacteria bifidobacteria and lactobacilli are obviously reduced, the content of proinflammatory bacteria desulfurization vibrio is obviously increased, the contents of the bifidobacteria and the lactobacilli in a broadleaf holly leaf treatment group are obviously increased, the specific gravity of the desulfurization vibrio is obviously reduced, and the intestinal flora disorder is obviously improved.
(4) After the mice were fed for 4 months, the mice were sacrificed by anesthesia, liver tissues and adipose tissues of the mice were fixed in 4% paraformaldehyde solution, paraffin sections were prepared, HE staining was performed, and the condition of lipid droplets in the liver tissues and the size of the adipose tissues were observed under an optical microscope (as shown in fig. 6 and 7).
As can be seen from fig. 6 and 7, compared with the control group, the lipid droplets in the liver tissue of the high-fat and high-cholesterol group are significantly increased, the area of fat cells is significantly increased, and after the broadleaf holly leaf is added to the high-fat and high-cholesterol mice, the lipid droplets in the liver tissue are significantly reduced, and the area of the fat tissue is also significantly reduced, which indicates that the broadleaf holly leaf has an obvious lipid-lowering effect on the mice with intestinal flora disorder induced by the high-fat and high-cholesterol diet.
TABLE 5 variation of key bacteria content in intestinal tract of each group of mice
(**P<The 0.01vs normal diet group was,##P<0.01vs high fat high cholesterol diet group)
Experimental groups/contents (%) Bifidobacterium Lactobacillus strain Desulfurization vibrio
Group of normal diet 12.89±1.26 17.66±2.98 0.18±0.02
Diet group with high fat and high cholesterol 0.11±0.01** 1.48±0.71** 25.34±2.84**
Tea group of normally added broadleaf holly leaves 9.87±1.22 21.34±1.90 1.26±0.82
High-fat and high-cholesterol broadleaf holly leaf group 8.56±1.3## 14.81±1.34## 2.37±1.72##
The invention researches the application of the aqueous extract of broadleaf holly leaf in adjusting the intestinal microecology through animal experiments. Dissolving broadleaf holly leaf powder with a certain concentration in animal sterilized drinking water, collecting fresh excrement of a mouse, discovering through 16S rRNA high-throughput sequencing that the diversity of intestinal flora of the mouse changes, detecting the change conditions of the weight, intestinal inflammation, intestinal mucoprotein, blood sugar and fat metabolism of the mouse, and discovering that the broadleaf holly leaf water extract can improve the diversity of the intestinal flora of the mouse, effectively adjusting the intestinal flora disorder and improving the barrier function of the intestinal tract, reducing the expression of inflammatory factors of intestinal tissue and promoting the secretion of the intestinal mucoprotein, thereby achieving the effects of reducing fat and losing weight, reducing blood sugar and reducing fat accumulation. The broadleaf holly leaf can be further developed to treat enteritis and other intestinal tract microecological disorders.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (6)

1. Application of Folum Ilicis water extract in preparing medicine for regulating intestinal microecology is provided.
2. The use according to claim 1, wherein said modulation of intestinal micro-ecology comprises modulation of intestinal flora disturbances and amelioration of impaired intestinal barrier function.
3. The use according to claim 1, wherein the aqueous extract of broadleaf holly leaf is administered in an amount of 1-500mg/kg body weight.
4. A pharmaceutical composition for regulating intestinal microecology is characterized by comprising an aqueous extract of broadleaf holly leaf, prebiotics and pharmaceutically acceptable auxiliary materials.
5. The pharmaceutical composition of claim 4, wherein the prebiotic is at least one of a xylooligosaccharide, a fructooligosaccharide, a chitooligosaccharide, a mannooligosaccharide, and a glucooligosaccharide.
6. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is in the form of capsules, tablets, pills, granules, syrups, powders, injections, solid drinks, beverages or baked foods.
CN201911075450.6A 2019-11-06 2019-11-06 Application of broadleaf holly leaf aqueous extract and pharmaceutical composition for regulating intestinal micro-ecology Pending CN110742915A (en)

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Publication number Priority date Publication date Assignee Title
CN112826876A (en) * 2021-02-02 2021-05-25 刘洪涛 Application of ampelopsis grossedentata aqueous extract in preparation of preparation for improving liver injury
CN114766672A (en) * 2022-05-25 2022-07-22 贵州泰和现代生态农业科技有限公司 Old lobular broadleaf holly leaf extract and application thereof in preparing food for relieving alcohol-induced liver injury
CN115919937A (en) * 2022-10-18 2023-04-07 美益添生物医药(武汉)有限公司 Preparation for regulating biological intestinal flora and preparation method and application thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112826876A (en) * 2021-02-02 2021-05-25 刘洪涛 Application of ampelopsis grossedentata aqueous extract in preparation of preparation for improving liver injury
CN114766672A (en) * 2022-05-25 2022-07-22 贵州泰和现代生态农业科技有限公司 Old lobular broadleaf holly leaf extract and application thereof in preparing food for relieving alcohol-induced liver injury
CN115919937A (en) * 2022-10-18 2023-04-07 美益添生物医药(武汉)有限公司 Preparation for regulating biological intestinal flora and preparation method and application thereof

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