CN114191457A - Application of abelmoschus manihot flower extract in regulating intestinal flora related to diabetic nephropathy - Google Patents
Application of abelmoschus manihot flower extract in regulating intestinal flora related to diabetic nephropathy Download PDFInfo
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Classifications
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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)
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- 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
Abstract
The invention provides an application of an abelmoschus manihot flower extract in regulating intestinal microorganisms related to diabetic nephropathy. The flos Abelmoschi Manihot flower extract contains quercetin-3-O-robioside, isoquercitrin, quercetin-3' -O-beta-D-glucoside, etc., and is mainly obtained by heating and extracting flos Abelmoschi Manihot flower with ethanol. The abelmoschus manihot extract can improve the relative abundance of different beneficial bacteria in the duodenum, ileum and colon of the small intestine to different degrees, reduce the relative abundance of harmful bacteria which can cause inflammatory reaction or damage intestinal barrier, effectively improve and/or regulate intestinal flora, and further effectively prevent or treat diseases such as chronic nephropathy, diabetic nephropathy and the like through the action on the intestinal flora.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to an application of an abelmoschus manihot flower extract in improving and/or regulating intestinal microorganisms related to diabetic nephropathy.
Background
The intestinal flora plays a very important role in the human body, and is regarded as another 'hidden organ' of the human body, carrying a 'second gene' of the human body, so that the human body is also described as a 'super organism'. The intestinal tract of an adult is roughly 1014The individual bacteria, which account for approximately 10 times their own cells, weigh approximately the weight of the liver. The human genome has approximately 2.3 ten thousand genes, while the genome of the intestinal flora has approximately 3.3 million genes, which is 150 times that of the human genome. The physiological conditions of different parts in the gastrointestinal tract are different, so that the composition of bacteria is different, the large intestine, particularly the colon part, is most suitable for the survival of microorganisms, the density of a microorganism system is highest, and the number of viable bacteria is as high as 1012-1014. Research shows that the health of human body is not only related to the genome of the human body, but also has a close and inseparable relation with environmental microorganisms in intestinal tract, the intestinal tract flora regulates and controls the intestinal tract homeostasis through the interaction between the intestinal tract flora and a host, and the imbalance of the intestinal tract homeostasis caused by various reasons can influence the health of the human body and cause various diseases.
In recent years, the kidney-intestine axis has been attracting attention in studies on the mechanism of Chronic Kidney Disease (CKD) and intestinal flora. The main physiological functions of the intestinal flora include: (1) the nutrition function, the flora can metabolize the nutrient substance ingested by the host into a substance which is beneficial to the absorption of the host; (2) biological antagonism, beneficial microorganism can inhibit growth and reproduction of pathogenic bacteria and prevent adhesion and damage to intestinal epithelial cells; (3) metabolism, and the beneficial microorganism metabolite short-chain fatty acid can inhibit the growth of pathogenic bacteria, stimulate the proliferation of intestinal epithelial cells, protect the kidney, and the like. However, disturbances of the intestinal flora can be caused by factors such as the environment, diet, viral infections, use of antibiotics and increased age. Researches prove that the intestinal dysbacteriosis is related to obesity, cardiovascular diseases, diabetes, inflammatory enteritis, multiple sclerosis, allergic diseases and other diseases, and multiple researches show that the intestinal dysbacteriosis is related to the occurrence of diabetes and diabetic nephropathy, the disordered flora can damage the epithelial cell barrier of the intestinal tract to cause the increase of intestinal permeability, pathogenic bacteria translocation, endotoxin accumulation and the like to trigger the inflammatory reaction of the organism, and can also accelerate the progress of the diabetic nephropathy by influencing lipid metabolism, short-chain fat metabolism and the like. In addition, researches show that the kidney injury of the diabetic patients cannot excrete metabolic wastes so that the metabolic wastes enter intestinal tracts, harmful flora utilizing the metabolic wastes in the intestinal tracts is greatly proliferated to cause flora imbalance, and toxic metabolites such as ammonia and indoxyl sulfate generated by the flora are also related to the kidney injury and the diabetes development. Certain toxic metabolites can also damage the intestinal mucosa, disrupting the intestinal epithelial barrier, allowing toxic metabolites to migrate into the blood, and promoting systemic inflammatory responses to accelerate the progression of diabetic nephropathy. Therefore, the imbalance of the intestinal flora is closely related to diabetic nephropathy.
Abelmoschus manihot (L.) Medic dried flower of Abelmoschus manihot (L.) Medic of Abelmoschus of Malvaceae has sweet, cold and smooth nature and taste, is nontoxic, has the effects of clearing heat, promoting diuresis, diminishing inflammation and detoxifying, and is mainly used for treating stranguria and edema by oral administration and treating carbuncle, cellulitis, pyogenic infections and scald caused by hot water by external use. Modern pharmacological research shows that the total flavone extract has the effects of resisting inflammation, protecting heart and brain ischemia injury and heart and brain anoxia injury, and is clinically used for treating chronic nephritis, diabetic nephropathy, oral ulcer, burn and the like. CN103610712B discloses an application of an abelmoschus manihot extract in preparing a medicine for preventing and treating inflammatory bowel diseases, CN1994337B discloses a total abelmoschus manihot extract in preparing a medicine for treating nephritis, and CN109481480A discloses an application of an abelmoschus manihot extract in preparing a medicine for preventing and treating intestinal dysbacteriosis caused by abuse of antibiotics, but only discloses the influence of the abelmoschus manihot extract on intestinal dysbacteriosis caused by abuse of antibiotics: wherein at the phylum level, antibiotic treatment results in a decrease in relative abundance of Firmicutes phylum, while a increase in relative abundance of Firmicutes phylum occurs after treatment with an extract of sunset abelmoschus flowers; antibiotic treatment resulted in an increase in relative abundance of Bacteroidetes phyla, whereas the bacteroides phyla decreased after treatment with sunset flower extract; at genus level, antibiotic treatment resulted in a decrease in the relative abundance of Alistipes, Lachnospiraceae, which returned to normal levels after treatment with an extract of sunset abelmoschus flowers; the relative abundance of bacteroidides in the antibiotic treatment group is increased, and the sunflower flower extract is reduced to a normal level after treatment. The effect of sunset abelmoschus flower extract on the types of intestinal microorganisms associated with diabetic nephropathy has not been reported.
Disclosure of Invention
In order to overcome the defects, the invention develops a new application of the abelmoschus manihot extract in preparing a medicament for improving and/or regulating intestinal microorganisms related to diabetic nephropathy by researching the influence of the abelmoschus manihot extract on the intestinal microorganisms related to the diabetic nephropathy.
The technical scheme of the application is as follows:
in one aspect, the invention provides an application of an extract of abelmoschus manihot in preparing a medicament for regulating intestinal microorganisms.
Specifically, the flos Abelmoschi Manihot flower extract contains at least one of quercetin-3-O-robioside 0.2%, isoquercitrin 0.5% and quercetin-3' -O-beta-D-glucoside 0.5%.
Further, the flos Abelmoschi Manihot flower extract contains at least one of quercetin-3-O-robioside 0.2-1.2%, isoquercitrin 0.5-2.0% and quercetin-3' -O-beta-D-glucoside 0.5-2.0%.
Further, the sunflower flower extract contains at least one of quercetin-3-O-robioside 0.4-0.8%, isoquercitrin 0.8-1.6% and quercetin-3' -O-beta-D-glucoside 0.8-1.6% by weight.
In particular, said intestinal microorganisms are selected from:
one or more of Bacteroides Bacteroidetes, Proteobacteria Proteobacteria, Firmicutes Firmicutes, Verrucomicrobia, Chloroflexi, Actinobacillus, Patescibacteria, Tenericutes;
or is selected from: one or more of Curvibacter of Comamonas, UCG-001Prevotella aceUCG-001 of Prevotella, Lachnospiraceae _ uncultured of Lachnospiraceae;
or is selected from: acinetobacter, Bradyrhizobium Bradyrhizobium, Allistipes, Bacteroides, Enterobacter Enterorhabdus, Aeromonas, Proteus 9Prevotella 9, Sphingobacterium, Streptococcus, Proteus 2Prevotella 2, Faecalibacterium Faecalis, Bacillus Sedimibacter, Phyllobacterium Phylobacter, Sphingomonas, Desulfovibrio, Vibrio Anaerobiospirillus, Aeromonas, Pseudomonas, Germinobacterium, Murilobacillus Norank, Gastrainers Norank, Pseudomonas, Methylobacterium, Lactobacillus paracasei, one or more of the genera Phyllobacterium, Eubacterium xylophilus [ Eubacterium ] xylophilum group, Clostridium ruminicola 5 Ruminicostium 5, Candidatussaccharans, GCA-900066225.
Further, said intestinal microorganism is selected from the group consisting of:
one or more of Bacteroides Bacteroidetes, Firmicutes Firmicutes, Verrucomicrobia, Actinobacteria actinomycetemcomitans, Chloroflexi Chloromyces, Proteobacteria proteorum, Patescibacteria, Tenericutes;
or is selected from: one or more of UCG-001Prevotella UCG-001 of Prevotella, Curvibacter of Comamonas, Lachnospiraceae _ uncultured of Lachnospiraceae;
or is selected from: desulfovibrio, Muronibacter _ norrank, Acinetobacter, Phyllobacterium, Sphingomonas, Streptococcus, Allorhizobium, Neoshizobium-Parashizobium-Rhizobium, Akkermansia, Aliskis, Geobacillus, Enterobacter Enterorhabdus, Bacteroides, Gastraerophilales _ norrank, Eubacterium xylophilus, Clostridium group, 9 ruminifloride 9, Lachnospiraceae NK4A group, Lactobacillus, Tucibacter, Clostridium, 5 Sarcophyllum, Clostridium, Pseudomonas sp, or Pseudomonas sp.
In another aspect, the invention provides an application of an extract of abelmoschus manihot in preparing a medicament for regulating intestinal microorganisms of a patient with diabetic nephropathy.
In another aspect, the invention provides an application of a sunset abelmoschus flower extract in preparing a medicament for treating diabetic nephropathy.
In another aspect, the present invention provides the use of a plant extract in the preparation of a medicament for modulating gut microbiota associated with diabetic nephropathy.
Specifically, the plant extract contains at least one of quercetin-3-O-robioside 0.2% or more, isoquercitrin 0.5% or more, and quercetin-3' -O-beta-D-glucoside 0.5% or more by weight.
Further, the plant extract contains at least one of quercetin-3-O-robioside 0.2-1.2%, isoquercitrin 0.5-2.0% and quercetin-3' -O-beta-D-glucoside 0.5-2.0% by weight.
Further, the plant extract contains at least one of quercetin-3-O-robioside 0.4-0.8%, isoquercitrin 0.8-1.6% and quercetin-3' -O- β -D-glucoside 0.8-1.6% by weight.
Specifically, the plant extract is extracted from a hibiscus plant and/or a malvaceae plant.
Further, the plant is one or more of abelmoschus manihot, hibiscus syriacus, fava sylvestris and millettia grass, preferably one or more of whole plant, corolla, root, stem, leaf and fruit of the plant; preferably corolla of said plant.
Furthermore, the plant extract is an extract of flower of abelmoschus manihot.
Specifically, the plant extract is a solvent extract, preferably an ethanol extract, and the ethanol may be absolute ethanol or aqueous ethanol, preferably an extract obtained by refluxing with 50-100% ethanol, and more preferably an extract obtained by refluxing with 80-100% ethanol.
Specifically, the preparation method of the abelmoschus manihot flower extract comprises the following steps: heating flos Abelmoschi Manihot with ethanol for extraction, concentrating the extractive solution, adjusting pH, standing, concentrating, and vacuum drying to obtain flos Abelmoschi Manihot extract.
Preferably, flos Abelmoschi Manihot extract is prepared by extracting flos Abelmoschi Manihot with 15-20 times of 80-100% ethanol under heating for 1-3 times (each for 0.5-2 hr), filtering, mixing filtrates, recovering ethanol, concentrating the filtrate to specific gravity of 1.10-1.35, adjusting pH to 5.6-6.4, standing at 0-10 deg.C for 24-60 hr, removing upper oil layer, concentrating, and vacuum drying.
More preferably, the flos Abelmoschi Manihot extract is prepared by extracting flos Abelmoschi Manihot with 16-19 times of 95-100% ethanol at a temperature higher than 80 deg.C for 1-2 times, filtering, mixing filtrates, recovering ethanol, concentrating the filtrate to specific gravity of 1.15-1.30, adjusting pH to 5.8-6.2, standing at 0-5 deg.C for 30-52 hr, removing upper oil layer, concentrating, and vacuum drying.
Preferably, the flos Abelmoschi Manihot extract is prepared by extracting flos Abelmoschi Manihot with 18 times of 95-100% ethanol under reflux for 1 time, each for 1 hr, filtering, mixing filtrates, recovering ethanol, concentrating the filtrate to specific gravity of 1.18-1.22, adjusting pH to 6.0, standing at 0-5 deg.C for 34-48 hr, removing upper oil layer, concentrating, and vacuum drying.
Specifically, the drying mode is thin layer quick drying or vacuum drying, and vacuum drying is preferred.
In still another aspect, the invention provides the use of the plant extract in the preparation of a medicament for regulating intestinal microorganisms.
In still another aspect, the invention provides the use of the plant extract in the preparation of a medicament for regulating intestinal microorganisms in a patient with diabetic nephropathy.
In another aspect, the invention provides the use of the plant extract in the preparation of a medicament for treating diabetic nephropathy.
Specifically, the administration dose of the plant extract to a patient is 4-400mg/kg, preferably 8-200mg/kg, preferably 14-160mg/kg, preferably 16-80 mg/kg.
Specifically, the plant extract is administered to the patient once to three times a day.
Specifically, the plant extract is administered to the patient three times a day, 30mg/kg each time.
In another aspect, the invention provides a use of a Chinese medicinal composition in improving and/or regulating intestinal microorganisms associated with diabetic nephropathy.
Specifically, the traditional Chinese medicine composition comprises the plant extract.
Specifically, the traditional Chinese medicine composition further comprises a pharmaceutically acceptable adjuvant, and the adjuvant comprises but is not limited to: diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents and binders.
Specifically, the traditional Chinese medicine composition can be prepared into granules, tablets, capsules or oral liquid preparations.
Compared with the prior art, the invention has the advantages that:
(1) the abelmoschus manihot flower extract provided by the invention can effectively reduce the biochemical indexes related to diabetic nephropathy, such as postprandial blood sugar, drinking water, food consumption, urine volume, 24h urine microalbumin excretion rate, kidney body ratio and the like of diabetic mice, and provides a new idea for treating and preventing diseases such as chronic nephropathy, diabetic nephropathy and the like.
(2) The invention provides the application of an abelmoschus manihot flower extract in improving and/or regulating intestinal microorganisms related to diabetic nephropathy. For diabetic nephropathy, after the sunset abelmoschus flower extract is dosed, the relative abundance of different beneficial bacteria in the small intestine duodenum, ileum and colon can be increased to different degrees, the relative abundance of harmful bacteria which can cause inflammatory reaction or damage intestinal barrier is reduced, the intestinal flora is effectively improved and/or regulated, and then diseases such as chronic nephropathy, diabetic nephropathy and the like are effectively prevented or treated through the effect of the intestinal flora.
(3) The application of the abelmoschus manihot extract is expanded.
Drawings
FIG. 1 shows the results of postprandial blood glucose tests. Wherein, 1A is the postprandial blood glucose test result after high dose administration, and 1B is the postprandial blood glucose test result after low dose administration.
FIG. 2 shows the results of measurements of drinking water, food intake and urine volume after high dose administration. Wherein, 2A is the drinking water detection result after high-dose administration, 2B is the food intake detection result after high-dose administration, and 2C is the urine volume detection result after high-dose administration.
FIG. 3 shows the results of the measurements of drinking water, food intake and urine volume after low dose administration. Wherein, 3A is the drinking water detection result after low-dose administration, 3B is the food intake detection result after low-dose administration, and 3C is the urine volume detection result after low-dose administration.
FIG. 4 shows the results of 24h urine microalbumin excretion assay. Wherein 4A is the result of detecting the urinary microalbumin excretion rate 24h after high dose administration, and 4B is the result of detecting the urinary microalbumin excretion rate 24h after low dose administration.
Fig. 5 shows the results of the measurement of the kidney weight-to-body weight ratio (kidney body ratio). Wherein, 5A is the detection result of the weight-to-weight ratio of the kidney (kidney body ratio) after high dose administration, and 5B is the detection result of the weight-to-body ratio of the kidney (kidney body ratio) after high dose administration.
FIG. 6 is a graph of the analysis of differences at the portal level for small intestine section A (duodenum) after low dose administration.
FIG. 7 is a graph of analytical differences at the genus level for small intestine segment A (duodenum) after low dose administration.
FIG. 8 is a graph of the differences analyzed at the portal level in the small intestine segment B (ileum) after low dose administration.
FIG. 9 is a graph of the analysis of differences at the genus level in the small intestine B segment (ileum) after low dose administration.
FIG. 10 is a graph of the differences analyzed at the portal level in the C-segment of the small intestine (colon) after low dose administration.
FIG. 11 is a chart of analysis of differences at the genus level in the C-segment (colon) of the small intestine after low dose administration.
FIG. 12 is a graph of the differences analyzed at the portal level for small intestine segment A (duodenum) after high dose administration.
FIG. 13 is a graph of analytical differences at the genus level for small intestine section A (duodenum) after high dose administration.
FIG. 14 is a graph of the differences analyzed at the portal level in the small intestine segment B (ileum) after high dose administration.
FIG. 15 is a graph of the genus level analysis of differences in small intestine B segment (ileum) after high dose administration.
FIG. 16 is a graph of the analytical difference at the portal level for the C-segment of the small intestine (colon) after high dose administration.
FIG. 17 is a graph of analytical differences at the genus level for the C-segment of the small intestine (colon) after high dose administration.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
The structures of the compounds involved in the examples of the present invention are:
quercetin-3-O-robinin:
isoquercitrin:
quercetin-3' -O- β -D-glucoside:
example 1 preparation of an extract of abelmoschus manihot
Taking 2000g of flower crown of Abelmoschus manihot, adding 18 times of anhydrous ethanol, heating and refluxing for 1 hour, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating the filtrate to specific gravity of 1.20, adjusting pH to 6.0, standing at 0-5 deg.C for 48 hours, removing the upper oil layer, concentrating, slowly adding into a vacuum belt drier, drying at 100 deg.C, pulverizing, and packaging into a clean double-layer plastic bag to obtain Abelmoschus manihot extract. Wherein the composition contains quercetin-3-O-robioside (6.0mg/g), isoquercitrin (12.1mg/g), and quercetin-3' -O-beta-D-glucoside (12.7 mg/g).
Example 2 preparation of an extract of abelmoschus manihot
Taking 3000g of abelmoschus manihot, extracting the abelmoschus manihot with 19 times of 95% ethanol under reflux for 2 times, each time for 1 hour, filtering, combining the filtrates, recovering ethanol, concentrating the filtrate to the specific gravity of 1.18-1.28, adjusting the pH value to 5.8-6.2, standing the concentrated solution at 0-5 ℃ for 40-48 hours, removing an oil layer of a cold storage solution, slowly adding the concentrated solution into a vacuum belt drier, drying at 80-100 ℃, crushing, and filling into a clean double-layer plastic bag to obtain the abelmoschus manihot extract. Wherein the flos Abelmoschi Manihot extract contains quercetin-3-O-robioside (5.6mg/g), isoquercitrin (10.1mg/g), and quercetin-3' -O-beta-D-glucoside (13.8 mg/g).
Examples of the experiments
1 materials of the experiment
1.1 Experimental animals
100 female NOD/LtJ mice (11-12 weeks old) purchased from Beijing Huafukang Biotech GmbH, license number: SCXK (Jing) 2014-. Feeding in SPF environment of pharmaceutical animal experiment center of Chinese university of pharmacy, and administering sufficient sterile drinking water and sterile common feed while maintaining 12h light and 12h night alternation.
1.2 medicine
An extract of abelmoschus manihot of example 1.
2 method of experiment
2.1 Experimental groups and dosing
After NOD mice are bred for one week and are adaptive to a new environment, postprandial blood sugar and body weight of all mice are measured once a week, and if the blood sugar is more than 11.1mmol/L for two consecutive days, the modeling of diabetes is successful. According to the principle that the blood sugar level and the body weight are similar, NOD mice with diseases after 22 weeks are divided into (1) a low-dose abelmoschus manihot extract administration group LH, (2) a disease low-dose control group LC, (3) a high-dose abelmoschus manihot extract administration group HH, (4) a disease high-dose control group HC and (5) a non-disease LN group. Each group was dosed as follows:
(1) low dose abelmoschus manihot extract administration group LH: the administration is flos Abelmoschi Manihot extract, the administration dosage is 1.08g flos Abelmoschi Manihot extract/kg body weight/day, and the administration is continuous for 4-5 weeks, and the administration mode is intragastric;
(2) diseased low dose control group LC: irrigating the stomach with ultrapure water;
(3) high dose abelmoschus manihot extract dosing group HH: the administration is the abelmoschus manihot extract, the administration dosage is 2.4g of the abelmoschus manihot extract per kg of body weight per day, and the administration mode is as follows: performing intragastric administration;
(4) onset high dose control HC: irrigating the stomach with ultrapure water;
(5) non-diseased LN group: and (5) irrigating the stomach with ultrapure water.
2.2 measurement of Biochemical indicators
2.2.1 postprandial blood glucose determination
Measuring postprandial blood sugar and body weight once per week before and after administration, collecting blood from tail vein, and measuring postprandial blood sugar with glucometer and blood sugar test paper according to instruction.
2.2.2 basic measurement of Water intake, urine volume, food intake, etc
Urine was collected 24 hours before and after administration every week, and the basic conditions such as water intake mL, diet intake g, urine output mL, etc. were recorded.
2.2.324 h determination of urinary microalbumin excretion Rate
Taking a proper amount of urine samples, operating according to the specification of the urine microalbumin kit, measuring the content of 24h urine microalbumin in each group, and calculating the excretion rate (UAER) of 24h urine microalbumin according to a calculation formula.
UAER urinary microalbumin concentration ng/mL 24h urine volume (mL)
2.2.4 Kidney weight and body weight determination
Before killing, the animal weight is weighed and the weight of the left kidney and the right kidney is weighed for calculating the kidney body ratio.
2.3 Collection of samples
After 4-5 weeks of dosing, animals were slaughtered and weighed before slaughtering. The duodenum, ileum and colon are respectively taken by adopting a surgical double ligation method, and each segment is about two centimeters. All samples are put into a freezing tube to be preserved for 5-60 minutes in liquid nitrogen, and then are preserved at minus 80 ℃ for detecting the intestinal flora.
2.4 sequencing of intestinal flora
2.4.1 genomic DNA extraction
Total genomic DNA in the samples was extracted using the CTAB/SDS method and DNA concentration and purity was monitored on a 1% agarose gel. The DNA was diluted to 1 ng/. mu.L using sterile water according to concentration.
2.4.2PCR amplification and purification
The V3-V4 variable region of the 16srRNA gene was PCR amplified according to the reaction system of Table 1, with universal primers of 341F (5 '-CCTAYGGGRBGCASCAG-3') and 806R (5 '-GGACTACNNGGGTATCTAAT-3').
Amplification reaction system for V3-V4 region of Table 116 Sr RNA
PCR reaction parameters: initial denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 27 cycles; finally 10 minutes at 70 ℃.
Repeating each sample for three times, mixing PCR products of the same sample, detecting by adopting 2% agarose gel electrophoresis, cutting a target band, and cutting according to the operation method of the gel recovery kit to recover PCR amplification products.
2.4.3 fluorescence quantitation
Referring to the preliminary quantification result of electrophoresis, the PCR product was quantified using QuantiFluorTMAnd (4) quantitative determination of ST blue fluorescence, and mixing according to the sequencing quantity requirement of each sample after detection and quantification.
2.4.4 sequencing library preparation and sequencing
NEB was used according to the instructions
The sequencing library was prepared by the UltraTMdnaLiabrary Prep Kit and the library was quality checked using the Qubit @2.0 fluorometer and Agilent bioanalyzer 2100 system. Finally, sequencing the library on the IlluminaMiSeq platform to obtain a 200-450bp double-end sequence.
2.4.5 bioinformatic analysis
After a high-quality sequence is obtained by primary screening and optimization of a sequence obtained by Illumina PE250 sequencing, analyzing the high-quality sequence by a bioinformatics method: 1) OTU (Operational taxomic Units, Operational classification unit) cluster analysis: classifying and dividing the obtained sequences according to 97% similarity by means of Usearch software, selecting the sequence with the highest abundance in each OTU as a representative sequence, and comparing the representative sequence with a database template sequence to obtain taxonomy information corresponding to each OTU; 2) alpha diversity analysis: analyzing The abundance and diversity of The microorganisms according to The obtained OTU, wherein The analysis comprises reflecting a Chano 1 index (The Chao1estimator), a Shannon index (Shannon diversity index) and a Simpson index (The Simpson index) which show The diversity of The community, and detecting whether The sequencing depth meets The requirement by using a dilution curve; 3) PCA Analysis (Principal Component Analysis): analyzing the similarity between each group of samples; 4) taxonomic composition analysis: according to the OTU division and classification status identification results, the composition and abundance distribution of each sample at different classification levels of phyla, class, order, family, genus and species can be obtained and clustered; 5) LEfSe differential analysis: species with significant differences between groups were analyzed.
2.5 statistical analysis of data
All data were plotted and statistically analyzed using Graphpad Prism 5.0 and SPSS23.0 software, and the significance between groups was compared using t-test, with P < 0.05 being statistically significant.
3 results of the experiment
3.1 Biochemical index results
3.1.1 postprandial blood glucose results
As shown in FIG. 1A, the blood glucose levels in HC group were elevated after onset and declined after the second week, while the blood glucose levels in HH group were elevated after administration and were not different from HC group. As shown in FIG. 1B, the blood sugar in the LC group increased after onset, while the blood sugar in the LH group was steadily increased after administration. Therefore, the low dose administration has a good hypoglycemic effect on the hyperglycemic mice, but the high dose administration does not have the hypoglycemic effect.
3.1.2 Water, food and urine measurements
As shown in fig. 2A, the water intake of HC group increased after onset, and decreased in the third week, but the water intake increased compared to 0week, and the water intake of HH group increased after onset, which was not different from HC group. As shown in FIG. 2B, the appetite increased after onset in HC group, but returned to an increased trend in the third week, and the appetite increased after onset in HH group, which was not different from HC group. As shown in FIG. 2C, the urine volume after the onset of HC was almost the same as that in HH group, and there was no difference therebetween.
As shown in FIG. 3A, the water intake of the LC group increased after onset of disease, but the water intake of the LH group did not increase and was always in a steady state. As shown in FIG. 3B, the food intake was increased all the time after onset in the LC group and decreased suddenly in the fourth week, which probably related to the long onset time and poor condition of the mice, while the food intake in the LH group was lower than that in the LC group. As shown in FIG. 3C, the urine volume of the LC group increased after onset, but the urine volume of the LH group was in a more steady state. Therefore, the low dose has a good improvement effect on the symptoms of 'more than three and less than three' in the hyperglycemic mice, while the high dose does not.
3.1.324 h determination of urinary microalbumin excretion Rate
As shown in FIG. 4A, UAER 1week increased more than 0week after onset in HC group, but 2-4week did not increase, and UAER increased more than HC group after onset in HH group. As shown in fig. 4B, UAER increased after onset in LC group, whereas UAER decreased in LH group starting in the second week, both lower than LC group. Thus, lower doses have a UAER-reducing effect, whereas higher doses do not.
3.1.4 Kidney weight to body weight ratio (Kidney body ratio)
As shown in fig. 5A, the renal body ratios of both the right and left kidneys increased after the onset of HC, and no difference was observed between the HH group and the HC group. As shown in fig. 5B, the renal body ratios of both the left and right kidneys increased after the onset of the LC group, and decreased to normal levels after the LH group administration. Thus, low doses improve kidney enlargement, while high doses do not.
3.2 sequencing results of intestinal flora
3.2.1 sequencing of intestinal flora with Low dose administration
Wherein A represents the duodenum, B represents the ileum, and C represents the colon.
Differential analysis of the relative abundance of the flora at the level of phylum and genus in duodenum is shown in fig. 6 and fig. 7, respectively. As can be seen from FIG. 6, the relative abundance of Bacteroides (Bacteroides) in duodenum increased after administration at the phylum level; the relative abundance of Firmicutes and Verrucomicrobia decreases. Analysis at the genus level as can be seen from FIG. 7, the relative abundance of Murivulariae _ norak, Allorhizobium-Parahizobium-Rhizobium, Desulfovibrio (Desulfovibrio) increased in the duodenum after administration; the relative abundance of Acinetobacter (Acinetobacter), Phyllobacterium (Phyllobacterium), Sphingomonas (Sphingomonas), and Streptococcus (Streptococcus) is reduced.
Differential analysis of the relative abundance of the flora at the level of the phylum and genus of ileum is shown in fig. 8 and fig. 9, respectively. As can be seen in fig. 8, the relative abundance of actinomycetemcomita (actinobacillia) and Bacteroidetes (bacteroidides) in the ileum increases after administration at the phylum level; the relative abundance of Microbacterium verruciformis (Verrucomicrobia) decreased. Analysis at the genus level as can be seen from fig. 9, the relative abundance of Akkermansia (Akkermansia), Alistipes (Alistipes), bacilli (Geobacillus), Phyllobacterium (Phyllobacterium), Sphingomonas (sphingamonas) decreased in the ileum after administration; increased relative abundance of Desulfovibrio (Desulfovibrio), Enterobacter (Enterobacter), and Murebacteriaceae _ norak.
The differential analysis of the relative abundance of the flora at the phylum and genus levels in the colon is shown in FIG. 10 and FIG. 11, respectively. As can be seen from FIG. 10, the relative abundance of Bacteroides (Bacteroides) and Microbacterium Verrucomicrobia (Verrucomicrobia) decreased after administration at the phylum level; the relative abundance of Actinomycetes (Actinobacilla) and Firmicutes (Firmicutes) has increased. Analysis at the genus level as can be seen from FIG. 11, the relative abundance of Exmanella incarnata (Akkermansia), Bacteroides (Bacteroides), Gastranaerophilles _ norank, Prevotella UCG-001(Prevotella UCG-001), Eubacterium xylanophilum group, Clostridium ruminicola 9 (Ruminostrobildium 9) decreased after administration; the relative abundance of Desulfosvibrio (Desulfovibrio), Lachnospiraceae NK4A136 group, Enterobacter (Enterobacter), Lactobacillus (Lactobacillus), and Turcibacter is increased.
3.2.2 sequencing of intestinal flora with high dose administration
Differential analysis of the relative abundance of the flora at the level of phylum and genus in duodenum is shown in fig. 12 and fig. 13, respectively. As can be seen from fig. 12, the relative abundance of actinomycetemcomita (actinobacillia), Bacteroidetes (Bacteroidetes), closterobacteroidetes (Chloroflexi) in the duodenum decreased after high dose administration at the phylum level. Analysis of this at the genus level, it can be seen from FIG. 13 that the relative abundance of Acinetobacter (Acinetobacter), Aeromonas (Aeromonas), Chromobium (Bradyrhizobium), Comamonas (Curvibacter), Pseudomonas (Pseudomonas), Sphingomonas, Muronibacter _ noran in the duodenum decreased after high dose administration.
Differential analysis of the relative abundance of the flora at the level of the phylum and genus of ileum is shown in fig. 14 and fig. 15, respectively. As can be seen in FIG. 14, the relative abundance of Bacteroides (Bacteroides) in the ileum decreased and that of Proteobacteria (Proteobacteria) increased after high doses at the phylum level. Analysis at the genus level it can be seen from figure 15 that the relative abundance of Acinetobacter (Acinetobacter), Sphingomonas (sphingamonas) increases in the ileum following high dose administration.
Differential analysis of the relative abundance of the bacterial population at phylum and genus levels in the colon is shown in FIGS. 16 and 17, respectively. As can be seen from fig. 16, at the phylum level, the relative abundances of Bacteroidetes (bacteroides) and Proteobacteria in the high-dose group C were low for the higher-dose group N, but the relative abundances of bacteroides (bacteroides) were not changed after administration, the relative abundances of Proteobacteria were increased, the relative abundances of Firmicutes (Firmicutes), Proteobacteria (Proteobacteria), and Tenericutes in the high-dose group C were all higher than those of the high-dose group N, and only the relative abundances of Firmicutes (Firmicutes) were slightly decreased after administration. The relative abundance of actinomycetales (actinobacilla) in the three groups was essentially the same. As can be seen from fig. 17, the Lachnospiraceae (Lachnospiraceae _ uncultured), ruminicostium 5, ruminicostium 9 were relatively more abundant in genus-level high-dose group C than in high-dose group N, and all decreased in relative abundance after administration; the relative abundance of Candidatus Saccharioninas, GCA-900066225 was lower in the high dose group C than in the HN group, and slightly increased after administration.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Jiangsu Chinese medicine industry group member company Jiangsu Chinese medicine industry research institute company Limited
Application of flower extract of abelmoschus manihot in regulating intestinal flora related to diabetic nephropathy
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Claims (10)
1. An application of a flower extract of abelmoschus manihot in preparing a medicine for regulating intestinal microorganisms related to diabetic nephropathy is characterized in that the flower extract of abelmoschus manihot contains at least one of quercetin-3-O-robioside more than 0.2%, isoquercitrin more than 0.5% and quercetin-3' -O-beta-D-glucoside more than 0.5% by weight.
2. The use as claimed in claim 1, wherein the sunflower flower extract comprises at least one of quercetin-3-O-robioside, isoquercitrin, 0.5-2.0% quercetin-3' -O- β -D-glucoside, all by weight.
3. The use as claimed in claim 2 wherein the sunflower extract comprises at least one of quercetin-3-O-robioside, isoquercitrin, 0.8-1.6% and quercetin-3' -O- β -D-glucoside, 0.8-1.6% by weight.
4. Use according to claims 1-3, wherein said intestinal micro-organisms are selected from the group consisting of:
one or more of Bacteroides Bacteroidetes, Proteobacteria Proteobacteria, Firmicutes Firmicutes, Verrucomicrobia, Chloroflexi, Actinobacillus, Patescibacteria, Tenericutes;
or is selected from: one or more of Curvibacter of Comamonas, UCG-001Prevotella UCG-001 of Prevotella, Lachnospiraceae _ uncultured of Lachnospiraceae;
or is selected from: acinetobacter, Bradyrhizobium Bradyrhizobium, Allistipes, Bacteroides, Enterobacter Enterorhabdus, Aeromonas, Proteus 9Prevotella 9, Sphingobacterium, Streptococcus, Proteus 2Prevotella 2, Faecalibacterium Faecalis, Bacillus Sedimibacter, Phyllobacterium Phylobacter, Sphingomonas, Desulfovibrio, Vibrio Anaerobiospirillus, Aeromonas, Pseudomonas, Germinobacterium, Murilobacillus Norank, Gastrainers Norank, Pseudomonas, Methylobacterium, Lactobacillus paracasei, one or more of the genera Phyllobacterium, Eubacterium xylophilus [ Eubacterium ] xylophilum group, Clostridium ruminicola 5 Ruminicostium 5, Candidatussaccharans, GCA-900066225.
5. The use according to claim 4, wherein said intestinal microorganisms are selected from the group consisting of:
one or more of Bacteroides Bacteroidetes, Firmicutes Firmicutes, Verrucomicrobia, Actinobacteria actinomycetemcomitans, Chloroflexi Chloromyces, Proteobacteria proteorum, Patescibacteria, Tenericutes;
or is selected from: one or more of UCG-001Prevotella UCG-001 of Prevotella, Curvibacter of Comamonas, Lachnospiraceae _ uncultured of Lachnospiraceae;
or is selected from: desulfovibrio, Muronibacter _ norrank, Acinetobacter, Phyllobacterium, Sphingomonas, Streptococcus, Allorhizobium, Neoshizobium-Parashizobium-Rhizobium, Akkermansia, Aliskis, Geobacillus, Enterobacter Enterorhabdus, Bacteroides, Gastraerophilales _ norrank, Eubacterium xylophilus, Clostridium group, 9 ruminifloride 9, Lachnospiraceae NK4A group, Lactobacillus, Tucibacter, Clostridium, 5 Sarcophyllum, Clostridium, Pseudomonas sp, or Pseudomonas sp.
6. Use of a plant extract for the preparation of a medicament for modulating gut microbiome associated with diabetic nephropathy, said plant extract comprising at least one of more than 0.2% quercetin-3-O-robioside, more than 0.5% isoquercitrin and more than 0.5% quercetin-3' -O- β -D-glucoside by weight; the plant extract contains at least one of 0.2-1.2% by weight of quercetin-3-O-robioside, 0.5-2.0% by weight of isoquercitrin and 0.5-2.0% by weight of quercetin-3' -O-beta-D-glucoside; the plant extract contains at least one of 0.4-0.8% by weight of quercetin-3-O-robioside, 0.8-1.6% by weight of isoquercitrin and 0.8-1.6% by weight of quercetin-3' -O-beta-D-glucoside.
7. The use as claimed in claim 6, wherein the plant extract is extracted from Hibiscus and/or Malvaceae; preferably one or more of Abelmoschus manihot, Hibiscus syriacus, Hibiscus esculentus, and Phyllanthus niruri; preferably one or more of the whole plant, corolla, root, stem, leaf and fruit of the plant; more preferably, corolla.
8. Use according to any one of claims 1 or 6, wherein the plant extract is an extract of Abelmoschus manihot, preferably an ethanol extract of Abelmoschus manihot, preferably an extract obtained by 50-100% ethanol reflux extraction, more preferably an extract obtained by 80-100% ethanol reflux extraction.
9. The use according to claim 8, wherein the preparation of the extract of sunset abelmoschus flower comprises the steps of: heating flos Abelmoschi Manihot with ethanol for extraction, concentrating the extractive solution, adjusting pH, standing, concentrating, and vacuum drying to obtain flos Abelmoschi Manihot extract;
preferably, taking flower of Abelmoschus manihot, adding 15-20 times of 80-100% ethanol, heating and extracting for 1-3 times, each time for 0.5-2h, filtering, combining filtrates, recovering ethanol, concentrating the filtrate to specific gravity of 1.10-1.35, adjusting pH to 5.6-6.4, standing at 0-10 deg.C for 24-60 hr, removing upper oil layer, concentrating, and vacuum drying to obtain extract of Abelmoschus manihot;
preferably, the flos Abelmoschi Manihot extract is prepared by extracting flos Abelmoschi Manihot with 16-19 times of 95-100% ethanol at a temperature higher than 80 deg.C for 1-2 times, filtering, mixing filtrates, recovering ethanol, concentrating the filtrate to specific gravity of 1.15-1.30, adjusting pH to 5.8-6.2, standing at 0-5 deg.C for 30-52 hr, removing upper oil layer, concentrating, and vacuum drying to obtain flos Abelmoschi Manihot extract;
preferably, the flos Abelmoschi Manihot extract is prepared by extracting flos Abelmoschi Manihot with 18 times of 95-100% ethanol under reflux for 1 time, each for 1 hr, filtering, mixing filtrates, recovering ethanol, concentrating the filtrate to specific gravity of 1.18-1.22, adjusting pH to 6.0, standing at 0-5 deg.C for 34-48 hr, removing upper oil layer, concentrating, and vacuum drying.
10. The use according to any one of claims 1 or 6, wherein the plant extract is administered in a dose of 4-400mg/kg, preferably 8-200mg/kg, preferably 14-160mg/kg, preferably 16-80 mg/kg; the administration frequency of the plant extract is from once to three times a day.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108567802A (en) * | 2018-07-25 | 2018-09-25 | 江苏苏中药业集团股份有限公司 | A kind of sunset abelmoschus flower flavones effective kind part and the preparation method and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108567802A (en) * | 2018-07-25 | 2018-09-25 | 江苏苏中药业集团股份有限公司 | A kind of sunset abelmoschus flower flavones effective kind part and the preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 郭建明: "黄蜀葵花调控肠道菌群中尿毒素代谢通路干预慢性肾病进展的作用机制", 中国药理学与毒理学杂志, vol. 33, no. 9, 30 September 2019 (2019-09-30), pages 665 * |
| 陆林玲: "黄葵胶囊主要成分PK、PD研究", 中国优秀硕士学位论文全文数据库 医药卫生科技辑, no. 8, 15 August 2016 (2016-08-15) * |
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