CN111494478B - Application of total flavone extract of wild jujube leaves in preparation of medicine for preventing and treating intestinal dysbacteriosis - Google Patents

Application of total flavone extract of wild jujube leaves in preparation of medicine for preventing and treating intestinal dysbacteriosis Download PDF

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CN111494478B
CN111494478B CN202010337850.6A CN202010337850A CN111494478B CN 111494478 B CN111494478 B CN 111494478B CN 202010337850 A CN202010337850 A CN 202010337850A CN 111494478 B CN111494478 B CN 111494478B
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wild jujube
flavone extract
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CN111494478A (en
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杜晨晖
闫艳
付彩
崔小芳
裴香萍
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Kampo Extract Biotechnology Hainan Co ltd
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Abstract

The invention provides an application of wild jujube leaf total flavone extract in preparing a medicament for preventing and treating intestinal dysbacteriosis. The invention takes a rat with D-galactose induced intestinal flora imbalance as a research object to research the intestinal flora regulation effect of the wild jujube leaf total flavone extract. The results of experiments in rats show that the supplement of the total flavonoids extract of wild jujube leaves can prevent the imbalance of intestinal flora in the aging process induced by D-galactose, such as increasing the diversity of intestinal flora, increasing bacteroidetes, decreasing the firmicutes, decreasing the lactobacilli, and making the ratio and the composition of the firmicutes and the bacteroidetes more tend to the normal control group.

Description

Application of total flavone extract of wild jujube leaves in preparation of medicine for preventing and treating intestinal dysbacteriosis
Technical Field
The invention belongs to the field of medicines, and particularly relates to an application of a total flavone extract of wild jujube leaves in preparation of a medicine for preventing and treating intestinal dysbacteriosis.
Background
The intestinal microorganisms are important 'microbial organs' of human bodies and closely related to various physiological functions such as digestion, immunity, nutrition, metabolism, cognition and the like, and the species and the quantity of the intestinal microorganisms are subtly changed, so that the physiological functions of the organisms are influenced.
At present, most of medicines for regulating intestinal flora are probiotics medicines such as bifidobacterium triple viable bacteria powder, compound lactobacillus acidophilus tablets, bacillus subtilis dual viable bacteria enteric-coated capsules and lactobacillus tablets, and the medicines have single regulating effect on the flora and are all used for directly supplementing normal physiological bacteria, regulating the intestinal flora and inhibiting bacteria and pathogenic bacteria which have potential harm to human bodies in the intestinal tract.
The recovery of the balance of the intestinal flora through dietary intervention is a difficult point of the current research, and the research researches the effect of the total flavonoids of the wild jujube leaves on the prevention and treatment of the imbalance of the intestinal flora from the perspective of dietary intervention.
Disclosure of Invention
The invention aims to provide a new application of a total flavone extract of wild jujube leaves.
The wild jujube leaf total flavone extract is prepared by the method comprising the following steps:
soaking folium Ziziphi Spinosae, extracting with ethanol under reflux, filtering, collecting filtrate, volatilizing solvent to obtain folium Ziziphi Spinosae extract solid, loading onto D101 macroporous resin, eluting, collecting eluate, and volatilizing solvent to obtain folium Ziziphi Spinosae flavone extract;
wherein, the concentration (volume concentration) of the ethanol for soaking can be 50%;
the soaking time can be 3 hours;
the proportion of the wild jujube leaves to the ethanol for soaking can be 1000 g: 12L;
the reflux extraction can be carried out for a plurality of times, specifically for 2 times, and each time lasts for 0.5 h;
when the D101 macroporous resin is applied, the sample application concentration can be 0.5 g/mL-1Passing through the resin column at a flow rate of 0.5 BV/h;
the elution operation was: the impurities are removed by washing with distilled water, and then 50% (volume concentration) ethanol is used for eluting at the flow rate of 1 BV/h.
The purity of total flavonoids in folium Ziziphi Spinosae extract is 40.95% by ultraviolet spectrophotometry.
The invention provides a new application of the total flavone extract of wild jujube leaves, and relates to an application of the total flavone extract in preparing products for preventing and treating intestinal dysbacteriosis.
In the 1), the intestinal dysbacteriosis refers to the reduction of the microbial diversity of the intestinal flora, the increase of the abundance of firmicutes, the reduction of the abundance of bacteroidetes, the increase of lactobacilli and the increase of the ratio (F/B) of the firmicutes to the bacteroidetes of the intestinal flora;
specifically, the new application of the total flavonoids of wild jujube leaves is the application of the total flavonoids in preparing any one of the following products:
1) products for the prevention and treatment of intestinal dysbacteriosis;
1a) products that increase the microbial diversity of enteric bacteria;
1b) products that increase the abundance of bacteroidetes phyla;
1c) products that reduce the abundance of firmicutes;
1d) products that reduce the abundance of lactobacilli;
1f) the ratio and the composition of the firmicutes and the bacteroidetes are more normal;
in the above application, the intestinal dysbacteriosis may be D-galactose induced intestinal dysbacteriosis.
In the application, the product can be a medicine or a health-care product.
The invention also provides a product for preventing and treating intestinal dysbacteriosis, which takes the wild jujube leaf total flavone extract as an effective active ingredient;
in the product for preventing and treating intestinal dysbacteriosis, the effective concentration of the total flavone extract of wild jujube leaves can be 100mg/kg-200 mg/kg.
The invention takes a rat with D-galactose induced intestinal flora imbalance as a research object to develop the intestinal flora regulating effect of the wild jujube leaf total flavone extract.
The results of experiments in rats show that the supplement of the total flavonoids extract of zizyphus jujube leaves can prevent D-galactose-induced intestinal flora imbalance, such as increase of diversity of intestinal flora, increase of bacteroides, decrease of firmicutes, decrease of lactobacilli, and the ratio and composition of firmicutes and bacteroides tend to normal control groups.
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FIG. 1 is a chromatogram of a reference sample and a sample for measuring 5 chemical components in wild jujube leaves under 254nm (note: No. 1 peak rutin, No. 2 peak hyperin, No. 3 quercetin-3-O-glucoside, No. 4 peak kaempferol-3-O-rutinoside; A, B, C, D is the reference peak, E is the sample peak).
Fig. 2 is a flora sparse curve analysis provided by the present invention.
FIG. 3 is a diversity index analysis of various sets of samples provided by the present invention.
FIG. 4 is the population abundance of five groups of rats at the phylum level provided by the present invention.
FIG. 5 is a graph showing differences in portal levels of rats provided by the present invention.
FIG. 6 shows the flora abundance of five groups of rats at the family level.
FIG. 7 is a graph showing the differences in rat levels provided by the present invention.
FIG. 8 shows the flora abundance of five groups of rats at the genus level.
FIG. 9 is a graph showing the difference in the level of the genus rat provided by the present invention.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of total flavone extract from leaves of Ziziphus jujuba Mill
Weighing 1000g of folium Ziziphi Spinosae, adding 50% (volume concentration) ethanol 12L, soaking for 3 hr, reflux extracting for 2 times, each time for 0.5 hr, filtering, mixing filtrates, volatilizing solvent to obtain folium Ziziphi Spinosae extract solid, loading onto D101 macroporous resin with sample concentration of 0.5 g.mL-1Passing through resin column at flow rate of 0.5BV/h, washing with distilled water to remove impurities, eluting with 50% (volume concentration) ethanol at flow rate of 1BV/h, collecting eluate, and volatilizing solvent to obtain 92g of folium Ziziphi Spinosae flavone extract powder. The purity of the total flavonoids in the wild jujube leaves in the obtained wild jujube leaf total flavonoids extract is 40.95 percent by ultraviolet spectrophotometry.
Example 2 determination of the Total Flavonoids extract of leaves of Zizyphus jujuba Mill
Experimental apparatus and materials:
an experimental instrument: the Waters 2695A liance system comprises a quaternary pump, a vacuum online degasser, an automatic sample injector, a Waters 2489 ultraviolet detector and a solid phase extraction column (Seisager science and technology Co., Ltd.).
Reagent: methanol (metallocene chemical reagent of Tianjin) is analytically pure, and acetonitrile (TEDIA USA) is chromatographic alcohol.
Medicine preparation: rutin (batch No. 100080-200707) is purchased from China food and drug testing research institute, eriocitrin (batch No. 20161216), hyperoside (batch No. 20161022), quercetin 3-O-acacia glucoside (batch No. 20161227), isoquercitrin (batch No. 20160830), kaempferol 3-O-rutinoside (batch No. 20170417) and Bingguan Biotech company of Baoji.
Weighing the total flavone extract powder of wild jujube leaves prepared in example 1 about 10mg, precisely weighing, placing in a conical flask with a plug, precisely adding 70% methanol 25mL, weighing, and ultrasonically extracting for 20 min. Standing at room temperature, weighing, adding 70% methanol to make up for loss, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain final product solution.
Precisely weighing appropriate amount of reference substance, adding 70% methanol to obtain single reference substance stock solution with eriocitrin, hyperoside, quercetin 3-O-locust glucoside, isoquercetin, kaempferol-3-O-rutinoside concentration of 1mg/mL
Chromatographic conditions are as follows:
a chromatographic column: agela Venusil MP C18Chromatography column (250nm × 4.6nm, 5 μm); mobile phase: 0.1% formic acid (A) -acetonitrile (B), flow rate 1.0mL min-1, gradient conditions: 0-5min, A90%; 5-15min, A80%; 15-25min, A80%; 25-35min, A70%; 35-45min, A55%; 45-65min, A35%; 60-65min, A90%. Detection wavelength: 254nm and 330 nm; column temperature: 30 ℃; sample introduction amount: 10 μ L and 20 μ L.
Compared with a reference substance, the 4 compounds with high content in the total flavonoids of the wild jujube leaves are respectively found as follows: rutin No. 1, hyperin No. 2, quercetin-3-O-glucoside No. 3, kaempferol-3-O-rutinoside No. 4; A. b, C, D is the control peak and E is the sample peak. As shown in figure 1.
Example 2 Regulation of D-galactose-induced intestinal dysbacteriosis
(1) Model building and drug delivery
60 SPF-grade healthy male SD rats with the weight of 180-200 g are taken, provided by Beijing Wintolite laboratory animal technology Limited, subjected to cage separation treatment according to 5 rats per cage, adaptively fed for 7D, and then randomly and averagely divided into 4 groups according to the weight, wherein each group comprises 6 rats, namely a blank control group, a D-galactose model group, a low-dose group of wild jujube leaf total flavone extract, a high-dose group of wild jujube leaf total flavone extract and a Vc group. Preparing D-galactose-induced intestinal flora imbalance models by adopting a D-galactose intraperitoneal injection method in the other 6 groups except the blank group, namely performing intraperitoneal injection of 300mg/kg/bw of D-galactose at regular time every day, and replacing the blank group with normal saline for continuous 36 days. At the same time of molding, 100 mg/kg/bw of the total flavone extract of wild jujube leaves and 200mg/kg/bw of the total flavone extract are administrated to all animals in a stomach irrigation mode, and distilled water with the same volume is administrated to a blank group and a model group.
(2) Stool collection and 16S rRNA sequencing analysis
Rats at night on weekend 5 18: 00 to 8 in the morning of the next day: 00 placing into a metabolism cage, fasting, collecting feces, and storing at-80 deg.C. Extracting total DNA of the microbiome by using the kit, carrying out PCR amplification on a target fragment, detecting a PCR amplification product by 2% agarose gel electrophoresis, carrying out gel cutting recovery on the target fragment, and recovering by using a gel recovery kit of AXYGEN company. Using Illumina MiSeq sequencing as an example, 16S rRNA sequencing analysis was performed using a sequencing Library prepared from TruSeq Nano DNA LT Library Prep Kit from Illumina.
Statistical analysis of the data was performed using SPSS16.0, mean and Standard Deviation (SD) calculated, and differences between groups were compared by one-way anova.
The experimental results are as follows:
(1) influence of total flavone extract (LZJSF) of Zizyphus jujube leaves on abundance and diversity of intestinal flora of rats with D-galactose induced intestinal flora imbalance
As shown in fig. 2, from the analysis result of the dilution curve, it was found that when the sequencing amount of the sample exceeds 1500, the dilution curve does not tend to be saturated, but the Shannon curve tends to be flat, which indicates that the sequencing data amount is large enough to reflect most of the microorganism information in the sample.
For microbial communities, there are a variety of indices to reflect Alpha diversity, and commonly used metrics include mainly the Chao1 index and the ACE index, which are focused on the abundance of the community (i.e., the number of microbial members, such as OTUs, in the community), and the Shannon index and the Simpson index, which are considered to be the uniformity of the community (i.e., the magnitude of the abundance difference between members).
As shown in fig. 3, the values of Ace, Chao, Shannon and Simpson index of the rat intestinal flora samples in the Control group are the highest compared with those in the other groups, indicating that the abundance and diversity of the flora are higher. After D-Gal induction, the indexes of Ace, Chao and Shannon and Simpson of each group of samples are reduced, and Ace and Chao are significantly changed (P <0.01 or P <0.001), so that D-Gal induction can be proved to cause significant change of rat intestinal flora structure and reduce flora diversity. In each administration group, D-gal + LZJSF (100mg/kg) and D-gal + LZJSF (200mg/kg) increased the diversity of intestinal flora Ace and Chao index in rats (P < 0.05).
(2) Influence of total flavone extract of Zizyphus jujube leaves on the composition of intestinal flora of rats with D-galactose induced intestinal dysbacteriosis
1. Influence on the phylum level of the flora
In order to obtain the classification information of the species corresponding to each OTU, the OTU sequence is subjected to taxonomic analysis by adopting an RDP classificator Bayesian algorithm, and the composition of each sample community is counted on the Phylum (Phylum) level. As shown in FIG. 4, the intestinal flora of rats after different administration mainly comprises 8 phyla such as Firmicutes (Firmicutes), bacteroides (Bacteroides), Verrucomicrobia (Verrucomicrobia), Actinomycetes (Actinobacillus), Tenericutes (Tenericutes), Proteobacteria (Proteobacteria), Cyanobacteria (Cyanobacter), and Trachidermobacteria (Elusimicrobia), wherein the Firmicutes and Bacteroides are the main dominant phyla, and the results are consistent with the results reported before. As shown in FIG. 5, in the Control group, Bacteroides accounted for 35%, and in the D-Gal group, Bacteroides accounted for 25% more than in the Control group, and the level of the Control group was restored after D-Gal + LZJSF (200mg/kg) was administered. For Firmicutes, the proportion of Firmicutes in the Control group is 60%, the proportion of Firmicutes in the D-gal group is remarkably increased (p <0.05) to 73% compared with the Control group, and the relative abundance of Firmicutes in the D-gal + LZJSF (200mg/kg) rat is remarkably reduced (p <0.05) compared with the D-gal group. Meanwhile, as shown in FIG. 5, the ratio of firmicutes/Bacteroides (F/B) was significantly increased in the D-gal group rats compared with the Control group rats, while the ratio of F/B in the D-gal + LZJSF (200mg/kg) rats was significantly decreased (200mg/kg) compared with the D-gal group rats. Meanwhile, compared with a blank group of rats, the relative abundance of Actinobacillus in a D-gal group of rats is remarkably increased (p is less than 0.05), and the LZJSF (100mg/kg) group and the LZJSF (200mg/kg) group have remarkable retroactive effect on Actinobacillus.
2. Influence on the level of the flora
As shown in FIG. 6, the family level of the flora of each group of rats mainly consisted of Lactobacillaceae (Lactobacillaceae), Ruminococcus (Ruminococcaceae), Prevoteriaceae (Prevotellaceae), Verrucomicrobiaceae (Verrucomicrobiaceae), Clostridiaceae (Clostridiaceae), Peptostreptococcaceae (Peptostreptococcaceae), Lachnospiraceae (Lachnospiraceae), Rhodostinidae (Corobacteriaceae), Erysipelotrichaceae and Fusarium order Uncapsasid _ Clostridiales without identifying the names of the families, Bacteroidales order bacteriodales-S24-7, and the like. These 11 flora represent more than 95% of the total flora. Analysis of variance of the relative abundances of the above subjects revealed that the relative abundances of Lactobacillus and Coriobacteriaceae in the D-gal group were significantly increased (p <0.05) compared to the blank group, and significant retrogradation (p <0.05) was observed after continuous administration of LZSF (200mg/kg), as shown in FIG. 6. The relative abundance of Bacteroidales-S24-7 in the D-gal group was significantly decreased compared to the blank group, and the relative abundance of Bacteroidales-S24-7 in the D-gal + LZJSF (200mg/kg) group was significantly increased compared to the D-gal group. The relative abundance of Erysipelotrichaceae in the D-gal group was increased and that of D-gal + LZJSF (200mg/kg) was decreased compared to the blank group. It has been reported that Erysipelotrichaceae increases the likelihood of gastrointestinal inflammation-related diseases and colorectal cancer. Meanwhile, the LZJSF (100mg/kg) group of Prevotellaceae, Ruminococcaceae and Lachnospiraceae was found to have an increased tendency compared with the other groups. Prevotellaceae and Ruminococcaceae have been reported to produce more Short Chain Fatty Acids (SCFAs) to prevent colonization of pathogenic bacteria and to modulate the immune system. Lachnospiraceae and Ruminococcaceae protect mouse models of colitis from colonic inflammation. Thus, the LZJSF (100mg/kg) has the regulation effect on flora.
3. Effect on the flora level
As shown in FIG. 7, the bacterial flora of each group of rats mainly included Lactobacillus (Lctobacillus), Proteus (Prevotella), Akkermansia, Clostridium (Clostridium), Ruminococcus (Ruminococcus), and Uncsaid _ Clostridiaceae, Uncsaid _ bacterioides, Bacteroides _ bacterioides S24-7, Uncsaid _ Ruminococcus of Ruminococcaceae, Uncsaid _ Peptostreptococcus of Peptostreptococcus, and Uncsaid _ Lachnospiraceae. As shown in FIG. 8, the relative abundance of Lctobacillus in the Control group is 23%, the relative abundance of Lctobacillus in the large genus of the D-gal group is significantly increased to 35% compared with the blank group, and the change trend is consistent with the literature report 8, the relative abundance of the lactobacillus in the D-gal + LZJSF (200mg/kg) group is significantly reduced (p <0.05) compared with the D-gal group, and organic acids, special enzyme systems, acidomycin and other substances generated by fermentation of the lactobacillus have special physiological functions. A large number of research data show that the lactobacillus can promote the growth of animals, regulate the normal flora of gastrointestinal tracts and maintain the microecological balance, thereby improving the gastrointestinal tract function, improving the food digestibility and the biological value, reducing the serum cholesterol, controlling the endotoxin, inhibiting the growth of putrefying bacteria in intestinal tracts, improving the immunity of organisms and the like. The research shows that LZJSF (200mg/kg) can regulate beneficial bacteria lactobacillus and generate a protection function on intestinal tracts. Uncsified _ S24-7 and Uncsified _ Lachnospiraceae bacteria showed significantly lower relative abundance in D-gal group (p <0.05), and D-gal + LZJSF (200mg/kg) group showed significant retrogradation of relative abundance of these two groups compared to blank group. The relative abundance of Uncsified _ Ruminococcaceae bacteria in the D-gal group was reduced compared to the blank group, and the D-gal + LZJSF (200mg/kg) group had a significant callback effect on the relative abundance of this group, but no significant difference.

Claims (4)

1. Application of total flavone extract of Zizyphus jujuba leaves in preparing product for preventing and treating intestinal dysbacteriosis;
the wild jujube leaf total flavone extract is prepared by the method comprising the following steps:
soaking folium Ziziphi Spinosae, extracting with ethanol under reflux, filtering, collecting filtrate, volatilizing solvent to obtain folium Ziziphi Spinosae extract solid, loading onto D101 macroporous resin, eluting, collecting eluate, and volatilizing solvent to obtain folium Ziziphi Spinosae flavone extract;
the method is particularly used for preparing products which can increase the microbial diversity of intestinal flora, increase the abundance of bacteroidetes, reduce the abundance of firmicutes and reduce the abundance of lactobacilli, so that the proportion and the composition of the firmicutes and the bacteroidetes tend to be normal.
2. Use according to claim 1, characterized in that: the volume concentration of the ethanol for soaking is 50 percent;
the soaking time is 3 hours;
the proportion of the wild jujube leaves to the ethanol for soaking is 1000 g: 12L;
the reflux extraction is carried out for a plurality of times, each time for 0.5 h;
when the D101 macroporous resin is applied, the sample application concentration is 0.5 g/mL-1Passing through the resin column at a flow rate of 0.5 BV/h;
the elution operation was: washing with distilled water to remove impurities, and eluting with 50% ethanol at a flow rate of 1 BV/h;
the purity of total flavonoids in folium Ziziphi Spinosae extract is 40.95% by ultraviolet spectrophotometry.
3. Use according to claim 1, characterized in that: the intestinal flora imbalance isDGalactose induces gut flora dysregulation due to aging.
4. Use according to claim 1, characterized in that: the product is a medicament.
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CN114869921B (en) * 2022-06-08 2023-06-30 佳木斯大学 Application of total flavone regulation adjuvant arthritis rat intestinal flora
CN115029273B (en) * 2022-06-22 2023-04-07 重庆市天友乳业股份有限公司 Lactobacillus fermentum TY-G03 and application thereof
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