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Abstract

Objective: This study aimed to assess the effect of Nelumbo nucifera alcoholic extract (LLAE) on the intestinal microbiota in high-fat diet-induced hyperlipidemia. Methods: Thirty male-specific pathogen-free Sprague–Dawley rats were randomly divided into 3 groups: the blank control group, the model control group, and the LLAE group. The body weight of the rats was recorded during the treatment period. The serum, liver, and intestinal content samples were collected for further analysis after sacrificing. Results: The results suggest that LLAE-treated hyperlipidemic rats showed a significant reduction in serum triglycerides, total cholesterols, and low-density lipoprotein cholesterol levels. LLAE could improve hepatocyte steatosis and ameliorate hepatocyte injury. The significant variation in gut microbial alpha diversity at multiple levels, including phylum and genus levels occurred throughout treatment in the LLAE group. The intestinal microbiota of hyperlipidemic rats were disordered, and there was a correlation between the amount of intestinal flora and the level of lipids. Conclusion: LLAE may improve hepatocyte function by changing the type and abundance value of intestinal microorganisms, reducing the content of total cholesterol, total triglyceride, and low-density lipoprotein cholesterol, and thus improving hyperlipidemia.

Keywords

dyslipidaemias hyperlipidemia intestinal microbiota

Introduction

Dyslipidemias, also called hyperlipidemia, are usually defined as increased levels of serum total cholesterol (TC), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), or a decreased serum high-density lipoprotein cholesterol concentration. According to the Chinese Guidelines on Prevention and Treatment of Dyslipidemia in Adults , the overall prevalence of dyslipidemia was 40.40%.¹ Previous studies have shown that dyslipidemia is positively associated with atherosclerosis, hypertension, coronary heart disease, stroke, and diabetes. The currently marketed drugs could significantly reduce blood lipid levels of hyperlipidemia. However, many studies reported about drug intolerance and impaired physical function. For example, abdominal pain, headache, dizziness, muscle pain, and abnormal liver function.² As expected with Niacin and Fibrates, the most common adverse drug reactions were skin reactions (mainly pruritus, rashes, and flushing), gastrointestinal symptoms (mainly nausea and diarrhea), and elevated liver enzymes.³

The medicinal value of Nelumbo nucifera has been recognized in traditional Chinese medicine according to the Compendium of Materia Medica. ⁴ N nucifera as a new agent for treating hyperlipidemia patients has potential advantages based on its multitarget effect, curative effect, and safety. Modern pharmacological studies have shown that the N nucifera alcoholic extract (LLAE) could improve dyslipidemia and obesity,⁵ though reduced body ability to digest, and decreased the energy intake as lipids and carbohydrates, and regulate systemic energy consumption.⁶ There are a few studies that have focused on the interaction between gut microbiota and hyperlipidemias, a model of diet-induced hyperlipidemias was established by imposing a high-fat diet on rats. Hematoxylin and eosin (HE) staining was conducted to observe the histopathological changes in the liver. The level of serum TC was measured by the CHOD-PAP method (Roche P-Modular), TG by the GPO-PAP method (Roche P-Modular), and LDL-C was calculated by the Friedewald formula. 16S ribosomal DNA (rDNA) sequencing was used to determine changes in the gut microbiome.

This study aimed to assess the associations between intestinal microbiota and high-fat-diet-induced hyperlipidemia and also proved that LLAE could improve dyslipidemia by modulating gut microbial abundance. The results of this study may provide a basis for the development of new drugs modulating hyperlipidemia.

Materials and Methods

Experimental Drug

N nucifera was purchased from TongRenTang Co., Ltd, washed with water, and dried, without other treatments.

Reagents and Experimental Animals

Thirty specific pathogen-free (SPF)-grade male Sprague–Dawley (SD) rats with 200 ± 20 g body mass (provided by the Animal Experiment Center of Harbin Medical University, license number SCXK (H) 2019-001). Handling and interventions involving experimental animals in this study were given humane care in accordance with the 3R principles of experimental animal use. Maintenance chow and high-fat chow (Beijing Keao Synergy Feed Co., Ltd). Lotus leaves (Beijing TongRenTang Harbin Pharmacy). HE staining kit (Wuhan Doctorate Biological Engineering Co., Ltd). TC assay kit, total TG assay kit, and LDL-C assay kit (Nanjing Jiancheng Institute of Biological Engineering).

Instruments

Paraffin Microtome (Model RM2235, LEICA Inc). Front-mounted white light photomicroscope (Model Eclipse Ci-L, Nikon (Japan) Inc.). Bio-radT100 gradient PCR instrument (Model T100USA, Bio-Rad Inc.). Universal DNA purification and recovery kit (Model DP214, TianGen Inc.). NovaSeq6000 Sequencer (Model IlluminaNovaseq 6000, Illumina Inc.).

Methods

Preparation of LLAE

A similar process as the study of Yi⁷ was used for the preparation of the extract. The conditions of LLAE were determined by single factor and orthogonal experimental tests. We extracted the LLAE using ultrasonic-assisted extraction, which was with 70% ethanol concentration, the solid–liquid ratio was 1:25, and the ultrasonic temperature and time were 35 °C and 2 h.

Animal Grouping and Feeding

In this experiment, 30 6-week-old SPF-grade male SD rats were selected and bred at a temperature of 18 °C–29 °C, relative humidity of 40%–70%, and a 12 h photo dark cycle.⁸ After 1 week of adaptive feeding, the rats were randomly divided into blank control (BC) group, model control (MC) group, and LLAE group according to body mass. The rats were given maintenance chow and gavage saline 1 mL/100 g every day in the BC group,^9,10 high-fat chow and gavage saline 1 mL/100 g every day in the MC group, and high-fat chow and lotus alcohol extract 1 mL/100 g every day in the lotus alcohol extract group for 20 weeks. During the experimental period, the rats in each group were fed and watered freely, and their body mass was measured and recorded each week. At the beginning of the intervention (0 month), at 1 month, 2 months, 3 months, 4 months, and 5 months. Then 1.5–2 mL of blood was collected from the inner canthus of each group, and the supernatant was centrifuged and stored as serum samples at −80 °C.

Animal Sample Collection and Processing

At 20 weeks of experimental intervention, after 12 h of fasting, rats in each group were administered an intraperitoneal injection of 10% chloral hydrate 350 mg/kg, liver tissues were isolated after dissection, and liver tissues of 1 cm³ volume were placed in 4% paraformaldehyde fixative and stored in 4 °C refrigerator, cecum contents were taken and stored in −80 °C refrigerator.

Indicator Testing

Serum Indicator Testing. Single reagent GPO-PAP method and double reagent direct method were used to detect rat serum samples expressing rat lipid levels (TC, TG, and LDL-C content) according to the operation instructions of the relevant kits.

Liver Histomorphometric Testing. Paraffin section dewaxing to water, sequentially put the section into xylene I for 20 min—xylene II for 20 min—anhydrous ethanol I for 5 min—anhydrous ethanol II for 5 min—75% alcohol for 5 min, tap water washing. Hematoxylin staining, slices were stained into hematoxylin staining solution for 3–5 min, washed with tap water, returned to blue with blue return solution, and rinsed with running water. Eosin staining, sections were dehydrated into 85% and 95% gradient alcohol for 5 min each in turn, and stained in eosin staining solution for 5 min. Dehydration and sealing, sections were placed into anhydrous ethanol I for 5 min—anhydrous ethanol II for 5 min—anhydrous ethanol III for 5 min—xylene I for 5 min—xylene II for 5 min transparently, and sealed with neutral gum. Microscopic examination and image acquisition were performed to analyze the histomorphology of rat liver. Five paraffin sections were observed in rat liver tissue, and the best field of view was selected for analysis, each section was observed with a 20× microscopic field map.

Intestinal Flora Testing. 16s rDNA sequencing was applied to detect intestinal flora changes, and the main steps of sequencing included extraction of genomic DNA, polymerase chain reaction (PCR) amplification, mixing and purification of PCR products, library construction, and up-sequencing. The amplified sequences (515F 5′-GTGCCAGCMGCCGCGGTAA→3′) and (806R 5′-GGACTACHVGGGTWTCTAAT→3′) were used to amplify the v3 region. The PCR amplification of the v4 region of the samples was performed, and the sequencing results were obtained by using NovaSeq6000 on the machine. The raw data obtained from sequencing had a certain proportion of interfering data, which needed to be spliced and filtered to obtain valid data, and then operational taxonomic units (OTUs) clustering and species classification analysis were performed based on the valid data.

Statistical Analysis

Statistical analysis was performed by t-test, and data were expressed as mean ± SD.

Ethical Approval

This study was reviewed by the Ethics Committee of the Heilongjiang Academy of Chinese Medicine Sciences (No. 2015-64) and the experiments were conducted by the Chinese Government's “Guidelines on the Care and Use of Laboratory Animals” to minimize their suffering.

Results

Index Test Results

The trend of body mass of rats in each group over time during the experiment is shown in Figure 1a and Table 1. At the beginning of the experiment (0 month), there was no significant difference in the body mass of rats in each group (P > .05). From the beginning of the experiment (0 month) to the 3rd month, the body mass of rats in each group showed a significant trend of increase. From the beginning of the 3rd month to the end of the experiment (5 months), the body mass of rats in the BC and MC groups showed a slow growth trend, and the body mass of rats in the LLEA group showed a significant decrease compared with the BC and MC groups (P < .01).

Figure 1.

(a) Trends in body mass of rats. (b) Trends in TC of rats. (c) Trends of TG in rats. (d) Trends of LDL-C in rats.

Table 1.

Average Body Mass of Rats.

	BC (g) (N = 10)	MC (g) (N = 10)	LLAE (g) (N = 10)
0 month	220.5 ± 13.41	219.2 ± 14.1	217.8 ± 14.41
1 month	392.5 ± 14.97	425.6 ± 14.95	428.7 ± 14.87
2 months	565 ± 19.41	570 ± 18.41	574 ± 19.47
3 months	551.1 ± 20.41	662 ± 20.31	659 ± 21.40
4 months	596.3 ± 21.41	733 ± 21.31	584 ± 21.11
5 months	637.2 ± 16.41	797 ± 16.88	534 ± 16.31

Abbreviations: BC, blank control; MC, model control; LLAE, Nelumbo nucifera alcoholic extract.

The trends of lipid levels in rats in each group over time are shown in Figure 1b to 1d. At the beginning of the experiment (0 month), there was no significant difference (P > .05) in the lipid levels (TC, TG, and LDL-C) of rats in each group. From the beginning of the experiment (0 month) to the 5th month, the lipid levels of the rats in the BC group remained stable, and the lipid levels of the rats in the MC group and the rats in the LLAE group showed an increasing trend with the change of time. At the end of the experiment (5 months), the lipid levels of rats in both the model group and the LLAE group were significantly different from those in the BC group, indicating that the high-fat diet could lead to an increase in the lipid levels of rats, the lipid levels of rats in the LLAE group showed a slowly increasing trend compared with those in the model group (P < .01). The lipid levels in the rats of each group showed that LLAE significantly reduced the elevated lipid levels caused by a high-fat diet.

Histomorphological Results of the Liver

The results of liver staining in each group are shown in Figure 2. In the HE-stained sections of the samples from the model group, hepatocyte steatosis was widely seen, with tiny round vacuoles visible in the cytoplasm (black arrows), a large number of hepatocytes with balloon-like changes, swollen cytosol and vacuolated cytoplasm (red arrows), and several small focal infiltrates of inflammatory cells visible in the lobules and around veins (yellow arrows). The HE-stained sections of the samples from the LLAE group showed a small amount of hepatocytes with mild steatosis and tiny round vacuoles in the cytoplasm (black arrows). The results of HE staining of liver tissues of rats in all groups showed that a high-fat diet could lead to hepatocyte steatosis and small focal inflammatory cell infiltration.

Figure 2.

Hematoxylin and eosin (HE) staining results of rat liver tissue in each group (20×).

Intestinal Flora Test Results

To investigate the species composition of each sample, the valid data of all samples were clustered by OTUs with a consistency of 0.97, and the sequences of OTUs were annotated with species. The following data analyses in the paper are based on the analysis of OTUs clustering results.¹¹

Species Abundance Clustering Heat Map

Based on the species annotation and abundance information of all samples at the 3 levels of phylum and genus, the top 35 genera in terms of abundance were selected and clustered at both species and sample levels according to their abundance values in each sample, which were plotted as heat maps to facilitate the discovery of species clustered more or less in the samples (Figure 3a and 3b). The horizontal side of the figure shows the sample information, the right side shows the species annotation information, and the clustering tree on the left side is the species clustering tree. The shades of color indicate the level of species abundance.

Figure 3.

(a) Plylum level abundance. (b) Genus level abundance clustering heatmap.

The clustering heat map indicates that at the phylum level, compared to the BC group, the MC group rats had significantly higher levels of intestinal flora Desulfobacterota, Thermoplasmatota, Campylobacterota, unidentified-Bacteria, Cyanobacteria, and Actinobacteria were significantly increased. Compared with the BC group and MC group, the rat intestinal flora of the LLAE contained significantly higher levels of Actinobacteria, Deferribacteres, Planctomycetota, Proteobacteria, Gemmatimonadota, Deinococcota, Nitrospirota, Myxococcota, Chloroflexi, Acidobacteriota, Entotheonellaeota, Verrucomicrobiota, Crenarchaeota, Halobacterota, and Nanoarchaeota. The clustering heat map indicated that at the genus level, the content of intestinal flora Lactobacillus, Pseudomonas, Alistipes, Limosilactobacillus, and Intestinimonas was significantly lower in the MC group and LLAE group of rats compared to the BC group. Compared with the MC group, the intestinal flora Colidextribacter, Coprococcus, Oscillospira, Desulfovibrio, Megamonas, Oscillibacter, Blautia, and Lachnoclostridium were significantly reduced. The content of intestinal flora Dubosiella, Roseburia, Allobaculum, Akkermansia, Streptococcus, and Bacteroides was significantly reduced in the BC and MC group rats compared to the LLAE group rats.

The results of the clustering heat map showed that the lotus alcohol extracts caused a significant decrease in the abundance of microorganisms Colidextribacter, Coprococcus, Oscillospira, Desulfovibrio, Megamonas, and Oscillibacter at the genus level by affecting the abundance of microorganisms at the phylum level. The abundance values of Desulfobacterota and Thermoplasmatota were changed. In addition, in Figure 3a, the clustered heat map results showed many similar results for the BC and model groups, and the reasons for this were not clearly stated after reviewing the literature and other studies. It was speculated that the association of these microorganisms with dyslipidemic diseases at the phylum level may be low or due to the existence of individual differences in experimental animals.

Comparative Analysis of Alpha Diversity

Alpha diversity, which is used to analyze the diversity of microbial communities within a sample, can reflect the richness and diversity of microbial communities within a sample by diversity analysis of a single sample.¹² Alpha diversity indices (Shannon, Simpson, Chao1, ACE, PD_whole_tree) were statistically analyzed for the 2 groups of samples at a consistency threshold of 0.97, as shown in Table 2.

Species accumulation box plot, an analysis to describe the increase of species diversity with the increase of sample size. It is an effective tool to investigate the species composition of samples and predict the abundance of species in samples. In biodiversity and community surveys, it is widely used to judge the adequacy of sample size and the estimation of species richness. Therefore, the species accumulation box chart can not only judge whether the sample size is sufficient, but also predict the species richness by means of the species accumulation box chart under the premise of sufficient sample size. The results are shown in Figure 4.

Figure 4.

The graph of species accumulation box plot.

Table 2.

Alpha Diversity index of Intestinal flora in two Groups of Rats.

Group	Diversity index
Group	Observed_species	Shannon	Simpson	Chao1	ACE	PD_whole_tree
MC	1058.86 ± 58.91	7.45 ± 0.38	0.98 ± 0.01	1144.23 ± 79.72	1157. 16 ± 78.48	47.67 ± 3.33
LLAE	1190 ± 112.56	7. 13 ± 0.95	0.96 ± 0.04	1316.86 ± 102.28	1330.39 ± 107.54	61.62 ± 11.36
P value	.016196658	.042416447	.0153521681	.003206919	.003806078	.028293395

Abbreviations: MC, model control; LLAE, Nelumbo nucifera alcoholic extract.

Beta Diversity Comparative Analysis

Principal coordinate analysis (PCoA), which is to extract the most dominant elements and structures from multidimensional data by a series of eigenvalues and eigenvectors sorting. PCoA was performed based on Bray–Curtis distance and the combination of principal coordinates with the largest contribution was selected for graphical presentation. It is generally considered that the closer the sample distances are, the more similar the species composition structure is indicated. Thus, samples with high similarity in community structure tend to cluster together, and samples with large community differences are far apart. The characterization results are shown in Figure 5, where significant differences in intestinal flora composition were observed between the high-fat diet-induced hyperlipidemic rats and the BC and LLAE groups.

Intergroup differential species analysis finds the different species between 3 groups at each taxonomic level, and a t-test between groups was conducted to find the species with significant differences (P < .01). The results of the default display of the gate level are shown in Figure 6. The left panel shows the abundance of species differing between groups, and each bar in the figure indicates the mean value of species differing significantly in abundance between groups in each group. The right panel shows the confidence of the difference between groups, the leftmost point of each circle in the figure indicates the lower limit of the 95% confidence interval of the mean difference, and the rightmost point of the circle indicates the upper limit of the 95% confidence interval of the mean difference. The center of the circle represents the difference of means. The group represented by the color of the circle is the group with a high mean. The rightmost point of the displayed results is the P value of the groups' significance test corresponding to the different species. The results of the analysis show that Firmicutes is the significantly different species between the LLAE group and the MC groups (P < .01). Euryarchaeota is the significantly different species between the LLAE groups and the BC group (P < .01). Bacteroidota is the different species between the BC group and the MC group (P < .05).

Figure 5.

Two-dimensional diagram of principal coordinate analysis (PCoA).

Figure 6.

The difference species between groups of t-test analysis graph.

Discussion

Experimental studies found that a high-fat diet increased body mass in SD rats and increased total TG, TC, and LDL-C cholesterol levels. Inflammatory factor infiltration in the liver and lesions in liver cell morphology, lead to abnormal lipid metabolism and hyperlipidemia. Dietary patterns have an important impact on the intestinal flora, and the improvement of the organism is closely related to the mediation of the intestinal flora. 16S rDNA macrogenome sequencing revealed that the intestinal microflora of SD rats with hyperlipidemia model differed from that of normal rats, and the relative proportions of these microorganisms changed with diet. Bacteroidota were significantly different between groups.

Experimental studies demonstrated that LLAE significantly inhibited the increase of body mass and blood lipid level caused by high-fat diet, and improved the morphological changes of liver cells and liver inflammatory factor infiltration caused by high-fat diet. The results of macrogenome sequencing showed that a high-fat diet could lead to the alteration of intestinal microflora in rats, and the Alpha diversity index was reduced, indicating a decrease in microflora species, flora stability, and anti-infection ability. The differences in intestinal flora between the model rats and the BC rats suggested that hyperlipidemic rats might have altered lipid metabolism and immune inflammation.

Data from one clinical study indicated that Blautia was significantly higher in stool samples from patients with hyperlipidemia than in healthy subjects, another study also indicated that the abundance of Blautia was significantly higher in patients with nonalcoholic fatty liver disease compared to healthy subjects. Data from two clinical studies demonstrated that Blautia abundance values were positively correlated with high-fat dietary intake.¹³ The experimental studies clearly correlated dyslipidemia caused by a high-fat diet with intestinal flora, and the results also demonstrated that the ethanolic extract of lotus leaves improved dyslipidemia by modulating the abundance of intestinal microorganisms. This study provides basic research data for drug treatment of hyperlipidemia and expands the scope of clinical medication.

Conclusion

In this experiment, we found that the abundance of Blautia in the intestinal flora of rats in the LLAE group was significantly reduced, and the morphological changes of liver cells and the infiltration of liver inflammatory factors were significantly improved, indicating that the liver injury caused by high-fat diet was closely related to the abundance of Blautia, and the LLAE could improve the liver injury by changing the abundance of Blautia. Therefore, LLAE may improve hepatocyte function by changing the type and abundance value of intestinal microorganisms, inducing the conversion of cholesterol into bile acids in the liver to secrete bile, promoting the digestion and absorption of fat, reducing the content of TC, total TG, and LDL-C cholesterol, and thus improving hyperlipidemia.

This study has some limitations.

The results demonstrated that the ethanolic extract of lotus leaves improved dyslipidemia by modulating the abundance of intestinal microorganisms. However, the correlation between the concentration of LLAE and the effect of improving dyslipidemia was not further clarified and needs to be demonstrated in follow-up experiments.

Microbial Latin Names in English

Microbial Latin names	Corresponding English name
Desulfobacterota	Vibrio desulfuricans
Thermoplasmatota	Tatum's bacterium
Campylobacterota	Campylobacter
Cyanobacteria	Cyanobacteria
Actinobacteriota	Actinomycetes
Actinobacteria	Actinomyces
Deferribacteres	Desferriobacterium
Planctomycetota	Mycobacterium floatum
Proteobacteria	Metamorphosis bacteria
Gemmatimonadota	Bacillus spp
Deinococcota	Squametococcus
Nitrospirota	Nitrospira
Myxococcota	Mucococcus
Chloroflexi	Green Bend Fungus
Acidobacteriota	Acidophilus
Verrucomicrobiota	Microbacterium warts
Crenarchaeota	Izumo Mushroom
Halobacterota	Salinobacter
Nanoarchaeota	Nagumycetes
Prevotellaceae	Prevotella
Lactobacillaceae	Lactobacillus
Pseudomonadaceae	Pseudomonas aeruginosa
Tannerellaceae	Tannerella
Rikenellaceae	Riken Fungus
Burkholderiaceae	Burkholderia cepacia
Peptococcaceae	Parrot coccus
Acidaminococcaceae	Acid Aminococcus
Erysipelotrichaceae	Salvia miltiorrhiza
Enterobacteriaceae	Enterobacteriaceae
Streptococcaceae	Streptococcus
Akkermansiaceae	Ackermania
Fusobacteriaceae	Clostridium perfringens
Sphingomonadaceae	Sphingomonas spp.
Lactobacillus	Lactobacillus
Pseudomonas	Pseudomonas aeruginosa
Alistipes	Another branching fungus
Limosilactobacillus	Lactobacillus royi
Coprococcus	Enterococcus faecalis
Blautia	Brucella abortus
Desulfovibrio	Vibrio desulfuricans
Oscillibacter	Mycobacterium tremens
Oscillospira	Treponema pallidum
Dubosiella	Bacillus thuringiensis
Roseburia	Rochesteria
Allobaculum	Easy leaf fungus
Akkermansia	Ackermania
Streptococcus	Streptococcus
Bacteroides	Mycobacterium phylum
Firmicutes	Phylum Thick-walled Bacteria
Bacteroidota	Mycobacterium spp.
Megamonas	Macrocystis aeruginosa

Footnotes

Acknowledgments

Not applicable.

Authors’ Contributions

Lu Zhenhua: Conceive ideas, complete experiments, and animal experiment implementation. Wei Gangjie: Data collection and arrangement. Cao Rui and Li Yangguang: Article revision and supportive work. Cai Xiaojun: Design and fund experiments.

Availability of Data and Material Statement

All relevant data are contained within the article: The original contributions presented in the study are included in the article/Supplementalal material, further inquiries can be directed to the corresponding author.

Consent for Publication

Written informed consent for publication of their clinical details and/or clinical images was obtained from the patient/parent/guardian/ relative of the patient. A copy of the consent form is available for review by the Editor of this journal.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethics Approval

This study was reviewed by the Ethics Committee of the Heilongjiang Academy of Chinese Medicine Sciences (No. 2015-64), Heilongjiang province, China. The experiments were conducted by the Chinese Government's “Guidelines on the Care and Use of Laboratory Animals” to minimize their suffering.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was partially supported by the Nature Science Foundation of Heilongjiang Province of China (Grant number: LH2023H077).

ORCID iD

Zhenhua Lu

Statement of Human and Animal Rights

All of the experimental procedures involving animals were conducted in accordance with the Chinese Government's “Guidelines on the Care and Use of Laboratory Animals”, and approved by the Ethics Committee of the Heilongjiang Academy of Chinese Medicine Sciences, Heilongjiang province, China.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

References

Junren

. Guidelines for the prevention and treatment of dyslipidemia in Chinese adults (2016 revised edition). Chin J Health Manage. 2017;11(1):7‐28.

Chengtian

Guanying

Wenying

. Analysis of 214 adverse reactions to statin lipid-lowering drugs. J Clin Ration Drug Use. 2018;11(20):90‐91.

Shijie

Ruhao

Lan

, et al. Application status and research progress of hypolipidemic drugs. Pharm Forum Mag. 2006(1):90‐92.

Shizhen

. Compendium of materia medica. Translated by Ma Songyuan. Cable Book Bureau; 2019.

Yue

Juan

Xinkai

, et al. Progress in the pharmacological effect and mechanism of lotus leaf alkali. Chin Mod Tradit Chin Med. 2021;23(01):164‐170.

Fengli

Ferce

Weihua

, et al. Overview of the pharmacological effects of lotus leaves. Global Tradit Chin Med. 2016;9(01):115‐118.

. Extraction and separation of lotus leaf alkali and blood lipid-lowering function of lotus leaf extract. Jiangnan University; 2019.

Zheng

. Practical manual of the latest experimental techniques for laboratory animals, surgical innovation and evaluation, and total quality management of laboratory animals. China Medical Science and Technology Electronic Publishing House; 2006.

Bingchun

. Fundamentals of medical animal experiments and basic technical methods. Heilongjiang People's Publishing House; 2008.

10.

Yanan

Jinhai

Weiming

. Effects and action mechanisms of lotus leaf (Nelumbo nucifera) ethanol extract on gut microbes and obesity in high-fat diet-fed rats. Front Nutr. 2023;10:1169843. doi: 10.3389/fnut.2023.1169843

11.

Zhao

Liang

Meng

, et al. 16s rDNA sequencing to study the changes of intestinal flora in hyperlipidemic rats induced by high-fat diet. Chin J Microecol. 2022;34(3):257‐261.

12.

Zhang

Guo

, et al. Characterization of tetracycline resistant bacterial community in saline activated sludge using batch stress incubation with high-throughput sequencing analysis. Water Res. 2013;47(13):4207‐4216.

13.

Wang

Tong

Shou

, et al. Gutmicrobiota-mediated personalized treatment of hyperlipidemia using berberine. Theranostics. 2017;7(9):2443‐2451.

The Effect of Nelumbo nucifera Ethanolic Extract on Gut Microbiota in Hyperlipidemic Rats Induced by a High-Fat Diet