Application of premna microphylla pectin in preparation of products for preventing and improving diet-induced obesity

Abstract

The invention discloses an application of premna microphylla pectin in preparation of a product for preventing and improving diet-induced obesity. The premna pubescens pectin is prepared from premna pubescens leaves as a raw material through extraction, purification and other processes. According to the present invention, the research on the high fat diet induced obese mouse model finds that the body lipid metabolism is regulated by remodeling the intestinal flora structure, particularly reducing the ratio of the phylum firmicutes to the phylum bacteroides, and specifically enriching the beneficial flora such as Muribacaceae and the like by using the premna microphylla pectin; experimental results prove that the premna microphylla pectin can significantly inhibit pathological hypertrophy of adipocytes, reduce visceral fat accumulation, effectively control weight gain and improve dyslipidemia and liver fatty degeneration, and has good biological safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and to new uses of *Bromhidrosis pilosula* pectin, particularly its application in the preparation of products for the prevention and improvement of diet-induced obesity. Background Technology

[0002] Obesity is a complex chronic metabolic disease caused by the interaction of multiple factors, including genetics, environment, and lifestyle. With improved living standards and changes in dietary structure, the prevalence of obesity is rapidly increasing, becoming a serious global public health problem. Obesity not only manifests as excessive accumulation of adipose tissue but also significantly increases the risk of type 2 diabetes, cardiovascular disease, non-alcoholic fatty liver disease, and various cancers. Existing anti-obesity drugs are generally limited by significant drawbacks such as gastrointestinal adverse reactions, high cost, easy rebound after discontinuation, and potential safety risks, making it difficult to meet the needs of long-term safe treatment. Therefore, developing novel intervention strategies that are naturally derived, have proven efficacy, and have extremely low side effects has become a key technical problem to be solved in the current medical field.

[0003] Premna puberula Pamb., a perennial shrub belonging to the genus Premna in the family Verbenaceae, is widely distributed in southwestern my country and is traditionally used in folk medicine for clearing heat and detoxifying. Currently, there is a significant gap in the systematic anti-obesity evaluation of Premna puberula pectin (PSAP), particularly regarding its mechanism of action in improving obesity through regulation of the gut microbiome-metabolome axis. This significantly limits the in-depth development and application of this plant resource in functional foods and medicines. Therefore, providing a method for improving diet-induced obesity based on Premna puberula pectin is urgently needed to explore its medicinal value and expand anti-obesity intervention strategies. Summary of the Invention

[0004] The technical problem this invention aims to solve is to provide the application of *Clerodendrum trichotomum* pectin in the preparation of products for the prevention and improvement of diet-induced obesity, thereby overcoming the limitations of existing chemical drug treatments for obesity. This invention, through a multi-dimensional biological evaluation system, confirms that *Clerodendrum trichotomum* pectin can significantly reduce body fat percentage and improve blood lipid levels and glucose tolerance in mice on a high-fat diet. Mechanistic studies show that this pectin improves the body's energy metabolism balance by specifically regulating the diversity and composition of the gut microbiota and remodeling related metabolic pathways. This invention reveals the potential mechanism by which *Clerodendrum trichotomum* pectin acts as a prebiotic to regulate metabolic health, providing experimental support for its clinical application.

[0005] This invention is achieved using the following technical solution: Application of *Bromhidrosis pilosula* pectin in the preparation of products for the prevention and improvement of diet-induced obesity.

[0006] The aforementioned method for preparing the *Bromhidrosis pilosula* pectin is carried out according to the following steps: (1) Pretreatment: Dry the leaves of *Clerodendrum trichotomum* at 35-45℃ for 20-28 hours, then grind them into fine powder of 360-400μm to obtain dry powder of *Clerodendrum trichotomum* leaves for later use; (2) Extraction: Weigh the dried powder of *Bungarus fasciatus* leaves and add tartaric acid. Extract at 90-100℃ for 50-70 min. The pH value of tartaric acid is 1.90-2.10. The ratio of dried powder of *Bungarus fasciatus* leaves to tartaric acid is 0.8-1.2g:50-70mL. After cooling to room temperature, filter to obtain filtrate. Concentrate the filtrate to 1 / 4-1 / 2 volume. Add 1.5-2.5 times the amount of 95% food ethanol and let stand at room temperature for 0.5-1.5h. Then centrifuge at 6000-10000min / r for 2-4min to separate the crude *Bungarus fasciatus* pectin for later use. (3) Purification: Wash the crude pectin of *Clerodendrum thomsoniae* 2-4 times with 95% edible ethanol. Each time, use 5-20 mL of 95% edible ethanol for 1 g of crude pectin of *Clerodendrum thomsoniae*. After each wash, centrifuge at 6000-10000 min / r for 0.8-1.2 min until the filtrate is colorless. Stop washing to obtain the purified pectin. Dry the pectin at 40-50℃ for 7-10 h, grind it in a mortar, pass it through an 80-120 mesh sieve, and collect the fine pectin powder to obtain *Clerodendrum thomsoniae* pectin.

[0007] In step (1) above, the pretreatment is as follows: the leaves of the *Clerodendrum trichotomum* are dried at 40°C for 24 hours and then ground into fine powder of 370-390 μm to obtain dry powder of *Clerodendrum trichotomum* leaves for later use.

[0008] In step (2) above, extraction: weigh dry powder of *Clerodendrum trichotomum* leaves, add tartaric acid and extract at 95℃ for 60 min, tartaric acid pH = 2.00, the ratio of dry powder of *Clerodendrum trichotomum* leaves to tartaric acid is 1g: 60mL, take it out and cool to room temperature, filter to obtain filtrate, concentrate the filtrate to 1 / 3 volume, add 2 times the amount of 95% food ethanol and let stand at room temperature for 1 h, then centrifuge at 8000 min / r for 3 min to separate crude *Clerodendrum trichotomum* pectin for later use.

[0009] In the aforementioned step (3), purification: wash the crude pectin of *Clerodendrum thomsoniae* 2-3 times with 95% edible ethanol. Each time, use 10-15 mL of 95% edible ethanol to wash 1 g of crude pectin of *Clerodendrum thomsoniae*. After each wash, centrifuge at 8000 min / r for 1 min until the filtrate is colorless, then stop washing to obtain the washed and purified pectin. Dry the pectin at 45℃ for 8-9 h, grind it in a mortar, pass it through a 100-mesh sieve, and collect the fine pectin powder to obtain *Clerodendrum thomsoniae* pectin.

[0010] The aforementioned products include pharmaceuticals or health foods.

[0011] The aforementioned drugs or health foods are added to conventional pharmaceutical carriers or excipients and processed according to conventional methods to produce corresponding formulations.

[0012] The aforementioned preparations include capsules, tablets, and granules.

[0013] The aforementioned *Bromhidrosis pilosula* pectin regulates lipid metabolism by reshaping the gut microbiota structure, reducing the ratio of Firmicutes to Bacteroidetes, and specifically enriching beneficial bacteria.

[0014] Beneficial effects of this invention: (1) The pectin of the bromhidrosis plant can regulate the gut microbiome, significantly inhibit the excessive accumulation of adipose tissue induced by a high-fat diet, improve insulin resistance and glucose and lipid metabolism disorders, thereby achieving a systematic intervention for obesity; (2) This invention reveals the specific biological function of *Bromhidrosis pilosula* pectin in improving obesity, and confirms its significant efficacy in controlling weight, reducing blood lipids and protecting liver function. It provides valuable information and scientific basis for the development and application of *Bromhidrosis pilosula* pectin as a functional food or dietary supplement for the prevention and treatment of obesity and metabolic syndrome. Attached Figure Description

[0015] Figure 1 : Statistical chart of average food intake of mice in each group during the experiment; Figure 2 : Trend graph of body weight change in each group of mice during the experiment; Figure 3 Statistical chart of wet weight of white adipose tissue (epididymis, perirenal region, retroperitoneum) in mice of each group; Figure 4 Images of H&E-stained sections of adipose tissue and quantitative analysis of the mean area of ​​adipocytes (A: NCD group; B: HFD group; C: HFD-PSAP group). Figure 5 Images of H&E staining and Oil Red O staining of liver tissue from mice in each group (A: NCD group; B: HFD group; C: HFD-PSAP group; D: NCD group; E: HFD group; F: HFD-PSAP group). Figure 6 Graphs showing blood glucose curves and area under the curve (AUC) for each group of mice in the oral glucose tolerance test (OGTT) (A is the blood glucose curve; B is the area under the curve). Figure 7 : HOMA-IR graphs of fasting serum insulin levels and insulin resistance index (A: fasting serum insulin level; B: insulin resistance index) in mice of each group. Figure 8 Statistical graph of plasma liver function indicators (ALT, AST) levels in mice of each group; Figure 9Statistical chart of plasma lipid levels (TC, TG, LDL-C, HDL-C) in mice of each group; Figure 10 Dilution curves and α-diversity analysis (Chao1 and Shannon indices) of gut microbiota sequencing data from each group of mice. Figure 11 PCoA analysis of gut microbiota β-diversity based on Bray-Curtis distance; Figure 12 Analysis of the community composition and relative abundance of gut microbiota at the phylum level in each group of mice; Figure 13 Evolutionary clade plot (Cladogram) of gut microbiota LEfSe analysis in each group of mice. Detailed Implementation

[0016] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention.

[0017] Example 1 Preparation process of pectin from *Bromhidrosis pilosula*: (1) Pretreatment: Dry the leaves of the broom-like leaves at 40℃ for 24 hours, then grind them into fine powder of 370-390μm to obtain broom-like leaf powder for later use.

[0018] (2) Extraction: Weigh the dried powder of *Bungarus fasciatus* leaves and add tartaric acid. Extract at 95℃ for 60 min. The pH value of tartaric acid is 2.00. The ratio of dried powder of *Bungarus fasciatus* leaves to tartaric acid is 1 g: 60 mL. After cooling to room temperature, filter to obtain filtrate. Concentrate the filtrate to 1 / 3 volume. Add 2 times the amount of 95% edible ethanol and let stand at room temperature for 1 h. Then centrifuge at 8000 min / r for 3 min to separate the crude *Bungarus fasciatus* pectin for later use.

[0019] 3) Purification: Wash the crude pectin of *Clerodendrum thomsoniae* three times with 95% edible ethanol. Each wash uses 15 mL of 95% edible ethanol for 1 g of crude pectin. After each wash, centrifuge at 8000 min / r for 1 min until the filtrate is colorless. Stop washing to obtain purified pectin. Dry the pectin at 45℃ for 9 h, grind it in a mortar and pestle, and collect the fine pectin powder after passing it through a 100-mesh sieve. This is the *Clerodendrum thomsoniae* pectin.

[0020] Example 2 (1) Pretreatment: Dry the leaves of the broom grass at 45℃ for 20h, then grind them into fine powder of 380-400μm to obtain broom grass leaf powder for later use; (2) Extraction: Weigh the dried powder of *Bungarus fasciatus* leaves and add tartaric acid. Extract at 100℃ for 50 min. The pH value of tartaric acid is 1.90. The ratio of dried powder of *Bungarus fasciatus* leaves to tartaric acid is 1.2 g: 50 mL. After cooling to room temperature, filter to obtain filtrate. Concentrate the filtrate to 1 / 2 volume. Add 2.5 times the amount of 95% edible ethanol and let stand at room temperature for 0.5 h. Then centrifuge at 6000 min / r for 2 min to separate the crude *Bungarus fasciatus* pectin for later use. (3) Purification: Wash the crude pectin of *Clerodendrum thomsoniae* with 95% edible ethanol 4 times. Each time, use 5 mL of 95% edible ethanol to wash 1 g of crude pectin of *Clerodendrum thomsoniae*. After each wash, centrifuge at 6000 min / r for 0.8 min until the filtrate is colorless. Stop washing to obtain the purified pectin. Dry the pectin at 40℃ for 10 h, grind it in a mortar, pass it through a 120 mesh sieve, and collect the fine pectin powder to obtain *Clerodendrum thomsoniae* pectin.

[0021] Example 3 (1) Pretreatment: Dry the leaves of the broom grass at 35℃ for 28 hours, then grind them into fine powder of 360-380μm to obtain broom grass leaf powder for later use; (2) Extraction: Weigh the dried powder of *Bungarus fasciatus* leaves and add tartaric acid. Extract at 90℃ for 70 min. The pH value of tartaric acid is 2.10. The ratio of dried powder of *Bungarus fasciatus* leaves to tartaric acid is 0.8 g: 70 mL. After cooling to room temperature, filter to obtain filtrate. Concentrate the filtrate to 1 / 4 volume. Add 1.5 times the amount of 95% food ethanol and let stand at room temperature for 1.5 h. Then centrifuge at 10000 min / r for 4 min to separate the crude *Bungarus fasciatus* pectin for later use. (3) Purification: Wash the crude pectin of *Clerodendrum thomsoniae* twice with 95% edible ethanol. Each time, use 20 mL of 95% edible ethanol to wash 1 g of crude pectin of *Clerodendrum thomsoniae*. After each wash, centrifuge at 10000 min / r for 1.2 min until the filtrate is colorless. Stop washing to obtain the purified pectin. Dry the pectin at 50℃ for 7 h, grind it in a mortar, pass it through an 80-mesh sieve, and collect the fine pectin powder to obtain *Clerodendrum thomsoniae* pectin.

[0022] Example 4 (1) Pretreatment: Dry the leaves of the broom-shaped wood at 40℃ for 22 hours, then grind them into fine powder of 370-390μm to obtain broom-shaped wood leaf powder for later use.

[0023] (2) Extraction: Weigh the dried powder of *Bungarus fasciatus* leaves and add tartaric acid. Extract at 95℃ for 55 min. The pH value of tartaric acid is 2.00. The ratio of dried powder of *Bungarus fasciatus* leaves to tartaric acid is 1 g: 60 mL. After cooling to room temperature, filter to obtain filtrate. Concentrate the filtrate to 1 / 3 volume. Add 2 times the amount of 95% edible ethanol and let stand at room temperature for 1.2 h. Then centrifuge at 8000 min / r for 3 min to separate the crude *Bungarus fasciatus* pectin for later use.

[0024] 3) Purification: Wash the crude pectin of *Clerodendrum thomsoniae* twice with 95% edible ethanol. Each wash uses 10 mL of 95% edible ethanol per 1 g of crude pectin. After each wash, centrifuge at 8000 min / r for 1 min until the filtrate is colorless. Stop washing to obtain purified pectin. Dry the pectin at 45℃ for 8 h, grind it in a mortar and pestle, and then collect the fine pectin powder through a 100-mesh sieve to obtain *Clerodendrum thomsoniae* pectin.

[0025] Example 5 Preparation of capsules: The pectin from *Bromhidrosis pilosula* prepared in Example 1 was added to a conventional pharmaceutical carrier or excipient and processed according to conventional methods to form capsules.

[0026] Dosage and administration: 1-2 capsules once or twice daily, each capsule containing 1.5g of *Clerodendrum trichotomum* pectin. (Conversion based on the body surface area (BSA) method recommended in the FDA's "Industry Guidance: Estimating the Maximum Safe Starting Dose for Clinical Trials in Healthy Adult Volunteers." According to the guidance, the equivalent dose conversion factor from mice to humans is 12.3. The dose in this study that did not show toxicity in animals is significantly higher than the proposed recommended human intake of *Clerodendrum trichotomum* pectin (1.5-3g / dose), indicating that this dose has extremely high safety.) Example 6 Tablet preparation: Take the pectin from *Bromhidrosis pilosula* prepared in Example 1, add a conventional pharmaceutical carrier or excipient, and process it according to conventional methods to make tablets.

[0027] Dosage and administration: 1-2 tablets once or twice a day, each tablet containing 1.5g of odor-causing pectin.

[0028] Example 7 Preparation of granules: The pectin from *Bromhidrosis pilosula* prepared in Example 1 was added to a conventional pharmaceutical carrier or excipient and processed into granules according to conventional methods.

[0029] Dosage and administration: 1-2 times a day, 1-2 packets each time, each packet contains 1.5g of body odor pectin.

[0030] This invention has verified the anti-obesity efficacy of PSAP (a type of pectin used to treat bromhidrosis) through systematic animal experiments. The following are the specific implementation methods and the research on its efficacy: 1. Preparation of pectin from *Bromhidrosis pilosula* (a type of herb) Leaves of *Clerodendrum trichotomum* were collected and dried in a laboratory oven at 40℃ with circulating air for 24 hours, then ground into a fine powder (380±10.0μm). The dried powder was weighed, and tartaric acid at pH 2.00 was prepared. 300mL of tartaric acid was added at a material-to-liquid ratio of 1g:60mL, and the mixture was extracted at 95℃ for 60 min. After cooling to room temperature, the mixture was filtered to obtain the filtrate. The filtrate was concentrated to 1 / 3 of its volume, and two times the volume of 95% edible ethanol was added to precipitate the pectin. The mixture was allowed to stand at room temperature for 1 hour, and then centrifuged at 8000 min / r for 3 min to separate the pectin. The pectin was washed with a small amount of 95% edible ethanol (each wash was performed using 15mL of 95% edible ethanol per 1g of crude *Clerodendrum trichotomum* pectin). The mixture was centrifuged at 8000 min / r for 3 min to obtain the pectin. The washing was repeated three times until the centrifuged filtrate was colorless. The washing was then stopped, and the pectin was dried at 45℃ and sieved through a 100-mesh sieve to obtain the pectin sample (i.e., PSAP).

[0031] 2. Experimental design and diet for animals Thirty-two male C57BL / 6J mice, 4 weeks old and weighing 16±2g, were obtained from the Animal Experiment Center of Guizhou Medical University. Seven mice were housed in each cage in a controlled environment with free access to food and water in a standard specific pathogen-free (SPF) facility at a temperature of 24±2°C, humidity of 60±5%, and a light cycle of 12:12h. After acclimatization for one week, the mice were randomly divided into three groups (n=7 per group): (1) normal control diet (NCD, D12450J, 10% of energy from fat, 3.85 kcal / g); (2) high-fat diet group (HFD, D12492, 60% of energy from fat, 5.24 kcal / g); (3) high-fat diet plus 30% PSAP group (HFD-PSAP, 60% of energy from fat). All animals were housed for 12 weeks. Food intake was monitored twice a week, and body weight was monitored once a week. Energy intake (kcal) is calculated by multiplying the food weight in grams by the energy in the diet (kcal / gram).

[0032] At week 12, fresh fecal samples were collected from sterile EP tubes of each mouse, immediately immersed in liquid nitrogen, and stored at −80°C for subsequent microbiome analysis. At the end of week 12, samples were collected after the mice were fasted for 12 hours and then dissected (cervical dislocation following mild pentobarbital anesthesia) to obtain organs and tissues. Blood samples were collected from the orbital vascular plexus, and serum was obtained after separation (3000 rpm, 10 min, 4°C) and stored at −80°C for further analysis.

[0033] 3. Biochemical analysis The concentrations of blood lipids, including total triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDLC), as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were determined using a 3100 automated biochemical analyzer (Tokyo, Japan).

[0034] 4. Oral glucose tolerance test and insulin resistance homeostasis model assessment Each mouse was orally administered glucose solution (2 g / kg body weight). Glucose levels were measured in tail vein blood samples at 0, 15, 30, 60, 90, and 120 min using a blood glucose meter (Bayer, Assen's Diabetes Care Shanghai Co., Ltd., China). The area under the curve (AUC) for each mouse's OGTT data was calculated. Fasting serum insulin levels were measured using a commercial mouse high-sensitivity insulin ELISA kit (ALPCO, USA).

[0035] 5. Histological analysis of liver and epididymal adipose tissue Fresh liver tissue was fixed in 4% paraformaldehyde for at least 24 hours. Liver tissue embedded at the optimal cutting temperature (OCT) was serially sectioned at 5 μm, mounted on glass slides, and allowed to dry. After drying, the slides were placed in propylene glycol for 2 minutes and then stained in 0.5% Oil Red solution for 30 minutes. Subsequently, the slides were washed twice with PBS and treated with hematoxylin and eosin. Section imaging was performed using an inverted microscope (Axio Observer A1; Carl Zeiss, Oberkochen, Germany), and white, cell-sized adipocytes were quantified using ImageJ software.

[0036] 6. DNA extraction and 19S rRNA gene sequencing Total genomic DNA was extracted from fecal samples using the OMEGA Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA). The quantity and quality of the extracted genomic DNA samples were monitored using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and agarose gel electrophoresis. The extracted genomic DNA was amplified based on the hypervariable region of the bacterial 16S rRNA gene V3-V4 using standard barcoded primer pairs 338F and 806R. The mixed PCR products were then extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, United Cities, CA, USA). After individual quantification, equal volumes of the obtained amplicones were pooled and sequenced (2 x 300 bps) on the Illumina MiSeq platform according to the standard protocol of Shanghai Majorbio Biomedical Technology Co., Ltd.

[0037] After sequencing, the raw data underwent a series of quality filtering, trimming, noise reduction, merging to eliminate adapter contamination, and removal of low-quality raw data. The remaining high-quality raw data were clustered into the same operational taxonomic units (OTUs) using Uparse (version 7.0, http: / / www.drive5.com / uparse / ). Based on the OTU clustering and annotation results, information on microbial species and their relative abundance was obtained. α- and β-diversity indices were calculated using the core diversity plugin of Mothur (V 1.30.2) and QIIME2 (version 1.7.0) and visualized using R software (version 2.15.3). Principal coordinate analysis (PCoA) was performed using the non-phylogenetic (Bray-Curtis) distance matrix to evaluate β-diversity. The ANOSIM statistic test was used to determine the significance of differences in β-diversity. Furthermore, the unweighted pairwise method using the arithmetic mean (UPGMA) was used to calculate hierarchical clustering of the samples. Linear discriminant analysis (LDA) and effect size analysis (LEfSe) with a threshold of 3.0 were used to identify biomarker groups with significant differences in relative abundance among different groups (P < 0.05).

[0038] 7. Bioinformatics Analysis After sequencing, the raw data was filtered and merged to obtain effective reads. Using Uparse (Version 7.0, http: / / www.drive5.com / uparse / ), these unique sequences were clustered into the same operational taxonomic unit (otu) with a 97% similarity threshold. Based on the otu clustering results, the corresponding microbial species information and their abundance distribution were obtained by labeling representative sequences for each OUT. α- and β-diversity were measured using Mothur (V1.30.2) and QIIME (V 1.7.0), respectively, and displayed using R software (V 3.3.1). Principal coordinate analysis (PCoA) was performed using the non-phylogenetic (Bray-Curtis) distance matrix to evaluate β-diversity. Significant differences in β-diversity were confirmed by the ANOSIM statistical test. Hierarchical clustering of the samples was calculated using UPGMA (Unweighted Pairgroup Method with ArithmeticMean). Furthermore, linear discriminant analysis (LDA) effect size (LEfSe) with a threshold of 3.0 was used to distinguish significantly important microbial groups among different groups.

[0039] 8. Statistical Analysis Comparisons between two or more groups were performed using unpaired t-tests and one-way ANOVA, followed by Tukey's multiple comparison test. All statistical analyses were performed using IBM SPSS Statistical Software V22.0, and graphs were generated using GraphPad Prism 8.

[0040] 9. Experimental Results 9.1 Weight The pectin in *Bromhidrosis pilosula* significantly inhibited high-fat diet-induced weight gain and body fat accumulation. During the experimental period, mice in the HFD group (model group) showed a sustained and significant increase in body weight, exhibiting a clear obesity phenotype. In contrast, the weight gain of mice in the HFD+PSAP group (treatment group) was significantly inhibited, and their final body weight was significantly lower than that of the HFD group. Furthermore, this weight change was not caused by reduced food intake (as can be seen). Figure 1 , Figure 2 ).

[0041] 9.2 Epididymal adipose tissue H&E staining revealed the following in epididymal adipose tissue: HFD group: Adipocytes showed significant pathological hypertrophy, with enormous cell volume and an average cell area as high as 10506.92±1073.37μm. 2 ; HFD+PSAP group: Pectin intervention significantly inhibited the increase in adipocyte volume, the morphology of adipocytes tended to be normal, and the average cell area was significantly reduced to 3105.65±239.57μm. 2 The difference was extremely significant compared to the HFD group, and approached the level of the NCD group (normal group) (1898.31 ± 130.82 μm). 2 This result directly confirms that pectin from *Bromhidrosis pilosula* can effectively inhibit the excessive expansion of adipose tissue (see...). Figure 3 , Figure 4 ).

[0042] 9.3 Liver adipose tissue The pectin in *Bromhidrosis pilosula* (a type of herb) improves hepatic steatosis and reduces lipid deposition in the liver. Histopathological analysis of liver tissue revealed that: H&E staining showed that: in the NCD group, the hepatic cords were neatly arranged and there were no lipid droplets or vacuoles; in the HFD group, a large number of round lipid droplets or vacuoles appeared in the hepatocytes, and the cell nuclei were squeezed to the edge, showing severe fatty degeneration; while in the HFD+PSAP group, the hepatocyte structure was relatively intact, and the number and size of lipid droplets or vacuoles were significantly reduced.

[0043] Quantitative analysis using Oil Red O staining showed that the proportion of red-stained lipid droplets in liver sections of the HFD group was as high as 37.87%, indicating severe lipid accumulation. After intervention with *Clerodendrum trichotomum* pectin, the proportion of lipid droplets in the HFD+PSAP group significantly decreased to 14.34%, a reduction of over 62%, significantly better than the HFD group. The results indicate that *Clerodendrum trichotomum* pectin can effectively reduce ectopic fat deposition in the liver and has a significant protective effect against fatty liver caused by obesity (see [link]). Figure 5 ).

[0044] 9.4 Blood lipids and liver function biochemical indicators Biochemical analysis of blood lipid and liver function indicators by pectin from *Bromhidrosis pilosula* showed that the HFD group mice had significantly elevated serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), and abnormal liver function indicators (ALT, AST). In contrast, the HFD-PSAP group showed significant improvement in these lipid metabolism and liver damage indicators, indicating that PSAP has good lipid-lowering and liver-protective activity. Furthermore, we evaluated the glucose metabolism capacity of the mice. OGTT and insulin detection results showed that PSAP intervention significantly reduced high-fat diet-induced hyperglycemia and fasting insulin levels, and improved the HOMA-IR index, indicating its potential to regulate glucose metabolism disorders (see [link to OGTT]). Figure 6 , Figure 7 , Figure 8 , Figure 9 ).

[0045] 9.5 Gene sequencing results The structure of gut microbiota in obese mice was reshaped by pectin from *Clerodendrum trichotomum*. Analysis of the fecal microbiome of mice using 16S rDNA sequencing technology was also performed. Dilution curves and α-diversity: The dilution curves tend to be flat, indicating sufficient sequencing depth. Figure 10 Box plots showed that HFD led to a decrease in gut microbiota richness (Ace / Chao1 index), while PSAP intervention restored the diversity and richness of the microbiota to some extent.

[0046] β-diversity analysis: PCoA analysis based on Bray-Curtis distance showed that the microbial community structures of the NCD group, HFD group, and HFD+PSAP group exhibited clear clustering and separation. Figure 11 The sample points of the HFD+PSAP group were significantly separated from those of the HFD group in terms of spatial distribution and moved closer to those of the NCD group, indicating that the pectin in the body odor-causing bacteria will restore the gut microbiota structure that was disrupted by a high-fat diet to a healthy state.

[0047] 9.6 Pectin-regulated relative abundance and specific species of key phyla in *Bromhidrosis pilosula* Phylogenetic analysis: Community composition analysis shows ( Figure 12 In the HFD group, an increase in the abundance of Bacillota / Firmicutes and a decrease in the abundance of Bacteroidetes / Bacteroidota were typically observed, leading to an increased F / B ratio. After PSAP intervention, the relative abundance of Bacteroidetes significantly recovered, while the abundance of Firmicutes decreased, thus improving metabolic disorders by regulating the F / B ratio.

[0048] LEfSe Differential Species Analysis: Evolutionary Branching Diagram ( Figure 13 Further analysis revealed the characteristic microbiota of each group. The PSAP group was significantly enriched with beneficial bacteria associated with metabolic health (e.g., [missing information]), while the HFD group was enriched with pathogenic bacteria associated with inflammation. This suggests that PSAP exerts its anti-obesity effect by specifically increasing beneficial bacteria and inhibiting harmful bacteria.

[0049] 10. Safety Assessment: Throughout the experimental period, mice ingesting 30% *Clerodendrum trichotomum* pectin exhibited normal diet, water intake, and activity levels, with no adverse reactions such as diarrhea, hair loss, or death. Post-dissection observation revealed no visible pathological damage to any major organs, indicating that this dose of *Clerodendrum trichotomum* pectin has good biocompatibility.

Claims

1. Application of *Bromhidrosis pilosula* pectin in the preparation of products for preventing and improving diet-induced obesity.

2. The application according to claim 1, characterized in that: The preparation method of the *Bromhidrosis pilosula* pectin is carried out according to the following steps: (1) Pretreatment: Dry the leaves of *Clerodendrum trichotomum* at 35-45℃ for 20-28 hours, then grind them into fine powder of 360-400μm to obtain dry powder of *Clerodendrum trichotomum* leaves for later use; (2) Extraction: Weigh the dried powder of *Bungarus fasciatus* leaves and add tartaric acid. Extract at 90-100℃ for 50-70 min. The pH value of tartaric acid is 1.90-2.

10. The ratio of dried powder of *Bungarus fasciatus* leaves to tartaric acid is 0.8-1.2g:50-70mL. After cooling to room temperature, filter to obtain filtrate. Concentrate the filtrate to 1 / 4-1 / 2 volume. Add 1.5-2.5 times the amount of 95% food ethanol and let stand at room temperature for 0.5-1.5h. Then centrifuge at 6000-10000min / r for 2-4min to separate the crude *Bungarus fasciatus* pectin for later use. (3) Purification: Wash the crude pectin of *Clerodendrum thomsoniae* 2-4 times with 95% edible ethanol. Each time, use 5-20 mL of 95% edible ethanol for 1 g of crude pectin of *Clerodendrum thomsoniae*. After each wash, centrifuge at 6000-10000 min / r for 0.8-1.2 min until the filtrate is colorless. Stop washing to obtain the purified pectin. Dry the pectin at 40-50℃ for 7-10 h, grind it in a mortar, pass it through an 80-120 mesh sieve, and collect the fine pectin powder to obtain *Clerodendrum thomsoniae* pectin.

3. The application according to claim 2, characterized in that: In step (1), the pretreatment is as follows: the leaves of the *Clerodendrum trichotomum* are dried at 40°C for 24 hours and then ground into fine powder of 370-390 μm to obtain dry powder of *Clerodendrum trichotomum* leaves for later use.

4. The application according to claim 2, characterized in that: In step (2), the extraction process is as follows: Weigh the dried powder of *Bungarus fasciatus* leaves, add tartaric acid, and extract at 95°C for 60 min. The pH value of tartaric acid is 2.00, and the ratio of dried powder of *Bungarus fasciatus* leaves to tartaric acid is 1 g: 60 mL. After cooling to room temperature, filter to obtain the filtrate. Concentrate the filtrate to 1 / 3 volume, add 2 times the amount of 95% edible ethanol, let stand at room temperature for 1 h, and then centrifuge at 8000 min / r for 3 min to separate the crude *Bungarus fasciatus* pectin for later use.

5. The application according to claim 2, characterized in that: In step (3), purification: wash the crude pectin of *Clerodendrum thomsoniae* with 95% edible ethanol 2-3 times. Each time, use 10-15 mL of 95% edible ethanol to wash 1 g of crude pectin of *Clerodendrum thomsoniae*. After each wash, centrifuge at 8000 min / r for 1 min until the filtrate is colorless, then stop washing to obtain the washed and purified pectin. Dry the pectin at 45℃ for 8-9 h, grind it in a mortar, pass it through a 100-mesh sieve, and collect the fine pectin powder to obtain *Clerodendrum thomsoniae* pectin.

6. The application according to any one of claims 1-5, characterized in that: The products include pharmaceuticals or health foods.

7. The application according to any one of claims 1-5, characterized in that: The medicine or health food is added to a conventional pharmaceutical carrier or excipient and processed according to conventional methods to produce the corresponding formulation.

8. The application according to claim 7, characterized in that: The formulations include capsules, tablets, and granules.

9. The application according to claim 1, characterized in that: The pectin from *Bromhidrosis pilosula* regulates lipid metabolism by reshaping the intestinal flora structure, reducing the ratio of Firmicutes to Bacteroidetes, and specifically enriching beneficial bacteria.

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