Patent Publication Number: US-2023142062-A1

Title: The application of aloesin in the preparation of products for preventing or treating obesity

Description:
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING 
     The contents of the electronic sequence listing (8629-0004 Sequence Listing.txt; Size: 3,318 bytes; and Date of Creation: Dec. 6, 2022) is herein incorporated by reference in its entirety. 
     FIELD OF THE INVENTION 
     The invention relates to the technical field of plant medicines, in particular to the application of aloesin in the preparation of products for preventing or treating obesity. 
     BACKGROUND OF THE INVENTION 
     Obesity refers to the accumulation of excess fat caused by calorie intake greater than consumption, and is currently one of the main health problems facing the world. In 2015, an estimated 609 million adults worldwide were obese, twice as many as in 1980. Obesity is a serious risk factor leading to insulin resistance, diabetes, atherosclerosis and cardiovascular disease. Changing dietary habits and increasing physical exercise are traditional methods of treating obesity. However, after controlling diet and increasing exercise, the fat reduction effect is temporary, and body weight will rebound. In addition, other methods such as bariatric surgery and medication also have their own risks and side effects such as alcoholism and acute kidney injury. 
     Aloe is a commonly used medicine in several traditional countries such as Greece, Egypt, India, Mexico, Japan and China. It is often used as a laxative to treat constipation in our country. Aloesin is an aromatic C-glycosylated 5-methylchromone compound, widely distributed in most aloe species and have a wide range of pharmacological effects. It is generally believed that it can directly inhibit the production of melanin and tyrosinase activity. In addition, aloesin can promote wound healing and improve diabetic syndrome by inhibiting oxidation and interfering with MAPK/Rho and Smad signaling pathways. Recently, new pharmacological effects of aloesin have been continuously reported, such as improving insulin resistance, inhibiting exacerbation of colitis in rats, inhibiting cell growth and metastasis of ovarian cancer SKOV3 cells, and inhibiting colorectal cancer cell growth by interfering with Wnt/β-catenin and Notch signaling pathways. However, whether aloesin has anti-obesity effects remains unclear. 
     BRIEF SUMMARY OF THE INVENTION hi view of this, the present invention provides the application of aloesin in the preparation of anti-obesity agents, obesity treatment agents and related products. 
     Application of aloesin in the preparation of the anti-obesity agent for preventing obesity. 
     The application of aloesin in the preparation of an obesity therapeutic agent for treating obesity. 
     The application of aloesin in the preparation of a UCP1 gene expression promoter for preventing or treating obesity. 
     The application of aloesin in the preparation of a PDRM16 gene expression promoter for preventing or treating obesity. 
     The application of aloesin in the preparation of the UCP-1 and the PDRM16 gene expression promoters for preventing or treating obesity. 
     The application of aloesin in the preparation of a transcription factor C-EBPa expression promoter for preventing or treating obesity. 
     Application of aloesin in the preparation of an adiponectin accelerator for preventing or treating obesity. 
     Application of aloein in the preparation of a IL-1β inhibitor for preventing or treating obesity. 
     Compared with the prior art, the beneficial effects of the present invention are: the research of the present invention found that aloesin can inhibit lipid accumulation, increase the expression of browning genes UCP1 and PRDM16 and transcription factor C-EBPa, and improve the expression and release of specific factor adiponectin, inhibit the release of non-specific factor IL-1β, indicating that aloesin can mediate the browning of 3T3-L1 adipocytes, and aloesin can be used to prepare products for preventing or treating obesity. Moreover, aloesin is derived from plants and has high safety, providing a better choice for obese patients, and at the same time, making aloesin to play a higher value. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG.  1    shows the chemical structure (A) and experimental design (B) of aloesin. The effect of aloesin on cell viability after the pretreatment therewith for 24 h (C) and 48 h (D). n=6-12. 
         FIG.  2    shows the effect of aloesin on the differentiation of preadipocytes. Oil red O staining image (A), the lipid accumulation after the pretreatment with aloesin for 24 h (B), the lipid accumulation after the pretreatment with aloesin for 48 h, n=6 (C). 
         FIG.  3    shows the effect of aloesin on lipid accumulation in adipocytes. Oil red O stained image (A). Lipid accumulation of adipocytes treated with aloesin for 24 hours (B). Lipid accumulation of adipocytes treated with aloesin for 48 h, n=6(C). 
         FIG.  4    show the effect of aloesin on the mRNA expression of UCP-1, PRDM16, PPAR-γ, C-EBPa and FABP in 3T3-L1 adipocytes. n=4-8. 
         FIG.  5    shows the effect of aloesin on the mRNA expression and release of adiponectin and IL-1β in 3T3-L1 adipocytes. n=3-8. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention. 
     Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods. 
     The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources unless otherwise specified. 
     3T3-L1 preadipocytes were originally derived from Swiss mouse embryonic tissue. Because of its potential to differentiate from fibroblasts into mature adipocytes, this cell line has been widely used in the study of mechanisms of obesity, diabetes and related diseases. Therefore, in this experiment, we will select 3T3-L1 pre-adipocytes and adipocytes to explore the effect of aloesin on adipocytes. 
     1. Materials and Methods 
     1.1 Cell Culture 
     Mouse 3T3-L1 preadipocytes were inoculated with high glucose culture medium (H-DMEM, Hyclon) containing 10% fetal bovine serum (Gibco) and antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) at a density of 1×10 4  /mL in an incubator at 37° C. and 5% CO 2  for 72 hours. In order to induce the differentiation of 3T3-L1 preadipocytes, when 80% cells are fused, on day 0, growth medium (GM) containing 10% fetal bovine serum, 10 μg/mL insulin (sigma), 1 μM dexamethasone (sigma), 0.5 mM isobutyl-1-Methylxanthine (sigma) was added therein. On the second day, the medium was replaced with a high-glycemic medium containing 10% calf serum and 10 μg/mL insulin. On the 10th day, a large number of lipid droplets accumulated during the differentiation of pre-adipocytes. 
     1.2 MTT Method 
     Cell viability was determined by MTT, 3T3-L1 preadipocytes were seeded in 96-well plates at a density of 1×10 4  /mL, cultured in an incubator for 24 hours, and then replaced with a medium respectively containing 10, 15, 30 and 60 μM aloesin to continuously cultivate for 24 h or 48 h. 0.5% MTT was added to the cells, after incubating for 1 hour at 37° C., the MTT solution was removed, and DMSO was added to dissolve. The absorbance at 570 nm was measured using an enzyme-labeled instrument (Molecular Devices, CA, USA). Cell viability (%) is expressed as a percentage relative to untreated control cells. 
     1.3 Oil Red Staining 
     The differentiated adipocytes were washed 3 times with PBS, fixed with 10% formaldehyde for 1 hour, then washed 3 times with PBS again, dried for 15 to 30 minutes and stained with Oil Red O working solution for 2 hours. Wash 2 times and 3 times with 60% isopropanol (IPA) and double distilled water respectively. A Zeiss fluorescence microscope (Tokyo, Japan) was used to collect images. 100% IPA extracts Oil Red O, and the absorbance at 490 nm was measured. 
     1.4 Real-Time Quantitative PCR Analysis 
     Total RNA extraction reagent (TIANGEN, China) was used to extract total RNA from 3T3-L1 adipocytes. Use 1 st  strand cDNA kit (Vazyme biotechnology) for reverse transcription into cDNA. SYBR Green PCR Master Mix Kit (Monad, China) reagent and CFX-1000 Touch instrument (Applied Biosystems, USA) were used to detect the mRNA level of adipocyte browning genes (Ucp-1 and PRDM16) and transcription factors (Fabp, Ppar-γ and C-EBPα). The following conditions were used for reverse transcription: 5 min at 25° C., 15 min at 50° C., and 5 min at 85° C. Amplification: initial denaturation at 95° C. for 30 seconds, followed by denaturation at 95° C. for 5 seconds and 59° C. for 30 seconds (40 cycles). The β-actin was used as a reference gene to standardize the qPCR reaction. The relative expression of genes was calculated based on the average Ct value between samples. Relative expression level=2{circumflex over ( )}{[Ct target gene −Ct (β-actin)] aloesin treatment group −[Ct target gene −Ct(β-actin)] 3T3-L1 adipocyte ]}. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Primer sequence 
               
            
           
           
               
               
               
               
            
               
                   
                 Gene 
                 Forward primer 
                 Reverse primer 
               
               
                   
                 name 
                 (3′-5′) 
                 (3′-5′) 
               
               
                   
               
               
                   
                 β-actin 
                 CCACAGCTGAG 
                 AAGGAAGGCT 
               
               
                   
                   
                 AGGGAAATC 
                 GGGAAAAGA 
               
               
                   
               
               
                   
                 UCP-1 
                 CCTGCCTCTCT 
                 GTAGCGGGGT 
               
               
                   
                   
                 CGGAAACAA 
                 TTTGATCCCA 
               
               
                   
               
               
                   
                 PRDM16 
                 CCCCACATTCC 
                 CTCGCAATCCT 
               
               
                   
                   
                 GCTGTGAT 
                 TGCACTCA 
               
               
                   
               
               
                   
                 PPAR-γ 
                 CAAGAATACC 
                 GAGCTGGGTC 
               
               
                   
                   
                 AAAGTGCGAT 
                 TTTTCAGAA 
               
               
                   
                   
                 CAA 
                 TA ATAAG 
               
               
                   
               
               
                   
                 C-EBPα 
                 AGCAACGAGT 
                 TGTTTGGCTTT 
               
               
                   
                   
                 ACCGGGTAC 
                 ATCTCGGCTC 
               
               
                   
               
               
                   
                 FABP 
                 AGTGAAAATT 
                 GCCTGCCACTT 
               
               
                   
                   
                 CGATGATTAC 
                 TCCTTGTG 
               
               
                   
                   
                 ATGAA 
                   
               
               
                   
               
               
                   
                 Adiponectin 
                 CAGGTCTTCT 
                 TCCACATTCTT 
               
               
                   
                   
                 TGGTCCTAAG 
                 CTTCCTGATAT 
               
               
                   
                   
                 GG 
                 G 
               
               
                   
               
               
                   
                 IL-1β 
                 TGCCACCTTTT 
                 GGAGCCTGTAG 
               
               
                   
                   
                 AGACAGTGTG 
                 TGCAGTTGT 
               
               
                   
               
            
           
         
       
     
     1.5 Enzyme-Linked Immunosorbent Assay (ELISA) 
     The 3T3-L1 adipocytes were treated with aloesin for 48 hours, then the supernatant was collected, and the levels of adiponectin and IL-1β in the supernatant were detected with an ELISA kit (Proteintech, China). The ELISA reagent operation was carried out according to the manufacturer&#39;s instructions. 
     1.6 Statistical Analysis 
     The result value is expressed as mean±standard error. Using GraphPad Prism 8 software to analyze statistical significance by one-way analysis of variance or two-sided T-test, P&lt;0.05 is considered to be of statistic difference. 
     2. Results 
     2.1 The Effect of Aloesin on the Viability of 3T3-L1 Preadipocytes 
     Effect of aloesin on the 3T3-L1 pre-adipocyte viability was evaluated by MTT assay. Pre-adipocytes were incubated with increasing concentration of aloesin (10 to 60 μM) for 24 h and 48 h. It was illustrated that pre-treated with aloesin (15 and 30 μM) for 24 h and 48 h did not influence the pre-adipocytes viability. Hence, the concentration of 15 and 30 μM was selected for further investigation ( FIGS.  1 C and  1 D ). 
     As shown in  FIG.  1   , the chemical structure (A) and experimental design (B) of aloesin. The effect on cell viability with aloesin pretreatment for 24 h (C) and 48 h (D). n=6-12. 
     2.2 Aloesin Does Not Affect the Differentiation of Preadipocytes 
     3T3-L1 pre-adipocytes were pre-incubated by the aloesin with concentration of 15 and 30 μM for 24 h and 48 h, respectively. Then pre-adipocytes were differentiated for 10 days. No influence was found after pretreated by 15 and 30 μM of aloesin for 24 h and 48 h ( FIG.  2 A  and  FIG.  2 B ) and 48 h ( FIG.  2 A  and  FIG.  2 C ) did not affect the differentiation of 3T3-L1 preadipocytes. 
     2.3 Aloesin Reduces Lipid Accumulation in 3T3-L1 Adipocytes 
     After 3T3-L1 preadipocytes were induced to differentiate into adipocytes, aloesin (15 and 30 μM) was added to the adipocytes and cultured for 24 h and 48 h respectively. The results showed that aloesin (15 and 30 μM) treatment for 24 hours did not affect the lipid accumulation in 3T3-L1 adipocytes ( FIG.  3 A  and  FIG.  3 B ). However, compared with the control group, treatment with 15 and 30 μM aloesin for 48 h could significantly reduce the lipid accumulation of adipocytes ( FIG.  3 A  and  FIG.  3 C ). 
     2.4 Aloesin Reduces Lipid Accumulation in Adipocytes by Inducing Browning 
     In order to further explore the mechanism of the influence of aloesin on lipid accumulation, we analyzed the mRNA expression of key genes related to adipocyte browning by real-time fluorescent quantitative PCR. It was found that aloesin could increase the expression of browning genes (UCP-1 and PDRM16). Further results showed that both 15 and 30 μM aloesin could increase the expression of the transcription factor C-EBPα, but did not affect FABP and PPAR-γ, indicating that the effect of aloesin on the transcription of UCP-1 and PDRM16 may be regulated by C-EBPα, but not related to FABP and PPAR-γ ( FIG.  4   ). 
     2.5 Aloesin Increases the Release of Specific Cytokines (Adiponectin) in the Adipocyte and Reduces the Expression of Non-Specific Cytokines (IL-1β) Therein 
     15 μM aloesin can enhance the mRNA expression and release of adiponectin in adipocytes, while 15 and 30 μM aloesin can reduce the release of IL-1β in adipocytes ( FIG.  5   ). 
     In this study, it is found that (1) Aloesin can reduce lipid accumulation by activating the browning of white adipocytes, and improve the specific and non-specific pro-inflammatory effects of adipocytes. 
     (2) Aloesin can reduce lipid accumulation in 3T3-L1 adipocytes and increase the mRNA expression of UCP1 and PRDM16, so it is indicated that aloesin may inhibit lipid accumulation by inducing browning of adipocytes. 
     (3) Aloesin can increase the expression and secretion of adiponectin mRNA, reduce the secretion of non-specific cytokine IL-1β, and consolidate the effect of aloesin in inducing the browning of 3T3-L1 adipocytes. 
     (4) Aloesin can enhance the fat to form the adipogenic transcription factor C-EBPα, but not the mRNA expression of FABP and PPAR-γ, so it is indicated that the mechanism of aloesin inducing browning is related to its enhancement of the expression of the transcription factor C-EBPα. 
     In summary, the present invention indicates that aloesin can inhibit lipid accumulation, increase the expression of browning genes UCP1 and PRDM16 and the transcription factor C-EBPa, increase the expression and release of the specific factor adiponectin, and inhibit the release of the non-specific factor IL-1β, it is illustrated that aloesin can mediate the browning of 3T3-L1 adipocytes. Aloesin can be used in the preparation of products for preventing or treating obesity and can be used in the preparation of anti-obesity agents for preventing obesity or obesity treatment agents for treating obesity, etc. 
     The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the present invention.