Application of hesperetin and hesperetin derivative in treatment of muscular atrophy

Abstract

The invention relates to an application of hesperetin or a derivative thereof, or a pharmaceutically acceptable salt thereof in preparation of a medicine for treating muscle atrophy. The invention accidentally finds that flavonoids compound hesperetin or derivatives thereof have beneficial effects on intestinal permeability and systemic inflammation, and hesperetin as a biomarker is expected to become a potential target spot and can be used for treating or improving clinical symptoms of patients with amyotrophy.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to a new application of hesperidin and its derivatives in treating muscular atrophy. Background Art

[0002] Sarcopenia is a progressive, systemic skeletal muscle disease that includes a decrease in muscle strength and muscle mass. Many factors are thought to be involved in the etiology of sarcopenia. These factors, including aging, fractures, obesity, cancer, or organ failure with cachexia, contribute to the development of sarcopenia in an individual or additive manner. Additional interventions targeting specific causes may enhance the therapeutic effect of sarcopenia on the basis of traditional treatments, including resistance training or exercise, nutritional interventions, protein supplementation, and drug therapy. Therefore, characterizing the different etiological indicators associated with sarcopenia can provide an effective way to develop targeted treatment and prevention strategies.

[0003] In recent years, studies have revealed the potential interactions between sarcopenia and the gut microbiota and gut metabolites. Characteristic changes in the gut microbiome, bile acid composition, and gut metabolome have been found in sarcopenia associated with cirrhosis. In addition, metabolites from the gut microbiota are expected to become accurate diagnostic markers for sarcopenia in the elderly. Both in clinical studies and animal models, liver cancer has been shown to be associated with gut microbiota and metabolites due to the existence of the gut-liver axis. Therefore, there are many reasons for the high incidence of sarcopenia in patients with liver cancer. Especially considering that 50%-90% of liver cancer patients are accompanied by cirrhosis, and studies have shown that the incidence of sarcopenia in patients with cirrhosis is as high as 23%-60%, which is much higher than the 7.5% of normal people. More importantly, studies have shown that sarcopenia is an independent prognostic factor for death after liver cancer resection. Therefore, exploring the changes in the gut microbiota and its metabolites in patients with liver cancer sarcopenia can provide empirical evidence for improving prognosis and new intervention pathways for sarcopenia. Summary of the invention

[0004] In response to the above problems, the inventors unexpectedly discovered the beneficial effects of flavonoids on intestinal permeability and systemic inflammation. Hesperidin, as a biomarker, is expected to become a potential target for treating or improving the clinical symptoms of patients with sarcopenia.

[0005] The inventors have been collecting feces from liver cancer patients with and without sarcopenia for a long time in the clinic to sequence and analyze the relevant intestinal flora and intestinal metabolites. While describing the differences in intestinal microbiota and metabolome between liver cancer patients with and without sarcopenia, the study found that liver cancer patients with sarcopenia had significantly less hesperidin in their intestinal metabolites than those without sarcopenia.

[0006] Hesperidin is a flavonoid compound mainly found in citrus fruits. At present, hesperidin has been proven to have a wide range of pharmacological effects, such as anti-inflammatory, free radical scavenging, antioxidant, antiviral, cardiovascular protection and neuroprotection. The inventors have verified through experiments that the differential metabolite hesperidin has an effect on liver cancer-related muscle loss symptoms, in order to improve the prognosis of patients with liver cancer and muscle loss symptoms, and also provide potential intervention targets for the treatment of sarcopenia.

[0007] In one aspect, the present invention relates to a use of hesperetin or its derivatives or pharmaceutically acceptable salts thereof represented by the following general formula in the preparation of a drug for treating muscular atrophy, wherein the hesperetin or its derivatives are selected from the compounds represented by formula (1) or formula (2):

[0008]

[0009] In another embodiment, the present invention relates to the use of hesperetin or its derivatives as shown above, or its pharmaceutically acceptable salts in the preparation of a medicament for treating muscular atrophy, wherein the hesperetin or its derivatives is hydrochloride, sulfate, bromate, fumarate, acetate or citrate, etc.

[0010] Hesperidin is one of the most important active ingredients in the orange peel of the Rutaceae Citrus plant. It is a dihydroflavonoid substance, mainly in the form of light yellow powder or needle-shaped crystals, and has multiple pharmacological effects such as anti-inflammatory response, antioxidant response, lowering blood lipids, anti-tumor effect, cardiovascular protection and immune system regulation. Hesperidin derivative-16 is a monomer compound derived from hesperidin, which has anti-inflammatory and liver-protective effects on a series of liver diseases.

[0011] In another embodiment, the present application discloses the use of hesperetin or its derivatives, or its pharmaceutically acceptable salts in the preparation of a drug for treating muscular atrophy, wherein the muscular atrophy is caused by muscle degeneration, decreased muscle strength or a decrease in the number of muscle fibers due to chronic disease consumption, mainly manifested as weight loss, low muscle strength and physical strength, and easy falls and fractures. This degeneration usually proceeds slowly over a long period of time, manifested as a decrease in the number and atrophy of fast muscle fibers.

[0012] In another embodiment, the present application discloses the use of hesperetin or its derivatives, or its pharmaceutically acceptable salts in the preparation of a medicament for treating muscular atrophy, wherein the muscular atrophy is a patient suffering from liver cancer and concurrently with muscle loss symptoms.

[0013] In order to confirm the therapeutic effect of hesperidin or its derivatives in muscle atrophy, the inventors conducted corresponding animal experiments based on the drug's mechanism of action. According to the experimental results, the grip strength test showed that the grip strength of the sarcopenia group was significantly reduced compared with the non-sarcopenia group (p < 0.001), while the grip strength of mice treated with hesperidin increased compared with the sarcopenia group (p < 0.05) (before the administration of hesperidin, there was no baseline difference in grip strength). Compared with the non-sarcopenia group mice, the values ​​of gastrocnemius (GAS) / body weight, soleus (SOL) / body weight, and tibialis anterior (TA) / body weight of the sarcopenia group mice were significantly reduced (p < 0.01). However, after supplementing with hesperidin, these parameters increased significantly (p < 0.05).

[0014] HE and Masson staining experiments were further used to perform histological analysis on the gastrocnemius muscle to observe the morphology and number of muscle fibers, as well as the distribution of collagen fibers. The results showed that the gaps between muscle fibers in the sarcopenia group increased, the irregularity was higher, the fragmentation or segmentation was more, and the cross-sectional area of ​​the muscle fibers was reduced. In contrast, the muscles of the sarcopenia group mice treated with hesperetin had more muscle fibers and denser fiber arrangement. Immunofluorescence staining experiments were used to compare the expression of myosin heavy chain (MyHC) in the muscles of each group. The results showed that the expression of MyHC in the sarcopenia group was significantly downregulated compared with that in non-sarcopenia mice, while this situation was reversed in the muscles of the sarcopenia group mice treated with hesperetin.

[0015] The present invention also provides a pharmaceutical composition comprising the above-mentioned hesperetin or its derivatives, wherein the pharmaceutical composition comprises a therapeutically effective amount of hesperetin or its derivatives, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0016] When hesperetin or its derivatives and pharmaceutically acceptable salts thereof are used as medicines, they can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1%-99%, preferably 1%-90%, 5%-80% or 10%-50% of hesperetin or its derivatives and pharmaceutically acceptable salts thereof, and the rest is pharmaceutically acceptable, non-toxic and inert pharmaceutical carriers and / or excipients to humans and animals or used in combination with other anticancer drugs.

[0017] The pharmaceutical composition of the present invention can be prepared into capsules, tablets, powders, granules, syrups or similar dosage forms for oral administration, or injection, ointment, suppository or similar dosage forms for parenteral administration. These pharmaceutical preparations can be prepared by ordinary methods using auxiliary agents well known in the art, such as adhesives, excipients, stabilizers, disintegrants, flavoring agents, lubricants, etc., and can also be prepared into controlled release dosage forms, sustained release dosage forms, and various microparticle delivery systems.

[0018] Although the dosage varies with the symptoms and age of the patient, the nature and severity of the disease or disorder, and the route and mode of administration, for oral administration to adult patients, hesperetin or its derivatives, or its pharmaceutically acceptable salts are normally administered at a total daily dose of 150 to 900 mg, preferably 100 to 300 mg, in a single dose or in divided doses; for example, twice a day.

[0019] The present invention provides a method for treating a disease, comprising administering a therapeutically effective amount of hesperidin or its derivatives, or a pharmaceutically acceptable salt thereof, to a subject in need of treatment. The term "subject" includes humans and non-human mammals, such as non-human primates, sheep, dogs, cats, cattle and horses, etc., and the preferred subject is a human patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Attached Figure 1 Animal experiments have verified that orange peel can alleviate muscle atrophy.

[0021] Attached Figure 2 Changes in intestinal flora composition and differences in intestinal permeability among the three groups of mice.

[0022] Attached Figure 3 Flow chart of overall clinical program design.

[0023] Attached Figure 4 Microbiome sequencing analysis of fecal samples. (AB) shows the distribution of dominant taxa calculated by the algorithm of liver cancer group and healthy group, and between sarcopenia group and non-sarcopenia group. (C) PLS-DA score diagram of fecal samples of ESI+ model. Sarcopenia patients are represented by blue, and non-sarcopenia patients are represented by red. (D) Cross-validation diagram of 200 permutation tests. (E) KEGG pathway enrichment analysis of metabolites significantly increased in sarcopenia patients. The x-axis represents the enrichment factor, which indicates the ratio of differentially expressed genes in a pathway to the total number of genes in the pathway. The y-axis represents the KEGG pathway. In the bubble chart, the bubble size corresponds to the number of genes, and the bubble color indicates the significance of the enrichment analysis. The smaller the p-value, the more significant the enrichment. (F) Stacked bar chart of the average proportion of taxonomic composition at the genus level between liver cancer group and control group, sarcopenia group and non-sarcopenia group.

[0024] Attached Figure 5 Metabolite sequencing analysis of fecal samples. (A) Cluster analysis of hierarchical differences in fecal metabolites between the sarcopenia group and the non-sarcopenia group and the relative abundance of selected metabolites. (B) Correlation analysis between flavonoids (|rho|>0.4) and clinical indicators. (CE) ELISA detection of fecal calcium protection, serum LPS and serum ZO-1 levels. DETAILED DESCRIPTION

[0025] Example 1 Animal Test

[0026] Hesperidin and its derivatives 16 were purchased from MedChemExpress, and their structural formulas are shown below:

[0027]

[0028] Animal experiments have shown that hesperidin can enhance the grip strength, muscle fiber cross-sectional area and mass of sarcopenic mice, thereby alleviating muscle atrophy. Animal experiments were used to verify that hesperidin can alleviate muscle atrophy. Three-month-old male C67BL / 6 mice were selected as the non-sarcopenic group, and 18-month-old male C67BL / 6 mice were selected as the sarcopenic group. The mice in the sarcopenic group were randomly divided into two groups, one was a sarcopenic group, and the other was a sarcopenic plus hesperidin treatment group. The hesperidin treatment group was given hesperidin solution by gavage at a dose of 150 mg / kg, and the sarcopenic group was given an equal amount of normal saline by gavage. After 2 weeks, the grip strength of the three groups of mice was measured using a handgrip dynamometer, and the body weight was weighed and fecal samples of the mice were collected. After handling the mice in accordance with animal ethics regulations, the mass of the gastrocnemius, tibialis anterior and soleus muscles of the mice were weighed respectively ( Figure 1 ). Among them, (A) Comparison of limb grip strength, SOL, TA, and GAS (normalized by body weight) of three groups of mice. (B) Comparison of morphology of GAS, SOL, and TA muscles of three groups of mice. (C) HE staining shows the cross-sectional area and interstitial tissue of mouse GAS muscle (100X and 200X). (D) Masson staining shows the distribution of muscle fibers (red) and collagen fibers (blue) in mouse GAS muscle (40X and 100X). (E) Immunofluorescence staining shows the expression of MyHC in muscle cells of three groups of mice. In the figure, green represents the distribution of basement membrane; blue represents the location of cell nuclei, and red represents the distribution of MyHC in muscle cells.

[0029] The feces of the three groups of mice were collected for intestinal flora sequencing, and the intestinal tissues of the three groups of mice were collected for Alcian blue staining to observe the changes in the intestinal chemical barrier of the mice, and the changes in the intestinal physical barrier of the mice were observed by electron microscopy ( Figure 2). Among them, (A) Beta diversity analysis described by PCoA using unweighted Unifrac index. P values ​​were calculated using similarity analysis (ANOSIM). (B) Relative abundance changes of intestinal flora at the genus level among the three groups of mice. (C-D) LEfSe analysis (LDA [log10 transformation]>3, P<0.05) showed differences in microbial markers between the non-sarcopenia group and the sarcopenia group (C), and between the non-sarcopenia group, sarcopenia group, and hesperidin group (D). (E) Representative images (100x) of intestinal mucin staining of the three groups of mice by Alcian blue staining. The bar graph represents the statistical comparison of the representative 100X field mucus staining counts for each mouse in each group. **, p<0.01; ****, p<0.0001. (F) Scanning electron micrographs of intestinal tissues of mice in each group. (G) Transmission electron micrographs of intestinal tissues of mice in each group, with representative tight junction structures marked (red arrows).

[0030] Example 2 Clinical Trial

[0031] A series of clinical trials and animal experiments have demonstrated the beneficial effects of flavonoids on intestinal permeability and systemic inflammation. Hesperidin, as a biomarker, is expected to become a potential target. Further clinical trials need to verify its effectiveness in intervening in patients with sarcopenia. This can not only provide a reference for the selection of treatment plans for clinical patients and the formulation of treatment plans by clinicians, but also greatly solve clinical needs and improve the survival prognosis of patients, which has important clinical practical significance. The design of the experimental plan can be found in Figure 3 A total of 161 participants were selected according to the inclusion and exclusion criteria, including 76 healthy individuals, 58 liver cancer patients without sarcopenia symptoms, and 27 liver cancer patients with sarcopenia symptoms.

[0032] Step 1: First, stool samples were collected from 105 patients in the liver resection cohort with liver cancer and 90 healthy people from the physical examination center from May 2020 to March 2022. The diagnosis of sarcopenia was evaluated, and the evaluation items included muscle strength, muscle mass, physical performance and questionnaires. The stool samples corresponding to the selected subjects were further screened according to the inclusion and exclusion criteria. Finally, a total of 161 participants were selected, including 76 healthy individuals, 58 liver cancer patients without sarcopenia symptoms, and 27 liver cancer patients with sarcopenia symptoms.

[0033] Inclusion criteria: (1) aged 18 years or older; (2) diagnosed with HBV-related liver cancer.

[0034] Exclusion criteria: (1) alcoholic liver cancer, nonalcoholic fatty liver cancer, HCB-related liver cancer, etc.; (2) intrahepatic bile duct cancer, mixed liver cancer; (3) use of antibiotics, proton pump inhibitors, or probiotics within 3 months before stool sample collection; (4) Crohn's disease, ulcerative colitis, and inflammatory bowel disease.

[0035] In the second step, before admission and any treatment intervention, the subjects were assessed by the researchers for sarcopenia parameters. The diagnostic criteria for sarcopenia were based on the EWGSOP2 algorithm (Find-Assess-Confirm-Severity), with a handgrip strength cutoff of 28 kg (male) and 18 kg (female), and a skeletal muscle index (SMI) cutoff of 51.1 cm 2 / m 2 (Male) and 37.1cm 2 / m 2 (Female). According to the critical value, liver cancer patients were divided into the muscle loss group (male handgrip strength <28kg or female handgrip strength <18kg, and male skeletal muscle index <51.1cm 2 / m 2 Or female skeletal muscle index <37.1cm 2 / m 2 alcohol) and non-sarcopenic group (grip strength >28 kg for men or >18 kg for women, and skeletal muscle index >51.1 cm for men 2 / m 2 Or female skeletal muscle index> 37.1cm 2 / m 2 ). Male grip strength > 28kg or female grip strength > 18kg, and male skeletal muscle index > 51.1cm 2 / m 2 Or female skeletal muscle index> 37.1cm 2 / m 2 Healthy participants were defined as the control group. The above cutoff values ​​have been shown to effectively distinguish between sarcopenic and non-sarcopenic individuals in previous studies.

[0036] Step 3: Subjects collect stool samples after admission and before treatment, verify and attach the subject's information label to the stool collection tube. After collecting the stool, use a stool collection spoon to collect the center of the stool and place it in the collection tube. Immediately afterwards, these samples are stored in a -80℃ refrigerator. Using EZNA RThe genomic DNA of stool samples was extracted using a stool DNA kit. The isolated DNA was subjected to two-step PCR amplification of the 16SrRNA gene V3-V4 region using slightly modified microbiome primers. The amplified products were purified using AMPure XT beads and quantified by Qubit. Subsequently, the amplified products were applied to sequencing, and the size and quantity of the amplified library were evaluated on the Agilent 2100 Bioanalyzer.

[0037] The raw reads were analyzed using QIIME2 software and quality filtered using fqtrim software to obtain high-quality clean tags. The sequence variation table and feature table of amplicon sequences were constructed using DABA2 software. BLAST classification analysis was performed on species annotation using SILVA and NT-16S databases. The dominant species in different groups and different sequence alignments were analyzed using MAFFT software. The Shannon index determined by QIIME2 was used to characterize the alpha diversity of the samples. The beta diversity was described using principal coordinate analysis and unweighted Unifrac index. The relationship between community composition and exogenous elements of the microbiome was analyzed using PERMANOVA and VEGAN R software packages. The Bonferroni q value method was used to avoid type I errors (false positives) caused by multiple tests of multivariate microbiome data. Wilcoxon-rank-sum analysis was used to evaluate the significant differences in microbial abundance, and the linear discriminant analysis effect size algorithm was used to determine the statistically significant enriched microorganisms with an LDA score greater than 3 (p < 0.05, q < 0.1). The Spearman algorithm was used to identify the microbial and metabolic correlations of biomarkers between the sarcopenia group and the non-sarcopenia group, and between the liver cancer group and the control group (correlation p < 0.05, rho > 0.5) ( Figure 4 PICRUSt analysis was used to predict the differences in MetaCyc pathways among the groups.

[0038] After screening, a total of 76 healthy subjects, 27 HCC patients with sarcopenia, and 58 HCC patients without sarcopenia were included in the study. Compared with individuals without sarcopenia, the SARC-F score in the sarcopenia group was significantly increased [3 (11.11%) vs 0 (0%), p = 0.001] ( Figure 4 A). Further observation of these muscle loss indicators between HCC patients and healthy people revealed similar significant differences, indicating that the severity of specific muscle loss indicators was higher when associated with comorbidities. Comparing the survival differences between the sarcopenia group and the non-sarcopenia group, it was found that the survival rate of sarcopenia patients was significantly lower than that of non-sarcopenia patients (p = 0.042) ( Figure 4B). The number of bacteria at the genus level in each group was obtained by 16S rRNA sequencing. The number of genera in the control group (619), non-sarcopenia group (504) and hyposarcopenia group (398) showed a decreasing trend. At the genus level, there were 307 genera in the three groups, 196 genera in the control group, 87 genera in the non-sarcopenia group, and 37 genera unique to the sarcopenia group ( Figure 4 C). There was no significant difference in Shannon index among the groups ( Figure 4 D). The HCC group had significant β-diversity compared with the control group (PCoA analysis, p = 0.001), while there was no difference between the sarcopenia group and the non-sarcopenia group ( Figure 4 E). Among the 30 genera with the highest relative abundance, the HCC group had higher abundance of Streptococcus (9.39% vs. 6.38%, P = 0.047) and Bacteroidetes (6.24% vs. 4.54%, P = 0.029), and lower abundance of Bifidobacterium (9.50% vs. 15.78%, P = 0.001) and Suberectiles (2.71% vs. 4.75%, P = 0.012) ( Figure 4 F).

[0039] In addition, the backup stool samples were taken out and thawed on ice, and the metabolites were extracted with 50% methanol buffer. 10ul of each extract was added to prepare mixed quality control samples. All chromatographic separations were performed using an ultra-high performance liquid chromatography system. The metabolites eluted from the column were detected using a high-resolution tandem mass spectrometer TripleTOF5600plus. Mass spectrometry data were acquired in IDA mode. During the acquisition process, the mass was accurately calibrated every 20 samples. In addition, in order to evaluate the stability of LC-MS throughout the acquisition process, a quality control sample (pool of all samples) was obtained after every 10 samples. The acquired MS data were preprocessed using XCMS software. The LC-MS raw data files were converted into mzXML format and then processed using XCMS, CAMERA, and metaX toolboxes. Metabolites were annotated using online KEGG and HMDB, the accurate molecular mass data (m / z) of the samples were matched with the data in the database, and an internal metabolite fragment library was used to verify the metabolite identification. t-tests were performed to detect differences in metabolite concentrations between the two phenotypes. The p-values ​​were adjusted for multiple testing using FDR. Supervised PLS-DA was performed using metaX to distinguish different variables between groups ( Figure 5 ).

[0040] To reveal the metabolic characteristics of patients with sarcopenia, we used hierarchical cluster analysis to compare the metabolite differences between patients with sarcopenia and those without sarcopenia (ratio >=2 or <=0.5, VIP>=1, p<0.05). We found that the differential metabolites mainly included lipids, amino acids and amino acid derivatives, carbohydrates and carbohydrate conjugates, organic acids and derivatives, benzene, phenols and substituted derivatives, and polyamines. The main phenotypes involved lipids, especially flavonoids ( Figure 5 A). To further understand the functions of these differential metabolites, we analyzed the correlation between clinical indicators and flavonoids. Notably, hesperidin was positively correlated with muscle mass (SMI) ( Figure 5 B).

[0041] Step 4: Blood samples and stool samples were collected from the subjects after admission and before treatment, and the information labels of the subjects were verified and affixed to the blood collection tubes and stool collection tubes. After collecting the blood, the blood collection tubes were placed in a centrifuge for centrifugation at 3000rpm for 20 minutes, and then the supernatant was collected as serum. The serum samples were immediately stored in a -80°C refrigerator. Considering that the potential biological functions of hesperidin may be related to inflammation suppression and maintenance of the intestinal barrier, we further used Elisa kits to detect the content of calprotectin in stool samples and the levels of ZO-1 and LPS in serum samples. The differences in intestinal permeability indicators between sarcopenia patients and non-sarcopenia patients were verified ( Figure 5 CE).

[0042] It can be observed that the levels of calprotectin in feces and ZO-1 and LPS in serum samples were significantly increased in the sarcopenic group compared with the non-sarcopenic group. However, compared with the healthy control group, the level of calprotectin in the non-sarcopenic group was significantly increased, while there was no significant difference in the levels of ZO-1 and LPS.

[0043] The advantages of the present invention over the prior art are:

[0044] 1. This study is based on a large sample of data from a medical center, with a high level of evidence and credible results;

[0045] 2. This study combined fecal flora sequencing and fecal metabolite sequencing in clinical samples, which is highly practical.

[0046] 3. This study describes the characteristics of HCC-related sarcopenia from the perspective of intestinal flora and metabolites. Through in vitro and in vivo experiments, we confirmed that hesperidin, as a metabolic biomarker, can alleviate skeletal muscle inflammation and thus improve muscle atrophy.

[0047] 4. Considering that a series of clinical trials and animal experiments have demonstrated the beneficial effects of flavonoids on intestinal permeability and systemic inflammation, hesperidin and its derivatives are expected to become potential targets as a biomarker and can be used in the clinical treatment of sarcopenia patients to improve clinical symptoms such as muscle atrophy.

Claims

1. Use of hesperetin or its derivatives, or its pharmaceutically acceptable salts in the preparation of a drug for treating muscular atrophy, wherein the hesperetin or its derivatives is selected from the compounds shown in formula (1) or formula (2):

2. The use according to claim 1, characterized in that: The hesperetin or its derivative is hydrochloride, sulfate, bromate, fumarate, acetate or citrate.

3. The use according to claim 1, characterized in that: The muscle atrophy is muscle degeneration, a decrease in muscle strength or a reduction in the number of muscle fibers due to consumption caused by chronic diseases.

4. The use according to claim 1, characterized in that: The muscular dystrophy patients are patients suffering from liver cancer and concurrently with muscle loss symptoms.

5. A pharmaceutical composition, comprising a therapeutically effective amount of hesperetin or its derivatives, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, wherein the hesperetin or its derivatives is selected from the compounds represented by formula (1) or formula (2):

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition contains 0.1%-99% of hesperetin or its derivatives and pharmaceutically acceptable salts thereof, and the rest is pharmaceutically acceptable, non-toxic and inert pharmaceutical carriers and / or excipients to humans and animals or is used in combination with other anticancer drugs.

7. The pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition contains 1%-90%, 5%-80% or 10%-50% of hesperetin or its derivatives and pharmaceutically acceptable salts thereof, and the rest is pharmaceutically acceptable, non-toxic and inert pharmaceutical carriers and / or excipients to humans and animals or is used in combination with other anticancer drugs.

8. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is prepared in the form of capsules, tablets, powders, granules, syrups for oral administration, or in the form of injections, ointments, suppositories or similar dosage forms for parenteral administration.

9. The pharmaceutical composition according to claim 5, characterized in that: The normal administration of hesperetin or its derivatives, or pharmaceutically acceptable salts thereof in the pharmaceutical composition is a total daily dose of 150-900 mg or 100-300 mg.

10. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is administered in the form of a single dose or divided doses.

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