Ganoderma lucidum triterpene composition and use thereof in clearing senescent cells and resisting tumor

Abstract

A Ganoderma lucidum triterpene composition and use thereof in the preparation of a drug and health-care food for clearing senescent cells and resisting tumors. The Ganoderma lucidum triterpene composition comprises ≥30% total triterpene, ≥1.8% ganodermanontriol, ≥0.1% ganoderic acid X, ≥0.7% ganodermanondiol, ≥0.7% ganoderol B, ≥2.5% ganoderol A, ≥0.2% ganoderal A, and ≥0% ergosterol.

Description

A Ganoderma lucidum triterpenoid component and its application in clearing senescent cells and anti-tumor activity

[0001] This invention claims priority to Chinese Patent Application No. 2024104906004, filed on April 23, 2024, entitled “A Ganoderma lucidum triterpenoid component and its application in clearing senescent cells and anti-tumor activity”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of traditional Chinese medicine components, characterization and application, specifically relating to a Ganoderma lucidum triterpenoid component and its application in clearing senescent cells and anti-tumor activity. Background Technology

[0003] Reishi (Ganoderma lucidum) is the dried fruiting body of the fungi Ganoderma lucidum (Leyss.Ex.Fr.) Karst or Ganoderma japonicum (Fr.) Lloyd, belonging to the Polyporaceae family. Also known as the "Immortal Herb," ​​"Auspicious Herb," ​​or "Lingzhi Grass," it is a traditional and precious tonic in Chinese medicine. Its health and medicinal value has a long history in my country; for example, the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) records that reishi has effects such as nourishing the body, protecting the liver and replenishing qi, strengthening the foundation and nourishing the essence, and prolonging life. Modern research shows that reishi contains polysaccharides, triterpenes, alkaloids, nucleosides, furan derivatives, polypeptides, amino acids, sterols, and lipids. Triterpenoids are among the most important active substances in reishi, possessing important biological activities such as anti-tumor and anti-inflammatory effects.

[0004] For example, Chinese patent CN108129541A discloses a method and application for extracting triterpenoids from Ganoderma lucidum, using dried Ganoderma lucidum fruiting bodies as raw materials, and involving ethanol extraction, concentration, ethyl acetate extraction, silica gel column chromatography separation and purification, filtration, and C2000. 18 A novel medicinal compound with pharmacological activity was extracted by high-pressure preparation and separation using reversed-phase chromatography packing material. It was named Ganosinensicacid C. This compound has potential applications in cytotoxic activity and provides a certain medicinal reference value for exploring the anti-tumor and other applications of Ganoderma lucidum triterpenoids.

[0005] Targeting senescent cells is a novel anti-aging strategy. Drugs available fall into two categories: one is senomorphics, which inhibit the functions of senescent cells, such as senescence-associated secretory phenotypes (SASPs). These drugs can inhibit, antagonize, or neutralize SASP components, thus requiring continuous administration to produce effects. The other category is senolyitics, a recently emerging class of drugs that can eliminate senescent cells. Through the senolytic strategy, these drugs can fundamentally address the pathological root cause of tissues, organs, and tumors—senescent cells—and can produce multiple benefits through intermittent administration (Chinese Journal of Medical Frontiers (Electronic Edition), 2023, Vol. 15, No. 10, pp. 18-25). Recent international research has found that conventional cancer treatments such as radiotherapy and chemotherapy can induce the formation of senescent cells in tumor cells. Senescent cells secrete inflammatory cytokines and growth factors into their microenvironment through SASP (Symptom-Related Apoptosis-Promoting Cell Microenvironment), creating a microenvironment that promotes tumor recurrence and metastasis. They also resist radiotherapy and chemotherapy by activating the anti-apoptotic pathways of senescent cells. Senescent cells are the root cause of tumor drug resistance, recurrence, and metastasis, and thus have become a new characteristic of tumors. Eliminating senescent cells is a new strategy for treating tumors (Cancer Discov. 2022; 12:31-46).

[0006] Currently, various triterpenoid compounds have been obtained from the fruiting bodies, mycelia, and spores of the Ganoderma genus, and numerous studies have shown that Ganoderma triterpenoids possess significant antitumor activity. However, the content of monomeric triterpenoid compounds obtained from Ganoderma is very low, and the preparation process is complex, which cannot meet the needs of pharmaceutical applications. Therefore, it is necessary to develop a method for extracting Ganoderma active triterpenoid components with high yield, stability, clear high-performance liquid chromatography fingerprint characteristics, and controllable quality, and to study their applications. Summary of the Invention

[0007] Based on the shortcomings of existing technologies, this invention screens the pharmacological activities of different components of the ethanol extract of Ganoderma lucidum fruiting body to obtain an active triterpenoid component with high extraction rate, clear high-performance liquid chromatography fingerprint characteristics, and controllable quality, named NT. It was also found that this component has a strong new function of clearing senescent tumor cells and anti-tumor activity.

[0008] This invention uses Ganoderma lucidum fruiting bodies as raw materials to isolate Ganoderma lucidum triterpenoid components (NT), and characterizes the high performance liquid chromatography fingerprint of NT and the compositional characteristics of the Ganoderma lucidum triterpenoids contained therein. It discloses for the first time that NT has the effects of clearing senescent cells and anti-tumor activity. It is a senolytic with anti-tumor activity extracted from Ganoderma lucidum and can be used to prepare drugs, health foods or food for the prevention and treatment of aging-related diseases, including tumors.

[0009] On one hand, the present invention provides a Ganoderma lucidum triterpenoid component, wherein the total triterpenoid content of the Ganoderma lucidum triterpenoid component is ≥30%, and the Ganoderma lucidum triterpenoid component includes ganoderic triol, ganoderic acid X, ganoderic diol, ganoderol B, ganoderol A, ganoderic aldehyde A and ergosterol, and by weight percentage, ganoderic triol ≥1.8%, ganoderic acid X ≥0.1%, ganoderic diol ≥0.7%, ganoderol B ≥0.7%, ganoderol A ≥2.5%, ganoderic aldehyde A ≥0.2%, and ergosterol ≥0%.

[0010] On the other hand, the present invention also provides a method for determining the above-mentioned Ganoderma lucidum triterpenoid components. Using ganoderic triol, ganoderic acid X, ganoderic diol, ganoderol B, ganoderol A, ganoderic aldehyde A, and ergosterol as standards, the analysis was performed using an Agilent 1290 Infinity II ultra-high performance liquid chromatograph. All reagents used were of chromatographic purity. The detection method is as follows: Agilent Eclipse Plus C18 column, flow rate 1 mL / min, column temperature 35℃, PDA detector, detection wavelength 254 nm, mobile phase A acetonitrile and B 0.04% formic acid in water, gradient elution: 0 min, A: 50%, B: 50%; 30 min, A: 65%, B: 35%; 65 min-80 min: A: 100%, B: 0%. The NT sample was dissolved in methanol at a concentration of 5 mg / mL, and the standards were dissolved in methanol at a concentration of 30 μg / mL, with an injection volume of 15 μL. The peak times of each standard on the UPLC chromatogram are shown in Table 1. The content (%) of each standard in NT was calculated according to the method of USP-NF Ganoderma Lucidum Fruiting Body in the 2020 edition of the United States Pharmacopeia. Based on multiple detection results of the active triterpenoid component NT from Ganoderma lucidum, combined with its pharmacological activity and quantitative production conditions, the basic requirements for the content of the above standards in NT were formulated (Table 1). In the UPLC fingerprint spectrum of NT, the sum of the peak areas with elution times between 1.7 and 8.1 min should account for ≤25% of the sum of the peak areas in the total spectrum (1.7-80 min).

[0011] Table 1. Content and elution time of each standard in the active triterpenoid components (NT) of Ganoderma lucidum.

[0012] Furthermore, the present invention provides a method for preparing the above-mentioned Ganoderma lucidum triterpenoid components, comprising the following steps:

[0013] 1. Raw material extraction: Take Ganoderma lucidum granules with a size of 1-2 cm, add 8-10 times the weight of the raw material in 95% ethanol, soak for 1 hour, then reflux extract 3-4 times, 1-3 hours each time, combine the extracts, filter, concentrate and dry under reduced pressure to obtain ethanol extract GE;

[0014] 2. Macroporous Resin Method: Dissolve GE in ethanol, then adjust the ethanol concentration to 50-60%, and the sample concentration to 0.2-0.4 g crude drug / mL. Allow to stand and precipitate, then centrifuge and filter. Reserve the clear liquid. Take D101 or AB-8 type macroporous resin, pretreat according to the manufacturer's product requirements, and pack into a column with a column diameter-to-height ratio of 1:10. Load the clear liquid at a flow rate of 1-1.5 BV / h, with a maximum loading volume of 1.4-1.8 g crude drug / mL resin. After loading, elute sequentially with 50%-65% ethanol and 85-95% ethanol at a flow rate of 2-3 BV / h, eluting 4-6 column volumes each time. Collect the eluent. Combine the 90-95% ethanol eluent, concentrate, and evaporate to dryness to obtain NT.

[0015] Furthermore, the present invention also provides the application of the above-mentioned Ganoderma lucidum triterpenoid components in the preparation of drugs for clearing senescent cells or anti-tumor drugs.

[0016] The removal of senescent cells includes, but is not limited to, inducing apoptosis to remove senescent cells, inhibiting the SASP of senescent cells, blocking the effect of senescent cells on promoting tumor cell proliferation, or inhibiting the damage of senescent cells to normal tissues and organs.

[0017] The tumors mentioned include, but are not limited to, liver cancer, breast cancer, lung cancer, cervical cancer, oral cancer, acute leukemia, chronic leukemia, lymphoma, nasopharyngeal carcinoma, and colon cancer.

[0018] The aforementioned Ganoderma triterpenoid components can be used alone or in combination with one or more chemotherapy drugs.

[0019] Specifically, in a senescent cell model induced by the chemotherapy drug doxorubicin, the active triterpenoid component NT obtained in this invention can eliminate senescent cells by inducing apoptosis, inhibit the SASP of senescent cells, block the effect of senescent cells on promoting tumor cell proliferation, and inhibit the damage of senescent cells to normal tissues and organs. In a mouse tumor model, the combined use of NT and chemotherapy drugs can enhance the anti-tumor efficacy of chemotherapy drugs and reduce the toxic side effects of chemotherapy drugs.

[0020] The effective dose of the Ganoderma lucidum triterpenoid components is 5 mg / kg-500 mg / kg.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention uses Ganoderma lucidum fruiting bodies as raw materials to isolate Ganoderma lucidum triterpenoid components (NT), and characterizes the high performance liquid chromatography fingerprint of NT and the compositional characteristics of the Ganoderma lucidum triterpenoids contained therein. It discloses for the first time that NT has the effects of clearing senescent cells and anti-tumor activity. It is a senolytic with anti-tumor activity extracted from Ganoderma lucidum and can be used to prepare drugs, health foods or food for the prevention and treatment of aging-related diseases, including tumors. Attached Figure Description

[0023] Figure 1 shows the UPLC spectra of the three NT samples and the standard.

[0024] In the diagram: 1-Ganoderic acid diol; 2-Ganoderic acid X; 3-Ganoderic acid triol; 4-Ganoderic alcohol B; 5-Ganoderic alcohol A; 6-Ganoderic aldehyde A; 7-Ergosterol;

[0025] Figure 2 shows that NT reduces the number of ADR-induced senescent liver cancer cells;

[0026] Figure 3 shows that NT inhibits the expression of senescence markers in ADR-induced senescent liver cancer cells;

[0027] Figure 4 shows how NT promotes ADR-induced apoptosis in senescent liver cancer cells;

[0028] Figure 5 shows the inhibition of ADR-induced SASP in senescent hepatocellular carcinoma cells by NT.

[0029] Figure 6 shows the inhibitory effect of NT on the tumor-promoting growth of senescent Sk-Hep-1 cells;

[0030] Figure 7 shows how NT enhances the in vivo antitumor effect of doxorubicin by clearing senescent tumor cells;

[0031] Figure 8 shows how NT reduces the in vivo toxicity of doxorubicin by clearing senescent cells from normal tissues. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. These embodiments are not intended to limit the scope of application of this patent.

[0033] Example 1: Preparation of Ganoderma triterpenoid fraction NT by separation of Ganoderma triterpenoid fraction from ethanol extract of Ganoderma lucidum fruiting body using AB-8 macroporous resin

[0034] Take Ganoderma lucidum granules with a size of 1-2 cm, add 95% ethanol at 10 times the weight of the raw material, soak for 1 hour, then reflux extract 3 times, 2 hours each time, combine the extracts, filter, concentrate and dry under reduced pressure to obtain ethanol extract GE.

[0035] Dissolve GE in ethanol, then adjust the ethanol concentration to 60%, bringing the sample concentration to 0.28 g crude drug / mL. Allow to stand and precipitate, then centrifuge and filter. Reserve the clear liquid. Take AB-8 macroporous resin, pretreat according to the manufacturer's product requirements, and pack it into a column with a resin diameter-to-height ratio of 1:10. Load the clear liquid with a flow rate of 1.2 BV / h and a maximum loading volume of 1.4 g crude drug / mL resin. After loading, elute sequentially with 55% ethanol and 95% ethanol at a flow rate of 3 BV / h, eluting for 6 column volumes each time, and collect the eluent. Combine the 95% ethanol eluent, concentrate, and evaporate to dryness to obtain NT.

[0036] Total triterpenes were determined using the method for triterpenoid determination of Ganoderma lucidum in Part I of the 2020 edition of the Chinese Pharmacopoeia. The three NT samples from Example 1 were quantitatively analyzed by ultra-high performance liquid chromatography (UPLC), and the results are shown in Table 2. Their characteristic UPLC spectra are shown in Figure 1. The sum of the peak areas of NT samples 1, 2, and 3 within the elution time range of 1.7–8.1 min accounted for 5%, 6%, and 5% of the sum of the peak areas within the elution time range of 1.7–80 min, respectively.

[0037] Table 2 shows the content of each standard and total triterpenes in the three NT samples of Example 1.

[0038] Example 2: Preparation of Ganoderma triterpenoid fraction NT by separation of Ganoderma triterpenoid fraction from Ganoderma lucidum fruiting body ethanol extract using D101 type macroporous resin

[0039] Take 1-2 cm Ganoderma lucidum granules, add 10 times their weight of 95% ethanol, soak for 1 hour, then reflux extract 4 times, 1.5 hours each time. Combine the extracts, filter, concentrate under reduced pressure and dry to obtain ethanol extract GE. Dissolve GE in ethanol, then adjust the ethanol concentration to 58%, and the sample solution concentration to 0.31 g crude drug / mL. Let it stand to precipitate, centrifuge and filter, and use the clear liquid for later use. Take D101 type macroporous resin, pre-treat according to the manufacturer's product requirements and pack it into a column, with a resin diameter-to-height ratio of 1:10. Load the clear liquid with a flow rate of 1 BV / h and a maximum loading amount of 1.6 g crude drug / mL resin. After loading, use 60% ethanol and 90% ethanol as eluents sequentially, with an elution flow rate of 2.1 BV / h, eluting 5 column volumes each time, and collect the eluent. Combine the 90% ethanol eluents, concentrate and evaporate to dryness to obtain NT. Total triterpenes were determined using the method for triterpenoid determination of Ganoderma lucidum in Part I of the 2020 edition of the Chinese Pharmacopoeia. Quantitative analysis of the three samples from Example 2 was performed using ultra-high performance liquid chromatography (UPLC). The results are shown in Table 3. The sum of the peak areas of samples NT1-3 with elution times between 1.7 and 8.1 min accounted for 5%, 5%, and 6% of the sum of the peak areas of the total spectrum between 1.7 and 80 min, respectively.

[0040] Table 3 shows the content of each standard and total triterpenoid in the three NT samples of Example 2.

[0041] Experimental Example 1: The in vitro scavenging effect of Ganoderma lucidum active triterpenoid component NT on senescent tumor cells.

[0042] Tumor cell lines: Human hepatocellular carcinoma HepG2 and SK-Hep-1 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, in 5% CO2. 2Incubated at 37°C. To induce senescence, after 24 h of incubation, the cells were treated with doxorubicin (ADR) for 72 h, and then administered NT (100, 150 μg / mL), 5 μM z-VAD-fmk, 20 μM CQ, 25 nM Baf, or 10 μM rapamycin, respectively.

[0043] Experimental methods: β-galactosidase staining: HepG2 and SK-Hep-1 cells in logarithmic growth phase were cultured for 24 h, then treated with 0.125 μg / mL doxorubicin (ADR, Sigma-Aldrich) for 72 h. After washing once with PBS, 1 mL of fixative from the β-galactosidase staining kit was added and fixed for 15 min at room temperature. After removing the fixative, the cells were washed three times with PBS for 3 min each time. After removing the PBS, 1 mL of staining working solution was added to each well and incubated overnight at 37 °C in a carbon dioxide-free incubator. The cells were photographed and the number of β-galactosidase-positive cells was counted under a regular optical microscope.

[0044] Detection of aging biomarkers: HepG2 and SK-Hep-1 cells in logarithmic growth phase were seeded into 6-well plates. After cell attachment, ADR was added for 72 h, the culture medium was discarded, and different concentrations of NT were added for 48 h. Cell proteins were extracted, and the expression of aging biomarkers γ-H2AX, P21, and P16 was detected by Western blotting.

[0045] Apoptosis detection: Flow cytometry was used to detect apoptosis using Annexin-V-APC / PI double staining. Logarithmically growing HepG2 and SK-Hep-1 cells were seeded in 6-well plates, 1.8 mL per well. Different concentrations of the drug were added to the experimental groups (200 μL each), while the control group received no drug. After culturing at 37°C and 5% CO2 for 48 h, cells were collected by trypsin digestion with EDTA-free enzyme, washed twice with pre-cooled PBS, resuspended in 1× loading buffer, and 5 μL of Annexin V-APC and 5-10 μL of PI were added. The mixture was gently mixed and incubated at room temperature in the dark for 15 min. Flow cytometry was then used for analysis. Live cells are not stained by Annexin V-APC and PI; cells in the early stage of apoptosis are stained only by Annexin V-APC and are negative for PI; necrotic cells and cells in the late stage of apoptosis can be stained by Annexin V-APC and PI simultaneously, and the proportion of cells in the early stage of apoptosis, necrotic cells and cells in the late stage of apoptosis can be determined by flow cytometry.

[0046] Detection of inflammatory factor levels: HepG2 and SK-Hep-1 human liver cancer cells in logarithmic growth phase were seeded in 6-well plates, designated as a control group, an ADR-induced senescence group, and an ADR-induced group plus NT treatment group, respectively. The supernatant of cell culture medium from each group was collected and centrifuged at 300g for 10 min. The supernatant was then stored at -80℃. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used. Antibodies were pre-coated onto high-affinity ELISA plates. Standards, the supernatant of the cell culture medium to be tested, and biotinylated detection antibodies were added to the wells of the ELISA plate. After incubation, inflammatory factors present in the sample bound to the solid-phase antibody and the detection antibody. After washing to remove unbound substances, horseradish peroxidase-labeled streptavidin (HRP) was added. After washing, the chromogenic substrate TMB was added, and the mixture was developed in the dark. The intensity of the color reaction was directly proportional to the concentration of inflammatory factors in the sample. The reaction was terminated by adding stop solution, and the absorbance was measured at 450 nm.

[0047] Results: A successful senescent cell model was established. HepG2 and SK-Hep-1 cells were induced for 3 days with 0.125 μg / mL ADR. β-galactosidase staining results showed that ADR successfully induced senescence in HepG2 and SK-Hep-1 cells, which were stained blue-green, and the proportion of β-galactosidase-positive senescent cells was significantly increased (Figure 2). To further verify that ADR induced the transformation of hepatocellular carcinoma cells into senescent hepatocellular carcinoma cells, the expression of senescence markers γ-H2AX, P21, and P16 was detected by Western blotting. Compared with the control group, the expression of senescence markers in ADR-induced senescent tumor cells was significantly increased (Figure 3). The experimental results indicate that ADR can induce the transformation of HepG2 and SK-Hep-1 hepatocellular carcinoma cells into senescent hepatocellular carcinoma cells.

[0048] NT effectively reduced the number of senescent cells and inhibited the expression of senescence markers: After inducing human hepatocellular carcinoma HepG2 and SK-Hep-1 cells with ADR for 3 days, we treated them with different concentrations of NT for 48 hours. β-galactosidase assays showed that the number of senescent cells treated with NT was significantly reduced compared to ADR-induced senescent cells (Figure 2). Immunoblotting results showed that the expression of senescence marker proteins γ-H2AX, P21, and P16 was increased in ADR-induced senescent cells, and the expression of these marker proteins decreased in a concentration-dependent manner after NT treatment compared to ADR-induced senescent cells (Figure 3). These experimental results indicate that NT can reduce the number of senescent human hepatocellular carcinoma cells and inhibit the expression of senescence markers in these cells.

[0049] NT-induced apoptosis in senescent cells: The effect of NT on ADR-induced apoptosis in human hepatocellular carcinoma cells HepG2 and SK-Hep-1 was investigated using Annexin V-APC / PI double staining. ADR alone showed a low apoptosis rate in senescent HepG2 and SK-Hep-1 cells, indicating an anti-apoptotic effect in senescent cells. The apoptosis rate induced by NT in ADR-induced senescent HepG2 and SK-Hep-1 cells was significantly higher than that induced by ADR alone (Figure 4). This suggests that NT eliminates senescent cells by inducing apoptosis.

[0050] NT inhibits SASP in senescent cells: ELISA was used to detect the expression levels of SASP-related cytokines in ADR-induced senescent human liver cancer cells HepG2 and SK-Hep-1, as well as in NT-treated senescent cells. The results showed that IL-6 and IL-1β expression was increased in ADR-induced senescent HepG2 and SK-Hep-1 cells, while NT treatment decreased both. These results suggest that NT may inhibit ADR-induced SASP in senescent cells, thereby mitigating the adverse effects of senescent cells, such as promoting cancer and organ damage (Figure 5).

[0051] Example 2: Senescent tumor cells promote tumor growth in vivo; Ganoderma lucidum active triterpenoid component NT clears senescent tumor cells and inhibits their tumor-promoting effect in vivo.

[0052] Experimental Methods: Senescent cells can promote tumor growth. NT (Nutrient Transplantation) cells, by clearing senescent cells, may inhibit the tumor-promoting effect of senescent cells. To verify this hypothesis, we used human Sk-Hep-1 cells to create a nude mouse model with xenograft tumors. Control Sk-Hep-1 cells were subcutaneously injected into the left upper quadrant of the mouse, senescent Sk-Hep-1 cells into the right upper quadrant, senescent Sk-Hep-1 cells + control Sk-Hep-1 cells into the right lower quadrant, and NT-pretreated senescent Sk-Hep-1 cells + control Sk-Hep-1 cells into the left lower quadrant. Tumor volume was measured using calipers. The estimated tumor volume was calculated using the following formula: Tumor volume = (a × b) 2 (a) / 2; where a is the long diameter of the tumor and b is the short diameter of the tumor, in mm. The mice were sacrificed 4 weeks later, the tumor was removed, and photographs were taken.

[0053] Results: As shown in Figure 6, ADR induced senescence in Sk-Hep-1 cells, and senescent Sk-Hep-1 cells lost their ability to form tumors subcutaneously in nude mice (ADR in Figure 6). Non-senescent control Sk-Hep-1 cells could form tumors (Control in Figure 6). However, co-inoculation of senescent Sk-Hep-1 cells with other Sk-Hep-1 cells subcutaneously in nude mice promoted Sk-Hep-1 cell proliferation and enhanced tumorigenesis (Control+ADR in Figure 6). Conversely, co-inoculation of NT-treated senescent Sk-Hep-1 cells with other Sk-Hep-1 cells subcutaneously in nude mice inhibited the tumor-promoting effect of senescent Sk-Hep-1 cells (Control+ADR+NT in Figure 6). These results indicate that NT can inhibit the tumor-promoting effect of senescent Sk-Hep-1 cells.

[0054] Experimental Example 3: The active triterpenoid component NT from Ganoderma lucidum enhances the in vivo antitumor effect of doxorubicin by clearing senescent tumor cells.

[0055] Experimental methods: Athymic nude mice (BALB / c-nu, 6-8 weeks old; male, weight: 20.0±2.0g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). A 20mm³ volume of thymic tissue was injected using a cannula. 3 HepG2 solid tumors were subcutaneously inoculated into the right axilla of mice to establish a liver cancer xenograft model. When the tumor reached 100 mm... 3Mice were randomly divided into 6 groups of 8 mice each, and subjected to different treatments, as detailed in Table 4: (a) Control group (0.9% saline, intraperitoneal injection once daily for 5 weeks after grouping), (b) NT (100 mg / kg daily, intraperitoneal injection once daily for 3 weeks starting in the 3rd week after grouping), (c) NT (250 mg / kg daily, gavage once daily for 3 weeks starting in the 3rd week after grouping), (d) A The mice were treated with different antitumor drugs: (e) ADR (6 mg / kg, once weekly via IP for 2 weeks after grouping) + NT (100 mg / kg, once daily via IP starting in week 3 after grouping) + NT (250 mg / kg, once daily via ig starting in week 3 after grouping). To induce aging in vivo, groups d, e, and f received intraperitoneal injections of ADR 6 mg / kg once weekly for 2 weeks after grouping. Groups b and c received the specified dose of NT for 3 weeks starting in week 3 after grouping, and groups e and f received the specified dose of NT for 3 weeks starting in week 3 after ADR treatment. Antitumor and tumor sensitivity activities were evaluated. One day after the last administration, mice were sacrificed, tumors were removed, photographed, and weighed. Frozen tissue sections were stained with 1xβ-gal staining solution to quantify aging. Proteins extracted from tumor tissue were used to detect aging markers using Western blotting.

[0056] Table 4 Treatment regimens for ADR and NT in tumor-bearing mice, either alone or in sequence.

[0057] Results: Figure 7a is a schematic diagram of the treatment scheme in Table 4. Human hepatocellular carcinoma HepG2 cells were subcutaneously inoculated into nude mice. When the tumor grew to 100 mm... 3Two weeks after ADR injection to induce tumor tissue senescence, some mice were sacrificed, and tumor masses were removed for β-gal staining. Other tumor-bearing mice were administered the drug according to the protocol in Table 4. Figure 7b shows that after two weeks of ADR injection, some mice were sacrificed, and tumor tissue was isolated for β-gal staining of tumor masses and tumor tissue sections. The positive rate of β-galactosidase staining in tumor tissue treated with ADR was significantly higher than that in the control group, indicating that ADR administration for 2 weeks can successfully induce tumor tissue senescence. Figures 7c, d, and e show that when ADR and NT were sequentially combined according to the treatment protocol in Table 1, with ADR+NT administered ip or ig, the tumor volume and weight in the combined group were lower than those in the ADR or NT single-administered groups, suggesting that the combined administration of NT and ADR can significantly enhance the in vivo antitumor effect of ADR. Figure 7f shows that the ADR group had the lowest body weight, indicating that ADR had the strongest toxicity to mice. The body weight of the NT group was no different from that of the saline control group, while the body weight of both ADR+NT groups was significantly higher than that of the ADR group, suggesting that NT can not only enhance the in vivo antitumor effect of ADR but also reduce the toxicity of DAR. Figure 7g shows that the β-galactosidase staining depth of tumor blocks in the ADR group was significantly higher than that in the control group, while the β-galactosidase staining depth of tumor blocks in the two ADR+NT groups was significantly lower than that in the ADR group. Figure 7h shows that proteins extracted from tumors of different treatment groups were analyzed using Western blotting to detect senescence markers P21 and P16. High expression of senescence markers was observed in ADR-treated tumor tissues, while the expression levels of senescence markers in the ADR+NT groups were significantly lower than those in the ADR group. These results indicate that although the ADR group has a strong in vivo antitumor effect, it can induce tumor cell senescence. Senescent tumor cells are known to be resistant to chemotherapy drugs and promote tumor growth. However, when ADR administration induces a large number of tumor cell senescence after two weeks, administration of NT, which has the effect of clearing senescent cells, can not only significantly enhance the in vivo antitumor effect of ADR by clearing the large number of senescent tumor cells induced by ADR and inhibiting the tumor-promoting effect of senescent tumor cells, but also reduce the toxicity of ADR to animals and reverse the weight loss in mice caused by ADR. This effect may be related to the fact that NT reverses the tissue damage caused by ADR-induced cell senescence.

[0058] Experimental Example 4: The active triterpenoid component NT from Ganoderma lucidum reduces the in vivo toxicity of doxorubicin by clearing senescent cells in normal tissues.

[0059] Experimental methods: In order to verify that the damage of ADR to mouse blood cells and heart is related to its induction of normal cell senescence, and that TN can counteract the toxicity of ADR by clearing ADR-induced senescent cells, the following experiment was designed: 33 mice (BALB / c, 6-8 weeks old; male, weight: 20.0±2.0g, purchased from Shanghai SLAC Experimental Animal Co., Ltd.) were divided into 3 groups, namely (1) saline control group of 6 mice, (2) ADR 6mg / kg ip once a week for 2 weeks of 21 mice, and (3) ADR 6mg / kg ip once a week × 2 followed by NT 100mg / kg / day ip for 3 weeks of 6 mice. (2) Three mice were collected from each group on days 0 (before administration) and days 6, 9, 12, and 15 after administration. Peripheral blood samples of 1 ml / mouse were collected, and mononuclear cells in the peripheral blood were isolated. The cells were stained with FITC-A C12FDG, and the proportion of β-galactosidase-positive senescent cells was detected by flow cytometry. The dynamic changes in peripheral blood mononuclear cell senescence at different time points after administration of ADR were observed. The remaining six mice served as the control group for the other two groups that received ADR alone. The mice were treated synchronously with the other two groups at the end of the experiment. After administration, blood samples were collected from the remaining mice in groups (1), (3), and (2) for routine blood tests and blood biochemistry tests. The animals were euthanized, and the hearts were routinely fixed, sectioned, and subjected to HE staining and β-galactosidase staining.

[0060] Results: Figure 8a shows that after administration of ADR 6 mg / kg / week, the proportion of β-galactosidase-positive senescent cells in mouse peripheral mononuclear cells gradually increased over time, peaking at 2 weeks, suggesting a time-dependent relationship between ADR-induced senescence in mouse peripheral blood mononuclear cells. Figure 8b shows that ADR-induced senescence in peripheral blood mononuclear cells peaked at 2 weeks, and NT treatment reduced the proportion of senescent cells in peripheral blood mononuclear cells. Figure 8c shows that ADR can deepen β-galactosidase staining in mouse cardiac tissue, suggesting that it can induce cardiac tissue cell senescence. Figure 8d shows that ADR-induced cardiac tissue structural disorder was observed in HE-stained cardiac tissue sections, suggesting age-related cardiac damage. NT treatment can reduce the depth of β-galactosidase staining in cardiac tissue and alleviate the changes in cardiac pathological structure caused by ADR.

[0061] As shown in Table 5, administration of ADR 6 mg / kg / week for 2 weeks significantly affected peripheral blood counts in mice. These effects may be related to ADR-induced senescence of nucleated cells in peripheral blood, thus causing a significant decrease in the number of nucleated cells such as leukocytes, lymphocytes, and monocytes. Administration of NT after modeling could partially reverse the ADR-induced decrease in leukocytes, lymphocytes, and monocytes. This effect may be related to NT's ability to clear senescent nucleated cells from peripheral blood. As shown in Table 6, administration of ADR 6 mg / kg / week for 2 weeks also caused increases in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen, and creatinine in mice, suggesting damage to liver and kidney function. Administration of NT after modeling could partially counteract ADR-induced liver and kidney damage.

[0062] Table 5. Effects of ADR on peripheral blood counts in mice and the antagonistic effect of NT.

[0063] Table 6. Effects of ADR on liver and kidney function in mice and the antagonistic effect of NT.

[0064] Experimental Example 5: Antitumor effect of Ganoderma lucidum active triterpenoid component NT on human cancer cells

[0065] Tumor cell lines: HepG2 liver cancer, MCF-7 breast cancer, A549 lung cancer, HeLa cervical cancer, KB oral cancer, HL60 acute leukemia, K562 chronic leukemia, CA46 lymphoma, CNE2 nasopharyngeal carcinoma, and SW620 colon cancer. These cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. The cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 saturated humidity incubator. Cells in the logarithmic growth phase were used for experiments. After culturing for 24 hours, different concentrations of NT were added, and cell viability was measured using the MTT assay. The concentration at which NT inhibited cell growth by 50% (IC50) was calculated. The results are shown in Table 7. NT showed significant antitumor activity against liver cancer (HepG2), breast cancer (MCF-7), lung cancer (A549), cervical cancer (HeLa), oral cancer (KB), acute leukemia (HL60), chronic leukemia (K562), lymphoma (CA46), nasopharyngeal carcinoma (CNE2), and colon cancer (SW620).

[0066] Table 7 IC50 (μg / mL) of NT against human tumor cell lines after 72 h of treatment.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Ganoderma lucidum triterpenoid component, characterized in that: The total triterpenoid content of the Ganoderma lucidum triterpenoid components is ≥30%, and the Ganoderma lucidum triterpenoid components include ganoderic triol, ganoderic acid X, ganoderic diol, ganoderol B, ganoderol A, ganoderic aldehyde A and ergosterol. By weight percentage, ganoderic triol ≥1.8%, ganoderic acid X ≥0.1%, ganoderic diol ≥0.7%, ganoderol B ≥0.7%, ganoderol A ≥2.5%, ganoderic aldehyde A ≥0.2%, and ergosterol ≥0%. The sum of the peak areas with elution times of 1.7-8.1 min in the fingerprint spectrum of the Ganoderma lucidum triterpenoid components accounts for ≤25% of the sum of the peak areas with elution times of 1.7-80 min in the total spectrum.

2. A method for determining the Ganoderma lucidum triterpenoid components according to claim 1, characterized in that: The following substances were analyzed using an Agilent 1290 Infinity II ultra-high performance liquid chromatograph as standards: ganoderic triol, ganoderic acid X, ganoderic diol, ganoderol B, ganoderol A, ganoderic aldehyde A, and ergosterol. The conditions were as follows: Agilent Eclipse Plus C18 column, flow rate 1 mL / min, column temperature 35℃, mobile phase A acetonitrile and B 0.04% formic acid in water, gradient elution: 0 min, A: 50%, B: 50%, 30 min, A: 65%, B: 35%, 65 min-80 min: A: 100%, B: 0%, using a PDA detector at a detection wavelength of 254 nm; injection concentration: sample dissolved in methanol 5 mg / mL, standard dissolved in methanol 30 μg / mL, injection volume 15 μL.

3. A method for preparing the Ganoderma lucidum triterpenoid component according to claim 1, characterized in that: (1) Take Ganoderma lucidum granules, add 8-10 times the weight of the raw material in 95% ethanol, soak for 1 hour, then reflux extract 3-4 times, 1-3 hours each time, combine the extracts, filter, concentrate and dry under reduced pressure to obtain ethanol extract GE. (2) Dissolve GE in ethanol, then adjust the ethanol concentration to 50-60% and the sample concentration to 0.2-0.4 g crude drug / mL. Let it stand to precipitate, centrifuge and filter, and use the clear solution for later use. (3) Take D101 or AB-8 type macroporous resin, pre-treat it according to the manufacturer's product requirements and then pack it into the column. The diameter-to-height ratio of the resin column is 1:

10. (4) Load the clarified liquid at a flow rate of 1-1.5 column volumes / hour and a maximum loading amount of 1.4-1.8 g crude drug / mL resin; (5) After loading the sample, use 50%-65% ethanol and 85-95% ethanol as eluents in sequence, with an elution flow rate of 2-3 BV / h, and elute for 4-6 column volumes each time. Collect the eluent. (6) Elute with 90-95% ethanol, collect and combine, concentrate and evaporate to dryness to obtain the Ganoderma triterpenoid components.

4. The use of the Ganoderma lucidum triterpenoid component according to claim 1 in the preparation of drugs for clearing senescent cells and / or antitumor drugs.

5. The application according to claim 4, characterized in that: The removal of senescent cells refers to inducing apoptosis to remove senescent cells.

6. The application according to claim 4, characterized in that: The aforementioned elimination of senescent cells refers to the inhibition of the senescence-associated secretory phenotype (SASP) of senescent cells.

7. The application according to claim 4, characterized in that: The aforementioned removal of senescent cells refers to blocking the effect of senescent cells on promoting the proliferation of tumor cells.

8. The application according to claim 4, characterized in that: The removal of senescent cells refers to inhibiting the damage of senescent cells to normal tissues and organs.

9. The application according to claim 4, characterized in that: The tumors mentioned include, but are not limited to, liver cancer, breast cancer, lung cancer, cervical cancer, oral cancer, acute leukemia, chronic leukemia, lymphoma, nasopharyngeal carcinoma, and colon cancer.

10. The application according to claim 4, characterized in that: The aforementioned Ganoderma triterpenoid components can be used alone or in combination with one or more chemotherapeutic drugs.

11. The application according to claim 10, characterized in that: The aforementioned Ganoderma lucidum triterpenoid components can enhance the anti-tumor efficacy of chemotherapy drugs.

12. The application according to claim 10, characterized in that: The aforementioned Ganoderma lucidum triterpenoid components can reduce the toxic side effects of chemotherapy drugs on normal tissues.

13. The application according to claim 4, characterized in that: The effective dose of the Ganoderma lucidum triterpenoid components is 5 mg / kg-500 mg / kg.

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