Cinnamomum theiferum polysaccharide, extraction method and application thereof

Abstract

The present application relates to a kind of extraction methods of tea fruit camphor polysaccharide, it is crushed after tea fruit camphor fruit with water at 65-90 ℃ heating extraction, water extract is reduced pressure concentration, concentrated liquid alcohol precipitation, after removing protein, again alcohol precipitation, freeze-drying, obtain tea fruit camphor polysaccharide A;Or, it is crushed after tea fruit camphor fruit, room temperature is respectively extracted with petroleum ether, ethyl acetate, solid-liquid separation, residue is extracted with water at 65-90 ℃ heating, water extract is reduced pressure concentration, concentrated liquid alcohol precipitation, after removing protein, again alcohol precipitation, freeze-drying, obtain tea fruit camphor polysaccharide B;Tea fruit camphor polysaccharide A and / or tea fruit camphor polysaccharide B is tea fruit camphor polysaccharide.The present application also finds a new use of tea fruit camphor polysaccharide, the extracted tea fruit camphor polysaccharide treatment effect is remarkable, and no toxic side effect, can be used in antioxidant stress medicine, especially liver protection medicine development and utilization.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and / or health product technology, specifically relating to a tea fruit camphor polysaccharide, its extraction method, and its application in the preparation of antioxidant stress drugs, especially liver-protective drugs. Background Technology

[0002] Under normal circumstances, the antioxidant-oxidation system is in a dynamic equilibrium within the body. However, when free radicals accumulate excessively, they can cause oxidative damage to proteins, lipids, and nucleic acids. With the overuse and increasing prominence of the side effects of synthetic antioxidants, there is growing interest in finding effective, natural antioxidants to reduce the damage caused by free radicals and antioxidants to our bodies.

[0003] As a vital metabolic organ, the liver actively participates in various life processes and energy metabolism within the human body, making it highly susceptible to damage from various endogenous or exogenous substances. Exogenous substances such as drugs, infections, alcohol, and chemicals can induce liver poisoning, leading to an overall decline in liver metabolic function. Carbon tetrachloride (CCl4) is a classic exogenously induced hepatotoxic substance, and increasing research indicates that oxidative stress is a crucial mechanism by which CCl4 induces hepatotoxicity. CCl4 is metabolized by the cytochrome P450 system to form trichloromethyl radicals (CCl3-) and trichloromethylperoxy radicals (CCl3OO-), thereby inducing membrane lipid peroxidation.

[0004] Tea fruit camphor ( Cinnamomun chago BSSun et HLZhao) is a species of the genus Cinnamomum in the family Lauraceae. Cinnamomum *Cinnamomum camphora* is an evergreen tree. It commonly grows on hillsides, along roadsides, and in shrublands. The wood is fragrant, and the fruit is a medium-sized berry rich in oil. The fruit can be eaten after being dried or roasted after removing the outer skin. In its distribution area, it is locally known as "tea fruit." Tea fruit is not only delicious but also has the effect of clearing heat and improving eyesight. However, no research has been reported on the activity of polysaccharides from *Cinnamomum camphora*. Exploring the liver-protective effect of *Cinnamomum camphora* is of certain research significance. Based on this, this application was developed. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the prior art and provide a tea fruit camphor polysaccharide. The prepared tea fruit camphor polysaccharide has significant therapeutic effects and no toxic side effects. It can be used in the development and utilization of anti-oxidative stress drugs, especially liver-protective drugs. In other words, this invention has discovered a new use for tea fruit camphor polysaccharide.

[0006] The present invention also provides a method for extracting the above-mentioned tea fruit camphor polysaccharide and its application in the preparation of antioxidant stress drugs, especially liver-protective drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for extracting polysaccharides from tea fruit camphor trees, comprising the following steps:

[0009] After crushing the fruit of the tea camphor tree, water was heated and extracted at 65-90 ℃. The water extract was concentrated under reduced pressure, and the concentrate was precipitated with alcohol and protein removed. After precipitating with alcohol again and freeze-drying, tea camphor polysaccharide A was obtained.

[0010] Alternatively, after crushing the fruit of the tea camphor tree, it is extracted with petroleum ether and ethyl acetate at room temperature, respectively. After solid-liquid separation, the residue is extracted with water at 65-90℃. The aqueous extract is concentrated under reduced pressure, and the concentrate is precipitated with alcohol and protein removed. After precipitating with alcohol again, it is freeze-dried to obtain tea camphor polysaccharide B.

[0011] The tea fruit camphor polysaccharide A and / or tea fruit camphor polysaccharide B are tea fruit camphor polysaccharides.

[0012] Specifically, water can be added at a solid-liquid ratio of 1g:20-40ml for heating and extraction. The heating and extraction time is 2-5 hours, and the extraction is repeated 1-4 times.

[0013] Furthermore, petroleum ether or ethyl acetate can be added at a solid-liquid ratio of 1g:10-30ml for extraction, with an extraction time of 8-15 hours, and the extraction can be repeated 1-5 times.

[0014] In the extraction method of this invention, after alcohol precipitation and protein removal of the concentrate, a second alcohol precipitation can be performed using conventional techniques in the art, such as the following steps:

[0015] Add ethanol to the concentrate to a final concentration of 75±5%, let stand, collect the precipitate, dissolve in water, remove protein using the Sevage method, take the supernatant, add ethanol to a final concentration of 75±5%, let stand, and freeze-dry the precipitate.

[0016] This invention provides tea fruit camphor polysaccharide extracted using the above extraction method.

[0017] This invention provides a compound preparation containing the above-mentioned tea fruit camphor polysaccharide, wherein the tea fruit camphor polysaccharide is compounded with conventional excipients in the art to form the compound preparation.

[0018] Furthermore, the dosage forms of the compound preparation include, but are not limited to, tablets, granules, pills, capsules, or injections.

[0019] This invention provides the application of the aforementioned tea fruit camphor polysaccharide or the aforementioned compound preparation in the preparation of drugs for treating or preventing oxidative stress.

[0020] Furthermore, the oxidative stress is cyclophosphamide-induced oxidative stress; the tea fruit camphor polysaccharide or compound preparation can effectively reduce the malondialdehyde (MDA) content in the liver induced by cyclophosphamide-induced oxidative stress, increase the levels of catalase (CAT) and total antioxidant capacity (T-AOC) in the liver, effectively enhance antioxidant capacity, and have a good protective effect against liver damage.

[0021] This invention also provides the application of the aforementioned tea fruit camphor polysaccharide or the aforementioned compound preparation in the preparation of drugs for treating or preventing liver damage, or in the preparation of liver-protective drugs.

[0022] Furthermore, the liver injury is acute liver injury; the tea fruit camphor polysaccharide or compound preparation can improve liver function indicators in Kunming mice, significantly reduce the levels of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, and at the same time significantly alleviate liver damage and effectively enhance the body's antioxidant capacity.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention utilizes *Camellia oleifera* polysaccharide administered to mice via gavage once daily for three weeks, followed by injection of 0.3% carbon tetrachloride olive oil solution to establish a mouse model of acute liver injury. Based on these conditions, a novel application of *Camellia oleifera* polysaccharide was discovered. The results showed that, compared to the model group, *Camellia oleifera* polysaccharide significantly reduced the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase in mice, enhanced their antioxidant capacity, and significantly alleviated liver damage, thus exhibiting a liver-protective effect. Therefore, *Camellia oleifera* polysaccharide of this invention can be used to prepare hepatoprotective drugs, possessing the potential and value for developing new hepatoprotective drugs and health products. Furthermore, *Camellia oleifera* polysaccharide can significantly reduce the malondialdehyde (MDA) content in mouse liver, increase the levels of catalase (CAT) and total antioxidant capacity (T-AOC) in mouse liver, and enhance the antioxidant capacity of mice. Therefore, *Camellia oleifera* polysaccharide of this invention can be used to prepare drugs against cyclophosphamide-induced oxidative stress, possessing the potential and value for developing new anti-oxidative stress drugs and health products. Attached Figure Description

[0025] Figure 1 To investigate the effects of tea fruit camphor polysaccharide on the morphology of mouse liver tissue, BC was the blank group, MC was the model group, PC was the biphenyl diester positive control group, and HD, MD, and LD were the high, medium, and low dose groups of tea fruit camphor polysaccharide, respectively, and the same applies below.

[0026] Figure 2 The effects of tea fruit camphor polysaccharide on the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AKP) in mouse serum were investigated. Data are expressed as mean ± standard deviation, n = 10. Compared with the control group,### P <0.001, ## P <0.01, # P <0.05. Compared with the model group, *** P <0.001,** P <0.01, * P <0.05. (The same applies below.)

[0027] Figure 3 The effects of tea fruit camphor polysaccharide on superoxide dismutase (SOD) activity (A), glutathione peroxidase (GSH-Px) activity (B), and malondialdehyde (MDA) content (C) in mouse liver;

[0028] Figure 4 The effects of tea fruit camphor polysaccharide on CAT(A), T-AOC(B), and MDA(C) activity in mouse liver (data are expressed as mean ± standard deviation, n = 8. Compared with the control group, ### P <0.001, ## P <0.01, # P <0.05. Compared with the model group, *** P <0.001,** P <0.01, * P <0.05). Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0030] Tea fruit camphor ( Cinnamomum chago The fruits (BS Sun & H. L. Zhao) were collected from Yangbi County, Yunnan Province. Unless otherwise specified, all ethanol concentrations in the following examples refer to volume percentage concentrations.

[0031] Example 1: Preparation of tea fruit camphor polysaccharide:

[0032] A method for preparing tea fruit camphor polysaccharide includes the following steps:

[0033] 5342 g of *Camellia sinensis* fruit was crushed and extracted with water at a solid-liquid ratio of 1 g: 30 ml at 80°C for 4 h each time, for a total of 2 extractions. The aqueous extracts were combined and concentrated under reduced pressure at below 60°C using a rotary evaporator. 95% ethanol was added to the concentrate until the final ethanol concentration reached 75%, and the mixture was allowed to stand for 24 h. After centrifugation, the precipitate was collected and dissolved in water. Protein removal was performed using the Sevage method (conventional techniques in the field can be used, or the following can be referenced: sample: Sevage reagent = 15:5, Sevage reagent is a chloroform-n-butanol mixture with a volume ratio of 4:1; shaking for 60 min, standing for 30 min, centrifuging at 7000 r / min for 6 min to remove denatured proteins outside the interface between the aqueous and chloroform layers. The upper sugar solution was collected, and the denatured protein removal process was repeated until all proteins were removed). Add 95% ethanol to the supernatant until the final ethanol concentration is 75%, let stand for 24 hours, centrifuge, collect the precipitate and freeze dry (-50 ℃, 12 h) to obtain 31.7 g of tea fruit camphor polysaccharide, with a yield of about 0.59%, for later use.

[0034] The following study investigates the protective effect of the tea fruit camphor polysaccharide prepared in Example 1 on carbon tetrachloride-induced acute liver injury in mice.

[0035] Application Experiment 1: Acute Liver Injury in Mice Induced by Carbon Tetrachloride (Animal Experiment)

[0036] The tea fruit camphor polysaccharide prepared in Example 1 was subjected to in vivo animal experiments.

[0037] I. Laboratory Animals

[0038] Kunming (KM) mice, Specific pathogen free (SPF) grade, male, 5–6 weeks old, 22–28 g; purchased from Henan Skebers Biotechnology Co., Ltd. Mice were acclimatized for one week under conditions of 25±2℃, 40–45% relative humidity, and 12h / d light, fed standard feed, and with free access to water.

[0039] II. Test Reagents and Instruments

[0040] Electronic balance (ME204, METTLER TOLEDO);

[0041] Rotary evaporator (N-1300, EYELA);

[0042] FMB40 Fully Automatic Snowflake Ice Maker (Shanghai Bilang Instrument Co., Ltd.);

[0043] MiniSpin benchtop centrifuge, manufactured by Shanghai Anting Scientific Instrument Factory;

[0044] Full-wavelength microplate reader, Thermo Fisher 1510;

[0045] KZ-Ⅲ-FP High-speed Low-temperature Tissue Grinder (Wuhan Saiwei Biotechnology Co., Ltd.);

[0046] Electric heating drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.);

[0047] Carbon tetrachloride (CCl4, analytical grade) (Tianjin Hongyan Chemical Reagent Factory);

[0048] Biphenyl diester drops (Zhejiang Wanbang Pharmaceutical Co., Ltd.);

[0049] Aspartate aminotransferase (AST / GOT) kit (Nanjing Jiancheng Bioengineering Institute);

[0050] Alanine aminotransferase (ALT / GPT) kit (Nanjing Jiancheng Bioengineering Institute);

[0051] Alkaline phosphatase (AKP) kit (Nanjing Jiancheng Bioengineering Institute);

[0052] Malondialdehyde (MDA) assay kit (Nanjing Jiancheng Bioengineering Institute);

[0053] Superoxide dismutase (SOD) assay kit (Nanjing Jiancheng Bioengineering Institute);

[0054] Glutathione peroxidase (GSH-Px) assay kit (Nanjing Jiancheng Bioengineering Institute);

[0055] BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd.)

[0056] III. Test Methods

[0057] Table 1 Experimental Groups and Corresponding Doses

[0058]

[0059] After one week of acclimatization, mice were randomly divided into six groups: a control group, a model group, a positive control group, a low-dose group, a medium-dose group, and a high-dose group, with 10 mice in each group. The corresponding drug dosages are shown in Table 1. Mice in each group were administered the drugs once daily by gavage at a volume of 10 mL / kg for three weeks. All drugs were dissolved in physiological saline before administration.

[0060] Mice were weighed every two days. The drugs were administered daily via gavage, with the volume adjusted according to changes in mouse weight. The control and model groups received physiological saline, the positive control group (PC) received biphenyl diester, and the treatment groups received tea fruit camphor polysaccharide. On the last day of the experiment, 12 hours after administration of the experimental samples, all mice except the control group (which received an intraperitoneal injection of olive oil at a volume of 10 mL / kg) were injected with the same dose of 0.3% CCl4 olive oil solution to induce acute liver injury.

[0061] Mice were used to establish the mouse model for 12 hours, during which they were fasted but allowed free access to water. Mice weight was recorded after 12 hours. The mice were pre-anesthetized with isoflurane, their eyeballs were removed, blood was collected, and the mice were euthanized. The blood was allowed to stand at 4 °C for 30 minutes, then centrifuged at 3500 rpm / min at 4 °C for 10 minutes. The supernatant was transferred to a clean centrifuge tube using a pipette to obtain serum. Simultaneously, mouse liver tissue was collected, washed with physiological saline, and surface water was removed. A portion of the liver tissue was accurately weighed and a 10% liver homogenate was prepared. Another portion of the liver tissue was fixed in 4% paraformaldehyde for 24 hours for use as pathological sections, and the remaining liver tissue was stored at -80 °C.

[0062] 1. Analysis of mouse liver histopathological sections

[0063] Mouse liver tissue was collected, fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining was used to observe changes in the liver tissue under a microscope.

[0064] 2. Determination of liver function levels in mice

[0065] The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AKP) in mouse serum were measured according to the kit instructions.

[0066] 3. Determination of antioxidant capacity in mice

[0067] Accurately weigh a portion of liver tissue and add physiological saline to prepare a 10% liver tissue homogenate. The activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA) in mouse liver tissue were determined using a kit.

[0068] 4. Statistical Analysis

[0069] Data were statistically processed using GraphPad Prism 8.0 software. One-way ANOVA was used for multiple groups, and results are expressed as mean ± standard deviation (X ± SD). P A value <0.05 indicates a statistically significant difference between groups.

[0070] IV. Effects of Tea Fruit Camphor Polysaccharide on Mice with Acute Liver Injury

[0071] 1. Effects of tea fruit camphor polysaccharide on pathological changes in mouse liver tissue

[0072] Figure 1 The effects of *Camellia sinensis* polysaccharide on the morphology of mouse liver tissue were presented. BC was the blank group, MC was the model group, PC was the biphenyl diester positive control group, and HD, MD, and LD were the high, medium, and low dose groups of *Camellia sinensis* polysaccharide, respectively. Figure 1 As shown, liver tissue sections in the blank group were normal, with clear cell nuclei and nucleoli, and central veins were visible. Furthermore, hepatic sinusoids were normal, and hepatic plates and sinusoids were regularly arranged. In contrast, hepatocytes in the model group were enlarged and irregularly arranged, hepatic plates were disordered, cell nuclei were atrophied, and hepatocytes showed extensive necrosis with vacuolation. Treatment with *Camellia sinensis* polysaccharide significantly improved liver lesions, and the liver tissue cell structure was normal, closely resembling that of a normal liver. These results indicate that *Camellia sinensis* polysaccharide has a significant protective effect against CCl4-induced liver injury in mice.

[0073] 2. Effects of tea fruit camphor polysaccharide on liver function in mice

[0074] Figure 2 The effects of tea fruit camphor polysaccharide on the activities of related enzymes in mouse serum were presented. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AKP) are important markers for clinical evaluation of liver damage, such as... Figure 2 As shown, the serum levels of AST, ALT, and AKP in the model group mice were significantly higher than those in the blank control group. P The concentrations <0.001 indicate that CCl4 induced liver injury in mice. After treatment, the levels in both the PC group and the treated group improved to varying degrees. These results suggest that AST, ALT, and AKP levels in mice with CCl4-induced liver injury can be improved by *Camellia sinensis* polysaccharide, and the reduction in liver function indicators in mice treated with *Camellia sinensis* polysaccharide showed a good dose-dependent effect.

[0075] 3. Effects of tea fruit camphor polysaccharide on antioxidant levels in mice

[0076] Figure 3 The effects of tea fruit camphor polysaccharide on the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) content in mouse liver were presented. Figure 3 As shown, compared with the blank group, the activities of SOD and GSH-Px in the serum of mice in the model group were significantly reduced ( P <0.05%, MDA content increased significantly ( P<0.001). Both the treated group and the PC group reversed this reduction to varying degrees in a dose-dependent manner. Furthermore, compared to the model group, the MDA content in the HD group and the MD group was significantly reduced. These results suggest that *Camellia sinensis* polysaccharide may exert its liver-protective effect by enhancing the antioxidant capacity of mice.

[0077] Example 2: Preparation of tea fruit camphor polysaccharide:

[0078] A method for preparing tea fruit camphor polysaccharide includes the following steps:

[0079] 254 g of *Camellia sinensis* fruit was crushed and extracted with petroleum ether and ethyl acetate at a solid-liquid ratio of 1 g: 20 ml at room temperature, for 12 h each time, for a total of 3 extractions. After extraction, the supernatant was filtered off, and the residue was retained. The residue was then heated with water at a solid-liquid ratio of 1 g: 30 ml at 80℃ for 4 h each time, for a total of 2 extractions. After combining the aqueous extracts, the mixture was concentrated under reduced pressure at below 60°C using a rotary evaporator. 95% ethanol was added to the concentrate until the final ethanol concentration reached 75%. The mixture was allowed to stand for 24 hours, then centrifuged. The precipitate was collected and dissolved in water. Protein removal was performed using the Sevage method (conventional techniques in the field can be used, or the following can be referenced: sample:Sevage reagent = 15:5, Sevage reagent is a chloroform-n-butanol mixture with a volume ratio of 4:1; shaking for 60 minutes, standing for 30 minutes, centrifuged at 7000 r / min for 6 minutes to remove denatured proteins outside the interface between the aqueous and chloroform layers. The upper sugar layer was collected, and the denatured protein removal process was repeated until all proteins were removed). 95% ethanol was added to the supernatant until the final ethanol concentration reached 75%. The mixture was allowed to stand for 24 hours, then centrifuged. The precipitate was collected and freeze-dried (-50°C, 12 hours) to obtain 18.158 g of *Camellia sinensis* polysaccharide, with a yield of approximately 7.15% and a sugar content of 49.86%, for later use.

[0080] The following study investigates the protective effect of the tea fruit camphor polysaccharide prepared in Example 2 on cyclophosphamide-induced oxidative stress in mice.

[0081] Application Experiment 2: Cyclophosphamide-induced oxidative stress in mice:

[0082] The tea fruit camphor polysaccharide prepared in Example 1 was subjected to in vivo animal experiments.

[0083] I. Laboratory Animals

[0084] Same as application experiment 1.

[0085] II. Test Reagents and Instruments

[0086] Electronic balance (ME204, METTLER TOLEDO)

[0087] Rotary evaporator (N-1300, EYELA);

[0088] FMB40 Fully Automatic Snowflake Ice Maker (Shanghai Bilang Instrument Co., Ltd.);

[0089] MiniSpin benchtop centrifuge, manufactured by Shanghai Anting Scientific Instrument Factory;

[0090] Full-wavelength microplate reader, Thermo Fisher 1510;

[0091] KZ-Ⅲ-FP High-speed Low-temperature Tissue Grinder (Wuhan Saiwei Biotechnology Co., Ltd.);

[0092] Electric heating drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.);

[0093] Cyclophosphamide for injection (Jiangsu Hengrui Medicine Co., Ltd.);

[0094] Lentinan tablets (Hubei Guangren Pharmaceutical Co., Ltd.);

[0095] Malondialdehyde (MDA) assay kit (Nanjing Jiancheng Bioengineering Institute);

[0096] Catalase (CAT) Assay Kit (Nanjing Jiancheng Bioengineering Institute);

[0097] Total antioxidant capacity (T-AOC) assay kit (Nanjing Jiancheng Bioengineering Institute);

[0098] BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd.)

[0099] III. Test Methods

[0100] Table 2 Experimental Groups and Corresponding Doses

[0101]

[0102] After one week of acclimatization, mice were randomly divided into six groups: a control group, a model group, a positive control group, a low-dose group, a medium-dose group, and a high-dose group, with eight mice in each group. The corresponding drug dosages are shown in Table 2. Mice in each group were administered the drugs once daily via gavage at a volume of 10 ml / kg for three weeks. All drugs were dissolved in physiological saline before administration.

[0103] Mice were weighed every two days. The drugs were administered daily via gavage, with the volume adjusted according to changes in mouse weight. The control and model groups received saline, the PC group received lentinan, and the treatment groups received camphor polysaccharide. On days 18, 19, 20, and 21 of drug administration, mice in the BC control group received intraperitoneal injections of saline, while mice in other groups were induced into the model by intraperitoneal injections of cyclophosphamide (CTX) at a dose of 70 mg / kg BW.

[0104] Mice were used to establish the model for 12 hours, during which they were fasted but allowed free access to water. Mice weight was recorded after 12 hours. They were pre-anesthetized with isoflurane, their eyeballs were removed, blood was collected, and the mice were euthanized. The blood was allowed to stand at 4 °C for 30 minutes, then centrifuged at 3500 rpm / min at 4 °C for 10 minutes. The supernatant was transferred to a clean centrifuge tube using a pipette to obtain serum. Simultaneously, mouse liver tissue was collected, washed with physiological saline, and surface water was removed. A portion of the liver tissue was accurately weighed to prepare a 10% liver homogenate. The remaining liver tissue was stored at -80 °C.

[0105] 1. Determination of antioxidant capacity in mice

[0106] Accurately weigh a portion of liver tissue and add physiological saline to prepare a 10% liver tissue homogenate. The levels of malondialdehyde (MDA), catalase (CAT), and total antioxidant capacity (T-AOC) in mouse liver tissue were determined using a kit.

[0107] 2. Statistical Analysis

[0108] Data were statistically processed using GraphPad Prism 8.0 software. One-way ANOVA was used for multiple groups, and results are expressed as mean ± standard deviation (X ± SD). P A value <0.05 indicates a statistically significant difference between groups.

[0109] IV. Effects of Tea Fruit and Camphor Polysaccharide on Cyclophosphamide-Induced Oxidative Stress in Mice

[0110] 1. Effects of tea fruit camphor polysaccharide on antioxidant levels in mice

[0111] Cyclophosphamide (CTX) is an alkylating cytotoxic drug commonly used to treat cancer; however, high doses can lead to immunosuppression and oxidative stress damage. Oxidative stress results in an imbalance between prooxidants and antioxidants, leading to damage to cellular DNA, proteins, and lipids. Antioxidant capacity in mice can be assessed by detecting malondialdehyde (MDA) levels, catalase (CAT), and total antioxidant capacity (T-AOC) in liver tissue.

[0112] Figure 4The effects of tea fruit camphor polysaccharide on CAT(A), T-AOC(B), and MDA(C) activity in mouse liver are presented. Figure 4 As shown, compared with the blank group, the activity of catalase (CAT) (Figure A) and the total antioxidant capacity (T-AOC) (Figure B) in the liver of mice in the model group were significantly reduced. P <0.001), malondialdehyde (MDA) content was significantly increased (Figure C). This decrease was significantly reversed in the high-dose group and PC group of the tea fruit camphor polysaccharide of this invention. Simultaneously, compared with the model group, the high-dose MDA content was significantly reduced. The results indicate that the tea fruit camphor polysaccharide of this invention may exert its effect by enhancing the antioxidant capacity of mouse liver to resist cyclophosphamide-induced oxidative stress.

[0113] In summary, using a mouse model of acute liver injury established with carbon tetrachloride, this invention found that *Camellia sinensis* polysaccharide can effectively reduce the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in the serum of mice with acute liver injury, improve liver tissue morphology, increase the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver, reduce malondialdehyde (MDA) content, and alleviate liver tissue damage in mice, demonstrating excellent hepatoprotective effects and the potential to be developed into hepatoprotective drugs, especially for the preparation of drugs that protect against acute liver injury. Furthermore, using a mouse model of oxidative stress established with cyclophosphamide, this invention found that *Camellia sinensis* polysaccharide can also effectively reduce the MDA content in the liver of mice, increase the levels of catalase (CAT) and total antioxidant capacity (T-AOC) in the liver, and has a good protective effect against oxidative stress damage in mice, demonstrating excellent anti-oxidative stress effects and the potential to be developed into anti-oxidative stress drugs.

Claims

1. A method for extracting polysaccharides from tea fruit and camphor tree, characterized in that, Includes the following steps: After crushing the fruit of the tea camphor tree, water was heated and extracted at 65-90 ℃. The water extract was concentrated under reduced pressure, and the concentrate was precipitated with alcohol and protein removed. After precipitating with alcohol again and freeze-drying, tea camphor polysaccharide A was obtained. Alternatively, after crushing the fruit of the tea camphor tree, it is extracted with petroleum ether and ethyl acetate at room temperature, respectively, and then the solid and liquid are separated. The residue is extracted with water at 65-90 ℃. The water extract is concentrated under reduced pressure, and the concentrate is precipitated with alcohol and protein is removed. After precipitating with alcohol again, it is freeze-dried to obtain tea camphor polysaccharide B. The tea fruit camphor polysaccharide A and / or tea fruit camphor polysaccharide B are tea fruit camphor polysaccharides.

2. The method for extracting polysaccharides from tea fruit camphor as described in claim 1, characterized in that, Add water at a solid-liquid ratio of 1g:20-40ml and heat to extract for 2-5 hours, repeating the extraction 1-4 times.

3. The method for extracting polysaccharides from tea fruit camphor as described in claim 1, characterized in that, Extraction is performed by adding petroleum ether or ethyl acetate at a solid-liquid ratio of 1g:10-30ml for 8-15 hours, repeated 1-5 times.

4. Tea fruit camphor polysaccharide extracted using any one of the extraction methods described in claims 1 to 3.

5. A compound preparation comprising the tea fruit camphor polysaccharide of claim 4, characterized in that, The tea fruit camphor polysaccharide is compounded with conventional excipients in the field to form a compound preparation.

6. The compound preparation according to claim 5, characterized in that, The dosage form of the compound preparation is tablets, granules, pills, capsules, or injections.

7. The use of the tea fruit camphor polysaccharide of claim 4 or the compound preparation of claim 5 in the preparation of drugs for treating or preventing oxidative stress.

8. The application as described in claim 7, characterized in that, The oxidative stress is cyclophosphamide-induced oxidative stress; the tea fruit camphor polysaccharide or compound preparation can effectively reduce the malondialdehyde content in the liver and increase the levels of catalase and total antioxidant capacity in the liver.

9. The use of the tea fruit camphor polysaccharide of claim 4 or the compound preparation of claim 5 in the preparation of drugs for treating or preventing liver damage.

10. The application as described in claim 9, characterized in that, The liver injury is acute liver injury; the tea fruit camphor polysaccharide or compound preparation can reduce the levels of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, and alleviate liver damage.

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