Application of aloe vera gel powder and its composition with stachyose

Abstract

The present invention belongs to the field of big health technology, and relates to the application of aloe gel powder and its composition with stachyose, and in particular to the application of aloe gel powder and the composition of aloe gel powder and stachyose in the preparation of anti-tumor, intestinal flora and immune-regulating foods, health products and medicines. The composition comprises aloe gel powder and stachyose, and the mass ratio of aloe gel powder to stachyose is 1:30-20:1. The aloe gel powder stachyose composition prepared by combining the aloe gel powder and stachyose of the present invention has a synergistic anti-tumor effect. It can significantly inhibit the tumor growth of CT26 tumor-bearing mice and improve the spleen index. Its possible mechanism is to exert anti-tumor effects through immunomodulation, as well as improving intestinal flora and increasing intestinal short-chain fatty acids.

Description

Technical Field

[0001] The invention belongs to the field of big health technology, and relates to the application of aloe gel powder (AGF) and a composition thereof with stachyose (STC), and in particular to the application of aloe gel powder and a composition of aloe gel powder and stachyose in the preparation of anti-tumor drugs and foods, health products and drugs for regulating intestinal flora and immunity. Background Art

[0002] Cancer is a leading cause of death worldwide, with nearly 10 million deaths in 2020 according to the World Health Organization. The most common cancers are breast cancer, lung cancer, colon cancer, rectal cancer, and prostate cancer. Some of the most common types of cancer, such as breast cancer, cervical cancer, oral cancer, and colorectal cancer, have a high chance of being cured if they are detected early and treated according to best practices (World Health Organization, 2020).

[0003] Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in the world. In 2020, there were 1.93 million cases of colorectal cancer and 916,000 deaths worldwide (World Health Organization, 2020). A healthy diet, including fruits and vegetables, can effectively prevent cancer, especially colorectal cancer.

[0004] Aloe vera gel powder (AGF) is obtained by a series of methods such as peeling, extraction, depigmentation, filtration and freeze-drying or spray-drying of Aloe vera L (Aloe vera L or Aloe barbadensis Miller), Aloe ferox Miller or other closely related plants of the lily family.

[0005] Aloe vera has unique therapeutic and healing properties and has been used for centuries. So far, more than 75 active ingredients have been identified from its internal gel, but the therapeutic effects are not well correlated with each individual ingredient. Although the polysaccharides in aloe leaf gel have some activity, it is believed that these biological activities should be attributed to the synergistic effects of the compounds contained therein, rather than a single chemical substance. Therefore, compared with the use of aloe polysaccharides alone, the activity of aloe vera gel powder that retains the aloe leaf gel extract deserves further in-depth study.

[0006] Aloe vera gel powder is mainly used in skin and hair protection products in cosmetics. It has the effects of moisturizing, sun protection, deodorization, anti-obesity, skin softening, anti-inflammatory and itching, analgesic, anti-acne, freckles, and lightening spots.

[0007] Stachyose (STC) is a naturally occurring tetrasaccharide, belonging to the galactoside class of raffinose, and is a non-reducing functional oligosaccharide. The pure product is a white powder, slightly sweet, and its sweetness is 22% of sucrose. Stachyose is not completely hydrolyzed by human digestive enzymes, but can be fermented and utilized by intestinal bacteria, thereby regulating intestinal flora and improving energy metabolism. However, because stachyose has a small molecular weight and is metabolized and decomposed quickly, it cannot take effect in the intestine for a long time. Combining it with aloe polysaccharides may prolong the effect of improving the intestinal environment and play a synergistic role.

[0008] There is no report in the prior art that aloe vera gel powder has anti-tumor effects, especially anti-colorectal cancer effects. There is also no report on the activity study of combining aloe vera gel powder with stachyose. We expect that the combination of aloe vera gel powder and stachyose will have a better synergistic anti-tumor effect. Summary of the invention

[0009] In order to overcome the defects of the prior art, the present invention firstly provides the use of aloe vera gel powder in the preparation of anti-tumor, especially anti-colorectal cancer drugs.

[0010] Secondly, the present invention also provides a composition of aloe gel powder and stachyose, which has anti-tumor, immune regulation and intestinal flora regulation effects, and compared with the use of aloe gel powder alone, the composition has better anti-tumor activity and immune regulation and intestinal flora regulation effects.

[0011] The present invention is achieved through the following technical solutions:

[0012] The invention provides application of aloe gel powder in preparing anti-tumor drugs.

[0013] Furthermore, the tumor includes but is not limited to colorectal tumor.

[0014] The invention provides a composition of aloe gel powder and stachyose, wherein the composition comprises aloe gel powder and stachyose, and the mass ratio of the aloe gel powder to the stachyose is 1:30-20:1;

[0015] Furthermore, the mass ratio of aloe vera gel powder to stachyose is 1:9-3:7. Under this ratio, the composition has a good inhibitory effect on colon cancer cells, has no significant toxicity to macrophages, and has a faster dissolution rate and a better taste.

[0016] Preferably, when the mass ratio of aloe vera gel powder to stachyose is 1:3-1:5, especially when the mass ratio of aloe vera gel powder to stachyose is 1:4, the composition has the strongest inhibitory effect on colon cancer cells and has no toxicity to macrophages.

[0017] The aloe vera gel powder of the present invention is derived from Aloe vera L. or Aloebarbadensis Miller, Aloe ferox Miller or other closely related plants of the same genus in the Liliaceae family. The aloe vera gel powder is prepared by decolorizing, freeze-drying or spray-drying the original aloe vera juice.

[0018] The aloe vera gel powder of the present invention can be prepared by an extraction method known in the industry, and the preparation process includes but is not limited to the following features:

[0019] Fresh aloe leaves are picked, washed, peeled, crushed, ground, centrifuged and filtered to prepare aloe gel liquid, food-grade pectinase is added for full enzymolysis, crushed, ground and centrifuged to prepare aloe juice, decolorized and filtered, and the aloe juice is concentrated by reverse osmosis technology, and the concentrated aloe juice is freeze-dried or spray-dried to prepare aloe gel powder.

[0020] The aloe vera gel powder of the invention has a polysaccharide content greater than or equal to 80 mg / g, and an O-acetyl content greater than or equal to 90 mg / g.

[0021] The molecular structure of stachyose is a tetrasaccharide of "galactose-galactose-glucose-fructose", and the molecular formula is: 24 H 42 O 21 The relative molecular weight is 666.59, and the stachyose is food grade or medical grade edible stachyose.

[0022] The present invention has the following technical effects:

[0023] The aloe gel powder of the invention is prepared by decolorizing, freeze-drying or spray-drying aloe original juice, and has a therapeutic effect on tumor-bearing mice.

[0024] Aloe vera gel powder and stachyose are combined to prepare an aloe vera gel powder stachyose composition (AS), which has a synergistic anti-tumor effect. It can significantly inhibit the tumor growth of CT26 tumor-bearing mice and improve the spleen index. Its possible mechanism is to exert anti-tumor effects through immune regulation, improving intestinal flora and increasing intestinal short-chain fatty acids (SCFAs).

[0025] Aloe vera gel powder has a peculiar sour taste and a poor taste. In addition, due to its high viscosity, the aloe vera gel powder dissolves slowly and easily clumps, which is not conducive to taking. Combining it with stachyose improves the above defects, increases the dissolution rate and improves the taste. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The toxic effects of different concentrations of aloe vera gel powder on RAW264.7 cells in Example 3;

[0027] ** :Compared with the control group, P<0.01.

[0028] Figure 2 The toxic effects of different concentrations of stachyose on RAW264.7 cells in Example 3;

[0029] ** : Compared with the control group, P<0.01; * :Compared with the control group, P<0.05.

[0030] Figure 3 The toxic effects of the composition of aloe vera gel powder and stachyose in different prescription ratios in Example 3 on RAW264.7 cells;

[0031] ** : Compared with the control group, P<0.01; ## : Compared with prescription 3, P<0.01.

[0032] Figure 4 The toxic effects of different concentrations of aloe vera gel powder on CT26-WT cells in Example 3;

[0033] ** : Compared with the control group, P<0.01; * :Compared with the control group, P<0.05.

[0034] Figure 5 The toxic effects of different concentrations of stachyose on CT26-WT cells in Example 3.

[0035] Figure 6 The toxic effects of the composition of aloe vera gel powder and stachyose in different prescription ratios in Example 3 on CT26-WT cells;

[0036] ** :Compared with the control group, P<0.01.

[0037] Figure 7 This is the tumor volume on the 7th day after tumor cell inoculation in Example 4.

[0038] Figure 8 The spleen index of the tumor mice in Example 4 on the 21st day after oral gavage;

[0039] ** :Compared with the control group, P<0.01.

[0040] Fig. 9 The tumor volume of each group of mice from day 1 to day 21 after oral administration in Example 4;

[0041] ** : Compared with the results of the normal saline group, P<0.01.

[0042] Fig.10 The tumor mass of each group of mice on the 21st day after oral administration in Example 4;

[0043] ** : Compared with the results of the normal saline group, P<0.01, # : Compared with the results of the combination group, P<0.05, ## : Compared with the combination group, P<0.01.

[0044] Fig.11 This is the HE staining image of the tumors of each group of mice in Example 4 on the 21st day after oral administration;

[0045] Fig.12 IL-1β, IL-10, IL-6, TNF-α, IgG, IgM, and IFN-γ in the serum of mice on the 21st day after oral administration in Example 4;

[0046] * : Compared with the results of the normal saline group, P<0.05, ** : Compared with the results of the normal saline group, P<0.01, # : Compared with the results of the combination group, P<0.05, ## : Compared with the combination group, P<0.01.

[0047] Fig.13 The flow cytometric analysis of the number of monocytes, DCs, and CD8 + T cells and Treg cells;

[0048] * : Compared with the results of the normal saline group, P<0.05, ** : Compared with the results of the normal saline group, P<0.01, # : Compared with the results of the combination group, P<0.05, ## : Compared with the combination group, P<0.01.

[0049] Fig.14 Analysis of Alpha diversity index in feces of mice on day 20 after oral administration in Example 5;

[0050] * : Compared with the results of the normal saline group, P<0.05, ** : Compared with the results of the normal saline group, P<0.01.

[0051] Fig.15 The OTU analysis of the bacterial flora in the feces of mice on the 20th day after oral gavage in Example 5;

[0052] * : Compared with the results of the normal saline group, P<0.05, ** : Compared with the results of the normal saline group, P<0.01.

[0053] Fig.16 Analysis of beneficial bacteria in feces of mice after intragastric administration on the 20th day in Example 5;

[0054] * : Compared with the results of the normal saline group, P<0.05.

[0055] Fig.17 Analysis of harmful bacteria in feces of mice after intragastric administration on the 20th day in Example 5;

[0056] * : Compared with the results of the normal saline group, P<0.05.

[0057] Fig.18 Analysis of short-chain fatty acids in feces of mice on day 20 after oral administration in Example 6;

[0058] * : Compared with the results of the normal saline group, P<0.05, ** : Compared with the results of the normal saline group, P<0.01, # : Compared with the results of the combination group, P<0.05, ## : Compared with the combination group, P<0.01. DETAILED DESCRIPTION

[0059] In order to better understand the essence of the present invention, the following embodiments are introduced, but the embodiments are only further explanations of the content of the invention and are not limitations of the present invention. The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should be equivalent replacement modes and are included in the protection scope of the present invention.

[0060] The aloe vera gel powder of the invention is food-grade aloe vera gel powder, wherein the polysaccharide content is 80 mg / g and the O-acetyl content is 90 mg / g.

[0061] Example 1: Effect of the ratio of aloe vera gel powder and stachyose composition on taste

[0062] (1) Composition formulation design and preparation of the composition

[0063] Weigh the corresponding mass fractions of aloe vera gel powder and stachyose according to Table 1, put them into a suitable dry and sealed container, shake them thoroughly to mix them evenly, and pack each 5g of the composition into a moisture-proof packaging bag. Store them in a transparent glass desiccator containing color-changing silica gel.

[0064] Table 1 Composition raw material ratio (mass ratio) prescription design table

[0065]

[0066] (2) Evaluators and evaluation methods

[0067] Evaluator standards: Healthy people aged 18-55 years were selected and signed the informed consent form for the taste experiment. According to the 13th Five-Year Plan textbook for colleges and universities "Food Sensory Evaluation" (Han Beizhong et al., ed., Food Sensory Evaluation (2nd edition), China Forestry Publishing House, 2016), the evaluators were preliminarily screened for ① interest and motivation ② availability of evaluators ③ attitude towards the evaluation object ④ knowledge and talent ⑤ health status ⑥ expression ability ⑦ personality characteristics, and the evaluators who met the requirements were examined for ① sensory function ② sensory sensitivity ③ ability to describe and express sensory reactions, and finally 13 evaluators were selected.

[0068] The taste evaluation of the composition was conducted by scoring method, using randomized block design scheme, and presenting samples one by one in a random order. The evaluator tasted the powder of the composition directly, and took it with 50 mL of purified water, and then scored it on an equal interval category scale of 0-15 points. The evaluator gave drinking purified water for gargling before the start of the experiment and when changing different prescriptions.

[0069] The scoring results are shown in Table 2.

[0070] Table 2 Overall evaluation of the taste of the prescription

[0071]

[0072] (3) Test on the difference of properties of each prescription

[0073] Table 3 Multiple comparison results of scores of each group (all pairs, Tukey-Kramer HSD)

[0074] level Mean Prescription 3 A 12.615385 Prescription 2 B 10.538462 Prescription 4 B 10.384615 Prescription 1 C 8.692308 Prescription 5 D 6.461538 Prescription 6 E 3.846154 Prescription 7 F 1.230769

[0075] Note: Levels not connected by the same letter are significantly different

[0076] The results show (Table 2, Table 3) that the order of taste scores of different prescriptions from high to low is: prescription 3, 2, 4, 1, 5, 6, 7, among which prescriptions 3, 2, and 4 have higher scores and better taste, and prescription 3 has the highest score for sweet and sour taste. If the proportion of stachyose in the composite prescription is high, the taste is too sweet, such as prescription 1; if the proportion of aloe vera gel powder is high, the sour taste is too heavy and there are other unpleasant tastes, such as prescriptions 5, 6, and 7. In summary, by performing Tukey-Kramer HSD multiple comparisons on prescriptions 1-7, prescription 3 (the mass ratio of aloe vera gel powder to stachyose is 1:4) has the best taste, followed by prescription 2 (1:9) and prescription 4 (3:7).

[0077] Example 2: Comparison of dissolution rates of compositions with different ratios

[0078] According to the prescription in Table 1 of Example 1, 5 g of the powder of prescription 1-7 was accurately weighed, 50 ml of pure water was measured and placed in a 100 mL beaker, heated to 40° C. under 50 rpm magnetic stirring, and then 5 g of the composition powder was added to the beaker, and the time for the powder to completely dissolve was recorded. Three experiments were performed for each prescription.

[0079] The experimental results are shown in Table 4. As the proportion of stachyose in the prescription increases, the dissolution time decreases and has a certain dose dependence, indicating that stachyose increases the solubility of aloe gel powder. Prescriptions 1, 2, and 3 can be dissolved quickly and have a good dissolution rate. Aloe gel powder has a large viscosity and is easy to stick to the container wall when dissolved in water, resulting in poor dissolution. Combining with stachyose can significantly improve the dissolution of aloe gel powder and increase the dissolution rate.

[0080] Table 4 The time for each prescription to completely dissolve at 40°C (min), n=3

[0081]

[0082] Example 3: Toxicity of stachyose, aloe vera gel powder, and combinations of aloe vera gel powder and stachyose in different ratios on RAW264.7 and CT26 cells

[0083] The cytotoxic effects of aloe vera gel powder (AGF), stachyose (STC), and the combination of aloe vera gel powder and stachyose (AS) on RAW264.7 and CT26 cells were investigated through in vitro cytotoxicity experiments on mouse mononuclear macrophages RAW264.7 and mouse colon cancer cells CT26.

[0084] 1. Experimental methods

[0085] (1) Related solution configuration

[0086] RPMI-1640 / DMEM complete medium: Add 50 ml of special grade fetal bovine serum and 5 ml of penicillin-streptomycin solution to 500 ml of RPMI-1640 / DMEM medium, mix well, and place in a 4°C refrigerator for later use.

[0087] AGF mother solution: Accurately weigh 400 mg of AGF into a 15 ml centrifuge tube, add 10 ml of PBS solution, mix well, filter through a 0.22 μm microporous membrane, and place in a 4°C refrigerator for later use.

[0088] STC mother solution: accurately weigh 160 mg of STC into a 15 ml centrifuge tube, add 10 ml of PBS solution, mix well, filter through a 0.22 μm microporous membrane, and place in a 4°C refrigerator for later use.

[0089] Prescription 1-7 mother solution: Accurately weigh 200 mg of each of Prescription 1-7 into a 15 ml centrifuge tube, add 10 ml of PBS solution, mix well, filter through a 0.22 μm microporous membrane, and place in a 4°C refrigerator for later use.

[0090] (2) Cell culture

[0091] Mouse colon cancer cell line CT26 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin. When the cells grew to 80%, they were digested with trypsin, centrifuged, and then subjected to experiments such as passage and plating.

[0092] Mouse mononuclear macrophage RAW264.7 cells were cultured in DMEM high glucose medium with 10% fetal bovine serum and 1% penicillin. When the cells grew to 80%, they were gently scraped with a cell scraper and centrifuged for passage and plating experiments.

[0093] (3) Cell viability determination

[0094] CCK-8 is an upgraded alternative to MTT. It is a rapid, highly sensitive and non-radioactive test based on WST-8 that is widely used in cell proliferation and cytotoxicity. Therefore, the CCK-8 method was used to investigate the effects of different concentrations of AGF, STC and the combination of the two on the cell survival rate of CT26 cells and RAW264.7 cells.

[0095] a. The inoculation concentration in a 96-well plate is 1×10 5 100 μl / well of cell suspension, and place the culture plate in a 37°C, 5% CO2 incubator for 12 h to allow the cells to adhere to the wall;

[0096] b. Dilute the mother solution of the relevant solution with complete medium according to the concentration in Table 5, add 100 μL to each well, set 6 parallel wells for each concentration, and place the culture plate in an incubator for 48 hours;

[0097] c. Add 10 μl of CCK-8 solution to the culture plate, taking care not to create bubbles, and place the culture plate in the incubator for 2 hours;

[0098] d. Use an ELISA reader to measure the absorbance at 450 nm, and calculate the cell survival rate and cell inhibition rate according to the following formula.

[0099]

[0100] As: absorbance of the experimental well (including cells, culture medium, CCK-8 solution and test solution)

[0101] Ac: Absorbance of control well (containing cells, culture medium, CCK-8 solution)

[0102] Ab: absorbance of blank well (containing culture medium, CCK-8 solution)

[0103] Table 5 In vitro study concentrations of stachyose, aloe vera gel powder and the combination

[0104]

[0105] (II) Experimental results

[0106] (1) Effects of different concentrations of aloe vera gel powder (AGF) on the proliferation of macrophages RAW264.7

[0107] Different concentrations of AGF affect the cell viability of RAW264.7 cells, such as Figure 1 As shown in A, compared with the control group, 24 hours after administration, when the concentration of AGF was 6.45-40 μg / ml, there was no significant effect on cell growth. When the concentration of AGF was greater than or equal to 60 μg / ml, the cell viability of RAW264.7 was reduced and the growth was significantly inhibited ( ** P<0.01). Figure 1 As shown in Figure B, compared with the control group, 48 hours after administration, when the concentration of AGF was less than 20 μg / ml, there was no significant effect on cell growth. When the concentration of AGF was greater than or equal to 20 μg / ml, the cell viability of RAW264.7 was reduced and the growth was significantly inhibited ( ** P<0.01), and the cell survival rate at 48 h was slightly decreased compared with that at 24 h. When the AGF concentration was greater than or equal to 190.5 μg / ml and the cells were acted on for 48 h, the cell survival rate was less than 50%.

[0108] (2) Effects of different concentrations of stachyose (STC) on the proliferation of macrophages RAW264.7

[0109] Different concentrations of STC had effects on the cell viability of RAW264.7 cells, such as Figure 2 As shown in A, compared with the control group, 24 hours after administration, when the concentration of STC was 9.5-100 μg / ml, there was no significant effect on cell growth. When the concentration of STC was greater than or equal to 160 μg / ml, the cell viability of RAW264.7 was significantly increased ( ** P<0.01), when the concentration was 140μg / ml, the cell viability was significantly increased compared with the control group ( * P<0.05). Figure 2 As shown in Figure B, compared with the control group, after 48 hours of administration, when the concentration of STC was 9.5-100 μg / ml, there was no significant effect on cell growth. When the concentration of STC was greater than or equal to 140 μg / ml, the cell viability of RAW264.7 was significantly increased ( ** P<0.01), and the cell survival rate at 48 h was slightly increased compared with that at 24 h.

[0110] (3) Effect of the ratio of aloe vera gel powder (AGF) and stachyose (STC) on the proliferation of macrophages RAW264.7

[0111] like Figure 3 As shown in A, compared with the control group, after 24 hours of administration, the AGF and STC ratios of different prescriptions all affected the cell viability of RAW264.7 cells. Among them, the cell viability of the STC group, prescription 1, prescription 2 and prescription 3 was generally significantly higher than that of the control group ( ** P<0.01), were 103.2%, 102.9%, 102.7% and 102.6% respectively. The cell viability of AGF group, prescription 4, prescription 5, prescription 6 and prescription 7 was significantly lower than that of control group ( ** P<0.01) and prescription 3 ( ## P<0.01), were 89.0%, 72.8%, 60.7%, 57.2% and 54.6% respectively. Figure 3 As shown in B, compared with the control group, after 48 hours of administration, the effects of STC and AGF and each prescription on RAW264.7 cells were more obvious than the 24-hour trend. This indicates that after 24 and 28 hours of administration, when the mass proportion of STC is greater than or equal to 80%, the proliferation of RAW264.7 cells can be promoted, and vice versa, the proliferation can be inhibited.

[0112] (4) Effects of different concentrations of aloe vera gel powder (AGF) on the proliferation of colon cancer cell CT26

[0113] Different concentrations of AGF affect the cell viability of CT26 cells, such as Figure 4 As shown in A, compared with the control group, 24 hours after administration, when the concentration of AGF was 0.032 mg / ml, there was no significant effect on cell growth, and when the concentration of AGF was 0.1 mg / ml, the cell viability of CT26 was significantly inhibited ( * P<0.05). When the concentration of AGF was greater than or equal to 0.2 mg / ml, the cell viability of CT26 was significantly inhibited ( ** P<0.01). Figure 4 As shown in Figure B, compared with the control group, after 48 hours of administration, when the concentration of AGF was 0.032 mg / ml, there was no significant effect on cell growth. When the concentration of AGF was greater than or equal to 0.1 mg / ml, the cell viability of CT26 was extremely significantly inhibited ( ** P<0.01), and the cell viability at 48 h was slightly decreased compared with that at 24 h.

[0114] (5) Effects of different concentrations of stachyose (STC) on the proliferation of colon cancer cells CT26

[0115] Different concentrations of STC had no effect on the cell viability of CT26 cells, e.g. Figure 5 As shown in A and B, there was no significant difference among the various drug administration groups, indicating that at a given STC concentration, the cell viability of CT26 cells was not affected.

[0116] (6) Effect of the ratio of aloe vera gel powder (AGF) and stachyose (STC) on the proliferation of mouse colon cancer cell CT26

[0117] like Figure 6 As shown in A and B, compared with the control group, the cell viability of CT26 cells in the AGF group, prescription 3, prescription 4, prescription 5, prescription 6, and prescription 7 was inhibited after 24h and 48h of administration ( ** P<0.01), the 24h cell survival rates were 89.7%, 84.4%, 71.7%, 68.6%, 64.7% and 58.9%, respectively, and the 48h cell survival rates were 85.1%, 76.3%, 69.7%, 65.6%, 54.2% and 51.4%, respectively. This shows that when the mass proportion of AGF is greater than or equal to 20%, the proliferation of CT26 cells can be inhibited, and the inhibitory effect increases with time.

[0118] In summary, when the mass ratio of aloe vera gel powder (AGF) and stachyose (STC) is 1:4 (prescription 3), it can inhibit the proliferation of mouse colon cancer cells CT26 (AGF: 0.2 mg / ml, STC: 0.8 mg / ml), exert an anti-tumor effect, and promote the proliferation of mouse mononuclear macrophages RAW264.7 (AGF: 40 μg / ml, STC: 160 μg / ml), exerting an immune-regulating effect, so this ratio was used for the CT26 tumor-bearing mouse experiment.

[0119] Example 4: Study on the anti-tumor effect of aloe vera gel powder, stachyose, and a combination of aloe vera gel powder and stachyose (1:4) on colon cancer in mice

[0120] 1. Experimental methods

[0121] (1) Cell culture

[0122] Mouse colon cancer cells CT26 were selected and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin. When the cells grew to 80%, they were digested with trypsin, centrifuged, and the cells were adjusted to 1.0×10 7 Pieces / mL.

[0123] (2) Establishment of colon cancer mouse model

[0124] Experimental animals: 24 SPF male BALB / c mice, 6-8 weeks old, weighing 20 (± 2) g, provided by Liaoning Changsheng Biotechnology Co., Ltd., animal certificate number: SYXK (Liao) 2020-0001. Breeding environment: standard SPF animal room, temperature: (23 ± 2) ° C, relative humidity: 50% to 60%, light and dark alternate for 12 hours each, mice free access to water and feed. All animals in this experiment were kept in the animal room of Shenyang Pharmaceutical University in accordance with the animal breeding regulations of Shenyang Pharmaceutical University, and animal experimental operations were performed under the guidance of the regulations of the Laboratory Animal Protection Association.

[0125] After 2 weeks of adaptive feeding, the mice were inoculated with CT26 wild-type cells. The lower abdomen of the mice was depilated and disinfected, and a 1 mL microsyringe was used to extract a CT26 cell suspension (1.0 × 10 7 / mL) were inoculated into the left lower abdomen of mice, with 0.1mL (1×10 6 pcs / piece).

[0126] like Figure 7 As shown, the day of injection was recorded as D0, and the tumor volume was 0. The mice were kept under SPF conditions for 7 days (D7). The results showed that the average tumor volume was no less than 62.5 cm 3 , the subcutaneous tumor model of colon cancer was successfully constructed.

[0127] (3) Grouping of mice

[0128] The model mice were divided into a normal saline group, a stachyose group, an aloe vera gel powder group, and a combination of aloe vera gel powder and stachyose group, with 6 mice in each group. 3 The time was recorded as the start of the experiment day 0, and gavage began. The corresponding solution or saline was gavaged once a day (9:00am) for 21 consecutive days. The dosage of each group was as follows:

[0129] 1.) The saline group was given 10 ml / kg / d of saline

[0130] 2.) Stachyose group was given 80 mg / kg / d of stachyose aqueous solution

[0131] 3.) Aloe vera gel powder group was given aloe vera gel powder aqueous solution at 20 mg / kg / d

[0132] 4.) The group composed of aloe vera gel powder and stachyose was given a combination solution at a dose of stachyose of 80 mg / kg / d and aloe vera gel powder of 20 mg / kg / d.

[0133] (4) Measurement of mouse tumor volume

[0134] Starting from the first day of tumor cell inoculation, the diameter (a) and short diameter (b) of the tumor were measured once a day (9:00 am) with a vernier caliper, and the tumor volume was calculated and the tumor growth curve was drawn. After the start of intragastric administration, the tumor size was measured daily, and the tumor volume was calculated and the tumor growth curve was drawn.

[0135]

[0136] (5) Mouse sampling

[0137] 24 hours after the last oral administration, the mice were killed by cervical dislocation, the tumors were carefully removed, washed in ice-cold saline, dried, the tumor weight was weighed and the tumor inhibition rate was calculated.

[0138]

[0139] On the 21st day, the mice were anesthetized, their eyes were removed and blood was collected, then they were killed by cervical dislocation. The liver and spleen were carefully removed, washed with ice-cold saline, dried, weighed, and the organ index was calculated. The mice were stored at -80°C for future use.

[0140]

[0141] On the 21st day, the mice were anesthetized, the eyeballs were removed and blood was collected, and the blood was kept at 37°C for 2 hours, centrifuged at 3500 rpm and 4°C for 20 minutes, and the pale yellow serum was carefully aspirated and stored at -80°C for later use. The kit was used to detect cytokines IgG (immunoglobulin G), IgM (immunoglobulin M), IFN-γ (Interferonγ, γ interferon), TNF-α (tumor necrosis factor α, tumor necrosis factor alpha), pro-inflammatory factors IL-1β (interleukin-1β, Interleukin-1β), IL-6 (interleukin-6, Interleukin-6) and anti-inflammatory factors IL-10 (interleukin-10, Interleukin-10).

[0142] (6) HE staining of mouse tumors

[0143] Fresh tumor tissue from mice was fixed in paraformaldehyde for 24 h, then the tumor tissue was sequentially placed in 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, and anhydrous ethanol II for dehydration for 0.5 h, then placed in anhydrous ethanol xylene solution (1:1) for 10 min, xylene I for 5 min, and xylene II for 5 min to make it transparent, finally placed in 58°C-60°C paraffin I and paraffin II for 1 h each, embedded, and sliced ​​after the paraffin solidified.

[0144] The cut tissue was placed in a 60°C oven for 1 h, and then placed in xylene, anhydrous ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and RO water for rehydration, and then HE staining was performed according to the kit.

[0145] (7) Flow cytometry detection of immune cells in mouse spleen and tumor

[0146] Spleen tissue pretreatment

[0147] 1.) Preparation: Prepare 1.5 mg / mL collagenase IV solution (prepared with HBSS), prepare a six-well plate and crushed ice, and place the six-well plate in ice.

[0148] 2.) Sampling: Place the removed spleen in a six-well plate (to keep the spleen fresh)

[0149] 3.) Crushing: Add 1.8 mL of collagenase IV solution to each spleen and cut it into pieces with small scissors (cut it as finely as possible, do not use a homogenizer or cell crusher), and wipe the scissors clean when operating on different samples.

[0150] 4.) Digestion: Use a rubber-tipped dropper to transfer the shredded tissue homogenate into a 4 mL EP tube, place in a 37°C water bath to digest for 1 hour, take out the sample and vortex for 30 minutes, and repeat the water bath and vortexing for about 2 times.

[0151] 5.) Make up the volume: Use the filter net to filter in small amounts multiple times, discard the large pieces of tissue at the bottom of the EP tube, add whole blood and tissue diluent to the filtered sample, and make up the volume to 2 mL.

[0152] Cell isolation and labeling

[0153] The fixed volume samples were taken and extracted using a mouse spleen tissue lymphocyte isolation kit (Beijing Solebow Technology Co., Ltd.).

[0154] CD8 marker + T cells, Treg cells: Add CD3, CD4, and CD8 antibodies, add 1 μL to each sample, vortex and incubate on ice for 20 minutes in the dark. After using the FOXP3 kit to disrupt the nucleus, add 1 μL of Foxp3 antibody and incubate on ice for 20 minutes (the working solution is prepared according to the instructions). Centrifuge at 4°C, 500g, 10 minutes, discard the supernatant, add 200 μL CellStaining Buffer to each sample, blow evenly,

[0155] Pass through the sieve, add 200μL Cell Staining Buffer (or add 400μL Cell Staining Buffer, blow evenly, and pass through the sieve), and test on the machine (keep away from light at 4℃). CD3, CD4 and CD8 antibodies are used to label CD8 + T cells, CD3, CD4, and Foxp3 antibodies were used to label Treg cells.

[0156] Labeling monocytes: centrifuge 500uL sample at 4℃: 500g, 10min, discard the supernatant, add 200μL Cell Staining Buffer, resuspend evenly, add 1μL CD14 antibody, vortex, incubate on ice for 20min away from light. Add 200μL Cell Staining Buffer to the sample, pipette evenly, pass through the sieve, and detect on the machine (place at 4℃ away from light).

[0157] Mark DC cells: Add CD8, CD86, and CD11c antibodies, add 1 μL to each sample, vortex and incubate on ice in the dark for 20 minutes. Centrifuge 500uL sample at 4°C: 500g, 10min, discard the supernatant, add 200μL Cell Staining Buffer, resuspend evenly, add 1μL of mixed antibodies, vortex, and incubate on ice in the dark for 20min. Add 200μL Cell Staining Buffer to the sample, pipette evenly, pass through the sieve, and detect on the machine (place at 4°C in the dark).

[0158] (8) Statistics

[0159] All data and plots were analyzed statistically using Graphpad 8.0 software. The quantitative data were expressed as ± SEM. One-way analysis of variance was used for comparison among multiple groups, and LSD-t test was used for pairwise comparison. * P<0.05 indicated that the difference was significant. ** P<0.01 indicates a very significant difference.

[0160] (II) Experimental results

[0161] (1) Stachyose and aloe vera gel powder-stachyose combination significantly improved the spleen index of CT26 tumor-bearing mice

[0162] like Figure 8 As shown in Figure 2, the spleen index represents the immune status of mice. After tumor inoculation, cancer cells stimulate the spleen, causing immune cells to overactivate and swell, resulting in increased spleen mass and spleen index. Studies have shown that the increase in spleen index is negatively correlated with the prognosis of colon tumors. After administration of stachyose, the spleen index was significantly reduced ( ** P<0.01), indicating that stachyose improved the swelling caused by tumors. The spleen index of the aloe vera gel powder group did not change significantly compared with the saline group. In the composition, the spleen index was significantly reduced compared with the saline group ( ** P<0.01). This indicates that the combination is superior to aloe vera gel powder alone in improving the spleen index of CT26 tumor-bearing mice.

[0163] (2) Aloe vera gel powder and its combination significantly inhibited tumor growth in CT26 tumor-bearing mice

[0164] like Fig. 9 As shown, at the initial stage of the experiment (0-7 days), there was almost no difference in the size and growth rate of the tumors in each group of mice. From 8-21 days, the tumors in the normal saline group grew rapidly, while the tumor growth in the stachyose group, the aloe gel powder group, and the combination group was inhibited.

[0165] On the 21st day, mice were euthanized and tumors were removed and weighed. Fig.10Compared with the saline control group, the tumor weights of each experimental group were significantly decreased ( ** P<0.01, the tumor weight of the combination group was the smallest, and the tumor inhibition effect was better than that of the stachyose group ( ## P<0.01) and aloe vera gel powder group ( # P<0.01).

[0166] (3) Effect of aloe vera gel powder and stachyose combination on tumor histopathology in CT26 colon cancer-bearing mice

[0167] like Fig.11 As shown in the figure, after HE staining of tumor tissue sections, observation under a 200-fold (20×10) light microscope showed that the tumor cells in the saline group were round or oval, polygonal, larger in size, with darker nuclei, larger nucleoplasm, more nuclear divisions, and less nuclear condensation. In the aloe gel powder group and the stachyose group, the tumor cells were partially necrotic, with uneven morphology and size. At the same time, the immune cells in the aloe gel powder group increased, indicating that both had a certain killing effect on tumor cells. The number of immune cells in the combination group increased, and the tumor cells were necrotic over a large area, almost losing the cell morphology, with blurred edges and loose arrangement, more nuclear fragmentation and nuclear condensation, crescent-shaped chromatin, reduced nuclear-cytoplasmic ratio, fewer cell division stages, and different degrees of swelling, vacuoles, and necrosis in the tumor tissue. This shows that the combination has a significant inhibitory effect on tumors in tumor-bearing mice, and its effect is better than that of the aloe gel powder group and the stachyose group.

[0168] (4) Effect of aloe vera gel powder and stachyose combination on cytokines in mouse serum

[0169] (4.1) IL-1β

[0170] IL-1β is mainly secreted by neutrophils infiltrating the lesion site. Solid tumors with upregulated IL-1β include breast cancer, colon cancer, lung cancer, head and neck cancer, and melanoma, and patients with IL-1β-producing tumors usually have a poor prognosis. In addition, interleukin IL-1β can promote the growth of colorectal cancer cells through the miR-181a / PTEN axis.

[0171] like Fig.12 As shown, when mice were given aloe gel powder, stachyose and the combination, the IL-1β levels in the serum of mice in the aloe gel powder group, stachyose group and combination group were significantly decreased ( ** P<0.01), and the combination group had the best effect. The mice given aloe vera gel powder, stachyose and the combination had reduced serum IL-1β levels, indicating that it had good anti-inflammatory and anti-tumor effects.

[0172] (4.2) IL-6

[0173] The pathological role of IL-6 in disease development has been shown in animal models of many diseases, and blocking IL-6 by gene knockout or administration of anti-IL-6 or anti-IL-6R Ab can lead to prevention or therapeutic inhibition of disease development. At the same time, IL-6 is also a core driver of cancer metastasis. In the tumor microenvironment, IL-6 can directly act on disseminated cancer cells (DCCs) of the primary lesion, promote the invasive ability of cancer cells through trans receptors on the surface of primary tumor cells, and induce interference with the immune system.

[0174] like Fig.12 As shown, when mice were given aloe gel powder, stachyose and the combination, the IL-6 levels in the serum of mice in the aloe gel powder group, stachyose group and combination group were significantly decreased ( ** P<0.01), and the combination group had the best effect. Mice given aloe vera gel powder, stachyose and the combination reduced the level of IL-6, a cytokine that suppresses immunity in serum, and reduced the invasive ability of cancer cells, among which the combination had the strongest effect in inhibiting IL-6.

[0175] (4.3) IL-10

[0176] In the treatment of tumors, the increase of IL-10 can inhibit the biological effects of anti-tumor immune response and make tumor cells immune tolerant. In addition, excessive IL-10 macrophage infiltration produces regulatory T cell infiltration and CD8 + Immune escape from the tumor microenvironment, characterized by T cell dysfunction, leads to metastasis, so low expression of IL-10 may indicate a certain inhibitory effect on tumor metastasis.

[0177] like Fig.12 As shown, when mice were given aloe gel powder, stachyose and the combination, the IL-10 levels in the serum of mice in the aloe gel powder group, stachyose group and combination group were significantly decreased ( ** P<0.01), and the combination group had the best effect. The mice given aloe vera gel powder, stachyose and the combination reduced the level of IL-10, a cytokine that suppresses immunity in the serum, suggesting that the anti-tumor immune response can be improved, and the combination has the strongest effect in suppressing IL-10.

[0178] (4.4) IgG and IgM

[0179] Immunoglobulin (Ig) is a type of globulin with antibody activity. It is an important component of the body's disease resistance and is widely present in blood, tissue fluid and exocrine fluid. According to traditional theory, only B lymphocytes and plasma cells can produce immunoglobulins. However, the latest research shows that various cancer cells (such as breast cancer, lung cancer, cervical cancer, colorectal cancer, etc.) can also express immunoglobulins, especially immunoglobulin G (IgG). Tumor-derived immunoglobulins are not conducive to the treatment of tumors, and mainly lead to tumor proliferation and metastasis, and tumor immune escape.

[0180] like Fig.12 As shown, when mice were given aloe gel powder, stachyose and the combination, the levels of IgG and IgM as a biomarker in the serum of mice in the aloe gel powder group, stachyose group and combination group were significantly decreased ( ** P<0.01), and the decrease was most significant in the combination group, indicating that the tumor in the combination group was inhibited.

[0181] (4.5) IFN-γ

[0182] Interferon-γ (IFN-γ) is a soluble dimeric cytokine. IFN-γ has antiviral, antitumor and immune regulatory effects. It can regulate the expression levels of 30 genes and produce a variety of cellular responses. It can promote NK cell activity, promote antigen presentation and increase macrophage lysosomal activity.

[0183] like Fig.12 As shown, when mice were given aloe gel powder, stachyose and the combination, the IFN-γ levels in the serum of mice in the aloe gel powder group, stachyose group and combination group were significantly increased ( ** P<0.01), with the combination group increasing the most significantly. Mice given aloe vera gel powder, stachyose and the combination increased the level of IFN-γ in serum, activated the immune system defense function, and exerted an anti-tumor effect. The combination increased IFN-γ better than stachyose or aloe vera gel powder alone.

[0184] (4.6) TNF-α

[0185] TNF is a cytokine that can cause hemorrhagic necrosis in tumor tissue. Tumor necrosis factor is divided into TNF-α and TNF-β according to its source and structure. TNF-α is mainly produced by monocytes and macrophages activated by lipopolysaccharide, and plays an important role in regulating immune response and inducing apoptosis of target cells.

[0186] like Fig.12 As shown, when mice were given aloe gel powder, stachyose and the combination, the TNF-α level in the serum of the mice in the combination group increased significantly ( **P<0.01), and there was also a significant difference compared with the aloe vera gel powder group ( # P<0.05). The combination increased the level of TNF-α in the serum of mice, induced apoptosis of target cells, and inhibited the growth of tumors.

[0187] (5) Flow cytometry detection of immune cells in the spleen of different groups of colon cancer mice

[0188] (5.1) Monocytes

[0189] Monocytes are the largest blood cells and the largest white blood cells in the blood. They are an important part of the body's defense system. Monocytes participate in immune responses and transfer the antigenic determinants they carry to lymphocytes after engulfing antigens, inducing specific immune responses in lymphocytes.

[0190] like Fig.13 As shown in Figure 2, after administration of aloe gel powder, stachyose and the combination, the proportion of monocytes in the spleen tissue of mice increased, and the proportion of monocytes in the stachyose group, aloe gel powder group and the combination was significantly higher than that in the normal saline group ( ** P<0.01), and the combination was still significantly higher than the aloe gel powder group and the stachyose group ( ## P<0.01). Oral administration of aloe vera gel powder, stachyose and the combination promoted the proliferation of monocytes in the spleen of mice.

[0191] (5.2) Dendritic cells

[0192] Dendritic cells (DC) are antigen-presenting cells that can take up, process and present antigens, and initiate T cell-mediated immune responses.

[0193] like Fig.13 As shown in the figure, after administration of aloe gel powder, stachyose and the combination, the proportion of DC cells in the spleen tissue of mice increased. The proportion of DC cells in the stachyose group, aloe gel powder group and the combination was significantly higher than that in the normal saline control group. Although the combination was lower than that used alone, it was still significantly higher than that in the normal saline group ( ** P<0.01). Oral administration of aloe vera gel powder, stachyose and the combination promoted the proliferation of DC cells in the spleen of mice, which can enhance the immune response mediated by T cells.

[0194] (5.3)CD8 + T cells

[0195] CD8 +T cells generally refer to cytotoxic T lymphocytes. Cytotoxic T lymphocytes (CTLs) are a subdivision of white blood cells and a specific T cell that specifically secretes various cytokines to participate in immune responses. They have a killing effect on certain antigenic substances such as viruses and tumor cells.

[0196] like Fig.13 As shown, after administration of aloe vera gel powder, stachyose and the combination, CD8 + The proportion of T cells increased, and the stachyose group, aloe gel powder group and the combination group were significantly higher than those in the saline group ( ** P<0.01), and the combination was still significantly higher than the aloe gel powder group and the stachyose group ( ## P<0.01). The mice were given aloe vera gel powder, stachyose and the combination by gavage, which increased the number of CD8 + The proportion of T cells can then increase the production of immune anti-tumor cytokines in the body.

[0197] (5.4) Treg cells

[0198] Regulatory T cells (Treg) are a type of cell subset with significant immunosuppressive effects. Treg cells can secrete inhibitory cytokines such as IL-10 to enable tumor cells to resist the apoptosis-induced effects of immune effector cells.

[0199] like Fig.13 As shown in the figure, after administration of aloe gel powder, stachyose and the combination, the proportion of Treg cells in the spleen tissue of mice decreased, but only the aloe gel powder group and the aloe gel powder stachyose group were significantly different from the saline group ( * P<0.05). The mice were given aloe vera gel powder, stachyose and the combination by gavage, which reduced the proportion of Treg cells in the spleen and the immunosuppressive factors in the mice, thereby enhancing the killing effect of the immune system on tumor tissues.

[0200] After the aloe gel powder and stachyose are combined in the present invention, the tumor inhibition rate is increased, and a synergistic effect is achieved. Compared with taking aloe gel powder alone, the composition of aloe gel powder and stachyose can significantly improve the spleen index of tumor-bearing mice and increase the positive factors for anti-tumor in the spleen of mice. When aloe gel powder, stachyose and the composition are administered, monocytes, CD8 + The proportions of T cells and DC cells increased, and the factors unfavorable to anti-tumor were significantly reduced, that is, the proportion of Treg cells was significantly reduced. Moreover, the effect of the combination was better than that of using stachyose alone or aloe vera gel powder alone.

[0201] Example 5: Effect of the composition on intestinal flora of mice with colon cancer

[0202] 1. Experimental methods

[0203] The modeling method, grouping and composition ratio of CT26 tumor-bearing mice were the same as those in Example 4. At 9:00 am the day before the mice were euthanized, they were wiped with alcohol cotton and placed in a clean bench. Sterile tweezers were used to stimulate the perianal area of ​​the mice. The collected feces were placed in sterile cryopreservation tubes, quickly frozen with liquid nitrogen, and stored at -80°C for sequencing of intestinal flora and determination of short-chain fatty acid content.

[0204] (II) Experimental results

[0205] α diversity analysis mainly includes species evenness analysis and species richness analysis. Among them, the Shannon diversity index and the Simpson index are the main indicators for measuring the evenness of bacterial community species. The Shannon diversity index represents the total number of individuals in the community and the number of each species, while the Simpson index represents the total number of species in the community and the proportion of a certain species in all species. The Chao index and the Ace index are the main indicators for measuring the species richness of bacterial communities. The Chao index is obtained by taking the quotient of the number of species containing 1 individual and the number of species containing 2 individuals, while the Ace (Abundance-based Coverage Estimator) index analyzes the number of species that are less than a certain number of individuals. In short, the Chao index and the Ace index pay more attention to the existence of rare species. Such as Fig.14 As shown, compared with the saline group, the Shannon diversity index of the combination group increased significantly ( * P<0.05), indicating that the composition can increase the species diversity of the bacterial flora in the feces of mice with colon cancer. Fig.14 As shown, compared with the saline group, the Chao index and Ace index indicating the abundance of bacterial species in the composition group and the aloe vera gel powder group increased significantly ( * P<0.05, and the combined group index was the highest ( ** P<0.01), indicating that the composition can increase the abundance of bacterial flora in the feces of mice with colon cancer.

[0206] OTU (Operational Taxonomic Units) is a unique marker artificially set for a taxonomic unit (strain, species, genus, grouping, etc.) in phylogenetic or population genetics studies to facilitate analysis.

[0207] like Fig.15 As shown in a, compared with the saline group, the number of OTUs in the feces of mice in the combination group increased significantly ( ** P < 0.01), and Fig.15 As shown in b, the number of OTUs unique to the combination group was the largest, indicating that the combination increased the types of intestinal flora in mouse feces. Fig.16As shown, compared with the saline group, the probiotics unclassified-Oscillatory Spirulina, unclassified-Lachnospira, Roseburia, Oscillatory Bacterium, Lachnospiraceae-NK4A136 and Segmented Filamentous Bacteria in the feces of the mice in the combination group increased significantly ( * P<0.05), increased by 4.3 times, 4.5 times, 8.6 times, 5.1 times, 8.8 times and 2.8 times respectively. (Chen H et al, Frontiers in immunology, 2020, 11: 588079; Chattopadhyay I et al, Applied biochemistry and biotechnology, 2021, 193: 1780-1799; Wolfe AE et al, PLoS one, 2020, 15(7): e0236595; Wang Q et al, Applied microbiology and biotechnology, 2020, 104: 5915-5928.) and as Fig.17 As shown in ab, compared with the saline group, the harmful bacteria Amoeba and the total amount of harmful bacteria in the feces of mice in the composition group were significantly reduced ( * The gut microbiota may modify the susceptibility and progression of CRC by regulating mechanisms such as inflammation and DNA damage and producing metabolites involved in tumor progression or suppression. Intestinal dysbiosis was observed in CRC patients, with a decrease in commensal bacterial species (butyrate-producing bacteria) and an enrichment in harmful bacterial groups (pro-inflammatory opportunistic pathogens).

[0208] The composition of aloe vera gel powder and stachyose of the present invention can significantly increase the number of intestinal probiotics, unclassified-Oscillatory Spirulina, unclassified-Lachnospira, Roseburia, Oscillatory Bacterium, Lachnospiraceae-NK4A136, segmented filamentous bacteria and the total number of beneficial bacteria, and reduce the number of harmful bacteria Amoeba and the total number of harmful bacteria.

[0209] Example 6: Effect of the composition on the content of short-chain fatty acids in mouse feces

[0210] 1. Experimental methods

[0211] The modeling method, grouping and composition ratio of CT26 tumor-bearing mice are the same as those in Example 4, and the fecal sample sampling is carried out according to Example 4. Weigh the frozen fecal sample, put the sample in a 2mL EP tube, add 1mL pure water, vortex for 10s; add steel balls, treat with a 40Hz grinder for 4min, ultrasonic for 5min (ice water bath), and repeat 3 times; centrifuge the sample at 4°C, centrifuge at 5000rpm for 20min; transfer 0.8mL of supernatant to a 2mL EP tube; add 0.1mL50% H2SO4, add 0.8mL of extract (containing internal standard 2-methylpentanoic acid, 25mg / L, methyl tert-butyl ether), vortex for 10s, oscillate for 10min, ultrasonic for 10min (ice water bath), centrifuge the sample at 4°C, centrifuge at 10000rpm for 15min; let stand at -20°C for 30min; take out the supernatant in an injection bottle, and detect it by GC-MS.

[0212] The instrument was a Shimadzu GC2030-QP2020 NX gas chromatograph-mass spectrometer. The system used an HP-FFAP capillary column; 1 μL of analyte was injected in split mode (5:1); helium was used as the carrier gas, and the front inlet purge flow rate was 3 mL min -1 The gas flow rate through the column was 1 mL min -1 The initial temperature was 80°C for 1 min, then increased at 10°C min -1 The temperature was raised to 200 °C at a rate of 100 °C and maintained for 5 min, then at 40 °C min -1 The temperature of the sample was raised to 240 °C at a rate of 1.5 °C and maintained for 1 min. The injection, transfer line, quadrupole and ion source temperatures were 240 °C, 240 °C, 150 °C and 200 °C, respectively. The energy was -70 eV in electron impact mode. Mass spectrometry data were acquired in scan / SIM mode after a solvent delay of 3.5 min with an m / z range of 33-150.

[0213] The detected fatty acids were acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, valeric acid, and caproic acid. The peaks of each component were determined by retention time, and the peak areas were calculated to obtain the concentrations of each fatty acid. The total sum was calculated to obtain the total amount of short-chain fatty acids in mouse feces.

[0214] (II) Experimental results

[0215] Short-chain fatty acids are metabolites produced by the fermentation of dietary fiber by colonic intestinal flora. They can not only maintain the proliferation of normal colon epithelial cells and promote the recovery of the intestinal barrier, but also significantly antagonize the proliferation of intestinal tumor cells and promote the apoptosis of tumor cells.

[0216] like Fig.18 As shown in the figure, the effect on the acetic acid content in mouse feces. Compared with the saline group, the acetic acid content in the feces of mice in the composition group increased significantly ( **P<0.01).

[0217] Effect on the content of propionic acid in mouse feces. Compared with the saline group, the content of propionic acid in the feces of mice in the aloe vera gel powder group and the combination group increased significantly ( * P < 0.05; ** P<0.01), and compared with the aloe vera gel powder group and the stachyose group, the propionic acid content in the feces of the mice in the combination group was significantly increased ( # P < 0.05; ## P<0.01).

[0218] Effect on the isobutyric acid content in mouse feces. Compared with the saline group, the isobutyric acid content in the feces of mice in the aloe gel powder group, stachyose group and combination group increased significantly ( * P<0.05).

[0219] Effect on butyric acid content in mouse feces. Compared with the saline group, the butyric acid content in the feces of mice in the aloe gel powder group, stachyose group and combination group increased significantly ( * P < 0.05; ** P<0.01), and the combination group was superior to the single drug group ( ## P<0.01).

[0220] Effect on the total content of SCFAs in mouse feces. Compared with the saline group, the total content of SCFAs in the feces of mice in the aloe vera gel powder group and the combination group increased significantly ( * P < 0.05; ** P<0.01), and the combination group was superior to the single drug group ( ## P<0.01). This suggests that oral administration of the composition can increase the amount of short-chain fatty acids in the intestine, which is beneficial to the integrity and reconstruction of the intestinal barrier and inhibits the growth and metastasis of intestinal tumors.

[0221] Therefore, the composition of aloe vera gel powder and stachyose of the present invention can significantly increase the amount of intestinal short-chain fatty acids including but not limited to acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, valeric acid and caproic acid.

[0222] In summary, the present invention can significantly improve the taste of aloe gel powder and the solubility of aloe gel powder by combining aloe gel powder with stachyose. Cell experiments unexpectedly found that the aloe gel powder and stachyose combination can inhibit the growth of colon cancer cells without significantly affecting the proliferation of macrophages.

[0223] Through animal experiments, it was unexpectedly discovered that this combination can better increase the number of intestinal probiotics, reduce the number of harmful bacteria and the amount of short-chain fatty acids, and has a better effect in improving immunity.

Claims

1. A composition of aloe vera gel powder and stachyose, characterized in that: The invention is composed of aloe gel powder and stachyose, wherein the mass ratio of the aloe gel powder to the stachyose is 1:3-1:5, and the aloe gel powder has a polysaccharide content greater than or equal to 80 mg / g and an O-acetyl content greater than or equal to 90 mg / g.

2. Use of the composition according to claim 1 in the preparation of anti-colorectal tumor medicines.

3. The use according to claim 2, characterized in that: The composition increases the infiltration of immune cells in colorectal tumor tissues, promotes the necrosis of colorectal cancer cells, and exerts an immune effect against colorectal tumors.

4. The use according to claim 2, characterized in that: The composition exerts an anti-colorectal tumor effect by increasing the anti-tumor cytokines TNF-α and IFN-γ and reducing the IL-1β, IL-6I and IL-10 cytokines that promote tumor deterioration.

5. The use according to claim 2, characterized in that: The composition improves CD8 + The number of T cells and DC cells can be reduced, and the number of immunosuppressive cells Treg cells can be reduced to exert anti-colorectal tumor effects.

6. The use according to claim 2, characterized in that: The composition exerts an anti-colorectal tumor effect by regulating intestinal flora, increasing the number of probiotics and reducing the number of harmful bacteria.

7. The use according to claim 2, characterized in that: The composition exerts an anti-colorectal tumor effect by increasing the content of intestinal short-chain fatty acids and improving intestinal health.

8. Use of the composition of claim 1 in the preparation of food, medicine or health product for regulating immunity, regulating intestinal flora or increasing intestinal short-chain fatty acids.

9. Use of the composition according to claim 1 in preparing food, medicine or health product for improving spleen index.

10. Use of the composition according to claim 1 in the preparation of medicines, foods or health products for improving the taste and dissolution rate of aloe vera gel powder.

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