Compound double-flower preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly provides a compound double-flower preparation which comprises a mixed water extract of honeysuckle, astragalus membranaceus, dendrobe, poria cocos, pericarpium citri reticulatae and liquorice. The active ingredients in the compound double-flower preparation provided by the invention have various pharmacological activities, and through the multi-component and multi-target synergistic effect, the compound double-flower preparation has the effects of improving thymus indexes, reducing spleen indexes, improving thymus and spleen tissue structures, reducing inflammatory factor levels, improving immune globulin levels and remarkably improving humoral immunity and cellular immunity functions. The compound can be used for preparing a product for improving humoral immunity and cellular immunity functions, or preparing a product for improving thymus and spleen injury caused by cyclophosphamide, or preparing a product for relieving in-vivo inflammation caused by cyclophosphamide, or preparing a product for regulating intestinal flora. The invention further provides a preparation method of the compound double-flower preparation, the preparation method has the advantages of simplicity and convenience in operation, low cost, low energy consumption and the like, the extraction process is accurate and reliable, and the yield of effective components is high.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a compound double flower preparation, a preparation method and an application thereof. Background Art

[0002] The immune system is the body's core defense system for resisting the invasion of external pathogens and maintaining homeostasis of the internal environment. Its functional level is directly related to the body's health level. More and more studies have shown that the occurrence of many diseases is closely related to abnormal immune system function. Long-term stressful and high-pressure emotions, bad living habits, lack of exercise, unreasonable diet and other lifestyle factors can easily lead to a decline in the body's immune function. With the continuous progress of modern society and the improvement of people's demand for health, immune-enhancing products have received widespread attention. Currently, commonly used immune-enhancing drugs such as thymosin and interferon are often accompanied by side effects such as fever, hepatotoxicity, or immune hyperactivity with long-term use. Traditional Chinese medicine and its compound prescriptions have better safety and effectiveness due to their multi-target action characteristics, and have become a research hotspot in recent years. Summary of the Invention

[0003] The purpose of the present invention is to overcome the problem that long-term use of commonly used immunopotentiating drugs in the prior art is often accompanied by side effects.

[0004] To this end, the present invention provides a compound double flower preparation, comprising a mixed aqueous extract of honeysuckle, astragalus, dendrobium, poria, tangerine peel and liquorice.

[0005] The present invention also provides a preparation method of the compound double flower preparation, comprising the following steps: mixing honeysuckle, astragalus, dendrobium, poria, tangerine peel and liquorice as raw materials; adding water to the raw materials for extraction, and centrifuging to obtain the supernatant to obtain the compound double flower preparation.

[0006] Specifically, the mass ratio of honeysuckle, astragalus, dendrobium, poria, tangerine peel and licorice in the above raw materials is 10:10:6:6:3:3.

[0007] Specifically, the material-liquid ratio of the above raw materials to water is 20-30 g / mL.

[0008] Specifically, the extraction temperature is 70-90° C., and the extraction time is 60-120 min.

[0009] The present invention also provides a compound Shuanghua preparation oral liquid, comprising citric acid, a sweetener, and the compound Shuanghua preparation. The sweetener is preferably honey. The compound Shuanghua preparation oral liquid comprises, by weight, 20% to 60% of the compound Shuanghua drink extract, 2% to 4% of honey, 0.008% to 0.012% of citric acid, and the remainder being water.

[0010] The present invention also provides the use of the above-mentioned compound double flower preparation or compound double flower preparation oral liquid in the preparation of products for improving humoral immunity and cellular immunity, preparing products for improving thymus and spleen damage caused by cyclophosphamide, preparing products for alleviating inflammation in the body caused by cyclophosphamide, and preparing products for regulating intestinal flora.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] The active ingredients in the compound huanghua preparation provided by the present invention possess multiple pharmacological activities. Through the synergistic action of multiple components and multiple targets, they increase the thymus index, decrease the spleen index, improve the tissue structure of the thymus and spleen, reduce the levels of inflammatory factors such as IL-6 and TNF-α, increase the levels of immunoglobulins IgM, IgG, and SIgA, and significantly enhance humoral and cellular immunity. The preparation can be used to prepare products for enhancing humoral and cellular immunity, or for ameliorating thymus and spleen damage caused by cyclophosphamide, or for alleviating cyclophosphamide-induced inflammation in the body, or for regulating intestinal flora. The preparation method of the compound huanghua preparation provided by the present invention has the advantages of simple operation, low cost, and low energy consumption, making it suitable for practical production use. The extraction process is accurate and reliable, with high yields of total polysaccharides, total phenolic acids, and total flavonoids. The finished product is clear and transparent, has a pleasant aroma, and a slightly sweet and slightly bitter taste. It combines good taste and efficacy with the advantages of ease of administration, rapid absorption, and easy dosing. Compound double flower preparations can also be used to prepare products for improving humoral immunity and cellular immunity, or to prepare products for improving thymus and spleen damage caused by cyclophosphamide, or to prepare products for relieving inflammation in the body caused by cyclophosphamide, or to prepare products for regulating intestinal flora, and have broad market prospects.

[0013] The present invention will be described in further detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the total ion current diagram of the compound double flower preparation in Example 3; wherein, A: positive ion mode; B: negative ion mode.

[0015] Figure 2 This is the effect of the compound double flower preparation in Example 4 on the body weight of mice.

[0016] Figure 3 The effect of the compound double flower preparation in Example 4 on the organ index of mice; compared with the control group, "*" represents a significant difference between the two groups (P < 0.05), "**" represents an extremely significant difference between the two groups (P < 0.01); compared with the model group, "#" represents a significant difference between the two groups (P < 0.05), and "##" represents an extremely significant difference between the two groups (P < 0.01).

[0017] Figure 4 This is the HE staining result of mouse spleen in Example 4 (×100).

[0018] Figure 5 This is the HE staining result of mouse spleen in Example 4 (×400).

[0019] Figure 6 This is the HE staining result of the mouse thymus in Example 4 (×100).

[0020] Figure 7 This is the HE staining result of the mouse thymus in Example 4 (×400).

[0021] Figure 8 The effect of the compound double flower preparation in Example 4 on mouse cytokines; compared with the control group, "*" represents a significant difference between the two groups (P < 0.05), "**" represents an extremely significant difference between the two groups (P < 0.01); compared with the model group, "#" represents a significant difference between the two groups (P < 0.05), and "##" represents an extremely significant difference between the two groups (P < 0.01).

[0022] Figure 9 The effect of the compound double flower preparation in Example 4 on mouse immunoglobulin; compared with the control group, "*" represents a significant difference between the two groups (P < 0.05), "**" represents an extremely significant difference between the two groups (P < 0.01); compared with the model group, "#" represents a significant difference between the two groups (P < 0.05), and "##" represents an extremely significant difference between the two groups (P < 0.01).

[0023] Figure 10 The effect of the compound double flower preparation in Example 4 on the hemolysin level in mice; compared with the control group, "*" represents a significant difference between the two groups (P < 0.05), "**" represents an extremely significant difference between the two groups (P < 0.01); compared with the model group, "#" represents a significant difference between the two groups (P < 0.05), and "##" represents an extremely significant difference between the two groups (P < 0.01).

[0024] Figure 11 The effect of the compound double flower preparation in Example 4 on the degree of delayed-type hypersensitivity in mice; compared with the control group, "*" represents a significant difference between the two groups (P < 0.05), "**" represents an extremely significant difference between the two groups (P < 0.01); compared with the model group, "#" represents a significant difference between the two groups (P < 0.05), and "##" represents an extremely significant difference between the two groups (P < 0.01).

[0025] Figure 12This is the OUT Venn diagram of the colon intestinal flora of each group of mice in Example 5.

[0026] Figure 13 Figure 5 is the change in the α-diversity index of the colon intestinal flora of mice in each group in Example 5; different lowercase letters indicate significant differences among the groups (P < 0.05).

[0027] Figure 14 This is the β diversity of the intestinal flora of each group of mice in Example 5.

[0028] Figure 15 The following are the distribution diagrams of the intestinal flora of each group of mice in Example 5 (A) and the F / B value (B); "***" indicates that there is a very significant difference between the two groups (P < 0.001).

[0029] Figure 16 This is the distribution diagram of the intestinal flora genus level of each group of mice in Example 5.

[0030] Figure 17 Figure 5 is a graph showing the differences in intestinal flora among the mice groups in Example 5; different lowercase letters indicate significant differences among the groups (P < 0.05).

[0031] Figure 18 Figure 5 is a graph showing the differences in intestinal flora among the mice groups in Example 5; different lowercase letters indicate significant differences among the groups (P < 0.05).

[0032] Figure 19 This is the intestinal flora function prediction of PICRUSt2 in Example 5.

[0033] Figure 20 This is the gas chromatogram of the reference solution (A) and the sample solution (B) in Example 5; wherein 1, acetic acid; 2, propionic acid; 3, isobutyric acid; 4, butyric acid; 5, isovaleric acid; 6, valeric acid; 7, 2-ethylbutyric acid.

[0034] Figure 21 The figure shows the effect of the compound Shuanghua drink on the content of short-chain fatty acids in colon contents in Example 5; *P < 0.05, **P < 0.01, compared with the control group; #P < 0.05, ##P < 0.01, compared with the model group.

[0035] Figure 22 The changes in short-chain fatty acids after 24 h of in vitro fermentation in Example 5; *P < 0.05, **P < 0.01, compared with the control group; #P < 0.05, ##P < 0.01, compared with the model group.

[0036] Figure 23This is a heat map of the correlation analysis between the genus-level intestinal flora and the inflammatory factor levels in Example 5; *P < 0.05, **P < 0.01, ***P < 0.001.

[0037] Figure 24 This is a heat map of the correlation analysis between genus-level intestinal flora and immunoglobulin levels in Example 5; *P < 0.05, **P < 0.01, ***P < 0.001.

[0038] Figure 25 This is a heat map of the correlation analysis between the genus-level intestinal flora and the short-chain fatty acid content in Example 5; *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0040] The effects of the compound double flower preparation, preparation method and application of the present invention are studied through specific examples below.

[0041] Example 1: This example provides 17 groups of compound Shuanghua preparations, prepared using the following steps: 10 g of honeysuckle, 10 g of astragalus, 6 g of dendrobium, 6 g of poria, 3 g of dried tangerine peel, and 3 g of liquorice were washed and extracted with water at a specific material-liquid ratio, extraction time, and temperature. The mixture was centrifuged at 5163 × g for 5 minutes, and the supernatant was collected to obtain the compound Shuanghua preparation. The material-liquid ratio, extraction time, and temperature for each group, as well as the test results for total polysaccharides, total phenolic acids, and total flavonoids in the compound Shuanghua preparations, are shown in Table 1.

[0042] Table 1 Preparation conditions and extraction results

[0043]

[0044] As shown in Table 1, the method provided by the present invention can obtain a compound double flower preparation with high contents of total polysaccharides, total phenolic acids and total flavonoids.

[0045] Example 2: This example provides a compound double flower preparation, which is prepared by the following steps: take 10 g of honeysuckle, 10 g of astragalus, 6 g of dendrobium, 6 g of poria, 3 g of tangerine peel, and 3 g of licorice, wash them, add water at a solid-liquid ratio of 27 g / mL, extract at 83 ° C for 100 min, centrifuge at 5163 × g for 5 min, and take the supernatant, which is the compound double flower preparation.

[0046] Three compound Shuanghua preparations were prepared using this method, with average yields of 32.6 mg / g of total polysaccharides, 15.4 mg / g of total phenolic acids, and 20.6 mg / g of total flavonoids. This method is accurate and reliable, with high yields of total polysaccharides, total phenolic acids, and total flavonoids, and is suitable for practical application.

[0047] Example 3: The compound double flower preparation prepared in Example 2 was freeze-dried, and about 1.0 g of the freeze-dried sample was accurately weighed and placed in a 100 mL centrifuge tube. 50 times 80% methanol was added and centrifuged at 12000 r / min for 10 min. The supernatant was taken and filtered through a 0.22 μm microporous filter membrane to obtain a test solution.

[0048] The chemical components of the test solution were analyzed using UPLC-Q-Exactive Orbitrap-MS technology, and positive and negative ion modes were used for mass spectrometry scanning. The structural formula of the compound was deduced by high-resolution mass spectrometry and secondary fragment ions. The mass spectrometry data were analyzed using Thermo XCalibur 4.1 software and Compound Discoverer 3.3 SP1 software. The compound was qualitatively compared with relevant literature and existing databases based on the retention time and secondary mass spectrometry characteristic fragment ions. The chemical components of the compound double flower preparation extract were identified. The total ion current is shown in the figure. Figure 1 shown.

[0049] A total of 150 chemical components were identified in the compound double-flower preparation, including 47 flavonoids (nobiletin, tangeretin, hesperidin, luteolin, rutin, wogonin, etc.), 47 organic acids (chlorogenic acid, caffeic acid, citric acid, ferulic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, cinnamic acid, tartaric acid, etc.), 12 sugars and their derivatives, 12 terpenoids (astragaloside IV, liquiritin, isoliquiritigenin, liquiritigenin, isoliquiritigenin, etc.), 8 alkaloids (stachydrine, synephrine, etc.), 6 nucleosides and their derivatives (adenosine, guanosine, uridine, etc.), 7 amino acids and their derivatives (L-norleucine, phenylalanine, oxoproline, DL-glutamic acid, methyltyrosine ester, etc.), 3 coumarins (7-hydroxycoumarin, 7-methoxycoumarin, esculin), and 8 other compounds. Among them, flavonoids and organic acid compounds are the most abundant, accounting for 62.7% of the total.

[0050] Example 4: This example uses a cyclophosphamide-induced immunosuppressive mouse model to study the immune function regulatory effect of the compound double flower preparation.

[0051] 1. Experimental Animals

[0052] Male Kunming mice, weighing 20–22 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. (License No.: SCXK (Beijing) 2019-0010).

[0053] Mice were maintained in an SPF animal room throughout the entire process, license number SYXK (Beijing) 2020-0033.

[0054] 2. Experimental methods

[0055] 2.1 Preparation of compound Shuanghua preparation

[0056] The same method as in Example 2 was used to prepare the compound Shuanghua preparation, which was concentrated under reduced pressure to 1 g crude drug / kg, and then packaged and stored in a -80°C refrigerator for later use.

[0057] 2.2 Establishment of immunosuppressive mouse model

[0058] After three days of adaptive feeding, 60 mice were randomly divided into two groups: a blank control group (10 mice) and a modeling group (50 mice). All but the blank control group received intraperitoneal injections of 80 mg / kg cyclophosphamide for three consecutive days to establish a cyclophosphamide-immunosuppressed mouse model. The blank control group received an equal dose of saline.

[0059] 2.3 Grouping and Dosing

[0060] After model establishment, 10 mice were excluded from the blank control group (BC). The remaining 50 mice were randomly divided into the model control group (MC), the positive drug (levamisole hydrochloride) group (PC) (25 mg / kg), the compound Shuanghua Decoction low-dose group (LD) (5.7 g crude drug / kg·d), the compound Shuanghua Decoction medium-dose group (MD) (11.4 g crude drug / kg), and the compound Shuanghua Decoction high-dose group (HD) (17.1 g crude drug / kg·d), with 10 mice in each group. Mice in each group were administered the drug by gavage. The blank control and model groups received an equal volume of deionized water (10 mL / kg). The mice were gavaged continuously for 21 days. After the last administration, they were fasted but not watered for 12 hours. Their eyes were removed to collect blood, and then they were killed by cervical dislocation.

[0061] 2.4 Body weight measurement

[0062] During the experiment, the behavioral status and appearance characteristics of the mice were observed, including limb activity, mental state, fur color, diet and water intake, and the weight of the mice was recorded every 4 days.

[0063] 2.5 Organ index measurement

[0064] Remove the mouse thymus and spleen, rinse with saline, remove surface water from the organs with filter paper, and weigh them. Calculate the organ index using the following formula: Organ index = organ weight (mg) / body weight (g).

[0065] 2.6 HE staining of thymus and spleen

[0066] (1) Paraffin-embedded sections

[0067] Fixation and Collection: Immediately after collection, thymus and spleen tissues were fixed in tissue fixative for at least 24 hours. After fixation, the thymus and spleen tissues were removed, trimmed, placed in embedding frames, and labeled accordingly. Dehydration and Paraffin Immersion: Dehydration was performed using the following solvents in sequence: 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, anhydrous ethanol I and II for 30 minutes each, benzene alcohol for 10 minutes, and xylene I and II for 10 minutes each. After dehydration, the tissues were placed at 65°C to melt paraffin for 1 hour, repeating three times. Paraffin Embedding: Embed the tissues using an embedding machine and label them accordingly. Cool the tissues in a -20°C freezer until the paraffin blocks solidify and then trim them. Paraffin Sectioning: Using a paraffin microtome, slice the paraffin blocks into 4 μm thick slices. Flatten the slices in 40°C warm water, remove them, and bake them at 60°C. Melt the wax, dry the water, and then remove them for later use.

[0068] (2) Thymus and spleen tissue staining

[0069] Rewarming fixation: Remove sections and return to room temperature. Fix sections in tissue fixative for 15 minutes, then rinse under running water. Dewaxing: Dewax using environmentally friendly dewaxing solutions I and II, anhydrous ethanol I and II, and 75% alcohol, then rinse under running water. Pretreatment: Treat with HD constant stain pretreatment solution for 1 minute. Hematoxylin staining: Stain with hematoxylin for 5 minutes, then rinse with tap water, differentiate with differentiation solution, rinse with tap water, bluing with bluing solution, and rinse under running water. Eosin staining: Dehydrate with 95% ethanol for 1 minute, then stain in eosin solution for 15 seconds. Dehydration and mounting: Dehydrate with anhydrous ethanol I, II, and III, n-butanol I and II, and xylene I and II, followed by mounting with neutral gum.

[0070] Microscope observation and image acquisition: Use a microscope to observe the sections and collect relevant images.

[0071] 2.7 Cytokine Assay

[0072] Twelve hours after the last administration, the eyeballs of mice in each group were removed for blood collection. After standing at room temperature for 1 hour, the blood was centrifuged at 3500 rpm for 15 minutes. The supernatant was collected as serum and stored at -80°C until further use. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in mouse serum were measured using ELISA kits for TNF-α and IL-6 according to the kit's instructions.

[0073] 2.8 Immunoglobulin Determination

[0074] Mouse serum was collected and the levels of mouse immunoglobulin A (IgM) and immunoglobulin G (IgG) in the serum were detected using ELISA kits.

[0075] The colon contents of each group of mice were extruded, the colons were flushed with normal saline, and the samples were frozen at -80°C until testing. The levels of mouse secretory immunoglobulin A (SIgA) in the mouse colonic mucosa were measured using an ELISA kit according to the kit's instructions.

[0076] 2.9 Serum hemolysin level determination

[0077] Four days before sampling, mice in each group were immunized with 0.2 mL of 2% packed red blood cells (RBCs) injected intraperitoneally. Twelve hours after the last dose, blood was collected from the eyeballs of each mouse. Care was taken to trim the whiskers and allow the centrifuge tube to naturally collect the blood to prevent hemolysis from interfering with experimental results. After standing at room temperature for 1 hour, the blood was centrifuged at 3500 rpm for 15 minutes, and the supernatant was obtained as serum.

[0078] A 96-well plate was set up with sample and blank wells. For each sample well, 50 μL of serum diluted 300-fold with SA buffer was added. For each blank control well, 50 μL of SA buffer was added. Then, 50 μL of complement (diluted 1:8 with SA buffer) and 25 μL of 10% (v / v) packed red blood cells were added to the sample and blank wells, respectively. The plates were incubated at 37°C for 30 minutes, followed by an ice-water bath for 10 minutes and a horizontal centrifugation at 1500 rpm for 10 minutes. Then, 50 μL of the supernatant from each well was precisely pipetted into a new 96-well plate, and 150 μL of Dulbecco's reagent was added to each well. Simultaneously, some blank wells were set up as half-hemolysis wells, and 12.5 μL of 10% (v / v) packed red blood cells were added to these half-hemolysis wells. All wells were then filled to 200 μL of Dulbecco's reagent, mixed thoroughly, and allowed to stand for 10 minutes. The optical density of each well was measured at 540 nm using a microplate reader. Calculate the half hemolysis value (HC 50 ). Half hemolysis value (HC 50 ) can be used to represent the hemolysin in serum. The HC of the test sample group is calculated according to the following formula. 50 Significantly higher than the control group HC 50 , the test result can be judged to be positive. 50 = (optical density of sample - optical density of blank) / (optical density of SRBC when half of the cells are hemolyzed - optical density of blank) × dilution factor.

[0079] 2.10 Delayed hypersensitivity reactions

[0080] Four days before sampling, mice in each group were immunized with 0.2 mL of 2% packed volume sheep red blood cells (RBCs) injected intraperitoneally. Three days later, the thickness of the left hind paw was measured, and then 20 μL of 20% packed volume sheep red blood cells were injected subcutaneously at the measurement site. On the fourth day, the thickness of the left hind paw was measured again. The difference in plantar thickness before and after RBC injection was used to indicate the degree of delayed-type hypersensitivity reaction. If the difference in the test sample group was significantly higher than that in the control group, the test result was considered positive.

[0081] 2.11 Data Processing

[0082] Excel was used for data collation and analysis, Graphpad Prism 7.0 software was used for data visualization, and IBM SPSS Satistics 23.0 software was used to perform analysis of variance (ANOVA) and independent sample t-test. When P < 0.05, the data between the two groups were considered to be statistically significant.

[0083] 3. Experimental results

[0084] 3.1 Effects of Compound Shuanghua Preparation on Mouse Body Weight

[0085] During the modeling and drug administration period, the mice in the blank control group were lively, alert, energetic, with thick fur, normal fur color, and normal diet. The mice in the model group had sparse fur, dull fur color, and bad temper. After oral administration of levamisole hydrochloride and compound double flower preparation groups, the fur of the mice gradually recovered, the fur color returned to normal, and the mental state was better than that of the model group. After modeling, except for the blank control group, the weight of the mice in the other groups decreased. After administration of levamisole hydrochloride and compound double flower preparation, the weight of the mice rebounded. Figure 2 shown.

[0086] 3.2 Effects of Compound Shuanghua Preparation on Organ Indexes in Mice

[0087] Thymus and spleen organ indexes are important indicators for quickly evaluating the basic immune function of mice. Figure 3As shown, compared with the blank control group, the model group showed a significant decrease in thymic index (P < 0.05) and a significant increase in spleen index (P < 0.05). Compared with the model group, the levamisole hydrochloride group and the low-, medium-, and high-dose groups of the compound Shuanghua preparation all increased the thymic index. The medium-dose compound Shuanghua preparation group had the strongest effect on the thymic index, followed by the low-dose group, which was superior to the levamisole hydrochloride and high-dose groups. Regarding the spleen index, the continuous administration of high-dose cyclophosphamide resulted in a severe immunosuppressive effect, leading to splenomegaly, presumably due to compensatory splenic hyperplasia. Compared with the model group, the levamisole hydrochloride group and the low- and medium-dose compound Shuanghua preparation groups all inhibited splenomegaly, while the high-dose group had no significant effect. The medium-dose compound Shuanghua preparation group showed the strongest effect, restoring the spleen index to normal levels. These results indicate that the compound Shuanghua preparation has the ability to protect against cyclophosphamide-induced thymic atrophy and splenomegaly in mice.

[0088] 3.3 Effects of the Compound Shuanghua Preparation on the Physiological Structure of the Spleen and Thymus in Mice

[0089] Spleen HE staining Figure 4-5 As shown, the spleen tissue structure of mice in the normal group was orderly, with normal morphology, tightly packed cells, and a clear distinction between the white and red pulp. The spleen structure of mice in the model group was disordered, with unclear boundaries between the white and red pulp, and many cells showing edema, indicating that cyclophosphamide inhibited the immune function of the spleen. Compared with the model group, the spleen structure of mice in the levamisole hydrochloride group and the low-dose compound double flower preparation group recovered to a certain extent, with gradually clear boundaries between the white and red pulp, and less cellular edema. Compared with the model group, the spleen tissue structure of mice in the medium- and high-dose compound double flower preparation groups was compact, with clear boundaries between the white and red pulp, and no or less cellular edema. Cellular edema was observed in the model group, consistent with the elevated spleen index in the model group.

[0090] Thymus HE staining Figure 6-7 As shown, the thymus tissue structure of the mice in the normal group was compact, with normal morphology, clear boundaries between the cortex and medulla, and dense thymocytes; the thymus tissue structure of the mice in the model group was disordered, with unclear boundaries between the cortex and medulla, and a decrease in thymocytes, indicating that cyclophosphamide inhibited the immune function of the thymus; compared with the model group, the thymus structure of the mice in the levamisole hydrochloride group and the low-dose group of the compound double flower preparation had a certain recovery, the boundaries between the cortex and medulla gradually became clear, and the thymocytes increased, but there was still a little tissue inflammation in the low-dose group; compared with the model group, the thymus tissue structure of the mice in the medium- and high-dose groups of the compound double flower preparation had a compact, clear boundaries between the cortex and medulla, and dense thymocytes.

[0091] 3.4 Effects of Compound Shuanghua Preparation on Cytokines in Mice

[0092] like Figure 8As shown, compared with the blank control group, the model group had a significantly increased IL-6 level (P < 0.01). Compared with the model group, the levamisole hydrochloride group and the low, medium, and high-dose groups of the compound Shuanghua preparation all had significantly decreased IL-6 levels (P < 0.05). The IL-6 levels in the medium-dose and high-dose groups of the compound Shuanghua preparation were comparable, and both were lower than those in the levamisole hydrochloride and low-dose groups. Compared with the blank control group, the model group had a significantly increased TNF-α level (P < 0.05). Compared with the model group, the levamisole hydrochloride group and the low, medium, and high-dose groups of the compound Shuanghua preparation all had significantly decreased TNF-α levels (P < 0.05). The TNF-α level in the medium-dose group of the compound Shuanghua preparation was lower than that in the other three groups. These results indicate that the compound Shuanghua preparation alleviates cyclophosphamide-induced inflammation in mice.

[0093] 3.5 Effects of Compound Shuanghua Preparation on Mouse Immunoglobulin

[0094] Immunoglobulins are important proteins in the human immune system and have multiple biological functions, including anti-infection, immune regulation and disease treatment. Figure 9 As shown, compared with the blank control group, the model group had significantly decreased IgM, IgG, and SIgA levels (P < 0.01). Compared with the model group, the levamisole hydrochloride group and the medium-dose compound double flower preparation group all had significantly increased IgM, IgG, and SIgA levels (P < 0.05). Compared with the model group, the low- and high-dose compound double flower preparation groups showed no significant increase in IgM levels, but significantly increased IgG and SIgA levels (P < 0.05). The medium-dose compound double flower preparation group had the best therapeutic effect, with IgM and SIgA levels comparable to those of the blank control group, and IgG levels significantly higher than those of the blank control group (P < 0.05). These results suggest that the compound double flower preparation enhances the immune function of mice by increasing the levels of immunoglobulins in the mice.

[0095] 3.6 Effects of Compound Shuanghua Preparation on Serum Hemolysin Levels in Mice

[0096] like Figure 10 As shown, compared with the blank control group, the serum hemolysin level in the model group was significantly reduced (P < 0.01). Compared with the model group, the levamisole hydrochloride group and the low-, medium-, and high-dose groups of the compound double flower preparation significantly increased the IL-6 levels (P < 0.05). Among them, the serum hemolysin level in the medium-dose compound double flower preparation group was the highest, and the serum hemolysin level in the high-dose compound double flower preparation group was the lowest. The serum hemolysin levels in the levamisole hydrochloride group and the low-dose compound double flower preparation group were comparable. The results indicate that the compound double flower preparation has the effect of increasing the serum hemolysin level in immunosuppressed mice and improving the humoral immune function of mice.

[0097] 3.7 Effect of Compound Shuanghua Preparation on the Delayed-Type Hypersensitivity in Mice

[0098] like Figure 11 As shown, compared with the blank control group, the degree of plantar swelling in the model group was significantly reduced (P < 0.05). Compared with the model group, the degree of plantar swelling in the levamisole hydrochloride group and the low-, medium-, and high-dose groups of the compound double flower preparation was significantly increased (P < 0.05). Among them, the high-dose compound double flower preparation group had the highest degree of plantar swelling, the levamisole hydrochloride group had the lowest degree of plantar swelling, and the low- and medium-dose compound double flower preparation groups had similar degrees of plantar swelling. The results indicate that the compound double flower preparation has the effect of increasing the degree of delayed-type hypersensitivity in immunosuppressed mice and improving the cellular immune function of mice.

[0099] In summary, the compound double-flower preparation significantly restored immune organs, increasing the thymus index and decreasing the spleen index. HE staining results also showed that the compound double-flower preparation effectively improved the histological structure of the thymus and spleen, with the medium-dose compound double-flower preparation group showing the best effect. Regarding cytokines, the compound double-flower preparation significantly reduced IL-6 and TNF-α levels, with the medium-dose compound double-flower preparation group showing the best effect. Regarding immunoglobulins, the compound double-flower preparation significantly increased IgM, IgG, and SIgA levels, with the medium-dose compound double-flower preparation group showing the best effect. Regarding serum hemolysin and delayed-type hypersensitivity, the compound double-flower preparation significantly increased serum hemolysin levels and the severity of delayed-type hypersensitivity, effectively enhancing the humoral and cellular immune functions of mice. The compound double-flower preparation primarily enhances immune function by improving the status of immune organs, reducing inflammatory factor levels, and increasing immunoglobulin levels. The medium-dose compound double-flower preparation group was superior to the low- and high-dose groups.

[0100] Example 5: This example is based on Example 4 and uses 16S rRNA high-throughput sequencing technology to study the regulatory effect of fecal intestinal flora in mice in the normal group, model group, and medium-dose group of Compound Shuanghua Drink.

[0101] 1. Experimental Materials

[0102] Intestinal flora analysis: Fresh feces were collected from each mouse the day before sampling and stored at -80°C for further analysis. Short-chain fatty acid analysis: Colon contents of dissected mice were collected on the same day and placed in test tubes and stored at -80°C for further analysis.

[0103] 2. Experimental methods

[0104] 2.1 Detection of intestinal flora in mouse feces

[0105] 2.1.1 Sample DNA extraction

[0106] According to the PF Mag-Bind Stool DNA Kit instructions, total genomic DNA from mouse fecal microbial communities was extracted, and the integrity, concentration, and purity of the extracted genomic DNA were tested.

[0107] 2.1.2 PCR amplification and sequencing library construction

[0108] 16S rRNA gene was amplified by PCR using the extracted DNA as template. 3 replicates per sample.

[0109] Using NextFlex The library was constructed using the Rapid DNA-Seq Kit, and sequencing was performed using the Illumina PE300 platform.

[0110] 2.1.3 High-throughput sequencing data analysis

[0111] Sequence quality control was performed using fastp (https: / / github.com / OpenGene / fastp, version 0.19.6) and assembly was performed using FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.11). UPARSE (http: / / drive5.com / uparse / , version 11) was used to cluster the quality-controlled assembled sequences into operational taxonomic units (OTUs) at 97% identity, and chimeras were removed. To minimize the impact of sequencing depth on subsequent alpha and beta diversity analysis, the number of sequences in all samples was truncated to 20,000. OTU taxonomic annotation was performed using RDPclassifier (http: / / rdp.cme.msu.edu / , version 2.13) against the Silva 16S rRNA gene database (v138) with a 70% confidence threshold. Community composition was calculated for each sample at different taxonomic levels. 16S gene function prediction analysis was performed using PICRUSt2 (version 2.2.0) software.

[0112] 2.2 Short-chain fatty acid detection

[0113] 2.2.1 Preparation of colonic content sample solution

[0114] Add 100 mg of colon contents to a centrifuge tube, add 1 mL of 0.2% formic acid aqueous solution, mix well, let stand for 1 h, centrifuge at 12000 r / min for 15 min, aspirate the supernatant into a 2 mL centrifuge tube, add 600 μL of ethyl acetate for extraction, centrifuge at 12000 r / min for 10 min, aspirate the ethyl acetate layer, and store at -20°C.

[0115] 2.2.2 Preparation of short-chain fatty acid reference working solution

[0116] Add 5 μL each of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid to a 5 mL brown volumetric flask and dilute to the mark with ethyl acetate to prepare the short-chain fatty acid reference stock solution (Table 2). Add 20 μL of 2-ethylbutyric acid to a 5 mL brown volumetric flask and dilute to the mark to prepare the internal standard reference stock solution. Take 1.5, 15, 30, 60, and 90 μL of the short-chain fatty acid reference stock solution, respectively, and add ethyl acetate to 300 μL to prepare the short-chain fatty acid reference working solution (Table 3). For the assay, add 200 μL of the test solution to the sample vial followed by 10 μL of the internal standard solution and vortex to mix thoroughly.

[0117] Table 2 Stock solutions of short-chain fatty acid reference substances

[0118]

[0119] Table 3 Gradient concentration of short-chain fatty acid reference solution (μg / mL)

[0120]

[0121] 2.2.3 Gas chromatography method parameters

[0122] Chromatographic system: Aglient 7890B gas chromatograph. Detector: Flame ionization detector. Chromatographic column: DB-FFAP capillary column, 30 m × 0.25 mm, coating film thickness 0.25 μm. Carrier gas: High-purity nitrogen. Combustion gas: Hydrogen. Supporting gas: Air. Temperature program: Initial column temperature 100°C, hold for 1 min, increase to 130°C at 3°C / min, hold for 1 min, and then increase to 190°C at 20°C / min. Detector and inlet temperature: 230°C. Gas flow rate: Hydrogen, air, and nitrogen mixed at flow ratios of 30, 400, and 25 mL / min, respectively. Flow rate: 1 mL / min. Injection volume: 1 μL. Split ratio: 1:10. Run time: 15 min. A standard curve was plotted with the concentration of the reference substance (μg / mL) on the horizontal axis and the peak area of ​​each reference substance relative to the internal standard on the vertical axis. The peak area of ​​each short-chain fatty acid in the sample relative to the internal standard was substituted into the regression equation to calculate the concentration.

[0123] 2.2.4 In vitro fermentation of compound Shuanghua decoction

[0124] Feces from the normal control group and the model group were collected and mixed to reduce the influence of individual differences on the experimental results. After mixing, the feces were diluted with PBS buffer at a ratio of 1:10 (g / mL). 0.5 mL of the fecal suspension was injected into a sealed fermentation bottle containing 5 mL of bacterial culture medium. Then, as shown in Table 4 below, different drugs were injected into the corresponding fermentation bottles. After constant temperature incubation at 37°C for 24 h, the fermentation broth was removed and centrifuged at 5000 × g for 5 min at 4°C to collect the supernatant, which was then determined according to the above chromatographic conditions.

[0125] Table 4 Drugs added to in vitro fermentation bottles

[0126]

[0127] 2.3 Statistical analysis

[0128] In this experiment, Excel was used for data collation and analysis, Graphpad Prism 8.0 software was used for data visualization, and IBM SPSS Satistics 23.0 software was used to perform analysis of variance (ANOVA) and independent sample t-test. When P < 0.05, the data between the two groups were considered to be statistically significant.

[0129] 3. Experimental results

[0130] 3.1 Effects of Compound Shuanghua Drink on the Intestinal Microflora of Mice

[0131] 3.1.1 OTU abundance of intestinal flora species

[0132] The number and abundance distribution of OTUs can reflect the species diversity of the intestinal flora. As shown in Table 5, the total number of OTUs in group Z mice was 1214, which ranked first among the three groups. Figure 12 As shown, the number of unique OTUs in group C was 280, the number of unique OTUs in group M was 232, and the number of unique OTUs in group Z was 415. The total number of OTUs in the three groups was 544. The number of OTUs in the model group decreased after modeling, but rebounded after treatment with a medium dose of Compound Shuanghuayin and was higher than that in the normal control group.

[0133] Table 5 OTU analysis of colonic intestinal flora in each group of mice

[0134]

[0135] 3.1.2 Alpha diversity

[0136] In the study of intestinal flora, α diversity is the core indicator for evaluating the diversity of microbial communities, reflecting the richness, evenness and comprehensive distribution characteristics of species, including two dimensions: species richness and species evenness. Figure 13 As shown in the figure, compared with the normal control group, the Chao, PD, Pielou-e, Observed species, Shannon and ACE indexes of the model group were significantly decreased (P < 0.05), indicating that after modeling, the richness, diversity and evolutionary differences of the intestinal flora of mice decreased. Compared with the model group, the Chao, PD, Pielou-e, Observed species, Shannon and ACE indexes of the medium-dose compound Shuanghuayin group were significantly increased (P < 0.05), indicating that after treatment with compound Shuanghuayin, the richness, diversity and evolutionary differences of the intestinal flora of mice increased, reaching the level of normal control group mice. Compound Shuanghuayin can effectively counteract the changes in the intestinal flora of mice caused by cyclophosphamide.

[0137] 3.1.3 β-diversity

[0138] According to the canberra algorithm, PCoA was used to analyze the β diversity of the mouse microbiome at the OTU level. Figure 14 As shown in the figure, the samples of the normal control group, model group, and medium-dose group of Compound Shuanghuayin clustered separately, indicating that the composition of the intestinal flora in each group was similar, and there were certain differences between the samples of each group. The ellipses of the normal control group and the model group did not overlap and were far apart, indicating that the flora of the two groups were quite different, suggesting that cyclophosphamide can cause intestinal flora imbalance in mice. After treatment with Compound Shuanghuayin, the intestinal flora of mice was significantly different from that of the model group and closer to the normal control group, indicating that Compound Shuanghuayin treatment can improve the intestinal flora of mice and approach the normal flora structure, which is consistent with the α diversity results.

[0139] 3.1.4 Differential microbial community analysis

[0140] 3.1.4.1 Analysis of differential bacterial communities at the phylum and genus levels

[0141] Phylum is a high-level unit of microbial classification. The composition at the phylum level reflects the overall structural characteristics of the microbiome and is the basis for the diversity and functional differentiation of the microbiome. The intestinal microbiota of mice in each group was divided into 11 phyla. In the phylum-level study of the intestinal microbiota, the ratio of the abundance of Firmicutes to Bacteroidetes (F / B) is often used as an important indicator for evaluating host metabolic diseases and health. Figure 15As shown in the figure, compared with the normal control group and the medium-dose Compound Shuanghuayin group, the F / B ratio in the model group was significantly decreased (P < 0.001), indicating that the intestinal flora structure of the model group mice was unbalanced at the phylum level and was at risk for metabolic diseases. The results showed that Compound Shuanghuayin could improve the imbalance of the intestinal flora structure of mice at the phylum level, with a significant effect on improving the ratio of Firmicutes and Bacteroidetes.

[0142] Genus is the intermediate unit of species classification, representing a group of microorganisms with common evolutionary characteristics and metabolic functions. Compared with the phylum level, the classification of genus is more detailed; compared with the species level, its annotation accuracy is higher. Figure 16 As shown, Lactobacillus, Muribaculaceae, Staphylococcus, Dubosiella, Bacteroides, Mucispirillum, and Candidatus_Saccharimonas were the predominant bacterial genera in all groups of mice. Lactobacillus was the dominant genus in the normal control group and the medium-dose Compound Shuanghuayin group, while Muribaculaceae was the dominant genus in the model group. Differences in bacterial community structure and abundance were observed among the groups at the genus level.

[0143] 3.1.4.2 Analysis of bacterial species differences

[0144] At the phylum level, based on the relative abundance of different bacterial groups, we can find the bacterial groups with significant differences between groups and conduct statistical analysis. Figure 17 Compared with the normal control group, the relative abundance of Firmicutes, Deferribacterota, and Campilobacterota in the model group was significantly decreased (P < 0.05), while the relative abundance of Bacteroidota was significantly increased (P < 0.05). Compared with the model group, the relative abundance of Firmicutes, Deferribacterota, and Campilobacterota in the medium-dose Compound Shuanghuayin group was significantly increased (P < 0.05), while the relative abundance of Bacteroidota was significantly decreased (P < 0.05). This indicates that after treatment with Compound Shuanghuayin, the intestinal flora of mice improved at the phylum level, becoming closer to that of the normal control group.

[0145] At the genus level, Figure 18Compared with the normal control group, the model group showed a significant decrease in Lactobacillus and Dubosiella (P < 0.05), while Muribaculaceae, Bacteroides, Staphylococcus, Saccharimonas, Mucispirillum, and Helicobacter (P < 0.05) were significantly increased. Compared with the model group, the medium-dose Compound Shuanghuayin group showed a significant increase in Lactobacillus and Dubosiella (P < 0.05), while Muribaculaceae, Bacteroides, Staphylococcus, Saccharimonas, Mucispirillum, and Helicobacter were significantly decreased (P < 0.05). This indicates that after treatment with Compound Shuanghuayin, the intestinal flora of mice improved at the genus level, becoming closer to that of the normal control group.

[0146] 3.1.4.3 PICRUSt2 Gut Microbiome Function Prediction

[0147] PICRUSt2 is a software that predicts the function of 16S amplicon sequencing results. It can perform COG and KEGG functional annotations on intestinal flora OTUs. Figure 19 As shown, the functions of the intestinal flora are mainly enriched in the following pathways: Translation, Ribosomal Structure and Biogenesis, Amino Acid Transport and Metabolism, Carbohydrate Transport and Metabolism, Cell Wall / Membrane / Envelope Biogenesis, Replication, Recombination and Repair, etc. This suggests that the mechanism of action of Compound Shuanghua Drink on mice may involve the above biological functions.

[0148] 3.2 Effects of Compound Shuanghua Drink on Short-Chain Fatty Acids in Mice

[0149] 3.2.1 Methodological validation

[0150] 3.2.1.1 Confirmation of SCFA GC Peaks

[0151] like Figure 20As shown in Table 6, the peak order of the short-chain fatty acid reference solution is acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid. The peak time of the short-chain fatty acids in the mouse colon content sample solution is basically consistent with that of the reference solution.

[0152] Table 6 Retention time of short-chain fatty acids (n = 6)

[0153]

[0154] 3.2.1.2 Precision test

[0155] The same short-chain fatty acid mixed reference solution was injected six times using the chromatographic conditions described in 2.2.3. The peak area of ​​each reference relative to the internal standard was recorded, and the relative standard deviation (RSD) of the relative peak area was calculated. The results are shown in Table 7. The RSD (%) of the relative peak area of ​​all references was less than 5%, indicating good precision of the method and instrument.

[0156] Table 7 Relative peak areas of short-chain fatty acid reference substances

[0157]

[0158] 3.2.1.3 Drawing of the standard curve

[0159] According to the chromatographic conditions in 2.2.3, the peak areas of the mixed solutions of short-chain fatty acid reference substances with different mass concentrations were measured. The mass concentration of the short-chain fatty acid was used as the horizontal axis, and the peak area of ​​each reference substance relative to the internal standard was used as the vertical axis to obtain the regression equation. The results are shown in Table 8. All six short-chain fatty acids showed good linearity, with R 2 All are greater than 0.99.

[0160] Table 8 Regression equation of short-chain fatty acids

[0161]

[0162] 3.2.1.4 Repeatability Experiment

[0163] The colonic contents of the same mouse were divided into six portions, each containing 50 mg. Test solutions were prepared according to the method in 2.2.1. The chromatographic conditions in 2.2.3 were used for analysis. The relative peak areas were recorded, and the RSD values ​​of the short-chain fatty acid content were calculated. The results are shown in Table 9. The RSDs (%) for all six short-chain fatty acids were less than 5%, indicating good reproducibility of the method.

[0164] Table 9 Repeatability experiment (μg / mg)

[0165]

[0166] 3.2.1.5 Stability test

[0167] The same sample solution was divided into five portions and assayed at 0, 4, 8, 12, and 24 hours using the chromatographic conditions outlined in 2.2.3. Calculate the RSD values ​​for the short-chain fatty acid content. As shown in Table 10, the RSD (%) values ​​for the six short-chain fatty acids were all less than 5%, indicating good stability of the sample solution over a 24-hour period.

[0168] Table 10 Stability test (μg / mg)

[0169]

[0170] 3.2.1.6 Accuracy Experiment

[0171] The same colonic contents were divided into seven aliquots, each 50 mg. One aliquot served as a control, and the remaining six aliquots were spiked with 100 μL of an aqueous solution of a short-chain fatty acid reference standard (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid concentrations of 104.9, 99.3, 94.0, 96.0, 93.0, and 94.0 μg / mL, respectively). Then, 900 μL of 0.2% formic acid was added. The test solutions were prepared according to the method in 2.2.1 and assayed according to the chromatographic conditions in 2.2.3. The RSD values ​​of the short-chain fatty acid content and the corresponding recoveries were calculated. The results are shown in Table 11. The recoveries ranged from 100% to 105%, with RSDs (%) less than 5%, indicating good accuracy of the method.

[0172] Table 11 Sample recovery

[0173]

[0174] 3.2.2 Determination of short-chain fatty acid content

[0175] The contents of six short-chain fatty acids in the colonic contents of mice were determined, e.g. Figure 21 As shown, compared with the normal control group, the model group showed a significant decrease in total short-chain fatty acid (SCFA) levels, as well as acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid (P < 0.05). Compared with the model group, the levamisole hydrochloride group and the low-, medium-, and high-dose Compound Shuanghua Drink groups showed a significant increase in SCFA levels (P < 0.05). The levamisole hydrochloride group showed increased acetic acid, propionic acid, and isovaleric acid levels (P < 0.05), while the medium-dose Compound Shuanghua Drink group showed increased acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid levels (P < 0.05), indicating that the medium-dose Compound Shuanghua Drink group was more effective than the low-, high-dose, and levamisole hydrochloride groups. The medium-dose Compound Shuanghua Drink group had the highest SCFA content, with no significant difference from the normal control group, indicating that the SCFA content in the medium-dose Compound Shuanghua Drink group had returned to normal levels.

[0176] 3.3 Intestinal microorganisms utilize Compound Shuanghua Drink through fermentation

[0177] In order to explore the metabolism of Compound Shuanghua Drink in the intestine, in vitro simulated fermentation was used to observe whether Compound Shuanghua Drink could directly improve the ability of intestinal microorganisms to produce short-chain fatty acids. The concentration of short-chain fatty acids in the fermentation broth reflects the activity of intestinal microbial metabolic capacity. Figure 22 As shown, there were no significant differences in total short-chain fatty acids (SCFAs), acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid between Groups 1, 4, and 5, indicating that cyclophosphamide and levamisole hydrochloride do not affect intestinal microbes and that their effects occur through other pathways in the body. Compared with Group 1, SCFAs, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid were significantly increased in Groups 2 and 3 (P < 0.05), indicating that Compound Shuanghua Decoction can affect intestinal microbes through fermentation, enhancing their ability to produce SCFAs. Within a certain concentration range, this was positively correlated with Compound Shuanghua Decoction concentration. There were also significant differences in SCFAs between Groups 1 and 6, possibly due to an imbalance in the intestinal flora structure in the model group, a decrease in beneficial bacteria, and a reduction in SCFA fermentation production. The trends in SCFA content in Groups 6, 7, and 8 were similar to those in Groups 1, 2, and 3, suggesting that Compound Shuanghua Decoction can enhance the metabolic activity of intestinal microbes.

[0178] 3.4 Correlation analysis between intestinal flora and pathological parameters in immunosuppressed mice

[0179] The Meiji BioCloud platform was used to analyze the correlation between bacterial genus abundance and serum inflammatory factor levels (TNF-α, IL-6), immunoglobulin levels (IgM, IgG, SIgA), and short-chain fatty acid content in colonic contents. P < 0.05 was considered statistically significant, and a heat map was drawn.

[0180] 3.4.1 Correlation Analysis between Intestinal Microbiota and Inflammatory Factor Levels

[0181] At the genus level, the abundance of intestinal flora and the levels of serum inflammatory factors were analyzed and a heat map was made. The results are as follows Figure 23As shown, a total of 15 bacterial genera were significantly correlated with inflammatory factor levels (P < 0.05), with 11 and 9 genera significantly correlated with TNF-α and IL-6 levels, respectively. Among them, Prevotellaceae_UCG-001, Helicobacter, Corynebacterium, Desulfovibrio, and Aerococcus were all positively correlated with inflammatory factors, indicating they were potentially harmful bacteria. Bacteroides, Erysipelatoclostridium, Parasutterella, Parabacteroides, and Lactobacillus were all significantly negatively correlated with inflammatory factors, indicating they were potentially beneficial bacteria.

[0182] Compared with the model group, the Lactobacillus genus, which was significantly enriched in the medium-dose group of Compound Shuanghua Decoction, was significantly negatively correlated with the levels of TNF-α and IL-6 (P < 0.05), and may be the key bacterial group for Compound Shuanghua Decoction to regulate the levels of inflammatory factors.

[0183] 3.4.2 Correlation Analysis between Intestinal Microbiota and Immunoglobulin Levels

[0184] At the genus level, the abundance of intestinal flora and immunoglobulin levels were analyzed and heat maps were made. The results are as follows Figure 24 As shown, a total of 17 bacterial genera were significantly correlated with immunoglobulin levels (P < 0.05), with 16, 14, and 14 genera significantly correlated with IgM, IgG, and SIgA levels, respectively. Among them, Gordonibacter, Bacteroides, Erysipelatoclostridium, unclassified_f__Prevotellaceae, Akkermansia, Prevotellaceae_UCG-001, Candidatus_Stoquefichus, unclassified_f__Eggerthellaceae, Parabacteroides, and Parasutterella were all significantly positively correlated with immunoglobulin levels, indicating they were potentially beneficial bacteria. Helicobacter, Jeotgalicoccus, and Corynebacterium were all significantly negatively correlated with immunoglobulin levels, indicating they were potentially harmful bacteria.

[0185] Compared with the model group, the Helicobacter genus, which was significantly reduced in the medium-dose group of Compound Shuanghua Drink, was significantly negatively correlated with the levels of IgM, IgG, and SIgA (P < 0.05), and may be the key bacterial group in regulating the levels of inflammatory factors in Compound Shuanghua Drink.

[0186] 3.4.3 Correlation Analysis between Intestinal Microflora and Short-Chain Fatty Acid Content

[0187] At the genus level, the abundance of intestinal flora and the content of short-chain fatty acids were analyzed and a heat map was made. The results are as follows: Figure 25 As shown, a total of 30 bacterial genera were significantly correlated with short-chain fatty acid content (P < 0.05), with 19, 17, 10, 10, 7, 7, and 10 genera significantly correlated with acetic acid, total short-chain fatty acid, isobutyric acid, propionic acid, valeric acid, butyric acid, and isovaleric acid, respectively. The highest number of bacterial genera was significantly correlated with acetic acid, indicating that acetic acid is most strongly influenced by the intestinal microbiota. Among them, Parasutterella and Prevotellaceae_UCG-001 both showed significant positive correlations with short-chain fatty acid content, indicating they are potentially beneficial bacteria.

[0188] Compared with the model group, the Mucispirillum and Helicobacter genera, which were significantly reduced in the medium-dose group of Compound Shuanghua Decoction, were significantly negatively correlated with the total short-chain fatty acid levels (P < 0.05), and may be the key bacterial groups for Compound Shuanghua Decoction to regulate the content of short-chain fatty acids.

[0189] In summary, to explore the effects of Compound Shuanghua Drink on intestinal flora and short-chain fatty acids, this example used 16S rRNA high-throughput sequencing technology to sequence and analyze the fecal flora of mice; and gas chromatography was used to determine the content of short-chain fatty acids in the colonic contents of mice. The results showed that Compound Shuanghua Drink can effectively counteract the reduction in intestinal flora richness and diversity caused by immunosuppression in mice, improve the flora structure of immunosuppressed mice, making it closer to that of blank control mice, significantly reduce the abundance of harmful bacterial genera such as Muribaculaceae, Bacteroides, Staphylococcus, Saccharimonas, Mucispirillum, and Helicobacterr, significantly increase the abundance of beneficial bacterial genera such as Lactobacillus and Dubosiella, and significantly increase the content of short-chain fatty acids in immunosuppressed mice.

[0190] Example 6: In this example, the compound Shuanghua preparation prepared in Example 2 was concentrated to a crude drug content of 1 g / mL, 0.3% chitosan was added for flocculation for 2 hours, and the mixture was filtered to obtain an oral liquid base. Honey, citric acid, and water were added to the base to adjust the sensory flavor of the oral liquid. The mixture was allowed to stand, filtered, sterilized, and canned. Nine groups of compound Shuanghua oral liquids were obtained. The proportions and sensory evaluation scores of each group are shown in Table 12.

[0191] Table 12 Ratio of raw materials for compound Shuanghua oral solution

[0192]

[0193] Three batches of compound Shuanghua oral liquid were prepared using the fifth formulation. This formulation resulted in a clear, fragrant, slightly sweet, and slightly bitter taste. The relative density, pH value, polysaccharide content, phenolic acid content, and flavonoid content were measured, as shown in Table 13.

[0194] Table 13 Index test of compound Shuanghua oral liquid

[0195]

[0196] The relative density of Compound Shuanghuayin oral liquid was 1.045 g / mL, the pH value was 5.5, the polysaccharide content was 45.26 mg / mL, the phenolic acid content was 13.17 mg / mL, and the flavonoid content was 17.79 mg / mL.

[0197] The above examples are merely illustrative of the present invention and do not limit the scope of protection of the present invention. Any design that is identical or similar to the present invention falls within the scope of protection of the present invention.

Claims

1. A compound double flower preparation, characterized in that: Includes a mixed aqueous extract of honeysuckle, astragalus, dendrobium, poria, tangerine peel and liquorice.

2. A method for preparing a compound double flower preparation, characterized in that: The following steps are involved: Honeysuckle, astragalus, dendrobium, poria, tangerine peel and liquorice are mixed as raw materials; water is added to the raw materials for extraction, and the supernatant is collected by centrifugation to obtain the compound double flower preparation.

3. The method for preparing the compound double flower preparation according to claim 2, wherein: The mass ratio of honeysuckle, astragalus, dendrobium, poria, tangerine peel and liquorice in the raw materials is 10:10:6:6:3:

3.

4. The method for preparing the compound double flower preparation according to claim 2, wherein: The material-liquid ratio of the raw material to water is 20-30 g / mL.

5. The method for preparing the compound double flower preparation according to claim 2, wherein: The extraction temperature is 70~90℃; the extraction time is 60~120 min.

6. A compound double flower preparation oral liquid, characterized in that: The invention comprises citric acid, a sweetener and the compound double flower preparation according to claim 1.

7. Use of the compound double flower preparation according to claim 1 or the compound double flower preparation oral liquid according to claim 6 in the preparation of a product for improving humoral immunity and cellular immunity.

8. Use of the compound Shuanghua preparation according to claim 1 or the compound Shuanghua preparation oral liquid according to claim 6 in a product for improving thymus and spleen damage caused by cyclophosphamide.

9. Use of the compound double flower preparation according to claim 1 or the compound double flower preparation oral liquid according to claim 6 in the preparation of a product for alleviating inflammation in the body caused by cyclophosphamide.

10. Use of the compound double flower preparation according to claim 1 or the compound double flower preparation oral liquid according to claim 6 in the preparation of a product for regulating intestinal flora.

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