Sanjiaofuzheng mixture, its preparation method and identification method

Abstract

This invention discloses a three-gelatin tonic mixture and its preparation and identification methods, comprising Astragalus membranaceus, Atractylodes macrocephala, Angelica sinensis, Salvia miltiorrhiza, Poria cocos, Psoralea corylifolia, deer antler glue, Lycium barbarum, red ginseng, Citrus aurantium, donkey-hide gelatin, tortoise shell glue, and prepared licorice root. Four reagent raw materials are obtained according to the required proportions and percolated to obtain a percolate. Three reagent raw materials are distilled, and the distillate residue is repeatedly decocted with the three reagent raw materials to obtain a decoction. The obtained donkey-hide gelatin, tortoise shell glue, and deer antler glue are dissolved in water. The decoction and percolate are combined, benzoic acid is added, stirred, and allowed to stand. After filtration and precipitation, the mixture is successively mixed with the distillate and the dissolved solution to obtain the three-gelatin tonic mixture. The obtained three-gelatin tonic mixture is identified using red ginseng and Astragalus membranaceus as control methods. Experiments show that it can inhibit the proliferation of tumor cells and alleviate chemotherapy-induced bone marrow suppression and immunodeficiency.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, and in particular to a three-coated tonic mixture and its preparation and identification methods. Background Technology

[0002] Currently, chemotherapy is commonly used to treat tumors. Although chemotherapy can slow down the proliferation of tumor cells, it also has the antagonistic effect of bone marrow suppression and weakened immune function. At present, there is no reagent that can slow down the proliferation of tumor cells while also alleviating the bone marrow suppression and weakened immune function caused by chemotherapy. Summary of the Invention

[0003] The purpose of this invention is to provide a three-coated tonic mixture and its preparation and identification methods, which can slow down the proliferation of tumor cells and alleviate the bone marrow suppression and immune dysfunction caused by chemotherapy.

[0004] To achieve the above objectives, in a first aspect, the present invention provides a three-ingredient tonic compound, which includes Astragalus membranaceus, Atractylodes macrocephala, Angelica sinensis, Salvia miltiorrhiza, Poria cocos, Psoralea corylifolia, deer antler glue, Lycium barbarum, red ginseng, Citrus aurantium, donkey-hide gelatin, tortoise shell glue, and prepared licorice root.

[0005] Secondly, the present invention provides a method for preparing a three-adhesive tonic compound, which, in order to obtain the three-adhesive tonic compound as described in the first aspect, includes the following steps:

[0006] Four reagent raw materials were obtained according to the required ratio, and after percolation, the ethanol in the percolate was drained to obtain the percolate.

[0007] The three reagent raw materials were distilled, and the distilled residue was boiled with the three reagent raw materials multiple times. After filtration and concentration, an aqueous decoction was obtained.

[0008] The obtained donkey-hide gelatin, tortoise shell gelatin, and deer shell gelatin are dissolved in water to obtain a dissolved liquid.

[0009] After combining the decoction and the percolate, benzoic acid was added, stirred, and allowed to stand for 48 hours. After filtration and precipitation, the mixture was then mixed with the distillate and the dissolved liquid in sequence to obtain the three-collagen tonic compound.

[0010] The process involves obtaining four reagent raw materials according to the required proportions, performing percolation, and then draining the ethanol from the percolate to obtain the percolate, which includes:

[0011] Four reagent raw materials were obtained according to the required proportions and then pulverized and filtered to obtain powder with a particle size of 100 mesh. The four reagent raw materials were red ginseng, psoralea corylifolia, salvia miltiorrhiza and poria cocos.

[0012] Based on the percolation method, four powders were percolated using 50% ethanol;

[0013] The ethanol in the collected percolate is removed using a reduced pressure recovery method to obtain percolate.

[0014] The process involves distilling three reagent raw materials, then repeatedly boiling the distilled residue with the three reagent raw materials, filtering and concentrating the liquid to obtain an aqueous decoction, which includes:

[0015] Three reagent raw materials were obtained and distilled to collect the distillate. The three reagent raw materials were Atractylodes macrocephala, Angelica sinensis and Citrus aurantium.

[0016] The dregs obtained after distillation were boiled three times with the three reagent raw materials. The decoctions obtained from each boiling were collected, mixed, filtered and concentrated in sequence to obtain a water decoction. The three reagent raw materials were roasted licorice, astragalus and wolfberry.

[0017] The content of benzoic acid is 3g.

[0018] The contents of the three decoctions were 4000ml for the first decoction, 3500ml for the second decoction, and 3000ml for the third decoction.

[0019] Thirdly, the present invention provides a method for identifying the Sanjiao Fuzheng compound, which is used to identify the Sanjiao Fuzheng compound prepared by the preparation method described in the second aspect, including a red ginseng reference identification method and an astragalus reference identification method.

[0020] The red ginseng identification method includes the following steps:

[0021] The prepared three-coated tonic mixture was mixed with ethanol, filtered and concentrated in sequence, and then chloroform was added for shaking extraction. After the chloroform reagent was removed, the evaporated residue was dissolved and filtered with n-butanol. The filtrate obtained was the test solution.

[0022] Red ginseng reference material was extracted with n-butanol by heating and reflux for 1 hour, and the solution of reference material was obtained after filtration and concentration.

[0023] The test solution and the reference medicinal material solution were tested by thin-layer chromatography to complete the identification.

[0024] The identification process involves performing thin-layer chromatography on the test solution and the reference medicinal material solution to achieve the following:

[0025] Based on thin-layer chromatography, the test solution and the reference medicinal material solution were respectively taken and spotted onto the same silica gel G thin-layer plate. After development with the developing solvent, the plates were removed and dried. A mixture of sulfuric acid and methanol was sprayed on the plates, and the plates were heated at 105°C until the spots were clearly visible. The plates were then examined under ultraviolet light to complete the identification.

[0026] The red ginseng identification method includes the following steps:

[0027] After the prepared three-coated stabilizing agent was evaporated to dryness, the residue was mixed with ethanol, filtered and evaporated to dryness in sequence. Then, it was dissolved in sodium hydroxide solution, and after adjusting the pH value, ethyl acetate solution was added and evaporated to dryness again. The residue was dissolved in ethyl acetate to obtain the test solution.

[0028] Astragalus membranaceus reference material was extracted with ethanol by heating and reflux for 20 minutes, and after filtration and evaporation of the filtrate, the reference material solution was obtained.

[0029] The test solution and the reference medicinal material solution were tested by thin-layer chromatography to complete the identification.

[0030] The identification process involves performing thin-layer chromatography on the test solution and the reference medicinal material solution to achieve the following:

[0031] Based on thin-layer chromatography, the test solution and the reference medicinal material solution were respectively taken and spotted onto the same silica gel G thin-layer plate. After development with developing solvent, the plate was removed, dried, and fumigated in ammonia vapor. Finally, it was examined under ultraviolet light to complete the identification.

[0032] This invention discloses a three-ingredient tonic compound, its preparation method, and its identification method. The three-ingredient tonic compound comprises Astragalus membranaceus, Atractylodes macrocephala, Angelica sinensis, Salvia miltiorrhiza, Poria cocos, Psoralea corylifolia, deer antler glue, Lycium barbarum, red ginseng, Citrus aurantium, donkey-hide gelatin, tortoise shell glue, and prepared licorice root. Four reagent raw materials are obtained according to the required proportions. After percolation, the ethanol in the percolate is removed to obtain the percolate. Three reagent raw materials are distilled, and the distilled residue is repeatedly decocted with the three reagent raw materials. After filtration and concentration, an aqueous decoction is obtained. The obtained donkey-hide gelatin... Tortoise shell glue and deer antler glue were dissolved in water to obtain a dissolved solution. The decoction and the percolate were combined, benzoic acid was added, and the mixture was stirred and allowed to stand for 48 hours. After filtration and precipitation, the mixture was successively mixed with distillate and the dissolved solution to obtain the Three-Glue Tonifying Compound. The Three-Glue Tonifying Compound was identified using the red ginseng and astragalus identification methods, and both methods met the requirements. At the same time, during the experiment, it was able to inhibit the proliferation of tumor cells and alleviate the problems of bone marrow suppression and low immune function caused by chemotherapy. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram of the preparation steps of a three-adhesive tonic mixture provided by the present invention.

[0035] Figure 2 This is a schematic diagram of the identification method of the Sanjiao Fuzheng Compound based on the red ginseng comparison identification method provided by the present invention.

[0036] Figure 3 This is a schematic diagram of the identification method of the Sanjiao Fuzheng Compound based on the Astragalus membranaceus comparison identification method provided by the present invention.

[0037] Figure 4 This is the TLC diagram of the Sanjiao Fuzheng Compound based on the red ginseng comparison identification method provided by the present invention.

[0038] Figure 5 This is the TLC diagram of the Sanjiao Fuzheng Compound based on the Astragalus membranaceus control identification method provided by the present invention.

[0039] Figure 6 This is a comparison chart of tumor volume in different groups of mice provided by the present invention.

[0040] Figure 7 This is a comparison chart of tumor volume in the blank group, low, medium and high concentration groups of the compound preparation, and the etoposide group provided by this invention. Detailed Implementation

[0041] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0042] In the description of this invention, it should be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, in the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0043] This invention provides a three-ingredient tonic compound, comprising Astragalus membranaceus, Atractylodes macrocephala, Angelica sinensis, Salvia miltiorrhiza, Poria cocos, Psoralea corylifolia, deer antler glue, Lycium barbarum, red ginseng, Citrus aurantium, donkey-hide gelatin, tortoise shell glue, and prepared licorice root.

[0044] Please see Figure 1 This invention provides a method for preparing a three-coated tonic mixture, comprising the following steps:

[0045] S101. Obtain four reagent raw materials according to the required ratio, and after percolation by percolation, remove the ethanol from the percolate to obtain the percolate.

[0046] Specifically, according to the required proportions, red ginseng, psoralea corylifolia, salvia miltiorrhiza, and poria cocos are pulverized and filtered through a 100-mesh sieve to obtain four powders with a particle size of 100 mesh. Then, following the percolation method for fluid extracts and extracts, 50% ethanol is used as a solvent for percolation. During percolation, the percolate is collected. After percolation, the collected percolate is subjected to reduced pressure to recover the ethanol from the percolate, thus obtaining the percolate.

[0047] S102. Obtain three reagent raw materials and distill them. Then, boil the distilled residue with the three reagent raw materials multiple times, filter and concentrate to obtain a decoction.

[0048] Specifically, Atractylodes macrocephala, Angelica sinensis, and Citrus aurantium are obtained based on the required proportions, mixed with water, and distilled. The distillate is collected, which is the liquid containing medicinal components that evaporates during the distillation process. The residue after distillation is decocted three times with separately obtained Glycyrrhiza uralensis, Astragalus membranaceus, and Lycium barbarum. The volume of the first decoction is 4000ml, the second is 3500ml, and the third is 3000ml. The decoction obtained from each decoction is collected. The three decoctions are mixed, filtered, and concentrated to obtain an appropriate amount of aqueous decoction.

[0049] S103. Add water to the obtained donkey-hide gelatin, tortoise shell gelatin and deer glue to dissolve them, and obtain a dissolved liquid.

[0050] Specifically, the donkey-hide gelatin, tortoise shell gelatin, and deer shell gelatin obtained according to the formula are mixed and then an appropriate amount of water is added to dissolve them, resulting in a dissolved liquid.

[0051] S104. After combining the decoction and the percolate, add benzoic acid, stir and let stand for 48 hours. After filtering and precipitating, mix with the distillate and the melting solution in sequence to obtain the three-collagen strengthening agent.

[0052] Specifically, the decoction and percolate are mixed, and 3g of benzoic acid is added and stirred thoroughly. After standing for 48 hours, the precipitate is removed, and the distillate and molten liquid are added and stirred again. Then water is added to 1000ml and mixed well to obtain the Sanjiao Fuzheng Mixture. The prepared Sanjiao Fuzheng Mixture is a brownish-brown viscous liquid with an aromatic odor and a slightly bitter, slightly sweet, and slightly spicy taste.

[0053] The prepared Sanjiao Fuzheng Mixture has a relative density of not less than 1.07 and a pH value of approximately 5.0-7.0. It nourishes the kidneys and replenishes essence, invigorates qi and blood, and strengthens the spleen. It is used for patients with leukopenia, weakened immune function, and mid-to-late stage tumors caused by radiotherapy and chemotherapy. It is generally taken orally, three times a day, 30-50 ml each time.

[0054] Please see Figure 2 This invention provides a method for identifying a three-coated tonic mixture, comprising the following steps:

[0055] S201. The prepared three-coated tonifying agent is mixed with ethanol in sequence, filtered and concentrated, then chloroform is added for shaking extraction, and after the chloroform reagent is removed, the evaporated residue is dissolved and filtered with n-butanol. The filtrate obtained is the test solution.

[0056] S202. Red ginseng reference material was heated and refluxed with n-butanol for 1 hour, and after filtration and concentration, the reference material solution was obtained.

[0057] S203. Based on thin-layer chromatography, the test solution and the reference medicinal material solution are tested respectively to complete the identification.

[0058] In this embodiment, 30 ml of the prepared three-coated tonic mixture was taken, 100 ml of ethanol was added, the mixture was shaken thoroughly, filtered, and the filtrate was concentrated to 10 ml. Then, 30 ml of chloroform was added, the mixture was shaken and extracted, the chloroform solution was separated, evaporated to dryness, and 5 ml of n-butanol was added to the residue obtained after evaporation to dissolve the residue. The mixture was filtered, and the filtrate was used as the test solution.

[0059] Take another 5g of red ginseng reference material, add 50ml of n-butanol, heat and reflux for 1 hour, filter, concentrate the filtrate to about 5ml, and use it as the reference material solution.

[0060] The experiment was conducted using thin-layer chromatography. 5 μl of each of the two solutions were spotted separately onto the same silica gel G thin-layer plate. Cyclohexane-acetone (2:1) was used as the developing solvent. After development, the plate was removed, dried, sprayed with sulfuric acid-methanol (1:1), and heated at 105℃ until the spots were clearly visible. The plate was then examined under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at positions corresponding to the color of the reference medicinal material. The comparison results are as follows: Figure 4 As shown.

[0061] Please see Figure 3 This invention provides a method for identifying a three-coated tonic mixture, comprising the following steps:

[0062] S301. After the prepared three-adhesive strengthening agent is evaporated to dryness, the residue is mixed with ethanol, filtered and evaporated to dryness in sequence. Then, it is dissolved with sodium hydroxide solution, and after adjusting the pH value, ethyl acetate solution is added and evaporated to dryness again. The residue is dissolved with ethyl acetate to obtain the test solution.

[0063] S302. Astragalus reference material was heated and refluxed with ethanol for 20 minutes, and after filtration and evaporation of the filtrate, the reference material solution was obtained.

[0064] S303. Based on thin-layer chromatography, the test solution and the reference medicinal material solution are tested respectively to complete the identification.

[0065] In this embodiment, 10 ml of the prepared three-coated tonic mixture was evaporated to dryness. 20 ml of ethanol was added to the residue to dissolve the evaporated three-coated tonic mixture. After standing for 20 minutes, the mixture was filtered. The filtrate was evaporated to dryness. 15 ml of 0.3% sodium hydroxide solution was added to the residue to dissolve it. The mixture was filtered. The pH of the filtrate was adjusted to 5-6 with dilute hydrochloric acid. Then, 15 ml of ethyl acetate was added, and the mixture was shaken to extract. The ethyl acetate solution was separated, and an appropriate amount of anhydrous sodium sulfate was added. The mixture was filtered, and the filtrate was evaporated to dryness. 1 ml of ethyl acetate was added to the residue to dissolve it, which was used as the test solution.

[0066] Take another 2g of Astragalus membranaceus reference material, add 30ml of ethanol, heat under reflux for 20 minutes, filter, evaporate the filtrate to dryness, and prepare the reference material solution in the same way.

[0067] The experiment was conducted using thin-layer chromatography. 10 μl of each of the two solutions were spotted onto the same silica gel G thin-layer plate using chloroform-methanol (10:1) as the developing solvent. After development, the plate was removed, dried, and fumigated in ammonia vapor. The chromatogram of the test sample was then examined under ultraviolet light (365 nm). Fluorescent main spots of the same color appeared at the corresponding positions as in the chromatogram of the reference medicinal material. The comparison results are as follows: Figure 5 As shown.

[0068] The test results obtained after testing this reagent are shown in Table 1.

[0069] Table 1. Test results of the three-coated tonic mixture

[0070]

[0071] As shown in Table 1, the three-coated tonic mixture prepared by the method defined in this invention meets the relevant national standards.

[0072] To test the efficacy of the three-coated tonic compound prepared by the method of the present invention, two experimental methods were used: the first was an experiment to antagonize the toxic side effects of radiotherapy and chemotherapy on mice, and the second was an experiment to test its anti-tumor effects.

[0073] In the first experiment, a low white blood cell model and an immunosuppression model were constructed using mice to observe whether the Sanjiao Fuzheng compound had the effect of antagonizing the toxic side effects of radiotherapy and chemotherapy, and the magnitude of their effects.

[0074] 1. Materials and Methods

[0075] 1.1 Instruments and Reagents

[0076] 1.1.1 Instruments

[0077] Model 722 UV-Vis Spectrophotometer

[0078] 1.1.2 Experimental drugs and animals

[0079] The Sanjiao Fuzheng Mixture is a brownish-brown extract, with each gram containing 5g of raw herbs. It is prepared with physiological saline to the required concentration before use. It also contains Compound Donkey-hide Gelatin Oral Solution, Cyclophosphamide for Injection, Levamisole Hydrochloride Tablets, and NIH mice, half male and half female, weighing 20-24g.

[0080] 1.2 Methods

[0081] 1.2.1 Experimental Grouping

[0082] Mice were randomly divided into 9 groups of 10 mice each. The hypoleukocyte model groups were: Group A (normal control), Group B (model control), Group C (CY+10ml / kg compound donkey-hide gelatin oral liquid), and Groups D, E, and F (CY+1.63g / kg). The hypoleukocyte model groups were: Group A (normal control), Group B (CY+6.5g / kg, CY+26g / kg No. 1 Sanjiao Fuzheng Heji group), and Groups G, H, and I (CY+1.25g / kg, CY+5.0g / kg, CY+20g / kg No. 2 Sanjiao Fuzheng Heji group). (The dosage was calculated by converting the human daily dose to mouse dosage as the medium dose, then decreasing the dosage by a factor of four to obtain the small dose, and increasing it by a factor of four to obtain the large dose). The immunosuppression model groups were: Group A (normal control), Group B (model control), and Group C (CY+0.025g / kg). LMS group, D, E, F groups CY+1.63g / kg, CY+6.5g / kg, CY+26g / kg; No. 1 Three-Glue Strengthening Mixture group, G, H, I groups CY+1.25g / kg, CY+5.0g / kg, CY+20g / kg; No. 2 Three-Glue Strengthening Mixture group.

[0083] 1.2.2 Effects on peripheral blood leukocytes, bone marrow, and CFU-S in CY-induced hypoleukocytosis model mice

[0084] Mice in all groups (except group A) were intraperitoneally injected with CY 0.1 g / kg once daily for 3 consecutive days to establish a mouse model of low white blood cell count. Two hours after the last administration, the drugs were administered by gavage once daily for 6 days. White blood cell counts were examined by collecting tail vein blood on days 3 and 6. Mice were then euthanized by cervical dislocation, and the right femur was dissected and flushed with saline to extract all bone marrow. A single-cell suspension was prepared, counted, and a certain amount was injected into the tail vein. 60 In mice irradiated with a lethal dose of 7.50 Gy of CO2 γ-rays, the spleens were harvested after 8 days, fixed with Bouin's solution, and the number of colonies per spleen was counted visually. Based on the number of nucleated cells per bone segment and the number of CFU-S produced by the number of intravenously injected nucleated cells, the number of femoral CFU-S in each group was calculated, and the colony survival rate was then determined.

[0085] The calculation formula is: Colony survival rate = Number of femoral CFU-S in the treatment group / Number of femoral CFU-S in the control group

[0086] 1.2.3 Effects on phagocytic function of the reticuloendothelial system in CY-induced immunosuppressed mice

[0087] Mice in all groups (except group A) were subcutaneously injected with CY 0.08 g / kg once daily for 5 consecutive days to induce an immunosuppressive model. Simultaneously, all groups were administered the drug via gavage once daily for 6 days. One hour after the last administration, each mouse was injected via tail vein with 10 ml / kg of Indian ink (diluted 5 times with physiological saline). At 1 and 5 minutes post-injection, 20 μl of blood was collected from the orbital vein and added to 2 ml of 0.1% solution. The absorbance was measured at 680 nm using a 722 spectrophotometer, and the clearance index K was calculated. After conversion based on body weight and liver / spleen weight, the phagocytic index a was determined.

[0088] 1.2.4 Acute toxicity study of mice administered via gavage

[0089] Forty NIH mice, half male and half female, were fed for 3 days under laboratory conditions (25°C) and fasted (but allowed water) for 8 hours before the experiment. They were randomly divided into two groups. Two solutions of the Sanjiao Fuzheng Heji (a traditional Chinese medicine formula) were prepared at a concentration of 5 g / ml (maximum solubility) using 0.9% physiological saline. The solutions were administered intragastric gavage (ig) at a dose of 0.1 mg / 10 g body weight every 2 hours for 3 consecutive times, for a total dose of 150 g / kg body weight (equivalent to 23 times the clinical human dose). Immediately after administration, the general condition of both groups of mice was observed, including activity level, gait, respiration, defecation, and fur. The number of deaths was recorded, and the mice were observed continuously for 14 days.

[0090] 1.2.5 Statistical Analysis

[0091] Statistical analysis and group-related t-tests were performed on all data using SAS 9.1 for Windows software.

[0092] 2 Results

[0093] 2.1 Effects on peripheral leukocytes, bone marrow, and CFU-S in mice

[0094] On the third day of administration, the peripheral blood leukocyte count in the model control group was significantly lower than that in the normal control group (P<0.01), indicating successful model establishment. The leukocyte count in the CY + Sanjiao Fuzheng Heji group was significantly higher than that in the model control group, indicating that the drug has a leukocyte-increasing effect (Table 2). The number of nucleated cells and CFU-S in the bone marrow of the model control group were significantly lower than those in the normal control group (P<0.05), indicating that CY inhibits bone marrow hematopoietic function. The number of nucleated cells, CFU-S, and survival rate in the bone marrow of the CY + Sanjiao Fuzheng Heji group were significantly higher than those in the model control group, indicating that the drug can restore bone marrow hematopoietic function (Table 3).

[0095] Table 2 Comparison of white blood cell counts in mice of different groups on days 3 and 6 after drug administration.

[0096]

[0097] Compared with the normal control group, ΔP<0.05, ΔΔP<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0098] Table 3. Effects of the Sanjiao Fuzheng Mixture on mouse bone marrow and CFU-S.

[0099]

[0100]

[0101] Compared with the normal control group, ΔP<0.05; compared with the model control group, *P<0.05, **P<0.01.

[0102] 2.2 Effects on phagocytic function of the mouse reticuloendothelial system

[0103] The carbon clearance capacity of the model control group was significantly lower than that of the normal control group (P<0.01), indicating that CY inhibits the body's immune function; the CY + Sanjiao Fuzheng Heji group significantly increased the K and a values, indicating that the drug can improve the reticuloendothelial system's ability to clear foreign bodies, that is, enhance non-specific immune function, as shown in Table 4.

[0104] Table 4. Effects of the Sanjiao Fuzheng Mixture on Phagocytic Function of the Reticuloendothelial System in Mice

[0105]

[0106]

[0107] Compared with the normal control group, ΔP<0.05, ΔΔP<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0108] 2.3 Results of acute toxicity test in mice administered via gavage

[0109] After administration, both groups of mice were in good general condition, with normal activity levels, even breathing, and no significant abnormalities in gait, muscle tone, urination, defecation, or fur. No convulsions occurred, and feeding was normal immediately after administration and thereafter. All tested mice survived the observation period. Therefore, the maximum tolerated dose for NIH mice in this experiment was 150 g / kg body weight.

[0110] In summary, the Sanjiao Fuzheng Mixture No. 1 and No. 2, as well as Compound Donkey-hide Gelatin Oral Liquid, all have the effect of increasing peripheral leukocytes, promoting the proliferation of nucleated cells and CFU-S in bone marrow, and enhancing the phagocytic function of the reticuloendothelial system in immunocompromised mice, with No. 1 showing the best effect. There was no significant difference in the enhancing effect of Sanjiao Fuzheng Mixture No. 1 and No. 2, and Levamisole Hydrochloride Tablets on the reticuloendothelial system function in mice; the medium dose is close to the optimal dose; the maximum tolerated dose in mice is 150 g / kg body weight. It can be concluded that the Sanjiao Fuzheng Mixture can antagonize the bone marrow suppression and immunocompromise induced by radiotherapy and chemotherapy in mice, and is safe and without toxic side effects, laying the foundation for clinical promotion.

[0111] In the second experiment, a low white blood cell model and an immunosuppression model were constructed using mice to observe whether the Sanjiao Fuzheng compound had the effect of antagonizing the toxic side effects of radiotherapy and chemotherapy, and the magnitude of their effects.

[0112] 1. Materials and Methods

[0113] 1.1 Experimental drugs.

[0114] The Sanjiao Fuzheng compound is a brownish-brown extract, 1.39 g / L, which should be diluted with physiological saline to the required concentration before use. Positive control group: Etoposide soft capsules, diluted with polyethylene glycol 400 to the required concentration before use.

[0115] 1.2 Laboratory animals.

[0116] KM mice, half male and half female, weighing 20-25g, were all housed in a constant temperature, constant humidity, and sterile purification barrier system.

[0117] 1.3 In vitro drug intervention trials for cancer cells

[0118] 1.3.1 MTT assay for detecting the anticancer effect of drugs

[0119] Human lung adenocarcinoma A549 cells, breast cancer MCF-7 cells, and liver cancer HepG2 cells in logarithmic growth phase were collected, and blank control groups were set up for each cell type. Drug sensitivity tests were performed on each cell type. The cell concentration was adjusted to 2 x 10⁻⁶ cells / cells. 4 mL -1 4 x 10 mm per hole 3 Cells were seeded in 96-well plates. After culturing at 37℃ and 5% CO2 for 4 hours, Sanjiao Fuzheng Mixture No. 1 and No. 2 were added at five different concentration gradients. A blank control group was also set up. Cell viability was measured by MTT assay at 24, 48, and 72 hours after drug administration in each experimental group, with a blank control group provided to clarify the inhibitory effect of Sanjiao Fuzheng Mixture on different tumor cell lines (as sensitive cells), the optimal drug concentration, and the optimal duration of efficacy. The cell proliferation inhibition rate was calculated.

[0120] 1.3.2 Tumor Cell Apoptosis Assay

[0121] A549 cells were loaded at a rate of 1 x 10 6 Cells were seeded at a concentration of 1 ml in culture flasks, with a culture medium volume of 1 ml. After 4 hours, 1 ml of the A / 3 concentration of the mixture No. 1 and No. 2 was added. After 72 hours, cells were digested, and apoptosis was detected by flow cytometry using Annexin V-FITC / PI double staining. Experimental groups: A. Blank control group (Simple0), B. Three-gelatin combination No. 1 group (Simple1), C. Three-gelatin combination No. 2 group (Simple2).

[0122] 1.4 Tumor-bearing KM mouse model experiment

[0123] 1.4.1 Tumor Inhibition Test of the Three-Glue Tonifying Mixture

[0124] Mice were randomly divided into four groups: ① blank control group, ② Sanjiao Fuzheng Heji No. 1 group, ③ Sanjiao Fuzheng Heji No. 2 group, and ④ etoposide group, with 5 mice in each group, for a total of 20 mice. The stock solution A was 1.39 g / ml. Three days before inoculation with sensitive tumor cells, mice in groups ② and ③ were administered Sanjiao Fuzheng Heji No. 1 and No. 2 (medium concentration, 0.46 g / ml) by gavage, respectively. The remaining groups were fed normally. Three days later, H22 tumor cells in the logarithmic growth phase were collected and the cell density was adjusted to 10-1. 7 mL -1 The drug was subcutaneously injected into the scapular region of mice in each group. After inoculation, mice were housed in a sterile, controlled environment with constant temperature (25±2℃), constant humidity (45%–50%), and a sterile purification barrier. Groups ② and ③ continued to receive the same concentration of the three-component tonifying compound via gavage after inoculation. The etoposide group received 4 mg / kg / day via gavage, once daily for 5 consecutive days. Mice were sacrificed after 30 days for observation. Observation indicators: histopathological confirmation of tumors (tumor tissue pathological sections, HE staining examination); tumor incidence rate in each group; average number of tumors in each group of tumor-bearing mice; average tumor volume in each group of tumor-bearing mice; metastasis and organ status (liver, lung, stomach tissue) in different treatment groups.

[0125] 1.4.2 Tumor Inhibition Test of Different Concentrations of No. 2 Three-Glue Tonifying Mixture

[0126] The animals were randomly divided into five groups: ① blank control group, ② low concentration of Sanjiao Fuzheng Heji No. 2 group, ③ medium concentration of Sanjiao Fuzheng Heji No. 2 group, ④ high concentration of Sanjiao Fuzheng Heji No. 2 group, and ⑤ ethoprofen group, with 10 animals in each group, for a total of 50 animals. Logarithmic growth phase H22 tumor cells were collected and the cell density was adjusted to 10⁻¹mL. These cells were then subcutaneously inoculated into the scapular region of mice in each group. After inoculation, mice were housed in a sterile environment with constant temperature (25±2℃), constant humidity (45%–50%), and a sterile purification barrier. On the day of inoculation, mice in groups ② and ③ were administered three different concentrations of No. 2 Sanjiao Fuzheng Heji (a traditional Chinese medicine formula) via gavage: low concentration A / 5, 0.28 g / ml; medium concentration A / 3, 0.46 g / ml; and high concentration 2A / 3, 0.92 g / ml, respectively. The remaining groups were fed normally. The etoposide group was administered 4 mg / kg / day via gavage once daily for 5 consecutive days. The remaining mice were sacrificed after 30 days for observation. Observational indicators and data analysis methods were the same as above.

[0127] 2 Results

[0128] After several experiments, the MTT results showed that the Sanjiao Fuzheng Mixture had a significant inhibitory effect on the growth of tumor cells, with Mixture No. 2 being slightly more effective than Mixture No. 1; the effect on HepG2 liver cancer cells was slightly better than the other two cell types.

[0129] Apoptosis detection experiments showed that early apoptosis and necrosis of tumor cells were significantly higher after the application of the compound preparation than in the control group.

[0130] In tumor-bearing mouse experiments, it was confirmed that the tumor formation rate in the drug-treated group was not significantly different from that in the control group, but the tumor volume and weight were significantly smaller than those in the blank control group, and the effect of compound No. 2 was better than that of compound No. 1. However, the tumor-suppressing effect of the compound group was worse than that of the etoposide group. Figure 6 As shown. The results of the tumor-suppressing experiment with three concentration groups of Compound No. 2 showed that the high concentration was more effective than the low concentration, but less effective than the etoposide group, as... Figure 7 As shown.

[0131] Regarding the NF-κB detection results, the NF-κB gene level in the combination therapy group was significantly lower than that in the control group, indicating that the drug can effectively inhibit the expression of intracellular NF-κB.

[0132] In conclusion, the Sanjiao Fuzheng compound has a significant inhibitory effect on the proliferation of tumor cells both in vivo and in vitro, as well as at the NF-κB gene level. It can be promoted and applied in clinical practice as an adjuvant anti-tumor drug.

[0133] The above description discloses only one preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for identifying a three-coated tonic mixture, used to identify the three-coated tonic mixture, characterized in that, The three-coated tonic compound is prepared from Astragalus membranaceus, Atractylodes macrocephala, Angelica sinensis, Salvia miltiorrhiza, Poria cocos, Psoralea corylifolia, deer antler glue, Lycium barbarum, red ginseng, Citrus aurantium, donkey-hide gelatin, tortoise shell glue, and prepared licorice root; the identification methods include the red ginseng reference identification method and the Astragalus membranaceus reference identification method; The red ginseng identification method includes the following steps: The prepared three-coated tonic mixture was mixed with ethanol, filtered and concentrated in sequence, and then chloroform was added for shaking extraction. After the chloroform reagent was removed, the evaporated residue was dissolved and filtered with n-butanol. The filtrate obtained was the test solution. Red ginseng reference material was extracted with n-butanol by heating and reflux for 1 hour, and the solution of reference material was obtained after filtration and concentration. Based on thin-layer chromatography, the test solution and the reference medicinal material solution were respectively taken and spotted on the same silica gel G thin-layer plate. After development with the developing solvent, the plate was removed and dried. Then, a mixture of sulfuric acid and methanol was sprayed on the plate, and the plate was heated at 105°C until the spots were clearly visible. The plates were then examined under ultraviolet light to complete the identification. The Astragalus membranaceus identification method includes the following steps: After the prepared three-coated stabilizing agent was evaporated to dryness, the residue was mixed with ethanol, filtered and evaporated to dryness in sequence. Then, it was dissolved in sodium hydroxide solution, and after adjusting the pH value, ethyl acetate solution was added and evaporated to dryness again. The residue was dissolved in ethyl acetate to obtain the test solution. Astragalus membranaceus reference material was extracted with ethanol by heating and reflux for 20 minutes, and after filtration and evaporation of the filtrate, the reference material solution was obtained. The test solution and the reference medicinal material solution were tested separately using thin-layer chromatography, including: Based on thin-layer chromatography, the test solution and the reference medicinal material solution were respectively taken and spotted onto the same silica gel G thin-layer plate. After development with developing solvent, the plate was removed, dried, and fumigated in ammonia vapor. Finally, it was examined under ultraviolet light to complete the identification.

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