Application of Yupingfeng granular polysaccharide in preparation of medicine for treating damage caused by cyclophosphamide

Abstract

The invention provides application of Yupingfeng granular polysaccharide, and particularly relates to application of the Yupingfeng granular polysaccharide in preparation of a medicine for preventing damage caused by cyclophosphamide. The Yupingfeng granule polysaccharide disclosed by the invention is used as an active ingredient of a medicine, can relieve chemotherapy injury caused by cyclophosphamide, such as weight loss, spleen injury and thymus injury, and can also play a role in relieving and even reversing the abnormal number of leukocytes.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical uses, and particularly to the use of polysaccharides from Yupingfeng granules in the preparation of a medicament for treating cyclophosphamide-induced injury. Background Art

[0002] Chemotherapy is one of the main and very effective means for treating cancer at present. Due to the broad-spectrum and non-selective nature of the action targets of chemotherapy drugs, while killing tumor cells, they will also kill some rapidly proliferating normal tissue cells, resulting in serious side effects such as immunosuppression, liver injury, gastrointestinal injury, myelosuppression, etc., causing damage to the patient's body. At present, there is no clear therapeutic drug for these chemotherapy-induced injuries, and usually patients can only relieve the pain through antiemetic, antidiarrheal, and liver-protecting drugs.

[0003] Yupingfeng granules are composed of three medicinal materials, Astragalus membranaceus, Atractylodes macrocephala, and Saposhnikovia divaricata, and are a classic prescription in traditional Chinese medicine for strengthening the healthy qi and consolidating the body surface, with the effects of replenishing qi, strengthening the spleen, and securing the exterior to stop sweating. Yupingfeng granules are widely used clinically and are involved in diseases such as the respiratory system, urinary system, digestive system, nervous system, as well as skin diseases and blood diseases.

[0004] The polysaccharides from Yupingfeng granules are obtained by dialysis of the extracts of the medicinal materials of Yupingfeng granules. In recent years, polysaccharide components have been proven to have various physiological activities, such as enhancing immunity, antioxidation, treating non-alcoholic fatty liver, liver fibrosis, and anti-intestinal mucosal injury, etc. Therefore, it is speculated that the polysaccharides from Yupingfeng granules have a certain improvement and alleviating effect on a series of adverse reactions caused by chemotherapy. Developing the polysaccharides from Yupingfeng granules as a medicament for treating post-chemotherapy injury has good prospects, but there is currently no report on the use of the polysaccharides from Yupingfeng granules for treating post-chemotherapy injury.

[0005] Therefore, there is an urgent need in this field to develop a Yupingfeng granule polysaccharide drug that has a certain improvement and alleviating effect on a series of adverse reactions caused by chemotherapy. Summary of the Invention

[0006] The object of the present invention is to develop a Yupingfeng granule polysaccharide drug that has a certain improvement and alleviating effect on a series of adverse reactions caused by chemotherapy, and particularly relates to the use of the polysaccharides from Yupingfeng granules in the preparation of a medicament for treating cyclophosphamide-induced injury.

[0007] In the first aspect of the present invention, there is provided a use of the polysaccharides from Yupingfeng granules for preparing a pharmaceutical composition for treating injury caused by administering a chemotherapy drug.

[0008] In another preferred example, the chemotherapy drug is a chemotherapy drug such as cyclophosphamide, dexamethasone, etc.

[0009] In another preferred example, the molecular weight of the polysaccharides from Yupingfeng granules is 1047 Da - 832332 Da.

[0010] In another preferred example, the injury has one or more characteristics selected from the group consisting of:

[0011] (i) Weight loss;

[0012] (ii) Spleen injury;

[0013] (iii) Thymus injury; and

[0014] (iv) Elevated white blood cell count.

[0015] In another preferred example, the total sugar content in the polysaccharide of Yupingfeng granules is > 85 wt%, preferably > 90 wt%, more preferably > 94 wt%, such as 92 - 98 wt%.

[0016] In another preferred example, the polysaccharide of Yupingfeng granules contains monosaccharides selected from the group consisting of: mannose, ribose, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, or a combination thereof.

[0017] In another preferred example, in the polysaccharide of Yupingfeng granules, the molar ratio of glucose to arabinose is 1:1 - 2.

[0018] In another preferred example, in the polysaccharide of Yupingfeng granules, the molar ratio of mannose to arabinose is 1:5 - 10.

[0019] In another preferred example, in the polysaccharide of Yupingfeng granules, the molar ratio of ribose to arabinose is 1:4 - 8.

[0020] In another preferred example, in the polysaccharide of Yupingfeng granules, the molar ratio of rhamnose to arabinose is 1:10 - 20.

[0021] In another preferred example, in the polysaccharide of Yupingfeng granules, the molar ratio of galacturonic acid to arabinose is 1:25 - 35.

[0022] In another preferred example, in the polysaccharide of Yupingfeng granules, the molar ratio of xylose to arabinose is 1:25 - 35.

[0023] In another preferred example, the total protein content in the polysaccharide of Yupingfeng granules is 3 - 5 wt%, preferably 4 - 4.5 wt%.

[0024] In another preferred example, the pharmaceutical composition is administered to a subject in need.

[0025] In another preferred example, the subject is a patient who has been administered cyclophosphamide.

[0026] In another preferred embodiment, the pharmaceutical composition is used to relieve or improve the side effects caused by chemotherapy drugs in the administered subject; preferably, the side effects include: weight loss, or damage to the spleen and thymus; more preferably, the alleviation means that compared with the patients in the control group (i.e., those administered cyclophosphamide but not the pharmaceutical composition), the weight of the subject increases or the indicators of spleen and thymus damage decrease, and the difference is significant.

[0027] In another preferred embodiment, the pharmaceutical composition can improve or reverse the decrease in the number of white blood cells in the administered subject, where the subject is a patient who has received chemotherapy drugs, and the improvement or reversal means that after administering the pharmaceutical composition to the subject, there is no significant difference in the white blood cell level compared with the normal population who have not received cyclophosphamide.

[0028] In another preferred embodiment, the pharmaceutical composition is used to maintain the expression level of intestinal tight junction proteins in the administered subject, where the subject is a patient who has received cyclophosphamide; preferably, the maintenance means that there is no significant difference in the expression level of intestinal tight junction proteins compared with the normal population who have not received cyclophosphamide.

[0029] In another preferred embodiment, the intestinal tight junction proteins are selected from the group consisting of: ZO-1, Occludin, E-cadherin.

[0030] In another preferred embodiment, the pharmaceutical composition is a preparation selected from the group consisting of: powder, granule, capsule, tincture, oral liquid, tablet, lozenge.

[0031] In another preferred embodiment, the pharmaceutical composition is a unit preparation.

[0032] In another preferred embodiment, the pharmaceutical composition is an oral preparation.

[0033] In another preferred embodiment, the polysaccharide of Yupingfeng granules is prepared by the following steps:

[0034] (1) Provide Yupingfeng extract, dilute it with water, and extract it 3-5 times with a mixed solution of chloroform and n-butanol to obtain an extract;

[0035] (2) Dialyze the extract obtained in step (1) with deionized water to obtain a dialysate;

[0036] (3) Dry the dialysate obtained in step (2) to prepare the polysaccharide of Yupingfeng granules.

[0037] In another preferred embodiment, the Yupingfeng extract described in step (1) is prepared by the following steps:

[0038] (a) Provide raw medicinal materials according to the mass ratio of Astragalus membranaceus: Atractylodes macrocephala: Saposhnikovia divaricata = 2.5 - 3.5: 0.5 - 1.5: 0.5 - 1.5;

[0039] (b) Extract Saposhnikovia divaricata in step (1) by steam distillation to obtain an extraction filtrate and medicinal residues;

[0040] (c) Mix the medicinal residues obtained in step (b) with Astragalus membranaceus and Atractylodes macrocephala in step (a) and decoct 2 - 3 times to obtain a decoction. Filter the decoction, take the filtrate, and concentrate to obtain a concentrated filtrate;

[0041] (d) Add ethanol to the concentrated filtrate obtained in step (c) until the alcohol content is 65 v / v% - 75 v / v%, stir, stand, and filter to obtain an alcohol-containing filtrate; and

[0042] (e) Recover ethanol from the alcohol-containing filtrate obtained in step (d) by vacuum evaporation, then mix it with the extraction filtrate obtained in step (b), stand, take the supernatant, and concentrate to prepare the Yupingfeng extract.

[0043] In another preferred example, the volume ratio of chloroform to n-butanol in step (1) = 3 - 5: 1.

[0044] In another preferred example, the dialysis bag in step (2) is 3000 Da.

[0045] In the second aspect of the present invention, a preparation is provided, including the pharmaceutical composition as described in the first aspect; and a pharmaceutically acceptable carrier.

[0046] In another preferred example, the preparation is used to relieve the damage caused by cyclophosphamide chemotherapy.

[0047] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0048] Figure 1 Shows the HPGPC chromatogram of the polysaccharide in Yupingfeng granules.

[0049] Figure 2 Shows the infrared spectrum of the polysaccharide in Yupingfeng granules.

[0050] Figure 3 Shows the monosaccharide composition map of the polysaccharide in Yupingfeng granules, where Figure 3 A in is the monosaccharide composition map of the standard product, Figure 3In B, it is the monosaccharide composition map of the sample, where 1 is mannose (Man), 2 is ribose (Rib), 3 is rhamnose (Rha), 4 is glucuronic acid (GlcA), 5 is galacturonic acid (GalA), 6 is glucose (Glc), 7 is galactose (Gal), 8 is xylose (Xyl), and 9 is arabinose (Ara).

[0051] Figure 4 It shows the average daily body weight and the body weight on the 16th day of mice in different groups. Among them, Figure 4 In A, it is the average daily body weight, Figure 4 In B, it is the body weight on the 16th day. Normal is the normal group, Model is the model group, YPFG is the Yupingfeng granule group, YPFP is the polysaccharide group, and LH is the positive drug group. indicates a highly significant difference, p < 0.05 (compared between the normal group and the model group), * indicates a highly significant difference, p < 0.05 (compared between the drug-administered group and the model group).

[0052] Figure 5 It shows the spleen and thymus organ indices of mice in different groups. Among them Figure 5 In A, it is the spleen organ index, Figure 5 In B, it is the thymus organ index. Normal is the normal group, Model is the model group, YPFG is the Yupingfeng granule group, YPFP is the polysaccharide group, and LH is the positive drug group. ## indicates a very significant difference, p < 0.01, indicates a highly significant difference, p < 0.005 (compared between the normal group and the model group), * indicates a significant difference, p < 0.05, *** indicates a highly significant difference, p < 0.005 (compared between the drug-administered group and the model group).

[0053] Figure 6 It shows the white blood cell counts of mice in different groups. Normal is the normal group, Model is the model group, YPFG is the Yupingfeng granule group, YPFP is the polysaccharide group, and LH is the positive drug group. # indicates a significant difference, p < 0.05, indicates a highly significant difference, p < 0.005 (compared between the normal group and the model group), *** indicates a highly significant difference, p < 0.005 (compared between the drug-administered group and the model group).

[0054] Figure 7 It shows the expression of intestinal tight junction proteins in the colon tissue of mice. Normal is the normal group, Model is the model group, YPFG is the Yupingfeng granule group, and YPFP is the polysaccharide group.

[0055] Figure 8The NF-κB expression in the colon tissues of each group of mice was shown. Normal was the normal group, Model was the model group, YPFG was the Yupingfeng granule group, YPFP was the polysaccharide group. ## indicated a very significant difference, p < 0.01 (compared between the normal group and the model group), *** indicated an extremely significant difference, p < 0.005 (compared between the drug-administered group and the model group). Detailed implementation manner

[0056] Through extensive and in-depth research and a large number of experimental screenings, the present inventors unexpectedly discovered for the first time the application of the polysaccharide components in Yupingfeng granules in the preparation of drugs for chemotherapy-induced damage caused by cyclophosphamide. In the present invention, Yupingfeng granule polysaccharide, as the active pharmaceutical ingredient, can relieve the damage after chemotherapy caused by cyclophosphamide, such as weight loss, spleen and thymus damage, and can also reverse the abnormal white blood cell count to play a relieving or even reversing role. On this basis, the present invention was completed.

[0057] Term description

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0059] As used herein, the terms "comprising", "including", "containing" can be used interchangeably, including not only closed definitions, but also semi-closed and open definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0060] As used herein, the components of the term "pharmaceutically acceptable excipients, carriers or diluents" refer to substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0061] As used herein, the term "effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the said "effective amount" may vary depending on the form of the pharmaceutical composition, the severity of the disease, and the combination with other drugs, etc.

[0062] The Yupingfeng extract of the present invention

[0063] In the present invention, the Yupingfeng extract is prepared by the following method:

[0064] (a) Take astragalus membranaceus: atractylodes macrocephala: ledebouriella divaricata = 2.5 - 3.5: 0.5 - 1.5: 0.5 - 1.5 to provide raw medicinal materials;

[0065] (b) Take the ledebouriella divaricata in step (1) and extract it by steam distillation to obtain an extraction filtrate and medicinal residues;

[0066] (c) Mix the medicinal residues obtained in step (b) with the astragalus membranaceus and atractylodes macrocephala in step (a), add water, and decoct 2 - 3 times to obtain a decoction. After filtration, obtain a filtrate, and after concentration, obtain a concentrated filtrate;

[0067] (d) Add ethanol to the concentrated filtrate obtained in step (c) until the ethanol content is 65 v / v% - 75 v / v%, stir, stand, and filter to obtain an ethanol-containing filtrate;

[0068] (e) Subject the ethanol-containing filtrate obtained in step (d) to vacuum evaporation to recover ethanol, then mix it with the extraction filtrate obtained in step (b), stand and take the supernatant, and concentrate the supernatant to obtain the said Yupingfeng extract.

[0069] In one embodiment of the present invention, the Yupingfeng extract is prepared by the following method:

[0070] (1) According to the method of the Chinese Pharmacopoeia 2020 edition, take astragalus membranaceus, atractylodes macrocephala, and ledebouriella seseloides at a feeding ratio of 3:1:1. Take the medicinal residues after extracting the volatile oil from ledebouriella seseloides by steam distillation method and astragalus membranaceus and atractylodes macrocephala, add water and decoct twice, 1.5 hours for the first time and 1 hour for the second time. Combine the decoctions, filter, and concentrate the filtrate to an appropriate amount to obtain a concentrated filtrate.

[0071] (2) Add ethanol to the concentrated filtrate until the ethanol content is 70%. Stir, stand, and filter this filtrate. Recover the ethanol in the filtrate by vacuum. Mix the filtrate after ethanol recovery with the aqueous solution after steam distillation of ledebouriella seseloides, stir, stand, take the supernatant, and filter to obtain a mixed filtrate.

[0072] (3) Concentrate the mixed filtrate to a clear paste with a relative density of 1.30 - 1.33 (70 °C), and after freeze-drying, obtain 39.0 g of the Yupingfeng extract.

[0073] The polysaccharide of Yupingfeng granules of the present invention

[0074] In the present invention, the molecular weight of the polysaccharide of Yupingfeng granules is 3000 Da - 3200 Da.

[0075] Preferably, the total sugar content in the polysaccharide of Yupingfeng granules is 94.90 ± 2.71%.

[0076] Preferably, the polysaccharide of Yupingfeng granules contains monosaccharides selected from the following group: mannose, ribose, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose.

[0077] Preferably, the molar ratio of glucose to arabinose in the polysaccharide of Yupingfeng granules is 1:1 - 2.

[0078] Preferably, the total protein content in the polysaccharide of Yupingfeng granules is 4.209 ± 0.023%.

[0079] In the present invention, the polysaccharide of Yupingfeng granules is prepared by the following steps:

[0080] Take the Yupingfeng extract, add 200 mL of water to dissolve it, extract 4 times with 100 mL of chloroform: n-butanol (4:1) (Sevage method), place it in a 3000 Da dialysis bag, dialyze with deionized water for 72 h, take out the solution in the dialysis bag, and freeze-dry to obtain 1.15 g of the polysaccharide of Yupingfeng granules. The content of polysaccharide in the obtained Yupingfeng granule extract is 2.95%.

[0081] Formulations and Administration

[0082] The present invention provides a formulation which contains the polysaccharide of Yupingfeng granules as an active ingredient, and pharmaceutically acceptable excipients, carriers or diluents. The said formulation may also contain carriers acceptable in pharmacy or health products.

[0083] There is no particular limitation on the dosage form of the formulation of the present invention, and it can be any dosage form suitable for mammals. Preferably, the dosage form may include tablets, capsules, granules, pills, mixtures, powders, oral liquids, buccal tablets, or aerosols. From the standpoint of being easy to prepare, administer or take, the preferred composition is a solid preparation. Oral administration is preferred.

[0084] Various conventional carriers and / or excipients required for preparing different dosage forms can be added to the formulation of the present invention. The excipients in the present invention are pharmaceutical-grade excipients, including dextrin, lactose, sodium bicarbonate, citric acid, sodium carboxymethyl starch, microcrystalline silicon dioxide, starch, soluble starch, polyethylene glycol or magnesium stearate. It can be prepared into any common dosage form by conventional methods of traditional Chinese medicine preparations, such as tablets, capsules, granules, capsules, pills, powders.

[0085] Some examples of pharmaceutically acceptable excipients are cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), buffers, chelating agents, thickening agents, pH regulators, transdermal promoters, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, bacteriostatic agents, pyrogen-free water, etc.

[0086] The preparation of the present invention can be directly prepared into a pharmaceutical composition for treating the damage caused by cyclophosphamide chemotherapy. The preparation of the present invention can also contain other drugs that are optionally effective for the damage caused by cyclophosphamide chemotherapy. Preferably, the preparation of the present invention can slow down the damage caused by cyclophosphamide chemotherapy.

[0087] Generally, the preparation of the present invention is administered at a dose of 15 - 20 mg / kg per day, preferably in 1 - 2 divided doses per day, or in a sustained-release form. Of course, the specific dose will also vary depending on the administration mode, dosage form, and severity of the disease being treated, which are all within the skills of a skilled physician. Administration can be carried out by conventional routes, including (but not limited to): oral, intramuscular, cutaneous, or topical administration. Oral administration is preferred.

[0088] The present invention also provides a method for slowing down the damage caused by cyclophosphamide chemotherapy, which includes the step of administering a pharmaceutically effective amount of the pharmaceutical composition or the pharmaceutical preparation of the present invention to a subject in need. The subject can be a human or a non-human mammal (such as a dog, pig, cat, monkey, sheep, horse, cow, etc.).

[0089] Use

[0090] In the present invention, the polysaccharide of Yupingfeng granules is used to prepare a pharmaceutical composition for treating the damage caused by cyclophosphamide chemotherapy.

[0091] Preferably, the damage has one or more characteristics selected from the following group:

[0092] (i) Weight loss;

[0093] (ii) Spleen;

[0094] (iii) Thymus damage; and

[0095] (iv) Elevated white blood cell count.

[0096] Preferably, the pharmaceutical composition is administered to a subject in need.

[0097] Preferably, the subject is a patient who has received cyclophosphamide.

[0098] In the present invention, the pharmaceutical composition can slow down the weight loss, spleen and thymus damage of the administered subject, where the subject is a patient who has received cyclophosphamide, and the slowdown is compared with the corresponding index decline of the patient who has received cyclophosphamide, and the difference is significant.

[0099] In the present invention, the pharmaceutical composition can maintain the white blood cell count of the administered subject to tend to be normal, where the subject is a patient who has received cyclophosphamide, and the normal is compared with the normal population who has not received cyclophosphamide.

[0100] Preferably, the pharmaceutical composition can maintain the expression of intestinal tight junction proteins in the administered subject, which is a patient administered with cyclophosphamide, and the "normal" is compared with the normal population not administered with cyclophosphamide.

[0101] Preferably, the intestinal tight junction proteins are ZO-1, Occludin, and E-cadherin.

[0102] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0103] 1. The present invention develops the application of polysaccharide components in Yupingfeng granules in the preparation of drugs for chemotherapy-induced damage caused by cyclophosphamide.

[0104] 2. The polysaccharide of Yupingfeng granules of the present invention, as a drug active ingredient, can relieve the damage after chemotherapy caused by cyclophosphamide, such as weight loss, spleen and thymus damage, and can also reverse the abnormal white blood cell count to play a relieving or even reversing role.

[0105] 3. The polysaccharide of Yupingfeng granules of the present invention, as a drug active ingredient, can increase the expression of intestinal tight junction proteins, reduce intestinal permeability, and achieve the purpose of relieving and / or treating the damage after chemotherapy caused by cyclophosphamide by significantly reducing the increased expression of inflammatory factors caused by intestinal mucosal damage after chemotherapy caused by cyclophosphamide.

[0106] 4. After administering the polysaccharide active ingredient of Yupingfeng granules, it is detected that the expressions of intestinal tight junction proteins ZO-1, Occludin, and E-cadherin are increased to varying degrees, and the expression of intestinal inflammatory factor NF-κB is decreased to varying degrees, which provides a basis and possibility for studying the mechanism of action of the polysaccharide components in Yupingfeng granules in playing a relieving role in the damage after chemotherapy caused by cyclophosphamide.

[0107] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight.

[0108] Instruments and reagents

[0109] Waters 2695 high performance liquid chromatograph (USA, Waters Technology Co., Ltd., equipped with Waters 2410 refractive index detector and Empower workstation), PerkinElmer FT-IR infrared spectrometer (USA, PerkinElmer Instruments Co., Ltd.), Thermo Ultimate3000 high performance liquid chromatograph (USA, Thermo Fisher Scientific Inc.), Shimadzu UV-2600 (Japan, Shimadzu Corporation), Azure multi-functional fluorescence imaging system (USA, Azure biosystems), protein electrophoresis apparatus, membrane transfer apparatus (USA, Bio-Rad Laboratories), microplate reader (USA, Thermo Fisher Scientific Inc.), automatic serum biochemical analyzer 7080 (Japan, Hitachi Ltd.), mouse scale (China, Nanjing Emmanuel Medical Instrument Co., Ltd.), high-speed centrifuge 5810 (Germany, Eppendorf), electronic analytical balance Sartorius ALC-210.3 (Germany, Sartorius AG).

[0110] Mannose (Jiangsu Yongjian Medical Science & Technology Co., Ltd., 200642), ribose (Shanghai Hongyong Biotechnology Co., Ltd., 130033-202009), rhamnose (National Institutes for Food and Drug Control, 1116830-200401), glucuronic acid (Adamas-beta Chemical Reagent Co., Ltd., P2282591), galacturonic acid (Adamas-beta Chemical Reagent Co., Ltd., P1434497), glucose (Shanghai Standard Technology Co., Ltd., 5459), galactose (Gal), xylose (Sinopharm Chemical Reagent Co., Ltd., F20060404), arabinose (Chengdu Dest Biotechnology Co., Ltd., DSTDB013302), extract of Yupingfeng granules (provided by Guangdong Global Pharmaceutical Co., Ltd., H220915), levamisole hydrochloride (Adamas-beta Chemical Reagent Co., Ltd., P1785704)

[0111] SPF-grade normal male BABLC mice (18-22 g), purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., raised in the animal house of Shanghai Institute of Pharmaceutical Industry, approved by the Animal Experiment Ethics Committee of Shanghai Institute of Pharmaceutical Industry, license number: SYXK (Shanghai) 2019-0027. Room temperature: 25±1°C, relative humidity: 50-70%, 12-hour day-night cycle, free access to food and water.

[0112] Example 1: Extraction and characterization of polysaccharides from Yupingfeng granules

[0113] 1.1 Preparation of polysaccharides from Yupingfeng granules

[0114] Take the Yupingfeng extract (prepared from Astragalus membranaceus, Atractylodes macrocephala, and Saposhnikovia divaricata at a feeding ratio of 3:1:1. According to the method in the Chinese Pharmacopoeia 2020 edition, after extracting the volatile oil from Saposhnikovia divaricata by steam distillation, the residue of the medicinal materials and Astragalus membranaceus, Atractylodes macrocephala are decocted twice with water. The first decoction is for 1.5 hours and the second for 1 hour. The decoction liquids are combined, filtered, the filtrate is concentrated to an appropriate amount, ethanol is added to make the ethanol content 70%, stirred, allowed to stand, filtered, the filtrate is concentrated under reduced pressure to recover ethanol, and then mixed evenly with the aqueous solution after the above steam distillation, allowed to stand, the supernatant is taken, filtered, and the filtrate is concentrated to a clear paste with a relative density of 1.30 - 1.33 (70 °C), and freeze-dried to obtain 39.0 g). Add 200 mL of water to dissolve it, extract 4 times with 100 mL of chloroform:n-butanol (4:1) (Sevage method), place it in a 3000 Da dialysis bag, dialyze with deionized water for 72 h, take out the solution in the dialysis bag, and freeze-dry to obtain 1.15 g of Yupingfeng granule polysaccharide. Therefore, the polysaccharide content in the Yupingfeng granule extract is 2.95%.

[0115] 1.2 Determination of relative molecular weight

[0116] The relative molecular weight of Yupingfeng granule polysaccharide was determined by high performance gel permeation chromatography (HPGPC). The chromatographic conditions are as follows: Waters 2695 high performance liquid chromatograph (equipped with Waters 2410 refractive index detector and Empower workstation); chromatographic column: Ultrahydrogel TM Linear 300 mm×7.8 mm id; mobile phase: 0.1 mol / L NaNO3; flow rate: 0.5 mL / min; column temperature: 40 °C; injection volume: 10 μL.

[0117] Accurately weigh different molecular weight Dextran reference standards (MW: 20000,000, 300,600, 135,030, 84,400, 64,600, 21,400, 9,750, 2,700, 180) and prepare a 5 mg / mL solution. After passing through a 0.22 μm filter membrane, inject for detection.

[0118] Weigh 50 mg of the sample into a 10 ml volumetric flask, dissolve it with the mobile phase, and make up the volume. After passing through a 0.22 μm filter membrane, inject for detection, and calculate its molecular weight according to its retention time.

[0119] Taking the retention time as x and the logarithm of the molecular weight as y, the regression equation y = -0.531x + 13.895 (R 2 = 0.9927) is obtained.

[0120] According to the regression equation and the retention time of this peak (see Figure 1 ), the molecular weight of Yupingfeng granule polysaccharide is 3020 Da.

[0121] 1.3 Infrared spectrum determination

[0122] Determined by a PerkinElmer FT-IR infrared spectrometer, with a scanning range of (4000 - 600 cm-1).

[0123] The infrared light absorption results of the polysaccharides in Yupingfeng granules are shown in Figure 2 . The infrared spectrum of the polysaccharides in Yupingfeng granules is a spectrum with typical infrared absorption characteristic peaks of polysaccharides: there is a relatively broad absorption peak near 3335 cm-1, which is the characteristic peak of the stretching vibration of O-H in sugars; there is a weak absorption peak at 2948 cm-1, which comes from the stretching vibration of C-H in -CH2- or -CH3; there is a strong absorption peak at 1634 cm-1, which comes from the stretching vibration of C=O; there is a strong absorption peak at 1423 cm-1, which is caused by the bending vibration of C-H; the strong absorption at 1027 cm-1 is caused by the stretching vibration of C-O-C in the pyran ring.

[0124] 1.4 Analysis of monosaccharide composition

[0125] Derivatization reaction was carried out using PMP (1-phenyl-3-methyl-5-pyrazolone), and high performance liquid chromatography (HPLC) was used to determine the composition and proportion of monosaccharides.

[0126] Precisely weigh 15 mg of each monosaccharide (glucose, galactose, arabinose, glucuronic acid, galacturonic acid) into a 10 mL volumetric flask, add distilled water to make up the volume to prepare a stock solution of 15 mg / mL. Add the standard solution to a new volumetric flask and dilute it with distilled water and mix evenly, so that the concentrations of xylose, galactose, and rhamnose are 0.3 mg / mL, and the concentration of each of the other monosaccharides is 1.5 mg / mL.

[0127] Precisely weigh the polysaccharides in Yupingfeng granules and add distilled water to prepare a solution of 2 mg / mL.

[0128] Take 1 mL of the prepared mixed standard solution and the polysaccharide solution of Yupingfeng granules and add them to a headspace vial, then add 1 mL of 0.4 mol / L trifluoroacetic acid solution and mix well. Tighten the bottle cap, seal it with high-temperature resistant tape, place it in an oven and react at 110 °C for 4 hours. Repeatedly add methanol and evaporate to dryness until there is no sour taste in the residue. Dissolve the residue in 200 μL of distilled water, transfer the solution to a new EP tube, centrifuge at 14000 rpm for 5 min, take 100 μL of the supernatant to a new EP tube, add 75 μL of 0.6 mol / L NaOH solution and mix well, then add 150 μL of 0.5 mol / L PMP methanol solution and mix well. Heat at 70 °C for 90 min and take it out. Add 150 μL of 0.3 mol / L HCl solution to terminate the reaction, make up the volume to 500 μL with distilled water, add an equal volume of chloroform, shake for 1 min, discard the chloroform layer, and continue to repeat the extraction 3 times to remove the excess PMP. Finally, filter through a 0.22 μm microporous membrane for HPLC analysis.

[0129] The liquid phase conditions were chromatographic column YMC Triart C 18 (250mm×4.6mm, 5μm); the mobile phase was potassium dihydrogen phosphate buffer (0.1mol / LKH2PO4, 0.045mol / LNaOH) and acetonitrile (16:84, V / V), isocratic elution, flow rate 1mL / min; column temperature 35℃; injection volume 5μL.

[0130] According to the peak time analysis ( Figure 3 ), Yupingfeng granule polysaccharide contains mannose, ribose, rhamnose, galacturonic acid, glucose, galactose, xylose, and arabinose. According to the ratio of the peak area of monosaccharides after hydrolysis and derivatization of polysaccharide to the peak area of the mixed standard, the molar ratio of each monosaccharide in Yupingfeng granule polysaccharide is 4.80:5.14:2.36:1.38:23.64:2.27:1:30.31.

[0131] 1.5 Determination of total sugar content by reaction of phenol and concentrated sulfuric acid

[0132] Preparation of reference solution

[0133] Based on the monosaccharide composition and ratio results, glucose and arabinose, with higher molar ratios, were selected as reference substances. After conversion, 4.8 mg of glucose reference substance and 5.2 mg of arabinose reference substance were accurately weighed and placed in a 50 mL volumetric flask. Dissolved with water, diluted to volume, and shaken to obtain a mixed standard stock solution with a concentration of 0.20 mg / mL. Accurately pipette 0.4, 1, 2.5, and 5 mL of the glucose standard stock solution into a 10 mL volumetric flask, diluted with water, and diluted to 10 mL to obtain a series of mixed standard solutions with concentrations of 8, 20, 50, and 100 μg / mL, respectively.

[0134] Preparation of test solution

[0135] Accurately weigh 3 portions of 6.5 mg of freeze-dried polysaccharide powder, place them in 100 mL volumetric flasks, add water to fully dissolve and adjust to volume, and mix well.

[0136] Content determination

[0137] Accurately pipette 2 mL each of the above-mentioned mixed standard solution and polysaccharide dilution into 20 mL test tubes, add 1 mL of 5% phenol solution respectively, mix well, and quickly add 5 mL of concentrated sulfuric acid, shake well, and keep warm in a 40°C water bath for 30 min. Take out and immediately place in ice water for 5 min. After returning to room temperature, use water as a running blank and measure the absorbance at 490 nm.

[0138] Content determination results

[0139] The results are shown in Table 1. The total sugar content in the polysaccharide of Yupingfeng granules measured was 94.90 ± 2.71%.

[0140] Table 1: Total sugar content in the polysaccharide of Yupingfeng granules for each sample

[0141]

[0142] 1.6 Determination of total protein content by Coomassie Brilliant Blue

[0143] Preparation of Coomassie Brilliant Blue solution

[0144] CBB G-250 dye reagent: Weigh 50 mg of CBB G-250 into a 500 mL volumetric flask, add 25 mL of 95% ethanol to dissolve, then add 50 mL of 85% phosphoric acid, add distilled water to volume, and mix well to obtain.

[0145] Preparation of reference solution

[0146] Precisely weigh 9.51 mg of bovine serum albumin (BSA) reference substance and place it in a 100 mL volumetric flask, add distilled water to dissolve and dilute to the mark, shake well to obtain a BSA reference solution with a concentration of 0.0951 mg / mL. Precisely pipette 1, 2, 4, 8 mL of the above solution into 10 mL volumetric flasks respectively, add distilled water to dilute to the mark, and mix well to prepare reference solutions with different concentrations.

[0147] Preparation of test solution

[0148] Precisely weigh about 15 mg of three portions of the polysaccharide of Yupingfeng granules into a 25 mL volumetric flask, add distilled water to dissolve and dilute to the mark, shake well, add activated carbon and let stand for 30 min for decolorization to obtain.

[0149] Content determination

[0150] Precisely pipette 1 mL of each of the above BSA standard solution and test solution into 20 mL test tubes respectively, then add 5 mL of Coomassie Brilliant Blue G-250 solution to each, mix well and let stand for 2 min, and detect at 595 nm.

[0151] Test results

[0152] The results are shown in Table 2. The total protein content in the polysaccharide of Yupingfeng granules measured was 4.209 ± 0.023%.

[0153] Table 2: Total protein content in the polysaccharide of Yupingfeng granules for each sample

[0154]

[0155] Example 2: Therapeutic effect of the polysaccharide of Yupingfeng granules on the chemotherapy-induced injury caused by cyclophosphamide

[0156] Forty mice were randomly divided into 5 groups. The mice were adaptively fed for 1 week and divided into a normal group, a model group, a polysaccharide group, and a positive drug group, with 8 mice in each group. The normal group and the model group were intragastrically administered 0.5% CMC-Na solution (10 mL / kg) daily; the Yupingfeng Granule group was given 2.3 g / kg of Yupingfeng Granule; the polysaccharide group was given 17 mg / kg of Yupingfeng Granule polysaccharide (dose converted according to the yield after dialysis of the extract based on the clinical dose); the positive drug group was given 40 mg / kg of levamisole. Yupingfeng Granule, polysaccharide, and the positive drug were all dissolved in 0.5% CMC-Na and continuously intragastrically administered for 15 days. The mice were weighed before each administration. On the 8th, 9th, and 10th days after administration, except for the normal group, all mice were intraperitoneally injected with 80 mg / kg of cyclophosphamide 2 hours after administration to establish a model. On the 0th, 8th (before modeling), 11th (after modeling), and 16th (after the end of administration) days, 100 μL of blood was collected from the mice in each group. The white blood cell count was detected using an automatic blood biochemical analyzer. On the 16th day, the mice were weighed, sacrificed after blood collection, and the spleen, thymus, and small intestine were taken and various indicators were detected.

[0157] 2.1 Body weight and organ index

[0158] As shown in the body weight change curve ( Figure 4 ), after cyclophosphamide was given on the 8th day, the body weight of the mice in all groups except the normal group decreased significantly. After the 13th day, the body weight of the mice in the polysaccharide group and the positive drug group showed a slow recovery trend. On the 16th day, it was found that the body weight of the mice in the model group was significantly lower than that of the normal group, and the body weight of the polysaccharide group and the positive drug group was significantly higher than that of the model group. The body weight of the mice in the Yupingfeng Granule group did not show a recovery trend after administration and decreased compared with the model group. This may be due to the poor solubility of the granule and the irritation of the excipient components in it to the gastrointestinal tract of the mice.

[0159] As shown in the organ index ( Figure 5 ), the spleen and thymus organ indices of the model group were significantly lower than those of the normal group. The organ indices of the administered groups were significantly higher than those of the model group. Among them, the data of the thymus organ index of the polysaccharide group were more uniform, so its significance was stronger. And since the organ index is the ratio of the organ weight to the body weight, the polysaccharide group was significantly higher than the model group and the Yupingfeng group. Therefore, the increase in the thymus organ index of the polysaccharide group was more significant than that of the Yupingfeng Granule group. And because the body weight of the mice in the Yupingfeng Granule group was significantly lower than that of the other administered groups, although its organ index increased significantly, in fact, the organ weight of the Yupingfeng Granule group was not significantly higher than that of the model group or the improvement effect of the other administered groups was more significant than that of the Yupingfeng Granule group.

[0160] 2.2 White blood cell count

[0161] From Figure 6It can be seen from the data that there were no significant changes in the white blood cell ratio of each group of mice before modeling after drug administration, and there were no significant differences among the groups. On the 11th day, it was found that the white blood cell count in the model group decreased significantly compared with the normal group, and the white blood cell count in the drug administration groups increased to varying degrees. On the 16th day, it was found that the white blood cell count in the model group showed an obvious rebound phenomenon compared with the normal group. This was mainly because cyclophosphamide would activate hematopoietic progenitor cells, which increased circulating hematopoietic stem cells by affecting the distribution of initial hematopoietic stem cells and mature progenitor cell subsets in the bone marrow and blood of mice. The white blood cell count in each drug administration group was significantly lower than that in the model group, showing an obvious trend of recovery to the normal group.

[0162] 2.3 Detection of the expression of intestinal tight junction proteins ZO-1, Occludin, and E-cadherin by Western Blot

[0163] Colon tissue homogenates were prepared using RIPA buffer containing protease inhibitors and phosphatase inhibitors. Protein quantification was performed using a BCA kit. Equal amounts of proteins from different treatments were separated using a 10% SDS-PAGE gel and then electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. Subsequently, the membrane was incubated with three primary antibodies against ZO-1, Occludin, and E-cadherin, followed by incubation with a secondary antibody conjugated to horseradish peroxidase to obtain the WB conditions for the target proteins. The expected proteins were detected using an ECL kit, and normalization was performed according to the expression level of the internal reference gene GAPDH.

[0164] The results were as Figure 7 shown. The relative expression levels of the three tight junction proteins (ZO-1, Occludin, and E-cadherin) in the small intestine of mice in the model group were decreased to varying degrees compared with the normal group. After intervention with Yupingfeng granules and Yupingfeng granule polysaccharides, different degrees of improvement effects on the expression of tight junction proteins were shown.

[0165] 2.4 Detection of the expression of intestinal inflammatory factor NF-κB by Elisa

[0166] The inflammatory factor NF-κB in the small intestine homogenate was measured using an Elisa kit, and the specific operation was referred to the instruction manual. The results showed ( Figure 8 ) that NF-κB in the intestinal tissue of the model group was significantly increased compared with the normal group, while after administration of Yupingfeng granules and Yupingfeng granule polysaccharides, the expression of NF-κB decreased significantly, and Yupingfeng granule polysaccharides had a better improvement effect on NF-κB than Yupingfeng granules.

[0167] In summary, not only can the polysaccharide of Yupingfeng granules alleviate the weight loss, and the reduction of spleen and thymus indices caused by the post-chemotherapy injury induced by cyclophosphamide, but it can also reverse the abnormal white blood cell count. This may be related to the fact that the polysaccharide of Yupingfeng granules can increase the expression of intestinal tight junction proteins, reduce intestinal permeability, and significantly reduce the increased expression of inflammatory factors caused by the post-chemotherapy intestinal mucosal injury induced by cyclophosphamide. Among them, in terms of the improvement effects on body weight, thymus organ index and the expression of inflammatory factor NF-κB, the polysaccharide of Yupingfeng granules is better than Yupingfeng granules at the same dose.

[0168] All documents mentioned in this invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of polysaccharide of Yupingfeng granules, characterized in that, For preparing a pharmaceutical composition for treating injuries caused by chemotherapy drugs after therapeutic administration.

2. The use according to claim 1, characterized in that, The total sugar content in the polysaccharide of Yupingfeng granules is > 85 wt%, preferably > 90 wt%, more preferably > 94 wt%, such as 92 - 98 wt%.

3. The use according to claim 1, characterized in that, The polysaccharide of Yupingfeng granules contains monosaccharides selected from the group consisting of mannose, ribose, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, or a combination thereof.

4. The use according to claim 1, characterized in that, In the polysaccharide of Yupingfeng granules, the molar ratio of glucose to arabinose is 1:1 - 2.

5. The use according to claim 1, characterized in that, The total protein content in the polysaccharide of Yupingfeng granules is 3 - 5 wt%, preferably 4 - 4.5 wt%.

6. The use according to claim 1, wherein The pharmaceutical composition is used for alleviating or improving side effects caused after a chemotherapy drug is administered to a subject; preferably, the side effects include: weight loss, or spleen and thymus damage; more preferably, the alleviation means that compared with the patients in the control group (i.e., those who received cyclophosphamide but did not use the pharmaceutical composition), their weight increases or the indexes of spleen and thymus damage decrease, and the difference is significant.

7. The use according to claim 1, characterized in that, The pharmaceutical composition can improve or reverse the decrease in the number of white blood cells of a subject, where the subject is a patient who has received a chemotherapy drug, and the improvement or reversal means that after the pharmaceutical composition is administered to the subject, there is no significant difference in the white blood cell level compared with the normal population who did not receive cyclophosphamide.

8. The use according to claim 1, characterized in that, The pharmaceutical composition is a preparation selected from the group consisting of powder, granule, capsule, tincture, oral liquid, tablet, and buccal tablet.

9. The use according to claim 1, wherein The polysaccharide of Yupingfeng granules is prepared by the following steps: (1) Provide an extract of Yupingfeng, dilute it with water, and extract it 3 - 5 times with a mixed solution of chloroform and n-butanol to obtain an extract solution; (2) Dialyze the extract solution obtained in step (1) with deionized water to obtain a dialyzate; (3) Dry the dialyzate obtained in step (2) to prepare the polysaccharide of Yupingfeng granules.

10. A preparation, characterized in that, Comprising the pharmaceutical composition according to claim 1; and a pharmaceutically acceptable carrier.

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