CN111617109B

CN111617109B - Application of five-cereal insect medicament, five-cereal insect medicament and preparation method thereof

Info

Publication number: CN111617109B
Application number: CN202010527167.9A
Authority: CN
Inventors: 戴靖宇
Original assignee: Hangzhou Yiketang Biotechnology Co ltd
Current assignee: Hangzhou Yiketang Biotechnology Co ltd
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2023-12-22
Anticipated expiration: 2040-06-11
Also published as: CN111617109A

Abstract

The invention relates to application of a five-cereal worm medicament, the five-cereal worm medicament and a preparation method thereof, and belongs to the technical field of biological medicines. Comprises the application of the five-cereal worm medicament for increasing the leucocyte density of a tumor patient and the application of the five-cereal worm medicament and CTX in combination. Also comprises a medicament of the five-cereal worm alcohol-water double-extract and a preparation method thereof. The common five-cereal worm medicament can effectively increase the white blood cell density in a tumor patient, and the five-cereal worm medicament prepared by an alcohol-water double-extraction method can fully exert the self efficacy of the five-cereal worm and can play a positive role in relieving/inhibiting the side effect of a chemotherapeutic medicament Cyclophosphamide (CTX).

Description

Application of five-cereal insect medicament, five-cereal insect medicament and preparation method thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an application of a five-cereal worm medicament, the five-cereal worm medicament and a preparation method thereof.

Background

In recent years, the incidence of malignant tumors has been increasing, becoming one of the diseases that seriously threatens health.

In the treatment of malignant tumors, particularly those treated by chemotherapy, a sharp decrease in the number of leukocytes in the patient is often involved. At this time, how to increase the density of leukocytes in a patient becomes a technical problem to be solved.

In the prior art, there is a technical scheme of increasing the number of white blood cells of a patient by adopting the Rui-Bai as an injection, but the Rui-Bai has a considerable side effect and is expressed in that:

musculoskeletal system: sometimes there are muscle soreness, bone pain, lumbago, chest pain; on the digestive system: sometimes, the phenomenon of inappetence or the rise of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase of liver occurs; some people can generate fever, headache, weakness and rash, and ALP and LDH rise; very few people will develop shock, interstitial pneumonia, adult respiratory distress syndrome and increased naive cells.

Another alternative solution is needed to increase the white blood cell density of a patient.

The current clinic treatment methods for malignant tumor mainly comprise surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy and the like. Because some tumor patients are in late stage when found, the chance of operation is lost, the radiotherapy technology has certain limitation, the targeted drug treatment is relatively expensive, the immunotherapy lacks specificity aiming at tumor cells, the effect is slow, the curative effect is not definite, and even the tumor can be increased to a certain extent. Chemotherapy is therefore still the primary method of treating tumors, but low immunity is one of its major side effects, which is also the primary reason for the patient's inability to complete treatment on time. Therefore, how to alleviate the immune deficiency caused by chemotherapy and leucopenia are urgent problems to be solved.

The dried larvae (maggots) of insect diptera chrysomya megacephala (Chrysomya megacephala, also known as chrysomyia megacephala and eastern toilet flies) and the nearby insects (such as Musca domastica) are a traditional Chinese medicine, and are also known as ' five-cereal insects ', also known as ' fagopyrum, and common names ' maggots '. According to the description of "Ben Cao Jiyuan" and "Ben Cao Bian Ji, five cereal worms are cold in nature and nontoxic, enter spleen and stomach meridians, and after being dried and ground, the Chinese medicinal composition is applied externally, so that the Chinese medicinal composition can treat infectious surgical diseases such as ecthyma, lip furuncle and carbuncle. The five cereal insects are originally carried in forty volumes of insect parts of Ben Cao gang mu, have the effects of clearing heat and removing food retention, treat infantile malnutrition, abdominal distention and infantile malnutrition, and are the first and only medical insects authenticated by the FDA in the United states. However, clinical treatment of leucopenia by five cereal worms has been reported so far.

Leukopenia is common in clinical radiotherapy and chemotherapy, and granulocyte colony stimulating factor medicines are mostly adopted in treatment. Cyclophosphamide (CTX) has a wide clinical application, belongs to a cytotoxic chemotherapy drug, is an alkylating agent, has no anticancer activity in vitro, enters into the body to be hydrolyzed by phosphatase existing in liver or tumor to become activated phosphamide nitrogen mustard to act, the substance is crossly connected with DNA to inhibit the synthesis thereof, the synthesis of RNA is interfered, and metabolites can be combined with macromolecules such as protein, RNA and DNA to cause DNA damage, bone marrow suppression and leucopenia as one of side effects thereof.

In the prior art, related technical schemes related to the anti-tumor of the five-cereal worms are disclosed in Chinese patent application publication No. CN 103271947A, and the novel application refers to the application of the five-cereal worms in preparing anti-tumor drugs or health care products, and the application takes whole worms of the five-cereal worms as effective parts to prepare medicaments. Experiments prove that the five-cereal insect can effectively inhibit the growth of tumor cells, has obvious killing effect on the tumor cells, has good medicinal and health care values, and does not relate to the technical effect of increasing the density of white blood cells.

Moreover, the conventional single or compound preparation of the five-cereal worm has limited efficacy, the due efficacy of the five-cereal worm is not fully exerted, and the prior art does not relate to the limitation/alleviation of the side effect of the high-efficiency dosage form of the five-cereal worm on the Cyclophosphamide (CTX) serving as a chemotherapeutic agent.

Disclosure of Invention

In view of the above problems, the present invention provides one of the purposes of increasing the white blood cell density of a tumor patient by using a five-cereal worm, the purpose of preparing a medicament capable of fully exerting the efficacy of the five-cereal worm, and the purpose of providing a five-cereal worm medicament capable of alleviating/inhibiting the side effects of Cyclophosphamide (CTX), which is a chemotherapeutic medicament, the third purpose of providing a technical scheme that a general five-cereal worm medicament can effectively increase the white blood cell density of a tumor patient, and the five-cereal worm medicament prepared by a special method can fully exert the efficacy of the five-cereal worm itself and can positively exert the side effects of Cyclophosphamide (CTX), which is a chemotherapeutic medicament.

The technical scheme for solving the problems is as follows:

the application of the five-cereal worm medicament is used for increasing the leucocyte density of a tumor patient.

As a preferable mode of the technical scheme, the five-cereal worm medicament is powder, tablet, capsule, pill, granule, oral liquid, microcapsule, injection, inhalant or powder of five-cereal worm.

Another application of the five-cereal worm medicament is: it is used in combination with CTX.

As the preferable choice of the technical proposal, the five-cereal worm medicament is alcohol-water double extract of five-cereal worms.

As the optimization of the technical scheme, the alcohol-water double extract of the five-cereal worms is extracted by adopting ethanol or an aqueous solution of the ethanol, then water is adopted for extraction, and then the extraction solution is mixed and concentrated to obtain the five-cereal worm medicament.

As the optimization of the technical scheme, the mass ratio of the five-cereal worm medicament to CTX is (57-230): 1.

the five-cereal insect medicament is alcohol-water double-extract of five-cereal insects.

The alcohol-water double-extract of the five-cereal worms is extracted by adopting ethanol or an aqueous solution of the ethanol, then water is adopted for extraction, and then the extraction solutions are mixed and concentrated to obtain the five-cereal worm medicament.

As a preferable mode of the above technical scheme, the alcohol-water double-extraction solution uses the powder obtained by crushing the collected five-cereal worms as an extraction object.

The preparation method of the five-cereal worm medicament comprises the following steps:

(1) weighing a proper amount of five-cereal worm medicinal material, wherein the mass of the five-cereal worm medicinal material is denoted as a;

(2) extracting the five-cereal worm medicinal material by adopting ethanol or ethanol aqueous solution for at least two times, marking the mass of ethanol or ethanol aqueous solution extracted each time as b, and collecting to obtain a first filtrate and medicinal residues;

(3) extracting the residues with water for at least two times, recording the quality of water extracted each time as c, and collecting to obtain a second filtrate;

(4) and mixing the first filtrate and the second filtrate to form a mixed solution, and concentrating the mixed solution to obtain the five-cereal worm medicament.

Firstly, alcohol extraction is adopted, so that the five-cereal worm medicinal material can be well dispersed, and the contact area between water and dregs in the step (3) is increased; if water extraction is adopted first, the five-cereal worm medicinal materials are easy to agglomerate into paste, and then when alcohol extraction is adopted, the contact area between the solvent and the dregs is greatly reduced, so that the extraction effect is greatly reduced.

The five-cereal worm medicament in the prior art is usually five-cereal worm powder, fat concentrate or protein concentrate, and no technical scheme for alcohol extraction and then water extraction of five-cereal worm medicinal materials has been disclosed yet.

The five-cereal insect is used as a traditional Chinese medicine, and contains a plurality of effective components, and the reason why the five-cereal insect medicament can achieve a better technical effect is probably as follows: the alcohol extraction and the water extraction are carried out firstly to fully extract various effective components with multiple targets, so that the effective components are in a dispersed and easily absorbed state, wherein the components can also have synergistic effect, and the efficacy of the five-cereal insects can be exerted as much as possible.

As the optimization of the technical scheme, the five-cereal worm medicinal material is the powder obtained by crushing the collected five-cereal worm.

As a preferred embodiment of the foregoing technical solution, in step (2), the five-cereal worm is extracted with 75% aqueous ethanol solution for three times, and the mass of the aqueous ethanol solution extracted each time is b=20×a.

Preferably, in the above-mentioned method, in the step (2), the extraction time is 1 hour each time.

As a preferred embodiment of the foregoing method, in step (3), the residue is extracted three times with water, and the mass of water extracted each time is c=20×a.

Preferably, in the above-mentioned method, in the step (3), the extraction time is 1.5 hours each time.

In the above aspect, in the step (4), the mixed solution is concentrated until the mass of the mixed solution becomes a.

In summary, the embodiment of the application has the following beneficial effects:

1) The five-cereal worm medicament can effectively increase the leucocyte density in a tumor patient.

2) The alcohol-water double-extraction liquid for the five-cereal worms can fully exert the effects of the five-cereal worms.

3) The five-cereal worm medicament is particularly suitable for relieving/inhibiting the side effects of CTX.

Drawings

FIG. 1 is a graph showing the trend of the effect of a five-cereal worm medicament on the white blood cell count of nude mice caused by chemotherapy;

FIG. 2 is a graph showing the trend of the effect of the five-cereal worm agent on the lymphocyte count of nude mice due to chemotherapy;

FIG. 3 is a graph showing the trend of the effect of five cereal worms on the level of the cytokine IL-2 in nude mice caused by chemotherapy;

FIG. 4 is a graph showing the trend of the effect of five cereal worms on the level of the cytokine TGF-beta in nude mice caused by chemotherapy;

fig. 5 is a graph showing the trend of the effect of five cereal worms on the spleen index of nude mice.

Wherein A in FIG. 1 is the white blood cell count level, B in FIG. 2 is the lymphocyte count level, A in FIG. 3 is the IL-2 cytokine level, and B in FIG. 4 is the TGF-beta cytokine level.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments of the invention, which are obvious to those skilled in the art without the exercise of inventive faculty, are intended to be within the scope of the invention.

The invention is described in detail below with reference to the accompanying drawings.

In a first class of embodiments, partial examples of the leukocyte increasing effect of a five-cereal agent on tumor patients:

patients Cao Mou in Tagao city, jiangsu province, had decreased appetite and poor mental state after surgery and chemotherapy with colon cancer in 2017, and had white blood cell values of (2-3) x10≡9/L. The five-cereal worms are taken twice a day, 3 g each time and continuously for 65 days in 8 months in 2019, and the test result of the hospital shows that the white blood cell number is increased to (6-7) 10 x 9/L. The normal range value is reached, the living state of the patient is better and better, and various body indexes reach the normal state.

The patients in Zhejiang province, changxing county, 16 years old, women, suffering from brain tumor, through Shanghai Huashan hospital operation treatment, chemotherapy is carried out for 10 times, white blood cells are reduced to (2-2.5) 10 x 9/L, and after 1 month in 2019, five-cereal worms are taken twice daily, 2 g is taken twice, 60 days are continuously taken, and the test in the hospital is carried out twice, so that the result shows that the white blood cell number is increased to (6-8) x10 x 9/L, the normal range value is reached, the current state of the patients is good, and the patients can return to school again.

The patients Wang Mou, 59 years old, women suffering from rectal cancer, after surgery, chemotherapy, hair loss, emaciation, bad appetite, leukopenia can not reach the normal value. And after the five-cereal worms are taken twice a day and 3 g each time in 2 months in 2018 and continuously taken for 30 days, the test results of a plurality of hospitals show that the number of the white blood cells is increased to (7-8) 10 x 9/L, the normal range value is reached, the current condition of the patient is good, and the patients can keep the housework and play outside.

Patients Yang Mou, 43 years old in Guangde county of Anmicro province, women suffer from gastric cancer, do tumor resection surgery, are subjected to chemotherapy for 6 times, and the white blood cells fall below a normal value, so that fatigue, inappetence and disease friends recommend that five cereal worms are taken three times a day, 2 g each time, 50 days later, and the white blood cell number is stabilized at (7.5-8.5) 10 x 9/L through multiple tests, so that the current state is good.

Patients Li Mou in Zhejiang province, xingcounty, age 50, men suffer from lung cancer, are subjected to chemotherapy for many times after surgical excision, leucopenia is reduced to (2-2.5) 10 x 9/L, physical condition is bad, five-cereal worms are taken in 5 months in 2018 twice a day, 3 g each time and three months after continuous taking, and the laboratory results of a hospital show that the leucopenia number is (6-7) 10 x 9/L, and the physical condition is good.

The patients, men and 71 years old, suffering from the colon hypodifferentiation adenocarcinoma, the tumor occupying the space and blocking the intestinal cavity, belong to the advanced stage and miss the operation treatment period. The direct chemotherapy uses capecitabine and folinic acid calcification medicines, which cause bone marrow hematopoietic failure, and the bone marrow can not be continued after three times of chemotherapy. Through the recommendation of the patient in the same village, seven grams of five-cereal worms are taken twice a day, after about 50 days of taking, the vital energy is slowly restored to the state before chemotherapy, the white blood cell number is increased from the bottommost (1.5-2) 10 x 9/L to the bottommost (6.3-6.9) 10 x 9/L, the physical strength is recovered, the mental state is good, the diet is normal, the life is self-care, and the five-cereal worms are taken all the time.

Qian Mou in the Changxing county of Zhejiang province, female, 63 years old, patients with gastric cancer, postoperative chemotherapy, poor appetite, debilitation, blood three systems decrease, white blood cells decrease to (2-2.5) 10 x 9/L, five-cereal worms are taken from 10 months in 2018, 6 g are taken every day, and twice are taken. After one month of physical strength is increased and the stomach is well turned, through examination, the three systems of blood are improved, the white blood cells are increased to (9-9.5) 10 x 9/L, and the patients feel good by themselves. Can be used for planting vegetables and holding housework, and is not different from healthy people.

The method is characterized in that the grain and trade bureau in the Changxing county of Zhejiang province, kong Mou, suffering from colon cancer, is operated by using capecitabine and oxaliplatin as chemotherapeutics, the leucocyte is always (2.5-3) 10 x 9/L, the mental state of a patient is poor, the stomach opening is poor, the five-cereal worm is taken in the beginning of 2019, the five-cereal worm is taken twice a day, 2.5 g each time, and after 3 months of continuous taking, the leucocyte is increased to (8-9) 10 x 9L through the examination result of a hospital, the current state of the patient is good, and the life quality is continuously improved.

Xu Mou for patients in Taihu street in Yangxing county of Zhejiang province, 45 years old, colon cancer of the shape of B, postoperative chemotherapy, leucocyte number 1.4 x10 x 9/L, and the patients recommend five-cereal insects to be taken for seven g each day, and the patients take the five-cereal insects twice per day, and after four months of continuous taking, the leucocytes rise to 4.4 x10 x 9/L through hospital examination, so that the patients are in good state.

The method is characterized in that urban residents in the forest of the Yangxing county of Zhejiang province, chen Mou, women, 74 years old, lung cancer, postoperative chemotherapy, one-time life dangers, leucocytes are reduced to 1.9 x10 x 9/L, seven grams of five-cereal worms are taken every day through friend introduction, the five-cereal worms are taken twice, the two-cereal worms are taken for three months continuously, and then go to a hospital for examination, the leucocytes are increased to 5.3 x10 x 9/L, and the life is self-care and mental state is good.

The method is characterized in that Zhou Mou of patients in Yang Wancun of Xingxing county, zhejiang province, 68 years old and 2016 year old are subjected to confirmed diagnosis of intestinal cancer, after operation treatment in a Changxing middle-aged hospital and intravenous infusion of chemotherapeutics, white blood cells are reduced to 2x10 x 9/L, five-cereal worms are taken from 3 months, twice a day and 3 g each time, and after three months of continuous taking, the results of hospital tests show that the white blood cells are increased to normal level, the whole feeling of the patients is good, and the patients are healthy.

The patients Jiang Mou, 76 years old, men and 2014 are diagnosed with gastric cancer in 12 months of Zhejiang province, after the treatment of the operation in Changxing Zhongxing Zhonghua street, the medicines omeprazole, pantoprazole and the like are used for 6 times of chemotherapy, the white blood cells are reduced to 3x10 x 9/L, the patients have no appetite and are hypodynamia in the period, the patients are introduced by sick friends, the five-cereal insects are taken for 3 g each time, twice a day, 75 days later, the total white blood cells reach 7x10 x 9/L after the examination in the middle hospital, and the patients are recovered to be normal and self-care in life.

The patients Yang Mou on the Taihu street in Zhejiang province, the men, 74 years, 9 months in 2015 are diagnosed with gastric cancer, 7 times of postoperative chemotherapy are carried out in hospitals in Zhejiang province, leucopenia is serious in period, doctors in the traditional Chinese medicine museum recommend that the patients begin to take the decoction pieces of the five-cereal insects in one month after operation, twice daily, 2 g and 5 g each time, and after four months of continuous taking, the patients are examined by the county people hospitals, leucopenia is increased to 8x10 x 9/L, and the patients are good in physical and mental states and self-care in life.

The patients on the Zhejiang Changxing Taihu street, yan Mou, 58 years old men, gastric cancer, 4 months Shanghai Hospital in 2014 are diagnosed and treated by surgery, people are emaciated, tired and hair is lost after domestic chemotherapeutics are used, white blood cells are reduced to 2x10 x 9/L, since the five-cereal insects are taken twice a day for 3 g each time, after four consecutive months, the white blood cell number is increased to 8x10 x 9/L through the test result of the county people hospital, and the patients are still on duty in factories at present.

In a second class of embodiments, some examples of the effects of the five-cereal agent on the inhibition of tumors are as follows:

the city and town patients Zhou Mou, 48 years old and women in Zhejiang Changxing county forest, who have undergone hyperplasia of mammary glands and excision, have double breast distention, such as breast pain of women during lactation, are hard to touch, and recommended by friends, purchase the five-cereal insect oral treatment by the TCM museum twice daily, 3 g each time, after three months of administration, the swollen breast is gradually reduced, pain is obviously relieved, and the current situation is better and better after 6 months of continuous administration.

Sian town patients in Changxing county of Zhejiang province, wu Mou, women, 41 years old, due to distending pain in the left breast during menstruation, examination by the Sian's hospital in county, ultrasonic examination shows that hypoechos of about 7x 9mm are visible at 2 o ' clock of the left breast, oral treatment of the five-cereal worms is purchased in a TCM hall twice daily, 3 g each time, pain is relieved when menstruation is carried out the next month, and after 70 days of continuous administration, ultrasonic examination results show that nodules at two o ' clock of the left breast are reduced to 3.5 x 4mm.

In the south Xuezhejiang province, the south Xuezhejun district patient, chen Mou, women, 39 years old, bilateral hyperplasia of mammary glands has been for years, axillary lymphatic vessels are affected, and secondary breast appears on both sides, so that the chest is painful at ordinary times, the pain before menstrual period is more serious, and the oral treatment of the five-cereal insects is purchased in a TCM hall by friends recommendation, twice daily, each time for 2.5 g, the pain is obviously relieved after taking for one month, and the secondary breast disappears after four months.

Patients with the symptoms of the star-like bridge town in the county of Changxing, zhejiang province, xia Mou, women, 46 years old, the self-checking double breast has a plurality of lumps with different sizes, and the pain feeling before menstruation is strong. Through friend recommendation, the treatment of the five cereal worms is purchased from the department of Chinese medicine, 6 g of the five cereal worms are taken twice a day, pain is gradually disappeared after two months of continuous taking, and obvious nodular lumps are not found after four months of ultrasonic examination.

Patients Chen Mou in Huzhou, zhejiang, women, 35 years old, found 6 x 5mm hypoechos at 11 o 'clock on the left and about 4 x 3mm hypoechos at 10 o' clock on the right on the 2018 6 month physical examination. Several lymph node echoes are visible in bilateral axillary scanning, 14mm on the left side is 4mm, 13mm on the right side is 4mm, five cereal worms are taken twice a day, 2.5 g of the five cereal worms are taken each time, breast distention pain is obviously improved after 70 days of continuous taking, ultrasonic examination is carried out again after 6 months of taking, 3x 2.6mm hypoechogenic nodules are arranged at 11 o' clock of left breast, the right breast nodules disappear, and obvious enlarged lymph node echoes are not seen.

In a third class of embodiments, the alcohol-water double extract of wubi is used as a wubi agent and is used to alleviate/inhibit the side effects of the chemotherapeutic agent Cyclophosphamide (CTX) as follows:

in the embodiment, a nude mice leucopenia animal model is established through cyclophosphamide, clinical medicine Rui Bai is taken as a positive medicine, the influence of five cereal worms on leucopenia nude mice leucopenia, lymphocyte count, spleen index and cytokine IL-2, TGF-beta is observed, the influence of five cereal worm alcohol water double extract on cyclophosphamide-induced immune function of nude mice is discussed, and the clinical medicine can be expected to be guided.

Materials and methods:

experimental animals:

the experiment is completed in animal experiment research center of Zhejiang university of traditional Chinese medicine. Clean grade 5 week old BALB/c nude mice were selected for 36, supplied by Shanghai Laek laboratory animal Co. The mass of the body is 18-20 g. Mice are fed into a barrier system of animal experiment research center of Zhejiang university, the temperature is 18-24 ℃, and the relative humidity is 60-80%.

Main drugs, reagents:

recombinant human granulocyte stimulating factor injection (G-CSF, trade name: rui Bai; lot number: 18Z03H; product family: produced by Kyowa Kagaku Co., ltd., kyowa Kagaku Co., fermented kylin (China) pharmaceutical split charging);

cyclophosphamide (production lot number: 8K272A; manufacturer: baxter Oncology GmbH);

five cereal worms (lot number: 20191013);

normal saline (production lot number: A1901152; manufacturer: hangzhou civil pharmaceutical Co., ltd.).

Modeling, grouping animals and administration:

after 7 days of BALB/c nude mice were fed adaptively, they were divided into the following 6 groups:

group A: normal control group (lavage saline, 0.1ml/10 g);

group B: model group (intraperitoneal injection of CTX 20 mg/Kg+lavage saline, 0.1ml/10 g);

group C: positive drug group (i.p. CTX 20 mg/Kg+Tail vein injection of Bai Zhen. Mu.g/Kg);

group D: high dose group of five-cereal worms (intraperitoneal injection of CTX 20 mg/Kg+46.0 mg/10g of gastric five-cereal worm extract);

group E: dose group of five-cereal worms (intraperitoneal injection CTX 20 mg/Kg+23.0 mg/10g of gastric-lavage five-cereal worm extract);

group F: five-cereal insect low dose group (intraperitoneal injection CTX 20 mg/Kg+intragastric five-cereal insect extract 11.5. 11.5 mg/10 g).

1 time per day for 14 consecutive days.

The preparation method of the five-cereal worm extract comprises the following steps:

(1) weighing a proper amount of five-cereal worms, and crushing to obtain five-cereal worm powder;

(2) extracting powder of five-cereal insects with 75% ethanol water solution of 20 times of mass for three times, extracting for 1 hour each time, and collecting to obtain a first filtrate and residues;

(3) extracting the residue with 20 times of pure water for three times for 1.5 hr, and collecting to obtain second filtrate;

(4) mixing the first filtrate and the second filtrate to form a mixed solution, and concentrating the mixed solution until the mass of the mixed solution is equal to that of the five-cereal worm powder to obtain the five-cereal worm extract.

Detecting the index:

blood convention: after the last dose for 1h, the orbital vein of the mice was sampled, EDTA-Na2 was anticoagulated, and the white blood cell count (WBC) and lymphocyte count (LYMPH) were read by a cytometer.

Serum LI-2, TGF- β content: after the last administration for 1h, the orbital vein of the mice is sampled, and the mice are centrifuged for 20min at a centrifugation rate of 3000r/min, and serum is taken. The OD value is measured at 450nm of an enzyme-labeled instrument by referring to the ELISA kit instruction operation of the mouse IL-2 and TGF-beta, and a standard curve is drawn to calculate the content of the mouse serum LI-2 and TGF-beta.

Calculation of immune organ index: mice were sacrificed 1h after the last dose, spleens were dissected, their wet mass was weighed with an electronic analytical balance, and spleen indexes (spleen mass/body mass) were calculated, respectively.

Statistical methods:

experimental data were processed using SPSS17.0 software. The normal distribution of the single factor comparison among multiple groups is analyzed by the single factor, and then the statistics is carried out by an LSD method. P <0.05 is statistically significant for the differences.

Results and analysis:

the effect of five cereal worms on the white blood cell and lymphocyte count levels of nude mice caused by chemotherapy is shown in table 1:

TABLE 1 influence of five cereal worms on the white blood cell count and lymphocyte count levels of nude mice due to chemotherapy，n=5）

In comparison with the normal control group, ^** P<0.01， ^* P<0.05; in comparison with the set of models, ^## P<0.01; in comparison with the positive drug, the preparation method, ^▲▲ P<0.01。

the effect of five cereal worms on the white blood cell count level of nude mice caused by chemotherapy is shown in fig. 1, and the effect of five cereal worms on the lymphocyte count level of nude mice caused by chemotherapy is shown in fig. 2.

The white blood cell results showed that the white blood cell count of the model group was extremely significantly reduced (p=0.002 < 0.01) compared to the normal control group, indicating that the modeling was successful. The number of white blood cells in the positive drug group is extremely obviously increased (P=0.001 < 0.01), which indicates that the Rui white injection can restore the number of white blood cells to normal level or even higher level. The white blood cell numbers of the high-dose group and the low-dose group of the five-cereal worms are obviously lower than that of a normal control group (P <0.05, P < 0.01), the dose group of the five-cereal worms is not different from the normal control group (P=0.748 > 0.1), and the white blood cell numbers of nude mice can be restored to normal level when the dose group of the five-cereal worms is in dose, but no positive effect is obvious. Compared with the model group, the dosage of the positive medicine and the five-cereal worms has significantly increased leukocyte count (P <0.01 and P < 0.01). Compared with the positive medicine, the five-cereal worm has significantly reduced white blood cell number at high, medium and low doses (P <0.01 ). The results show that: the five-cereal insects with medium dosage can treat nude mice leukopenia caused by cyclophosphamide chemotherapy, raise the number of white blood cells, and recover the function of differentiated white blood cells of bone marrow hematopoietic stem cells so as to improve immunity.

Analysis of lymphocyte results showed that the model group showed extremely significant drop in lymphocyte count (P < 0.01) compared to the normal control group, indicating successful modeling. The positive drug group, the five-cereal worm high-dose group and the medium-dose group had increased lymphocyte numbers compared with the model group, but no significant differences (P >0.1 ). The result shows that the high dose and the medium dose of the five-cereal worms can relieve the bone marrow suppression of the nude mice caused by cyclophosphamide chemotherapy to a certain extent, and the effect is equivalent to that of a positive medicine.

The effect of five cereal worms on the levels of the nude mouse cytokine IL-2, TGF-beta, caused by chemotherapy is shown in Table 2:

TABLE 2 influence of five cereal worms on the level of the nude mouse cytokine IL-2, TGF-beta caused by chemotherapy，n=5）

P <0.01, P <0.05 compared to normal control; compared to the model group, #p <0.01, #p <0.05; in comparison with the positive drug, the preparation method, and P <0.01, ++p <0.05.Δp <0.01, Δp <0.05 compared to the dose in wubi.

The trend of the effect of the five-cereal worms on the level of the nude mouse cytokine IL-2 caused by the chemotherapy is shown in figure 3, and the trend of the effect of the five-cereal worms on the level of the nude mouse cytokine TGF-beta caused by the chemotherapy is shown in figure 4.

IL-2 belongs to anti-inflammatory factors, and can mediate T, B cell proliferation and differentiation, stimulate hematopoietic stem cells to produce immune cells such as leucocytes and the like, and improve the immunity of organisms. IL-2 cytokine level results showed a significant decrease in IL-2 levels in the model group (P < 0.01) compared to the normal control group. The IL-2 levels were significantly elevated in the positive drug group, the five-cereal worm high, medium and low dose groups (P <0.01, P < 0.05) compared to the model group. Compared with the positive medicine group, the high and medium dose groups of the five-cereal insects have no difference (P > 0.1), and the low dose group of the five-cereal insects is significantly lower than the positive medicine group (P < 0.05). The results show that: the high, medium and low doses of the five-cereal insects can stimulate lymphocytes to secrete anti-inflammatory factors IL-2, relieve the symptom of low immunity of the nude mice caused by the chemotherapeutic drugs, and improve the immunity of the nude mice, and the high, medium dose of the five-cereal insects has the drug effect equivalent to that of positive drugs.

TGF-beta belongs to immunosuppressive factors and inhibits the immune function of the body. TGF- β cytokine level results showed significant increases in TGF- β levels (P < 0.01) in the model group compared to the low dose group of wubi. Compared with the model group, the positive drug group, the five-cereal worm high and medium dose group are obviously reduced (P <0.01, P <0.01 and P < 0.01). Compared with the positive medicine, the high and medium dose groups of the five-cereal insects have no difference (P > 0.1), and the low dose group of the five-cereal insects is obviously lower than the positive medicine group (P < 0.01). The low dose group had significantly reduced TGF- β water (P < 0.01) compared to the dose in the five-cereal worm. The results show that: the high and medium doses of the five-cereal worms can lower the level of the immunosuppressive factor TGF-beta, improve the body immunity of nude mice and have the drug effect equivalent to that of positive drugs.

The effect of five cereal worms on spleen index of nude mice is shown in table 3:

TABLE 3 influence of five cereal worms on spleen index of nude mice ，n=5）

In comparison with the normal control group, ^** P<0.01; in comparison with the set of models, ^## P<0.01; in comparison with the positive drug, the preparation method, ^▲▲ P<0.01. compared with the dosage of the five-cereal worms, ^△ P<0.05。

the trend of the effect of five cereal worms on the spleen index of nude mice is shown in fig. 5.

The spleen index results showed that the spleen index was significantly reduced (P < 0.01) in the model group compared to the normal control group, indicating successful modeling. Compared with the model group, the spleen index of the positive medicine group and the high-dose group of the five-cereal worms is obviously increased (P <0.01 and P < 0.01), and the spleen index of the middle-dose group and the low-dose group of the five-cereal worms is also increased, but no obvious difference exists. Compared with the positive medicine, the spleen index of the high, medium and low dose groups of the five-cereal worms is obviously reduced (P <0.01, P <0.01 and P < 0.01). The spleen index was significantly increased (P < 0.05) in the high dose group compared to the middle dose group of the five-cereal worms, and there was no significant difference in the low dose group. The results show that: the high dose of the five-cereal insects can obviously raise the spleen index of the nude mice with low immunity, and the immunity of the nude mice is improved, but the efficacy is inferior to that of positive drugs, and the medium dose and the low dose of the five-cereal insects also have the efficacy of raising the spleen index of the nude mice with low immunity, but the efficacy is inferior to that of high dose groups.

Discussion:

the white blood cell number and the lymphocyte number of the model group are obviously reduced (P <0.01 ) compared with the normal control group, the IL-2 level of the anti-inflammatory factor is obviously reduced (P < 0.01), the TGF-beta of the immunosuppressive factor is obviously up-regulated (P < 0.01), and the spleen index is obviously reduced (P < 0.01), thus indicating that the modeling is successful.

Secretion of leukocytes, lymphocytes and cytokines is an important index for evaluating cellular immune functions [12]. The results of this study showed that the number of white blood cells in the positive drug, five-cereal worm, was significantly increased in doses compared to the model group for white blood cells (P <0.01 ). Compared with the positive medicine, the five-cereal worm has significantly reduced white blood cell number at high, medium and low doses (P <0.01 ). The five-cereal insects can treat nude mice leukopenia caused by cyclophosphamide chemotherapy, increase the number of white blood cells, and recover the function of differentiated white blood cells of bone marrow hematopoietic stem cells so as to improve immunity. But the efficacy is inferior to that of the positive medicine Ruibai injection. For lymphocytes, the positive drug group, the five-cereal worm high and medium dose group had elevated lymphocyte numbers compared to the model group, but no significant differences (P >0.1 ). The result shows that the high and medium doses of the five-cereal worms can relieve the bone marrow suppression of the nude mice caused by cyclophosphamide chemotherapy to a certain extent, the effect is equivalent to that of a positive medicine, but the main action mechanism is probably not to promote the proliferation of lymphocytes.

IL-2 belongs to an anti-inflammatory factor, and can mediate T, B cell proliferation and differentiation, stimulate hematopoietic stem cells to produce immune cells such as leucocytes, and improve the body immunity [13]. The IL-2 cytokine level results show that the IL-2 level of the positive drug group, the five-cereal worm high, medium and low dose groups is obviously increased (P <0.01, P < 0.05) compared with the model group. Compared with the positive medicine group, the high and medium dose groups of the five-cereal insects have no difference (P > 0.1), and the low dose group of the five-cereal insects is significantly lower than the positive medicine group (P < 0.05). Description: the high, medium and low doses of the five-cereal insects can stimulate lymphocytes to secrete anti-inflammatory factors IL-2, relieve the symptom of low immunity of the nude mice caused by the chemotherapeutic drugs, and improve the immunity of the nude mice, and the high, medium doses of the five-cereal insects have the same drug effect as the positive drugs.

TGF-beta belongs to immunosuppressive factors and inhibits the immune function of the body. The TGF-beta cytokine level results show that the positive drug group, the five-cereal worm high and medium dose group are significantly reduced (P <0.01 ) compared with the model group. Compared with the positive medicine, the high and medium dose groups of the five-cereal insects have no difference (P > 0.1), and the low dose group of the five-cereal insects is obviously lower than the positive medicine group (P < 0.01). The low dose group had significantly reduced TGF- β water (P < 0.01) compared to the dose in the five-cereal worm. The high and medium doses of the five-cereal insects can be used for down regulating the level of the immunosuppressive factor TGF-beta, improving the organism immunity of the nude mice, and the drug effect is equivalent to that of a positive drug. The low dose effect of the five cereal worms is not obvious.

Thymus and spleen are important immune organs of animals. The thymus has very important functions in establishing immunity and reconstructing lost immunity. The spleen is the largest lymphoid organ in the body, and has a filtering effect on pathogens, immune complexes and other substances, and is also the place where mature lymphocytes reside and the place where immune responses to blood-derived antigens are generated. It is believed that a decrease in immune organ weight is due to immunosuppression, while an increase in immune organ weight is indicative of an immune enhancement. Since the experiment adopts nude mice as the study object, the nude mice have no thymus, so the experiment is discussed by the index of spleen. The spleen index results show that compared with the model group, the spleen index of the positive medicine group and the high-dose group of the five-cereal worms is obviously increased (P <0.01 and P < 0.01), and the spleen index of the middle-dose group and the low-dose group of the five-cereal worms is also increased without obvious difference. Compared with the positive medicine, the spleen index of the high, medium and low dose groups of the five-cereal worms is obviously reduced (P <0.01, P <0.01 and P < 0.01). The spleen index was significantly increased (P < 0.05) in the high dose group compared to the middle dose group of the five-cereal worms, and there was no significant difference in the low dose group. The results show that: the high dose of the five-cereal insects can obviously raise the spleen index of the nude mice with low immunity, and the immunity of the nude mice is improved, but the efficacy is inferior to that of positive drugs, and the medium dose and the low dose of the five-cereal insects also have the efficacy of raising the spleen index of the nude mice with low immunity, but the efficacy is inferior to that of high dose groups.

In conclusion, the five-cereal worm alcohol water double-extract can up-regulate the level of anti-inflammatory factor IL-2, down-regulate the level of immunosuppressive factor TGF-beta by increasing the number of white blood cells and lymphocytes, and increase spleen index to enhance the immune function of an immunosuppressive mouse.

Claims

1. The five cereal worm medicament is characterized in that: when the compound is matched with CTX, the mass ratio of the five-cereal worm medicament to the CTX is (57-230): 1, a step of;

the alcohol-water double-extraction liquid of the five-cereal worms takes the powder obtained by crushing the collected five-cereal worms as an extraction object;

2. Use of the five-cereal agent of claim 1 for the manufacture of an agent for alleviating/inhibiting the side effects of the chemotherapeutic agent cyclophosphamide.

3. The preparation method of the five-cereal worm medicament is characterized by comprising the following steps:

4. A method of preparing a five-cereal insect medicament according to claim 3, wherein: in the step (2), the five-cereal worm medicinal material is extracted by adopting 75% ethanol water solution for three times, and the mass of the ethanol water solution extracted each time is b=20×a.

5. A method of preparing a five-cereal insect medicament according to claim 3, wherein: in step (3), the residue is extracted three times with water, the mass c=20×a of each extracted water.

6. A method of preparing a five-cereal insect medicament according to claim 3, wherein: in the step (4), the mixed solution is concentrated until the quality of the mixed solution becomes a.