CN101721612B - Chinese medicinal composition for raising leucocytes and preparation method and application thereof - Google Patents

Abstract

The invention discloses a traditional Chinese medicine composition for increasing white blood cells, which is mainly prepared by weight ratio of the following raw materials: 6-60 parts of astragalus, 6-60 parts of Polygonatum, 6-60 parts of trichosanthin and 3 parts of epimedium -30 servings. The invention also discloses the preparation method of the traditional Chinese medicine composition of the invention and its use in the preparation and treatment of leukopenia; improving the immune function of the body; promoting the proliferation of bone marrow hematopoietic stem/progenitor cells and protecting the hematopoietic microenvironment; repairing the effects of radiation on bone marrow hematopoietic tissue. damage and damage; prevention and treatment of myelosuppression caused by radiotherapy and chemotherapy; promotion of colony formation of myeloid-monoline hematopoietic progenitor cells; improvement and improvement of TCM symptoms and quality of life in cancer patients;

Description

A traditional Chinese medicine composition for raising white blood cells and its preparation method and application

technical field

The invention relates to a traditional Chinese medicine composition for increasing white blood cells, a preparation method and application thereof, and belongs to the field of traditional Chinese medicines.

Background technique

The most common side effect of tumor radiotherapy and chemotherapy is myelosuppression, which is mainly manifested as inhibition of proliferation of hematopoietic stem cells. Due to the short lifespan of leukocytes, leukopenia is common in clinical manifestations. Bone marrow suppression limits the consistency of radiotherapy and chemotherapy, thereby affecting the efficacy of radiotherapy and chemotherapy. In recent years, hematopoietic cytokines, such as G-CSF and GM-CSF, have been researched and developed at home and abroad, which play a good auxiliary role in chemotherapy. However, these hematopoietic factors are expensive, have a rebound phenomenon, and can cause side effects such as bone pain and fever. Traditional Chinese medicine has a certain effect on the prevention and treatment of myelosuppression caused by radiotherapy and chemotherapy, but most of them are used for newly diagnosed patients or patients with mild myelosuppression.

Contents of the invention

The technical problem to be solved by the present invention is to provide a traditional Chinese medicine composition capable of increasing white blood cells and effectively promoting the recovery of hematopoietic function, its preparation method and application. The finished medicine of the present invention promotes the expression of GM-CSF protein in splenocytes, increases the generation of endogenous GM-CSF, increases the activity of GM-CSF, and then promotes the differentiation and proliferation of leukocytes and bone marrow cells, which can increase the leukocytes of patients undergoing radiotherapy and chemotherapy, and prevent and treat Bone marrow suppression in patients with radiotherapy and chemotherapy can improve and improve TCM symptoms and life quality of cancer patients; for patients with qi deficiency, it can improve immunity and promote the recovery of patients with qi deficiency by nourishing qi and nourishing yin; and has no toxic side effects.

The traditional Chinese medicine composition of the present invention is mainly prepared from the following raw material Chinese medicinal materials in weight ratio: 6-60 parts of Astragalus membranaceus (Fisch.) Bge, 6-60 parts of Polygonatum sibiricum Red., 6-60 parts of Polygonatum sibiricum Red. kirilowii Maxim.) 6-60 parts and Epimedium (Epimedium brevicornum Maxim.) 3-30 parts.

Preferably, the raw Chinese medicinal materials also include 3-30 parts of Ligustrum lucidum Ait. and 6-60 parts of Drynaria fortunei (Kunze) J.Sm..

The traditional Chinese medicine composition of the present invention is mainly prepared from the following raw Chinese medicinal materials in parts by weight: 10-40 parts of Radix Astragali, 10-40 parts of Rhizoma Polygonatum, 10-40 parts of Trichosanthes trichosanthis, 5-20 parts of Ligustrum lucidum, and broken bone It is prepared from 10-40 parts of supplement and 5-20 parts of epimedium.

The best one is mainly prepared from the following raw materials by weight: 30 parts of astragalus, 30 parts of sealwort, 30 parts of trichosanthemi, 15 parts of epimedium, 15 parts of Ligustrum lucidum and 30 parts of drynaria.

The traditional Chinese medicine composition of the present invention can adopt the conventional method of Chinese medicine preparation to make any conventional preparation, preferably oral liquid preparation, granule, drop pill, powder, tablet, capsule (comprising hard capsule and soft capsule), fat capsule Plastids, sustained-release preparations, controlled-release preparations or injections (including small needles, infusion solutions and freeze-dried powders), more preferably oral liquid preparations, granules, drop pills, tablets, capsules or injections.

The present invention further relates to the preparation process of the pharmaceutical composition of the present invention: the above-mentioned raw materials can be directly dried and pulverized or extracted and refined by conventional extraction techniques in the field to obtain active pharmaceutical ingredients that can increase white blood cells and improve immunity. Three preferred preparation methods are:

Method 1: weighing the raw Chinese medicinal materials according to the weight ratio; drying and pulverizing the above-mentioned Chinese medicinal materials into fine powders to obtain fine powders of active ingredients.

Method 2: Weigh raw Chinese medicinal materials according to the weight ratio; decoct Astragalus, Rhizoma Polygonatum and Epimedium with water, filter, take the filtrate, centrifuge to remove solid matter, take the supernatant and concentrate it into a clear ointment. The rest of the raw medicinal materials are dried and pulverized into powder, and mixed evenly with the above clear ointment to obtain the active ingredient clear ointment.

Method 3: Weigh the raw Chinese medicinal materials according to the weight ratio; add water to decoct, filter, take the filtrate, centrifuge to remove solid matter, take the supernatant and condense it into a clear paste to obtain the active ingredient clear paste.

In the above method two or three, in the step of adding water to decoct, the amount of water added each time is preferably 8-16 times the weight of the crude drug, the decoction temperature is preferably the temperature at which the decoction liquid boils, and the decoction time is preferably 8-16 times the weight of the crude drug. Preferably it is 2-4 hours, and the number of times of decocting is preferably 1-4 times. When the number of decoctions is more than 2 times, filter after each decoction, combine the filtrates, and then perform centrifugation to remove solid substances, and take the supernatant and concentrate it into a clear paste. The relative density of the clear paste is preferably 0.8-1.5 (the temperature is below 60°C).

Further, the active ingredient fine powder prepared by method one, or the active ingredient clear paste prepared by method two or three, or the active ingredient fine powder made by drying and pulverizing the clear paste, and excipients according to the art Various pharmaceutical dosage forms are prepared by conventional methods, such as powder, oral liquid, granule, capsule, pill, tablet or injection and the like. Wherein, the excipients are selected according to the conventional knowledge in the field according to different dosage forms.

The preparation method of the oral liquid preparation of the traditional Chinese medicine composition of the present invention can be: add sweet taste element and potassium sorbate again to the active ingredient clear cream that the aforementioned method makes, and consumption can be conventional quantity, add the water of conventional dosage, stir , adjust the pH value to weakly acidic, preferably pH 6-7, which can be adjusted with NaOH aqueous solution, and then filtered, filled, and sterilized. The advantages of this dosage form are: rapid onset of action and high bioavailability.

The preparation method of the granule preparation of the traditional Chinese medicine composition of the present invention can be: add dextrin and sweetener again in the clear cream of active ingredient that the aforementioned method makes, and the consumption of dextrin is preferably the equal amount of clear cream quality, The dosage of the sweetener can be a conventional amount, generally below 10% by mass; after drying, granulating and sizing, the granule is obtained.

The preparation method of the capsule of the traditional Chinese medicine composition of the present invention can be: add the starch of conventional amount again to the clear ointment of the active ingredient that aforesaid method makes and mix, granulate, sieve, dry, granulate, fill, i.e. Get capsules.

The preparation method of the tablet of the traditional Chinese medicine composition of the present invention can be: dry the clear paste of the active ingredient prepared by the aforementioned method, pulverize, granulate, and press into tablets; or granulate the fine powder of the active ingredient prepared by the aforementioned method, Pressed into tablets.

The reagents and raw materials used in the present invention are all commercially available.

The traditional Chinese medicine composition of the present invention is especially suitable for treating leukopenia; improving the immune function of the body; promoting the proliferation of bone marrow hematopoietic stem/progenitor cells and protecting the hematopoietic microenvironment; repairing the damage and damage to bone marrow hematopoietic tissue caused by radiation; preventing and treating bone marrow suppression caused by radiotherapy and chemotherapy; Promote the formation of granule-monoline hematopoietic progenitor cell colonies; improve and improve the TCM symptoms and quality of life of cancer patients; it is also suitable for promoting the recovery of patients with Qi and Yin deficiency.

The therapeutic dose of the traditional Chinese medicine composition of the present invention can be: the daily dosage for adults is equivalent to 0.06-1.5 grams of crude drug/kg body weight. In the present invention, the "crude drug" refers to the quality of raw materials of Chinese medicinal materials.

The positive and progressive effect of the present invention is that the Chinese medicine composition of the present invention has been found through clinical and animal experiments: the Chinese medicine composition of the present invention can increase the white blood cells of cancer patients, prevent and treat severe bone marrow suppression in cancer patients, and improve and improve the quality of traditional Chinese medicine in cancer patients. symptoms and quality of life.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples.

Embodiment 1 Preparation of oral liquid preparation

1. Weigh raw Chinese medicinal materials: Astragalus 6kg, Polygonatum 6kg, Trichosanthes 6kg, Drynaria 6kg, Ligustrum lucidum 3kg, Epimedium 3kg.

2. Add 15 times the quality of the above medicinal materials to decoct for 3 hours, filter, add water to the filter residue and decoct once, filter, combine the filtrate twice, centrifuge to remove solid matter, take the supernatant and concentrate to obtain clear Paste (relative density 0.8, temperature 59°C).

3. Add 1kg of sweetener and 2kg of potassium sorbate, stir and add 30L of water, adjust the pH value to 6-7 with 10wt% NaOH aqueous solution, filter, fill and sterilize.

The preparation of embodiment 2 granules

1. Weigh raw Chinese medicinal materials: Astragalus 30kg, Polygonatum 30kg, Trichosanthum Fen 30kg, Rhizoma Drynariae 30kg, Ligustrum lucidum 15kg, Epimedium 15kg.

2. Add 14 times the quality of the above medicinal materials to decoct for 3 hours, filter, add water to the filter residue and decoct once again, filter, combine the filtrate twice, centrifuge to remove solid matter, take the supernatant and concentrate to obtain clear Paste (relative density 1.2, temperature 59°C).

3. Add dextrin with the same quality as the clear paste and 5% sweetener by mass, and then dry, granulate and granulate.

The preparation of embodiment 3 capsule preparation

1. Weigh raw Chinese medicinal materials: Astragalus 60kg, Polygonatum 60kg, Trichosanthes 60kg, Drynaria 60kg, Ligustrum lucidum 30kg, Epimedium 30kg.

2. Add 15 times the quality of the above medicinal materials to decoct for 3 hours, filter, add water to the filter residue and decoct once, filter, combine the filtrates twice, centrifuge to remove solid substances, and concentrate the supernatant to obtain clear Paste (relative density 1.5, temperature 40°C).

3. Add 8kg of starch and mix evenly, granulate, sieve, dry, granulate, and fill.

The preparation of embodiment 4 tablet

1. Weigh raw Chinese herbal medicines: Astragalus 10kg, Polygonatum 10kg, Trichosanthum Fen 10kg, Rhizoma Drynariae 10kg, Ligustrum lucidum 5kg, Epimedium 5kg.

2. Add 15 times the quality of the above medicinal materials to decoct for 3 hours, filter, add water to the filter residue and decoct once, filter, combine the filtrates twice, centrifuge to remove solid substances, and concentrate the supernatant to obtain clear Paste (relative density 1.1, temperature 59°C).

3. Drying, crushing, granulating, and pressing into tablets.

The preparation of embodiment 5 tablet

1. Weigh raw Chinese medicinal materials: Astragalus 40kg, Polygonatum 40kg, TCS 40kg, Drynaria 40kg, Ligustrum lucidum 20kg, Epimedium 20kg.

2. Add 15 times the quality of the above medicinal materials to decoct for 3 hours, filter, add water to the filter residue and decoct again, filter, and combine the two filtrates. The solid matter was removed by centrifugation, and the supernatant was concentrated to obtain a clear paste (relative density 1.1, temperature 50° C.).

3. Drying, crushing, granulating, and pressing into tablets.

The preparation of embodiment 6 tablet

1. Weigh raw Chinese medicinal materials: Astragalus 12kg, Polygonatum 12kg, Trichosanthum Fen 12kg, Rhizoma Drynariae 12kg, Ligustrum lucidum 6kg, Epimedium 6kg.

2. Add 15 times the quality of the above medicinal materials to decoct for 3 hours, filter, add water to the filter residue and decoct again, filter, and combine the two filtrates. The solid matter was removed by centrifugation, and the supernatant was concentrated to obtain a clear paste (relative density 1.2, temperature 50° C.).

3. Drying, crushing, granulating, and pressing into tablets.

Embodiment 7 preparation of active ingredient clear ointment

1. Weigh raw Chinese medicinal materials: Astragalus 6kg, Polygonatum 6kg, Trichosanthin 6kg, Epimedium 3kg.

2. Boil astragalus, sealwort and epimedium with 15 times the mass of water for 3 hours, filter, add water to the filter residue and repeat decoction once, filter, combine the filtrate twice, centrifuge to remove solid matter, and take the supernatant The liquid was concentrated to obtain a clear paste (relative density 0.8, temperature 50°C). Dry and pulverize the trichosanthemi powder into powder, mix evenly with the above clear paste to obtain the active ingredient clear paste.

Embodiment 8 preparation of active ingredient clear ointment

1. Weigh the raw Chinese medicinal materials:. Astragalus 60kg, Polygonatum 60kg, Trichosanthum Fen 60kg, Epimedium 30kg.

2. Add 8 times the quality of the above medicinal materials to decoct for 2 hours, filter, add water to the filter residue and decoct repeatedly 3 times, filter, and combine the filtrates of the two times. The solid matter was removed by centrifugation, and the supernatant was concentrated to obtain a clear paste (relative density 1.2, temperature 50° C.).

The preparation of embodiment 9 tablet

1. Weigh the raw Chinese medicinal materials:. Astragalus 20kg, Polygonatum 20kg, Trichosanthum Fen 20kg, Epimedium 10kg.

2. Add 16 times the mass of the above medicinal materials to decoct for 4 hours, filter, centrifuge the filtrate to remove solid matter, take the supernatant and concentrate to obtain a clear paste (relative density 1.4, temperature 59°C).

3. Drying, crushing, granulating, and pressing into tablets.

The preparation of embodiment 10～41 active ingredient fine powder

The raw Chinese medicinal materials were weighed according to the formula in Table 1; the above-mentioned Chinese medicinal materials were dried and pulverized into fine powders to obtain the active ingredient fine powders of Examples 10-41.

Example Astragalus (kg) Polygonatum (kg) Trichosanthes (kg) Epimedium (kg) Ligustrum (kg) Drynaria (kg) 10 6 60 60 30 \ \ 11 60 6 6 3 \ \ 12 6 60 6 3 \ \ 13 60 6 60 30 \ \ 14 6 6 60 3 \ \ 15 60 60 6 30 \ \ 16 6 6 6 30 \ \ 17 60 60 60 3 \ \ 18 6 60 60 30 30 60 19 60 6 6 3 3 6

20 6 60 6 3 3 6 twenty one 60 6 60 30 30 60 twenty two 6 6 60 3 3 6 twenty three 60 60 6 30 30 60 twenty four 6 6 6 30 3 6 25 60 60 60 3 30 60 26 60 60 60 30 3 60 27 6 6 6 3 30 6 28 6 6 6 3 3 60 29 60 60 60 30 30 6 30 10 40 40 20 20 40 31 40 10 10 5 5 10 32 10 40 10 5 5 10 33 40 10 40 20 20 40 34 10 10 40 5 5 10 35 40 40 10 20 20 40 36 10 10 10 20 5 10 37 40 40 40 5 20 40 38 40 40 40 20 5 40 39 10 10 10 5 20 10 40 40 40 40 20 20 10 41 10 10 10 5 5 40

Effect Example 1

1. Clinical data:

1.1 Criteria for case collection: All patients diagnosed with lung cancer, breast cancer, gastric cancer, and intestinal cancer by X-ray, CT, cytology, pathology, etc., with syndrome differentiation of Qi and Yin (blood) and Qi deficiency; age ≥ 18 years old, gender Limit; Karnofsky's score standard is more than 50 points, it is expected to complete the whole course of treatment and agree to receive chemotherapy patients. Exclude 1) patients who used other whitening drugs during chemotherapy; 2) patients with severe heart, liver, kidney, and blood system diseases; 3) patients with infection and pregnancy; 4) patients with a history of drug and food allergy.

1.2 Case selection: 112 patients with malignant tumors were observed, 95.5% of whom were re-treatment patients. They were randomly divided into 4 groups by opening envelopes. The treatment group of the traditional Chinese medicine composition of the present invention is divided into treatment group 1 and treatment group 2, and the control group is also divided into control group 1 and control group 2.

Treatment group 1: 28 patients, 19 males and 9 females. Aged 41-77 years old. Among them, there were 20 cases of lung cancer and 8 cases of other cancers.

Treatment group 2: 27 patients, 17 males and 10 females. Aged 42 to 75 years old. Among them, there were 24 cases of lung cancer and 3 cases of other cancers.

Control group 1: 33 patients, 21 males and 12 females. Aged from 37 to 77 years old. Among them, there were 23 cases of lung cancer and 10 cases of other cancers.

Control group 2: 24 patients, 13 males and 11 females. Aged 38-76 years old. Among them, there were 20 cases of lung cancer and 4 cases of other cancers.

The average course of chemotherapy in the two groups was 2.54 courses in the treatment group and 2.86 courses in the control group. There was no statistical difference in gender, age, disease type, and course of chemotherapy between the two groups, and they were comparable.

2. Treatment method

Chemotherapy regimens vary with disease types and pathological types, and they are all effective chemotherapy regimens with obvious myelosuppressive effects. MVP program for non-small cell lung cancer; L-MFP program for gastric cancer; NV program for breast cancer; same program for intestinal cancer as gastric cancer.

Treatment group 1: take the granules prepared by the method of Example 2 at the same time as chemotherapy, 2 times a day, 2 bags each time (0.4-0.8g crude drug/kg body weight/time) for 2-3 weeks as a course of treatment.

Treatment Group 2: After chemotherapy, WBC<4.0×10 ⁹ /L began to take the granules prepared by the method of Example 2, twice a day, 2 bags each time (0.4-0.8g crude drug/kg body weight/time), 2 weeks course of treatment.

Control group 1: taking rubia di-ester at the same time as chemotherapy, 4 tablets each time, 3 times a day, 2-3 weeks as a course of treatment.

Control group 2: After chemotherapy, WBC<4.0×10 ⁹ /L began to take rubia di-ester, 4 tablets each time, 3 times a day, for 2 weeks.

During the observation period, blood transfusions are not allowed, and any other drugs that affect white blood cells cannot be used.

3. Observation items: clinical whitening effect, clinical symptoms, improvement of symptoms, peripheral blood white blood cell count, urine and feces routine examination before and after treatment, X-ray, electrocardiogram, liver function, kidney function, and liver and spleen ultrasound examination.

4. Curative effect standard

4.1 TCM curative effect standard:

Classification of TCM symptoms: use (—), (+), (++), (+++) respectively according to none, mild, moderate, and severe

express.

Scoring method: None (—) is 0 points; light (+) is 1 point; medium (++) is 2 points; heavy (+++)

3 points.

Efficacy criteria: after treatment, the clinical symptom score decreased by ≥ 2/3, it was obviously improved; after treatment, the clinical symptom score decreased by ≥ 1/3, but < 2/3 was partially improved; after treatment, the clinical symptom score decreased by < 1/3 to no improvement.

4.2 Efficacy standard of western medicine According to the curative effect standard of increasing white blood cell (WBC) in the "Guiding Principles of Drug Clinical Research" formulated by the Ministry of Health:

Significantly effective: the total number of WBC > 4.0×10 ⁹ /L, and can maintain the curative effect during the course of treatment until 1 week after the drug is stopped, and the clinical symptoms are significantly reduced or disappeared;

Effective: total WBC <4.0×10 ⁹ /L, but increased (0.5-1.0×10 ⁹ /L) than before treatment and can maintain curative effect during the course of treatment until 1 week after drug withdrawal, and clinical symptoms improved;

Ineffective: the total increase of WBC <0.5×10 ⁹ /L, no improvement in clinical symptoms.

2. Research results

1.1 Curative effect of traditional Chinese medicine

Symptom changes in the treatment group and the control group before and after treatment:

Treatment group: 25 cases were significantly improved, 19 cases were partially improved, 11 cases were not improved, and the total effective rate was 80%;

Control group: 10 cases were significantly improved, 19 cases were partially improved, 28 cases were not improved, and the total effective rate was 50.88%;

There was a significant difference in symptom improvement between the treatment group and the control group.

2.1 Efficacy of western medicine

Treatment Group 1: 17 cases were markedly effective, 4 cases were effective, and 7 cases were ineffective, with a total effective rate of 75.00%;

Treatment group 2: 9 cases were markedly effective, 15 cases were effective, and 3 cases were ineffective, with a total effective rate of 88.89%;

Control group 1: markedly effective in 9 cases, effective in 9 cases, and ineffective in 15 cases, with a total effective rate of 54.55%;

Control group 2: 1 case markedly effective, 13 cases effective, 10 cases ineffective, the total effective rate was 58.33%;

The treatment group 1 was compared with the control group 1, and the treatment group 2 was compared with the control group 2. The differences were significant.

1.2 Changes of WBC in the two groups of patients before and after treatment: see Table 2. After 3 weeks of treatment, there was a significant difference in WBC difference between the treatment group and the control group (P<=0.0239<0.05). The recovery of WBC in the treatment group was significantly higher than that in the control group.

Table 2 Changes of WBC before and after treatment in the two groups (×10 ⁹ /L)

group Number of cases Before treatment 1 week after treatment 2 weeks after treatment 3 weeks after treatment therapy group 54 5.82±1.38 3.75±1.58 3.55±1.62 5.07±1.34 control group 57 6.30±1.42 3.69±1.62 3.36±1.65 4.35±1.31 P 0.7206 0.8114 0.3032 0.0239

Effect Example 2 Animal Experimental Research

1. Experimental animals and grouping: C ₅₇ BL/6J pure line mice, female, body weight 18-20g, 8-10 weeks old, 144 were randomly divided into 4 groups, 36 in each group, respectively for the traditional Chinese medicine composition of the present invention group, rubidin group, normal saline group, normal group.

2. Administration method:

Chinese medicine composition group: the granules prepared by the method of Example 2 are formulated into liquid, and each mouse is fed with 0.4ml (equivalent to crude drug 40g/kg) every day;

Rubia diester group: 0.4ml was given to each mouse in the stomach every day, equivalent to 100mg/kg crude drug;

Normal saline group: 0.4ml normal saline was given to each mouse by gastric feeding every day;

Normal group: no treatment.

3. Experimental method:

On the 2nd, 4th, 6th, and 8th days of administration, 9 mice in each group were sacrificed by cervical dislocation, the muscles were bluntly separated, the femurs were taken out, and quickly soaked in 2.5% glutaraldehyde. After fixing with 1% osmic acid for 1.5 hours, acetone dehydrated step by step, embedded in EPON 812 epoxy resin, sectioned, stained, and the ultrastructural changes of the mice in each group on the 2nd, 4th, 6th, and 8th day were observed under the electron microscope.

4. Results:

Under the electron microscope, the bone marrow of the mice in the normal group showed granulocytic and erythroid hematopoietic islands at different stages of differentiation.

On the 4th day of the experiment, the bone marrow cells of the mice in each medication group were significantly reduced, the granulocytes were seriously damaged, and a small amount of erythroid hematopoietic islands could be seen under the microscope. In the traditional Chinese medicine composition group, the cell membrane was complete, the mitochondria in the cytoplasm were slightly swollen, and the chromosomes in the nucleus had no major changes; the cell membrane in the rubia diester group was relatively complete, and the mitochondria were also swollen; the normal saline group was barren under the microscope, and bone marrow granulocytes were rarely seen , only a small amount of erythroid early hematopoietic cells.

On the 6th day of the experiment, the bone marrow cells in the traditional Chinese medicine composition group were densely packed with clear borders, and blood cells of different stages could be seen. In the rubia diester group, a small number of bone marrow blood cells, cell membrane rupture, chromatin leakage, sinus endothelial cell cytoplasm distorted and broken, and mitochondrial swelling were seen; in the normal saline group, the number of blood cells in the bone marrow was small, the cell membrane was irregular, and heterochromosomal agglomeration in the nucleus was aggregated .

On the 8th day of the experiment, the degree of mitochondrial swelling in the Chinese medicine composition group was reduced; under the microscope in the rubia diester group, hematopoietic cells increased, the cytoplasmic endoplasmic reticulum expanded significantly, and even cystic, and some cells had incomplete nuclear membranes. Chromosomes were condensed into plaques, and the distribution of autosomes was uneven. In the normal saline group, blood cells in the bone marrow increased, the cell membranes were irregular, the cytoplasmic endoplasmic reticulum expanded, and the mitochondria swelled significantly.

5. Summary

Chemotherapy is one of the main means of cancer treatment, but some chemotherapy drugs have severe bone marrow suppression, leading to interruption of chemotherapy, and death due to severe infection. In recent years, oncologists and manufacturers at home and abroad have developed hematopoietic cytokines, such as G-CSF and GM-CSF, which play a good auxiliary role in tumor treatment, but these hematopoietic cytokines are relatively expensive and have a rebound phenomenon. Can cause bone pain, fever and other side effects. In recent years, some progress has been made in the clinical research of traditional Chinese medicine on the leucocyte-increasing effect on cancer chemotherapy patients. The traditional Chinese medicine composition of the present invention not only has obvious curative effect on increasing leukocytes, improves body immunity, but also can improve symptoms and symptoms of traditional Chinese medicine. Although hematopoietic factors do not have.

Most patients undergoing chemotherapy are manifested as fatigue, dry mouth, soreness of the waist and knees, loss of appetite, soft and weak pulse and other Qi and Yin deficiency syndromes. The balance of yin and yang and the balance of qi and blood in the patient's body.

Traditional Chinese medicine believes that qi and blood come from the viscera, and the two breed and depend on each other and cannot be separated. There is blood in it, and there is qi in the blood, qi is used to generate blood and nourish blood, and blood is used to nourish qi to carry qi. Without qi, blood will not grow, and blood without qi will not grow. Invigorating qi can generate blood, yin and blood have the same source, and generating blood can also generate yin. Therefore, the traditional Chinese medicine composition of the present invention is also clinically applicable to patients with qi deficiency, and has achieved satisfactory results.

Animal experiments showed that on the 4th day of this experiment, observed under the electron microscope, the sinusoids of the bone marrow of the mice in the rubia diester group and the normal saline group were severely damaged, and the endothelial cells were distorted and ruptured. On the 6th day, they still did not return to normal. On the contrary, The hematopoietic function of the traditional Chinese medicine composition group of the invention recovers quickly; protects and improves the hematopoietic microenvironment of bone marrow; increases white blood cells and improves body immunity.

Effect Example 3 Animal Experimental Research

1. Experimental animals, administration methods and grouping

ICR mice, male, weighing 18-22 g. Seventy-eight mice were randomly divided into 6 groups, 13 in each group. Except for the normal control group, they were all irradiated with Se-137 radioactive source. The WBC was detected 1 day after the irradiation (1d), and then they were randomly divided into groups according to the WBC. : (1) normal control group (distilled water 20ml/kg/d); (2) positive model group with less leukocytes (distilled water 20ml/kg/d); (3) Likejun group (20mg Likejun/kg/d ); (4) large dose group of Chinese medicine composition (9.2g crude drug/kg/d); (5) medium dose group of Chinese medicine composition (4.6g crude drug/kg/d); (6) small dose group of Chinese medicine composition ( 2.3g crude drug/kg/d), intragastric administration after grouping, once a day.

2. Leukopenia model group

The mice were irradiated with Se-137 radioactive source at a dose rate of 0.87Gy/min, and the total dose was 4Gy. The white blood cells were detected 1 day after irradiation (1d), and the leukopenia model was defined as the white blood cells decreased to 4×10 ⁹ /L.

3. Experimental method

3.1 Peripheral blood test

On the 4th, 7th, and 10th day of administration, blood was taken from the tail vein of the mice in each group, and the contents of white blood cells, red blood cells, platelets, and hemoglobin were measured on a blood cell analyzer.

3.2 Bone marrow nucleated cell count (BMNC)

On the 10th day of administration, the animals in each group were weighed and sacrificed by dislocation. The right femur was taken, and all bone marrow was washed out with 10 mL of 3% acetic acid solution to form a single cell suspension, and BMNC were counted under a microscope.

3.3 Spleen nodule count (CFU-S)

On the 10th day of administration, the mice in each group were killed by dislocation, and the spleens were weighed and stored in Bouin's solution. On the second day, the number of splenic convex nodules was counted, which was the number of endogenous splenic nodules.

3.4 Thymus and spleen index: Weigh the thymus and spleen of mice in each group, and their ratio to body weight is the thymus and spleen index.

3.5 Preparation method of drug-containing serum

The normal ICR mice were randomly divided into 3 groups, and the Chinese medicine composition, Likejun or equal volume of normal saline (NS) were administered orally, twice a day for 3 consecutive days. Take blood from mouse eyeballs, store in a 1.5ml centrifuge tube, let stand at room temperature for 3 hours, centrifuge at 3000r/min for 10min, separate serum, inactivate at 56°C for 30min, and set aside.

3.6 Bone marrow cell proliferation assay (MTT method)

Six ICR mice were taken and received Se-137 radiation source irradiation with a total dose of 3.5Gy. On the second day after irradiation, the bilateral femurs of the mice were aseptically removed, and the bone marrow was flushed out with RP-MI1640 solution, and passed through a No. 4 needle. Make a single cell suspension, wash the cells twice, discard the supernatant, adjust the cell content to 5×10 ⁵ /ml, add 100 μl of cell suspension to each well of a 96-well culture plate, and then add drug-containing or blank serum, each well 30 μl, of which, 20 and 10 μl of drug-containing serum were added to each well of the medium-dose and low-dose groups of _traditional Chinese medicine composition, and the volume was reduced to 30 μl with blank serum. Add 20μl of 0.5% (5mg/ml) MTT, continue to cultivate for 4h, centrifuge at 1000r/min for 15 minutes, discard the supernatant, add 200μl of dimethyl sulfoxide (DMSO) to each well, mix well, let it stand for several minutes, and put it in a microplate reader The absorbance (A) value was detected at a wavelength of 570 mm, and this value represented the degree of bone marrow cell proliferation.

3.7 Colony culture of hematopoietic progenitor cells

The mice were sacrificed by dislocation (6 irradiated models). Soak in 75% ethanol for 3 minutes, take the complete femur, wrap it with sterile gauze, pick out the muscle, use a syringe to draw RPMI1640 solution to flush out the bone marrow cells, and filter through a No. 4 needle to make a single cell suspension.

Under sterile conditions, take a centrifuge tube with a capacity of 10ml, add 3-4ml of lymphocyte separation medium, load the bone marrow cells on the layering medium, centrifuge at 2000r/min for 15min, take out the yellow interface layer in the middle, and place it in another centrifuge tube. Add culture medium containing 10% calf serum to wash 2 to 3 times, filter through a No. 4 needle, count nucleated cells to make the content 2×10 ⁵ /ml, add 0.9ml of this bone marrow cell suspension to each culture dish (repeat 2 dishes for each mouse cell suspension), and add 100 μl of drug serum to each dish. GM-CSF10ng, add 100μl of 3% agar, shake gently to make it fully paved, send it to 5% CO ₂ , 37°C saturated humidity incubator for 7 days, count the number of colonies under an inverted microscope, if more than 40 The clonal unit of a cell is a colony.

3.8 CD34 ⁺ cell detection

On the 2nd day after irradiation, the model mice were randomly divided into groups, administered, and on the 7th day of administration, the mouse femoral bone marrow cells were washed out with phosphate buffered saline (PBS) containing bovine serum leukocyte concentration of 0.2%, and 1×106 Add 30 μl normal mouse serum to each cell to block the non-specific binding site, add 10 μl FITC-labeled rat anti-mouse CD34 antibody, add the corresponding control antibody to the control tube, react in the dark at 4°C for 30 minutes, add 2ml red blood cell lysate, After 5 minutes of action, the cells were washed twice, and propidium iodide (PI) staining solution with a final concentration of 3 μg/ml (microgram/ml) was added, detected by flow cytometry, and the green fluorescence was recorded.

4. Results

4.1 Effect on mouse WBC

Irradiation can reduce the WBC of the mice, and the difference is significant compared with the normal group. The traditional Chinese medicine composition can significantly increase the WBC of the model mice. Compared with the model group, there was a significant difference (P<0.05). On the 7th and 10th day of administration, the WBC of each dose group of the traditional Chinese medicine composition was increased compared with the model group, and there was a significant difference (Table 3).

Table 3 The influence of traditional Chinese medicine composition on mouse WBC

4.2 Effects on mouse spleen nodule count and bone marrow nucleated cell count

Administration of traditional Chinese medicine composition can increase CFU-S and BMNC of model mice, and the difference is significant compared with model group (Table 4).

Table 4 The influence of traditional Chinese medicine composition on mouse spleen nodule, bone marrow nucleated cell number, thymus index and spleen index

4.3 Effects on mouse bone marrow cell proliferation

The experimental results showed that the serum of the traditional Chinese medicine composition had a promoting effect on the proliferation of bone marrow cells in the control model mice (Table 5).

Table 5 Effect of Drug Serum on Mouse Bone Marrow Cell Proliferation

4.4 The effect of drug serum on the formation of myeloid-monolineage hematopoietic progenitor cell colony (CFU-GM)

The serum of the traditional Chinese medicine composition can promote the proliferation of mouse CFU-GM, and the difference is significant compared with the control group (P<0.001), indicating that the traditional Chinese medicine composition has a promoting effect on the proliferation of granuloid-monoline hematopoietic progenitor cell colonies (Table 6).

Table 6 Effect of drug serum on the formation of myeloid monolineage hematopoietic progenitor colony (CFU-GM)

group Dose/g/kg Amount of serum added/μl CFU-GM/unit normal - 100 21.8±4.7 Chinese medicine composition 9.2 100 43.8±7.5 Li Kejun 0.02 100 34.5±6.6

4.5 Effects on mouse CD34 ⁺ cells

CD34 hematopoietic cells include hematopoietic stem cells and hematopoietic progenitor cells. The proportion of CD34 ⁺ cells in the mouse bone marrow can reflect the number of hematopoietic stem and progenitor cells in the bone marrow. The results of this experiment show that the drug can increase the proportion of CD34 ⁺ cells in the irradiated model mice , suggesting that the drug has the effect of promoting the proliferation of bone marrow hematopoietic stem/progenitor cells (Table 7).

Table 7 Effects of Drugs on Mouse Bone Marrow CD34 ⁺ Cells

Conclusion: Chinese medicine composition of the present invention can obviously increase the white blood cell (WBC) of model mouse; Can increase the bone marrow nucleated cell count of model mouse and promote bone marrow cell proliferation; Can increase spleen nodule count, improve granule list It can increase the yield of colony forming units, increase the proportion of bone marrow CD34 cells, and promote the proliferation of hematopoietic stem/progenitor cells; it can protect the hematopoietic microenvironment of bone marrow in model mice; it can increase the thymus index of model animals, and has a certain immune enhancement effect.

Claims (7)

1. but the Chinese medicine composition of a leukocyte increasing, it is characterized in that: described Chinese medicine composition is to be made by following materials of weight proportions Chinese crude drug: 30 parts of the Radixs Astragali, 30 parts of Rhizoma Polygonatis, 30 parts of Radix Trichosanthis, 15 parts of Herba Epimedii, 15 parts of Fructus Ligustri Lucidi and 30 parts of Rhizoma Drynariae.

2. Chinese medicine composition as claimed in claim 1 is characterized in that: the dosage form of described Chinese medicine composition is oral liquid, granule, drop pill, powder, tablet, capsule, liposome, slow releasing preparation, controlled release preparation or injection.

3. the method for preparing of a Chinese medicine composition as claimed in claim 1 is characterized in that being selected from following three kinds of methods any:

Method one:

(1) takes by weighing raw materials of traditional Chinese medicinal materials according to described weight proportion;

(2) the above-mentioned raw materials Chinese crude drug is dried and crushed into fine powder, gets the active component fine powder;

Method two:

(1) takes by weighing raw materials of traditional Chinese medicinal materials according to described weight proportion;

(2) with the Radix Astragali, Rhizoma Polygonati and Herba Epimedii decocte with water, filter, get filtrating, solid matter is removed in centrifugalize, gets supernatant concentration and becomes clear paste, and all the other raw materials of traditional Chinese medicinal materials are being dried and crushed into powder, with above-mentioned clear paste mix homogeneously, gets the active component clear paste;

Method three:

(1) takes by weighing raw materials of traditional Chinese medicinal materials according to described weight proportion;

(2) with the decocte with water of above-mentioned raw materials Chinese crude drug, filter, get filtrating, solid matter is removed in centrifugalize, gets supernatant concentration and becomes clear paste, gets the active component clear paste.

4. method for preparing as claimed in claim 3 is characterized in that: in the method two or three, in the step of described decocte with water; The amount that at every turn adds water is 8-16 a times of crude drug weight; The temperature that decocts is the ebullient temperature of decoction liquor, and the time of decoction is 2-4 hour, and the number of times of decoction is 1-4 time; The described supernatant concentration of getting becomes in the step of clear paste, and the relative density of clear paste is 0.8～1.5.

5. method for preparing as claimed in claim 3; It is characterized in that: the active component fine powder that described method one is made; Or the active component clear paste that makes of described method two or three; Or dry by this clear paste, pulverize the active component fine powder of processing, process regular dosage form with excipient by this area conventional method.

6. method for preparing as claimed in claim 5 is characterized in that: described regular dosage form is oral liquid, granule, drop pill, powder, tablet, capsule, liposome, slow releasing preparation, controlled release preparation or injection.

7. Chinese medicine composition as claimed in claim 1 is in preparation treatment leukopenia; The human body immunity improving function; Promote bone marrow hematogenesis ancestral cells propagation, protection hematopoieticmicroenviron-ment; Repair damage and the damage of lonizing radiation to the bone marrow hematogenesis tissue; The bone marrow depression of control chemicotherapy; Promote grain-monosystem hemopoietic progenitor cell colony to form; Improve cancer patient's traditional Chinese medical science disease and quality of life; Or the application in the medicine of promotion deficiency of both QI and YIN patient's recovery.