阅读说明：本技术 一种增强机体免疫力的中药组合物 (Traditional Chinese medicine composition for enhancing immunity of organism ) 是由 伍正会 伍洪立 于 2021-02-08 设计创作，主要内容包括：本发明公开了一种增强机体免疫力的中药组合物,其可以作为各中药配方药的基础药物,用于增强人体免疫力,从而达到抗病效果。一种增强机体免疫力的中药组合物,原料药包括：人参、丹参、蒲公英。通过上述技术方案,便可很好的解决现有技术中的问题。(The invention discloses a traditional Chinese medicine composition for enhancing immunity of organisms, which can be used as a basic medicine of various traditional Chinese medicine formulas for enhancing immunity of human bodies so as to achieve a disease-resistant effect. A traditional Chinese medicine composition for enhancing immunity of organisms comprises the following raw material medicines: ginseng, salvia miltiorrhiza, dandelion. Through above-mentioned technical scheme, alright fine solution problem among the prior art.)

1. A traditional Chinese medicine composition for enhancing immunity of organisms is characterized in that raw material medicines comprise: ginseng, salvia miltiorrhiza, dandelion.

2. The traditional Chinese medicine composition for enhancing immunity of organisms according to claim 1, wherein the raw material medicines comprise: 5-20 parts of ginseng, 5-20 parts of salvia miltiorrhiza and 10-30 parts of dandelion.

3. The traditional Chinese medicine composition for enhancing immunity of the organism according to claim 2, wherein the raw material medicines comprise: 10 parts of ginseng, 10 parts of salvia miltiorrhiza and 20 parts of dandelion.

Technical Field

The invention relates to the field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composition for enhancing body immunity.

Background

The traditional Chinese medicine is a traditional medicine in China, mainly adopts herbaceous plants as raw materials after being processed, and has the advantages of nature and difficult generation of drug resistance. The action mechanism of the traditional Chinese medicine is different from that of western medicines, and the action mechanism of the traditional Chinese medicine is mainly to improve the disease resistance of a human body by improving the immunity of the human body, so that the recovery of a patient is accelerated, and the curative effect is achieved.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition for enhancing the immunity of organisms, and provides a basic medicine composition which can achieve the effect of enhancing the immunity; meanwhile, on the premise of taking the traditional Chinese medicine as a basis, medicinal materials can be added according to different specific symptoms so as to meet the requirements of different symptoms.

A traditional Chinese medicine composition for enhancing immunity of organisms comprises the following raw material medicines: ginseng, salvia miltiorrhiza, dandelion.

As a preferred mode, the traditional Chinese medicine composition for enhancing the immunity of the organism comprises the following raw material medicines: 5-20 parts of ginseng, 5-20 parts of salvia miltiorrhiza and 10-30 parts of dandelion.

As a preferred mode, the traditional Chinese medicine composition for enhancing the immunity of the organism comprises the following raw material medicines: 10 parts of ginseng, 10 parts of salvia miltiorrhiza and 20 parts of dandelion.

The Chinese medicinal composition for enhancing immunity can be made into decoction, pill, powder, etc. by conventional method.

Detailed Description

The present invention aims at providing the traditional Chinese medicine composition for enhancing the immunity of the organism, and the present invention is further described in detail with reference to the following embodiments.

Example 1

A traditional Chinese medicine composition for enhancing immunity of organisms comprises the following raw material medicines: 10 parts of ginseng, 10 parts of salvia miltiorrhiza and 20 parts of dandelion.

Example 2

A traditional Chinese medicine composition for enhancing immunity of organisms comprises the following raw material medicines: 5 parts of ginseng, 5 parts of salvia miltiorrhiza and 10 parts of dandelion.

Example 3

A traditional Chinese medicine composition for enhancing immunity of organisms comprises the following raw material medicines: 20 parts of ginseng, 20 parts of salvia miltiorrhiza and 30 parts of dandelion.

Example 4

A traditional Chinese medicine composition for enhancing immunity of organisms comprises the following raw material medicines: 20 parts of ginseng and 20 parts of dandelion.

Example 5

A traditional Chinese medicine composition for enhancing immunity of organisms comprises the following raw material medicines: 20 parts of salvia miltiorrhiza and 20 parts of dandelion.

Example 6

A traditional Chinese medicine composition for enhancing immunity of organisms comprises the following raw material medicines: 20 parts of ginseng and 20 parts of salvia miltiorrhiza.

Examples of the experiments

1. Sample handling and administration mode

The decoction prepared in the examples 1 to 3 and the comparative examples 1 to 3 is extracted for 2 times at normal pressure and 80 to 90 ℃ for 30 to 60min, wherein the water amount is more than 10 times of the volume of the sample to be tested, and the water is combined and concentrated to the required concentration of 2 times.

2. Animal experiments

(1) Animal selection: inbred mice of 20g + -2 g, single sex, 10-15 mice per group are recommended.

(2) Dose grouping: three dose groups and a blank control group are set in the experiment, one dose group is 10 times of the recommended amount of a human body, and the other two dose groups are set.

(3) Time of sample administration: all experimental animals eat the complete nutrition compound feed, and the animals can freely eat and take water. The gavage volume for the test substance was 20 ml/(kg · Bw). The test samples were given for 30 days.

3. Index detection

ConA-induced splenic lymphocyte transformation experiment (MTT method) for mice

1) Reagent preparation

I: complete culture solution: the RPMI1640 culture solution is filtered and sterilized, 10% calf serum, 1% Pen/Strep/Glutamine (100 x, liquid) and 5 x 10 < -5 > mol/L beta-mercaptoethanol are added before use, and the pH is adjusted to 7.0-7.2 by using sterile 1mol/L HCl or 1mol/L NaOH to obtain the complete culture solution.

ii: ConA liquid: preparing 100 μ g/ml solution with double distilled water, filtering for sterilization, and storing in low temperature refrigerator (-20 deg.C).

iii: sterile Hank's solution: sterile Na HCO 3.5% before use₃Adjusting the pH value to 7.2-7.4.

Iv: MTT solution: 5mg of MTT was dissolved in 1ml of PBS pH7.2 and used as it is.

v: acidic isopropanol solution: to 96ml of isopropyl alcohol, 4ml of 1mol/L HCl was added and prepared just before use.

2) Preparation of spleen cell suspension: the spleen was aseptically removed and placed in a dish containing an appropriate amount of sterile Hank's solution, and the spleen was gently ground with forceps to make a single cell suspension. The spleens were filtered through a 200 mesh screen or triturated with 4 layers of gauze and washed 2 times with Hank's solution and centrifuged 10min (1000 r/min) each time. Then, the cells were suspended in 1ml of complete culture medium, and the number of viable cells (which should be 95% or more) was counted by staining with Trypol blue to adjust the cell concentration to 3X 10⁶One per ml.

3) Lymphocyte proliferation reaction: the cell suspension was added to a 24-well plate in two wells, 1ml per well, 75. mu.l ConA solution (equivalent to 7.5. mu.g/ml) was added to one well, and 5% CO was added to the other well as a control ₂Culturing for 72h in a 37 ℃ CO incubator. 4 hours before the end of the culture, 0.7ml of the supernatant was gently aspirated from each well, and 0.7ml of calf serum-free RPMI1640 culture medium was added thereto together with 50. mu.l/well of MTT (5 mg/ml), and the culture was continued for 4 hours. After the culture is finished, 1ml of acidic isopropanol is added into each hole, and the mixture is uniformly blown and beaten to ensure that the purple crystals are completely dissolved. Then subpackaging the cells into a 96-well culture plate, subpackaging 3-6 wells of each well as a parallel sample, and measuring the optical density value by using an enzyme linked immunosorbent assay detector at the wavelength of 570 nm. The solutions can also be transferred directly into a 2ml cuvette and the OD measured at 570nm on a model 721 spectrophotometer. If the control group is compared with the corresponding examples, P is less than 0.05, the control group is positive.

Examples 1 to 3 were positive, and examples 4 to 6 were negative.

Antibody-producing cell detection (Jerne modified slide method)

1) SRBC (step A): collecting sheep jugular vein, placing sheep blood into sterilized conical flask with glass beads, shaking in one direction to remove fiber, and storing in refrigerator at 4 deg.C for 2 weeks.

2) Preparation of complement: collecting guinea pig blood, separating serum (at least 5 guinea pig mixed serum, adding 1ml packed SRBC into 5ml guinea pig serum, standing in refrigerator at 4 deg.C for 30min, shaking frequently, centrifuging to obtain supernatant, subpackaging, storing at-70 deg.C, and diluting with SA buffer solution at a ratio of 1: 8-15.

3) Film coating on glass slides: a thin layer of agarose (0.5 g agarose added with double distilled water to 100ml, heated and dissolved) is brushed on the cleaning slide, and after the agarose is dried, the slide box can be stored for a long time for later use.

4) Immunizing animals: collecting sanguis Caprae Seu Ovis, washing with normal saline for 3 times, centrifuging (2000r/min) for 10min, counting cells, and injecting SRBC 5 × 10 into abdominal cavity or vein of each mouse⁷~2×10⁸And (4) respectively. The packed SRBC can also be prepared into 2% (V/V) cell suspension by using normal saline, and 0.2ml of the cell suspension is injected into the abdominal cavity of each mouse.

5) Preparation of spleen cell suspension: killing mice cervical vertebra dislocated after SRBC immunization for 4-5 days, taking out spleen, placing in a small plate containing Hank's solution, slightly shredding spleen to prepare cell suspension, filtering through a 200-mesh screen or grinding spleen with 4 layers of gauze, centrifuging (1000 r/min) for 10min, washing with Hank's solution for 2 times, finally suspending cells in 5ml RPMI1640 culture solution, counting cells, and adjusting cell concentration to 5 × 10⁶One per ml. Cells were also suspended in 8ml Hank's solution and the number of plaque formations in the spleen was determined.

6) Determination of plaques: heating and dissolving a surface layer culture medium (1 g of agarose and double distilled water to 100 ml), placing the mixture into a 45 ℃ water bath for heat preservation, mixing the mixture with 2 times concentration of Hank's solution with equal amount of pH 7.2-7.4, subpackaging small test tubes, wherein 0.5ml of each tube is added, 50 mu l of 10% SRBC (V/V prepared by SA solution) and 20 mu l of spleen cell suspension (5 multiplied by 106/ml) or 25 mu l of spleen cell suspension are added into the tubes, quickly and uniformly mixed, poured onto a glass slide with an agarose thin layer to be made into parallel sheets, after the agar is solidified, horizontally buckling the glass slide on a sheet frame, placing the glass slide into a carbon dioxide incubator for incubation for 1-1.5 h, adding complement (1: 8) diluted by SA buffer solution into a groove of the glass slide frame, continuing incubation for 1-1.5 h, and counting the number of hemolytic plaques. If the control group is compared with the corresponding examples, P is less than 0.05, the control group is positive.

Examples 1 to 3 were positive, and examples 4 to 6 were negative.

Determination of the median hemolysis value (HC50)

The principle is as follows: after an animal is immunized by the SRBC, an anti-SRBC antibody (hemolysin) is generated, the anti-SRBC antibody and the SRBC are incubated together, a hemolysin reaction can occur in the presence of complement, hemoglobin is released, and the content of the hemolysin in the serum of the animal is reflected by measuring the content of the hemoglobin;

instruments and materials: 721 spectrophotometer, centrifuge, thermostatic water bath, SRBC, complement (guinea pig serum), SA buffer, Japanese reagent (sodium bicarbonate 1.0g, potassium ferricyanide 0.2g, potassium cyanide 0.05g, distilled water to 1000 mL);

the experimental steps are as follows:

1) blood is taken from jugular vein of SRBC sheep, the sheep blood is put into a sterilized conical flask with glass beads and shaken in one direction to remove fiber, and the sheep blood is put into a refrigerator at 4 ℃ for storage for 2 weeks.

2) Preparation of complement the complement is prepared by collecting guinea pig blood, separating serum (mixed serum of at least 5 guinea pigs), adding 1mL packed SRBC into 5mL guinea pig serum before use, placing in refrigerator at 4 deg.C for 30min, shaking frequently, centrifuging to collect supernatant, packaging into small bottles, and storing at-70 deg.C. When in use, the solution is diluted by SA liquid according to the ratio of 1: 10.

3) Immune animal and serum separation: sheep blood was collected and washed 3 times with physiological saline, and centrifuged (2000r/min) for 10min each time. The packed SRBC was made into 2% (v/v) cell suspension with physiological saline, and each mouse was immunized by intraperitoneal injection of 0.2 mL. And after 4-5 days, removing the eyeball, taking blood, placing the blood in a centrifugal tube for about 1 hour, peeling the coagulated blood from the tube wall to fully separate out serum, centrifuging at 2000r/min for 10min, and collecting the serum.

4) And (3) hemolysis reaction: serum was diluted with SA buffer (typically 200-500 fold). 1mL of the diluted serum was placed in a test tube, and 0.5mL of 10% (v/v) SRBC and 1mL of complement (diluted 1: 10 with SA solution) were added in this order, and a control tube without serum (substituted with SA solution) was provided. And (5) preserving the temperature in a constant-temperature water bath at 37 ℃ for 15-30 min, and terminating the reaction in an ice bath. Centrifuging at 2000r/min for 10 min. Taking 1mL of supernatant, putting 3mL of Dushi reagent in a test tube, simultaneously taking 0.25mL of 10% (v/v) SRBC, adding the Dushi reagent to 4mL, fully mixing in another test tube, standing for 10min, blanking a control tube at 540nm, and respectively determining optical density values.

5) Data processing and result judgment: data statistics were generally performed by anova, and the amount of hemolysin was expressed as a half hemolysis value (HC50) and calculated by the following formula, and if P < 0.05 in the control group compared to the corresponding examples, it was positive.

The formula:

examples 1 to 3 were positive, and examples 4 to 6 were negative.

Carbon clearance test of mice

1) Solution preparation

I: ink for injection: diluting the India ink stock solution by 3-4 times with physiological saline.

ii: 0.1g of Na2CO3 was added to the Na2CO3 solution to make 100 ml.

2) Injecting ink: weighing, and injecting diluted India ink from mouse tail vein, wherein the diluted India ink is calculated as 0.1ml per 10g body weight. And immediately timing when the ink is injected.

3) And (3) determination: 20 μ l of blood was collected from the inner canthus venous plexus 2min (t 1) and 10min (t 2) after the injection of ink, and immediately added to 2ml of 0.1% Na₂CO₃In solution. The Optical Density (OD) values were measured at a wavelength of 600nm with a model 721 spectrophotometer, over Na₂CO₃The solution was used as a blank control. Mice were sacrificed, livers and spleens were removed, blood stains on the surfaces of the visceral organs were blotted with filter paper and weighed.

Calculated according to the following formula:

phagocytosis index:

k =(lgOD ₁-lgOD ₂)/(t2-t1)；OD ₁OD value for t1 blood sample; OD ₂The OD value was t 2. If the control group is compared with the corresponding examples, P is less than 0.05, the control group is positive.

Examples 1 to 3 were positive, and examples 4 to 6 were negative.

Cell Activity assay (lactate dehydrogenase assay)

1) Passage of target cells (YAC-1 cells): the target cells were subcultured 24h before the experiment. Hank's wash 3 times before application and complete medium in RPMI1640 was used to adjust the cell concentration to 4X 105 cells/ml.

2) Preparation of spleen cell suspension (effector cells): the spleens were aseptically removed and placed in a small dish containing a suitable amount of sterile Hank's solution, and the spleens were gently ground with forceps to make a single cell suspension. The spleens were filtered through a 200 mesh screen, or the spleens were triturated with 4 layers of gauze, or washed 2 times with Hank's solution, and centrifuged each time for 10min (1000 r/min). Discarding supernatant to bounce the cell pulp, adding 0.5ml of sterilized water for 20s, cracking erythrocytes, adding 0.5ml of 2-fold Hank's solution and 8ml of Hank's solution, centrifuging at 1000r/min for 10min, re-suspending with 1ml of RPMI1640 complete culture solution containing 10% calf serum, diluting with 1% glacial acetic acid, counting (the number of living cells should be more than 95%), staining with Taifenglan, counting (the number should be more than 95%), and finally adjusting the cell concentration to 1640 × 107/ml with the RPMI complete culture solution.

3) And (3) detecting the activity of NK cells: taking 100 mul of target cells and effector cells respectively (the effective target ratio is 50: 1), and adding the target cells and the effector cells into a U-shaped 96-hole culture plate; target cell natural release holes are filled with 100 mul of target cells and culture solution respectively, and target cell maximum release holes are filled with 100 mul of target cells and 1% NP40 or 2.5% Triton respectively; each of the above items was plated with three multiple wells, incubated for 4 hours in a 5% CO2 incubator at 37 ℃ and then centrifuged at 1500r/min for 5min, 100. mu.l of supernatant was aspirated from each well and placed in a flat-bottomed 96-well plate, 100. mu.l of LDH matrix solution was added simultaneously, the reaction was carried out for 3min, 30. mu.l of 1mol/L HCl was added to each well, and the Optical Density (OD) was measured at 490nm using a microplate reader. Calculating the NK cell activity according to the following formula; if the control group is compared with the corresponding examples, P is less than 0.05, the control group is positive.