RU2302879C2 - Pharmaceutical composition for treatment of rheumatism, method for its preparing and using - Google Patents

Abstract

FIELD: medicine, phytotherapy, pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to a composition prepared from the following raw medicinal plants: Tripterygium hypoglaucum (Levl.) Hutch, barrenwort (Epimedium), box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) taken in the following mass ratio: 1-4; 1-4; 1-4 and 1-4, respectively. Method involves the following procedures: weighing and milling of Tripterygium hypoglaucum (Levl.) Hutch and barrenwort (Epimedium), their separate boiling in water 3 times, wetting box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) in warm water at temperature 80-95°C 1-3 times, separate decantation of decocts and liquid, their separate passing through adsorption column, washing out with water up to clearing eluate, elution with 30-99.3% ethyl alcohol and mixing eluate with alcoholic liquid. The total weight of eluate is 1-8-fold more of weight of parent raw medicinal agents. Then method involves recycling each eluent of the raw medicinal agent, concentrating to specific gravity 1.1, drying extracts and their mixing. Pharmaceutical composition is used in treatment of rheumatism, rheumatic arthritis and chronic nephritis. Invention provides realization of indicated designation.

EFFECT: improved preparing method, valuable medicinal properties of pharmaceutical composition.

8 cl, 21 tbl, 13 ex

Description

This invention relates to an anti-rheumatic agent, in particular, to a pharmaceutical composition, a method for its preparation and use.

Rheumatism and rheumatoid arthritis (RA) is an intractable disease, about 18,000,000 patients became disabled due to RA. Research on new anti-RA drugs has been going on for about a hundred years; aspirin is the first commonly used anti-RA drug. Anti-RA drugs can mainly be divided into two types: non-steroidal anti-inflammatory drug (NSAIDs) and immunosuppressant. NSAIDs include DICLOFENAC SOD or indomethacin and adrenocorticogormone. Clinical research shows that NSAIDs are an effective remedy; an immunosuppressant and a cytotoxic agent include ametopterin, cyclofamide, penicillamine, and so on. In recent years, immunological regulation has been used in the treatment of rheumatic disease. All anti-rheumatic drugs have a relatively serious side effect, to date, a highly effective drug with hypotoxicity has not yet been obtained.

There are three areas in the research and development of antirheumatic drugs. The first area includes NSAIDs and a cell factor antagonist, for example, a recombinant soluble necrotic tumor factor antagonist, an interleukin-1 inhibitor, and an active platelet factor. The second direction is a new immunosuppressant or immunomodulator, for example cyclosporin A. The third direction is a complex medication.

In the field of Chinese traditional medicine, for the treatment of Bi-Syndrome (obstruction) (RA), we used "Ma-xing-shi-gan-tan (a decoction of Chinese ephedra, apricot ordinary, gypsum and Ural licorice)", "Fan-chi-huan-qi -tan (decoction of Stephanie Chetyretychinkova and astragalus) "and" U-tou-tan (decoction of aconite Sichuan) "by the medical and pharmaceutical specialist Zhang Zhong-ching of ancient China. “Torch-Flowers” is a wild plant in Sichuan, which, when tested in clinical practice in the regions (Sichuan), has proven to some extent effective for rheumatics. Unfortunately, many uncontrolled effects and a serious side effect on the human reproductive system have been identified.

There is a long history in the Chinese traditional treatment for Bi-Syndrome, with the Chinese traditional treatment for Bi-Syndrome showing high efficacy, and there are many drugs. In the Dictionaries of Chinese Pharmacy of 1995 and 2000, no less than 80 folk remedies and 29 finished medicines are mentioned. But there are still a number of problems, mainly: the effectiveness of the treatment for severe Bi-syndromes, for example rheumatoid arthritis, has not yet been high enough; the dosage form of the pharmaceutical product does not meet the requirements of modern life, some drugs have a high treatment efficiency, but with serious side effects, for example, a pharmaceutical preparation from Tripterygium wilfordii.

Known pharmaceutical composition for the treatment of autoimmune diseases, such as rheumatoid arthritis, inflammation and pain, including Tripterygium wilfordii (Patent CN 1098933 from 02.22.95).

The composition has a high treatment efficiency, but has a serious side effect.

Therefore, the development of an anti-rheumatic drug with high efficiency and hypotoxicity, the dosage form of which corresponds to the forms of use of the medicine and modern life, especially the anti-rheumatic drug, the effectiveness of treatment of which is close to synthetic anti-rheumatic drugs, but with a weak toxic side effect, is necessary.

The aim of the present invention is to develop a complex antirheumatic composition with high efficiency and hypotoxicity and ease of use.

Another objective of this invention is to develop a method of preparing an anti-rheumatic composition.

The purpose of the claimed invention is achieved by selecting raw medicinal materials from which the pharmaceutical composition is prepared in various dosage forms, including:

Tripterygium hypoglaucum (Levl.) Hutch

and at least one of materials such as

Goryanka, large-flowered (Epimedium brevicornum Maxim.),

Berry berries (Lycium barbarum L.),

Seeds of dodder (Cuscuta chinensis Lam., Cuscuta australis R. Br.)

or a mixture thereof.

The pharmaceutical composition is obtained from the above raw medicinal materials taken in various mass ratios.

The claimed composition may consist of Tripterygium hypoglaucum (Levl.) Hutch and at least one of the three mentioned medicinal materials.

The optimal mass ratio of these raw medicinal materials according to this invention is:

Tripterygium hypoglaucum (Levl.) Hutch 1 ~ 4 Goryanka large-flowered 1 ~ 4 Dereza Berries 1 ~ 4 Dodder seeds 1 ~ 4

Also proposed is a composition consisting of an optimal mass ratio of raw medicinal materials according to the invention:

Tripterygium hypoglaucum (Levl.) Hutch 2 Goryanka large-flowered 2 Dereza Berries one Dodder seeds one

Also proposed is a composition prepared from two raw medicinal materials in a mass ratio:

Tripterygium hypoglaucum (Levl.) Hutch 1 ~ 4 Goryanka large-flowered 1 ~ 4

The composition is proposed to be prepared from the named raw medicinal materials in the following weight ratio:

Tripterygium hypoglaucum (Levl.) Hutch 2 Goryanka large-flowered 2

A composition is prepared from raw medicinal materials taken in a mass ratio:

Tripterygium hypoglaucum (Levl.) Hutch 1 ~ 4 Goryanka large-flowered 1 ~ 4 Dereza Berries 1 ~ 4

The optimal mass ratio of raw medicinal materials for the preparation of the claimed composition may be:

Tripterygium hypoglaucum (Levl.) Hutch 2 Goryanka large-flowered 2 Dereza Berries one

The optimal mass ratio of raw medicinal materials for the preparation of the claimed composition may be:

Tripterygium hypoglaucum (Levl.) Hutch 1 ~ 4 Goryanka large-flowered 1 ~ 4 Dodder seeds 1 ~ 4

The optimal mass ratio of raw medicinal materials for the preparation of the claimed composition may be:

Tripterygium hypoglaucum (Levl.) Hutch 2 Goryanka large-flowered 2 Dodder seeds one

The total content of icariin C ₃₃ H ₄₀ O _{15 of the} above medicinal formulations is not less than 2.0 mg.

The optimal mass ratio of raw medicinal materials for the preparation of the claimed composition may be:

Tripterygium hypoglaucum (Levl.) Hutch 1 ~ 4 Dereza Berries 1 ~ 4 and / or Dodder seeds 1 ~ 4

The optimal mass ratio of raw medicinal materials for the preparation of the claimed composition may be:

Tripterygium hypoglaucum (Levl.) Hutch 2 Dereza Berries 1 and / or Dodder seeds one

Using the above raw medicinal materials in the above ratio and using conventional pharmaceutical technology, any dosage forms acceptable in clinical practice are obtained, e.g., pill, powder, paste, tablet, capsule, e.g. hard capsule and soft capsule, granule, injection and so on.

Another objective of the present invention is achieved by the use of a method for preparing the above pharmaceutical composition, comprising the following operations: weighing the raw medicinal materials and grinding Triptervgium livpoglaucum (Levl.) Hutch and called Goryanka krupnotsvetkovaya, separate boiling them in water 3 times, soaking the named Berries of derez, Seeds of dill at a temperature of 80-95 ° C 1-3 times, separate transmission of the resulting broths and obtained liquids through a wide-necked resin adsorption column, washing with water a molar column before clarification of the effluent, eluting with 30-99.3% ethyl alcohol until a dark-colored liquid flows out, collecting the eluent before its color changes from dark to light, removing the alcoholic liquid from the column with water, mixing the eluate with alcoholic liquid, the total weight of the eluate is 1-8 times heavier than the weight of the named raw medicinal products, recycling the eluent of each of the raw medicinal material to extract alcohol, concentration to a specific gravity of 1.10, drying the extracts of each raw l Medicinal materials individually or in combination, mixing them uniformly or mixing in proportion to obtain a dosage form acceptable in clinical practice.

Another objective of the present invention is achieved by that. what is proposed is a method of preparing the named pharmaceutical composition consisting in that Tripterygium hypoglaucum (Levl.) Hutch is crushed into small pieces, extracted three times separately in 13-, 10-, 10-fold waters for one hour. Goryanka, large-flowered, is crumbled into segments, extracted separately three times in 15-, 10-, 10-fold waters for one hour. Dereza berries are crushed into coarse material and soaked in 20-fold waters at a temperature of 80-95 ° C for one hour. Grapefruit seeds are crushed into coarse powder, soaked in 31-fold water at 80 ° C for one hour, the extracts or soaking liquids of the four raw medicinal materials are filtered separately, then passed through a wide-necked resin adsorption column, eluted with 70% ethanol to the outflow of the eluent of a dark color, which is collected before its color changes from dark to light, the eluent of each of these raw materials is recycled to extract alcohol, concentrate comfort, dried derived medicinal extract powder is mixed uniformly or in proportion to give a dosage form that is acceptable in clinical practice.

A study of the pharmacodynamics of this invented agent (Fenshipin capsule from the named pharmaceutical compositions for the treatment of rheumatism) suggests that the use of Fenshipin by perfusion can significantly inhibit the primary and secondary injuries of arthritis from the adjuvant (AA) of a large mouse; markedly inhibit the delayed hypersensitive reaction (DTH) of the ears of a small mouse caused by 2,4-dinitrofluorobenzene (DNFB); markedly inhibit the antibody formation of hemolysin of a small mouse and the activity of macrophage, splenocyte 1L-1, 1L-2, 1L-6, TNF of a small mouse. Fenshipin can markedly inhibit ConAom-induced lymphocyte transformation. Fenschipin can also significantly inhibit CD4, CD8 cells, in particular noticeable for CD4, and does not significantly affect the CD4 / CD8 ratio. The above actions of Fenshipin have a visible linear dose-response relationship. The minimum effective dose is 12 ~ 18 (medicinal raw materials) g / kg. Fenshipin can also markedly inhibit NK cell activity. However, with an effective dose, Fenshipin does not cause atrophy of the thymus, spleen and other immune organs, nor does it inhibit macrophage activity.

Fenshipin can markedly inhibit the inflammatory response, it can inhibit the increased capillary permeability of the small mouse abdomen caused by acetic acid, inhibit the small mouse ear swelling caused by croton oil, inhibit small mouse pleurisy and leukocyte aggregation in the mouse mouse CMC capsule caused by gelatin from eye chondrus However, Fenshipin has weak inhibition for swelling of the legs and nail caused by gelatin from eye chondrus, and granulation tampon hyperplasia Ani big mouse. In addition, Fenshipin can markedly inhibit the body twist of a small mouse caused by acetic acid.

Experimental Example 1

Effect on arthritis from adjuvant (AA)

1.1 Prophylactic effect on AA of a large mouse

по изменению периметров, измеряемых до инъекции FCA и после инъекции FCA, результаты представлены в таблице 1.1, 1.2. По истечении срока взвешивают вес основных органов животных разных групп, результаты представлены в таблице 1.3, 1.4.72 large SD mice of the same litter were selected, including half female and half male, body weight 180 ~ 220 g, randomly divided into 6 groups, in each group 12 mice. In each cage 6 mice live. The maximum perimeters of the legs and ankle joint of the left and right rear claw are measured with an accurate narrow measuring tape as a normal value. All mice are injected with the claimed means of the same volume of the appropriate concentration or they are injected with a solution of gummitragant (Xihuangqi) of the same volume. After 1 hour, an intradermal injection is carried out with Freund's complete adjuvant (Freund-FCA), 0.1 ml of each mouse in the left back sole. The agent is poured once a day, continuously for 30 days, every day the perimeters of the legs and ankle joint of the left and right hind claws of the large mouse are measured by the same method. In this experiment, the degree of swelling is counted.

for the change in perimeters measured before FCA injection and after FCA injection, the results are presented in table 1.1, 1.2. At the end of the term, the weight of the main organs of animals of different groups is weighed, the results are presented in table 1.3, 1.4.

1.1 Influence of Fanshipin on the degree of swelling of the ankle joint of the left claw and foot after FCA injection of a large mouse infected

Group dose (g / kg) degree of swelling

3d 9d 12d 14d 16d Id 2d control - 0.69 ± 0.17 0.69 ± 0.12 0.92 ± 0.18 0.84 ± 0.41 1.10 ± 0.30 1.65 ± 0.68 2.10 ± 0.55 Fan ship 7.5 0.74 ± 0.12 0.66 ± 0.074 0.83 ± 0.13 0.77 ± 0.27 1.11 ± 0.45 1.34 ± 0.53 1.91 ± 0.61 Fan ship fifteen 0.80 ± 0.24 0.62 ± 0.13 0.76 ± 0.18 0.49 ± 0.17 * 0.73 ± 0.34 * 1.00 ± 0.48 * 1.38 ± 0.67 * Fan ship thirty 0.75 ± 0.19 0.67 ± 0.19 0.87 ± 0.28 0.63 ± 0.22 0.73 ± 0.34 * 0.82 ± 0.43 ** 1.05 ± 0.53 ** Triplerygium hypoglalaucum (Levl.) 5 0.72 ± 0.11 0.68 ± 0.16 0.91 ± 0.18 0.66 ± 0.23 0.88 ± 0.29 1.03 ± 0.36 * 1.37 ± 0.33 * Hutch prednisone 0.01 0.64 ± 0.14 0.64 ± 0.16 0.50 ± 0.26 0.46 ± 0.25 0.72 ± 0.46 * 0.87 ± 0.46 ** 1.28 ± 0.69 * Group dose (g / kg) degree of swelling

22d 24d 26d 28d 18d 20d control - 2.18 ± 0.44 2.05 ± 0.46 2.00 ± 0.46 2.04 ± 0.57 1.92 ± 0.65 1.83 ± 0.67 Fan ship 7.5 1.74 ± 0.73 1.81 ± 0.55 1.81 ± 0.52 1.77 ± 0.55 1.65 ± 0.55 1.55 ± 0.49 Fan ship fifteen 1.32 ± 0.59 ** 1.28 ± 0.58 ** 1.34 ± 0.61 * 1.33 ± 0.67 * 1.20 ± 0.64 * 1.08 ± 0.58 ** Fan ship thirty 0.95 ± 0.50 ** 0.87 ± 0.51 ** 0.95 ± 0.54 ** 0.89 ± 0.59 ** 0.90 ± 0.57 ** 0.86 ± 0.51 ** Tripterygium hypoglaucum 5 1.47 ± 0.43 ** 1.50 ± 0.43 ** 1.49 ± 0.43 * 1.42 ± 0.53 * 1.40 ± 0.56 * 1.32 ± 0.57 (Levl.) Hutch prednisone 0.01 1.18 ± 0.7 ** 6 1.03 ± 0.67 ** 1.05 ± 0.69 * 0.90 ± 0.64 ** 0.86 ± 0.65 ** 0.85 ± 0.59 ** Compared with the control group * P 0.05, ** P <0.01 (the same in the following)

1.2 Effect of Fenshipin on the degree of swelling of the ankle joint of the left claw and foot after FCA injection for a large mouse that becomes ill

Group dose (r / kg) degree of swelling

14d 16d 18d 2d 9d 12d control - 0.14 ± 0.05 0.06 ± 0.10 0.34 ± 0.36 0.80 ± 0.52 1.43 ± 0.67 1.36 ± 0.61 Fan ship 7.5 0.18 ± 0.06 0.10 ± 0.14 0.26 ± 0.36 0.82 ± 0.52 1.31 ± 0.64 1.28 ± 0.71 Fan ship fifteen 0.15 ± 0.08 0.02 ± 0.06 0.13 ± 0.10 * 0.37 ± 0.31 * 0.90 ± 0.56 * 0.79 ± 0.60 * Fan ship thirty 0.18 ± 0.09 0.06 ± 0.06 0.16 ± 0.08 * 0.29 ± 0.20 ** 0.49 ± 0.41 ** 0.33 ± 0.29 ** Tripterygium hypoglaucum (Levl.) Hulch prednisone 5 0.16 ± 0.07 0.01 ± 0.07 0.11 ± 0.10 0.44 ± 0.19 ** 0.87 ± 0.56 * 0.84 ± 0.67 * 0.01 0.20 ± 0.06 0.08 ± 0.08 0.21 ± 0.16 0.44 ± 0.43 0.99 ± 0.63 0.84 ± 0.74 * Group dose (g / kg) degree of swelling

24d 26d 28d 20d 22d control - 1.28 ± 0.57 1.38 ± 0.64 1.35 ± 0.75 1.20 ± 0.78 1.12 ± 0.63 Fan ship 7.5 1.33 ± 0.71 1.31 ± 0.73 1.27 ± 0.73 1.16 ± 0.73 1.07 ± 0.65 Fan ship fifteen 1.74 ± 0.57 * 1.92 ± 0.61 * 0.95 ± 0.64 * 0.88 ± 0.58 * 1.83 ± 0.55 Fan ship thirty 0.27 ± 0.30 ** 0.34 ± 0.31 ** 0.32 ± 0.33 ** 0.31 ± 0.32 ** 0.34 ± 0.32 ** Tripterygium hypoglaucum (Levl.) Hulch 5 0.82 ± 0.65 * 0.89 ± 0.70 * 0.80 ± 0.67 * 0.83 ± 0.68 0.75 ± 0.69 prednisone 0.01 0.82 ± 0.72 * 0.79 ± 0.74 * 0.75 ± 0.67 ** 0.68 ± 0.64 * 0.71 ± 0.67

1.3 Influence of Fenshipin on the change in body weight of large mice that become ill

Group dose (g / kg) change in body weight (g) initial body weight body weight after 1 month of AA disease weight gain control - 228 ± 34 231 ± 52 3 7.5 229 ± 34 220 ± 46 -9 Fan ship fifteen 223 ± 40 232 ± 34 9 thirty 224 ± 37 256 ± 60 32 Tripterygium hypoglaucum (Levl.) Hutch 5 226 ± 45 230 ± 43 four prednisone 0.01 264 ± 55 244 ± 31 -21 1.4 Influence of Fenshipin on the scales of immune organs (preventive experiment) of a large mouse that becomes ill:

Group dose (g / kg organ ratio (g tissue / 100 g body weight) liver spleen thymus adrenal gland control - 3.92 ± 0.65 0.34 ± 0.10 0.098 ± 0.040 0.027 ± 0.01 Fan ship 7.5 3.73 ± 0.29 0.31 ± 0.09 0.078 ± 0.038 0.027 ± 0.008 Fan ship fifteen 3.48 ± 0.32 0.38 ± 0.10 0.100 ± 0.034 0.023 ± 0.005 Fan ship thirty 3.38 ± 0.28 * 0.44 ± 0.12 * 0.100 ± 0.032 0.022 ± 0.007 Tripterygium hypoglaucum (Levl.) Hutch 5 3.21 ± 0.30 ** 0.36 ± 0.05 0.052 ± 0.011 ** 0.026 ± 0.009 prednisone 0.01 3.04 ± 0.20 ** 0.32 ± 0.08 0.050 ± 0.060 ** 0.02 ± 0.004 *

1.2 therapeutic effect on AA of a large mouse

так: периметр на дату измерения минус периметр на текущий день начала вливания средства. Результаты представлены в таблице 1.5.1.6. весы основных органов представлены в таблице 1.7.Select 50 large male SD mice, randomly divide them into 5 groups, treat them with the same method, however, they begin to pour the agent into the mice 12 days after the inflammation caused by FCA adjuvant injection, once a day, continuously for 2 weeks, the degree is calculated swelling

so: the perimeter on the date of measurement minus the perimeter on the current day of the start of the injection of funds. The results are presented in table 1.5.1.6. the scales of the main organs are presented in table 1.7.

1.5 The therapeutic effect of Fenshipin on swelling of the ankle of the left claw after injection with FCA adjuvant for a large mouse that becomes ill

Group dose (g / kg) swelling

1d 2d 4d 6d control - 1.81 ± 0.27 1.92 ± 0.19 2.12 ± 0.22 2.16 ± 0.27 Fan ship 7.5 1.68 ± 0.50 1.64 ± 0.54 1.70 ± 0.57 1.82 ± 0.61 Fan ship fifteen 1.44 ± 0.41 * 1.51 ± 0.36 ** 1.65 ± 0.34 ** 1.74 ± 0.31 ** Fan ship thirty 1.50 ± 0.56 1.48 ± 0.41 ** 1.51 ± 0.44 ** 1.59 ± 0.51 ** prednisone 0.01 1.78 ± 0.51 1.70 ± 0.51 1.63 ± 0.50 * 1.58 ± 0.50 ** Group dose (g / kg) swelling

8d 10d 12d 14d control - 1.92 ± 0.32 1.87 ± 0.34 1.92 ± 0.39 1.78 ± 0.44 Fan ship 7.5 1.67 ± 0.68 1.60 ± 0.71 1.61 ± 0.77 1.58 ± 0.71 Fan ship fifteen 1.46 ± 0.37 ** 1.48 ± 0.30 * 1.28 ± 0.37 ** 1.22 ± 0.38 ** Fan ship thirty 1.29 ± 0.58 ** 1.29 ± 0.65 ** 1.26 ± 0.67 ** 1.20 ± 0.68 * prednisone 0.01 1.27 ± 0.46 ** 1.09 ± 0.54 ** 0.94 ± 0.50 ** 0.94 ± 0.42 **

1.6 The therapeutic effect of Fenshipin on swelling of the ankle joint of the right claw after injection with FCA adjuvant for a large mouse that becomes ill

Group dose (g / kg) swelling

2d 4d 6d 8d control - 0.36 ± 0.26 0.45 ± 0.25 0.55 ± 0.34 0.47 ± 0.29 Fan ship 7.5 0.12 ± 0.25 0.34 ± 0.32 0.48 ± 0.41 0.28 ± 0.38 Fan ship fifteen 0.21 ± 0.18 0.38 ± 0.27 0.44 ± 0.33 0.21 ± 0.33 * Fan ship thirty 0.10 ± 0.48 0.06 ± 0.28 ** 0.11 ± 0.24 ** 0.06 ± 0.27 ** prednisone 0.01 0.10 ± 0.13 * 0.15 ± 0.28 * 0.11 ± 0.25 ** -0.08 ± 0.34 ** Group dose (g / kg) swelling

10d 12d 14d control - 0.48 ± 0.25 0.46 ± 0.31 0.40 ± 0.36 Fan ship 7.5 0.35 ± 0.30 0.30 ± 0.29 0.30 ± 0.35 Fan ship fifteen 0.19 ± 0.45 * 0.06 ± 0.31 ** -0.06 ± 0.34 ** Fan ship thirty 0.02 ± 0.39 ** 0.05 ± 0.38 * -0.02 ± 0.41 ** prednisone 0.01 -0.13 ± 0.28 ** -0.26 ± 0.36 ** -0.33 ± 0.39 ** n = 10, compared with the control group, * P <0.05, ** P <0.01

1.7 Effect of Fanshipin on the weight of the body and immune organs (prevention) of a large mouse that becomes ill

Group dose (r / kg) organ ratio (g tissue / 100 g body weight) liver spleen thymus adrenal gland control - 0.35 ± 0.23 0.35 ± 0.061 0.073 ± 0.014 0.026 ± 0.0071 Fan ship 7.5 3.21 ± 0.52 0.33 ± 0.091 0.071 ± 0.026 0.024 ± 0.0085 Fan ship fifteen 3.40 ± 0.54 0.36 ± 0.014 0.067 ± 0.022 0.023 ± 0.0048 Fan ship thirty 2.79 ± 0.43 0.32 ± 0.083 0.069 ± 0.029 0.023 ± 0.0072 Tripterygium hypoglaucum (Levl.) Hutch 5 3.92 ± 0.59 0.35 ± 0.100 0.075 ± 0.034 0.027 ± 0.0060 prednisone 0.01 3.52 ± 0.35 0.28 ± 0.047 * 0.05 ± 0.011 * 0.02 ± 0.0043 *

Tables 1.1, 1.2, 1.3, 1.5, 1.6 show that for a large mouse that develops AA, Fenshipin can strongly inhibit primary joint damage after injection with FCA adjuvant and secondary damage to the joint of a large mouse after injection with FCA adjuvant. An infusion of the claimed agent was effective both simultaneously with the onset of inflammation and 2 weeks after inflammation. This indicates that Fenshipin has a significant prophylactic and therapeutic effect on arthritis AA of the large mouse. Analysis of the effect of Fenshipin on specific immune swelling of the ankle joint of the hind limbs and non-specific swelling of the claw of a large mouse shows that Fenshipin's effect on swelling of the ankle joint is stronger. This result indicates that the main effect of Fenshipin is to inhibit the immune inflammatory response.

The results in tables 1.3, 1.4 and 1.7 show that during the entire experiment, the body weight of a large mouse with AA did not increase significantly. In the group receiving the effective dose of Fanshipin, an increase in the weight of a large mouse was observed. During treatment with prednisone and in the prophylactic group, a decrease in the body weight of a large mouse was noted, a noticeable atrophy of the thymus and adrenal glands was revealed. Atrophy of the thymus gland was also observed in the Tripterygium hypoglaucum (Levl.) Hutch group, however, in the three groups taking different doses of Fenshipin, there was no noticeable effect on the thymus and adrenal gland.

1.3 Pathological change from AA in the treatment of the claimed means of a large mouse

Select 45 large SD mice and divide them into 6 groups, body weight 180 ± 20 g, after the formation of AA through the use of Freund's adjuvant, treatment is started by injection into the stomach of Fenshipin. Infusions continue for 5 days. 1 hour after the last use of the product, the joint parameters of the large mouse are evaluated and counted. The hind limb joint of the large mouse, which has undergone secondary damage, is fixed with formaldehyde. After staining tissues NOT under a microscope, changes in the composition of the synovial membrane and cartilage were studied. The results of the composition indices of different groups of the large mouse are presented in table 1.8.

1.8 Influence of Fanshipin on the joint performance of a large mouse that becomes ill

Group dose (g / kg) the number of mice (piece) joint index control group - 8 0 ** model group AA - 7 6.2 ± 0.49 Fan ship 7.5 9 4.86 ± 0.90 ** Fan ship fifteen 7 4.71 ± 0.95 ** Fan ship thirty 7 4.56 ± 1.13 ** Glucosidorum Tripterygll Totorum 0.006 7 4.57 ± 0.79 ** Compared to the model group ** P <0.01

According to the degree of swelling of each joint, we individually evaluate the joint parameters by 0-4 points, the joint index is determined by a combination of four points. The norm for evaluating four limbs and joints is as follows: 0 points - normal, 1 point - only red, 2 points - red and slight swelling, 3 points - severe swelling, 4 points - joint deformation and stiffness.

When examined under a microscope, hyperplasia of the synovial layer of the hind limb joint of the large mouse of the model group, an increase in collagen fiber, the formation of a noticeable granuloma due to infiltration of lymphocytes and plasmocytes; synovial cell denaturation, redness of the cytoplasm and pycnosis of the cell nucleus, exfoliation of the epithelium of the synovial membrane in some areas; cartilage atrophy, its surface is rough and uneven, slight chondrocyte hyperplasia. After treatment with Fenshipin of a different dose group, we can notice a relief of inflammation of the synovial tissue of the joint, an increase in the formation of collagen fiber; exfoliation of part of the synovial cell; hyperplasia of the surface cells of the cartilage, its surface becomes smooth, the cartilage is in a state of recovery.

In a large mouse of the control group, we can notice hyperplasia of the synovial layer, an increase in collagen fiber, the formation of a noticeable granuloma due to infiltration of lymphocytes and plasmocytes, denaturation of the synovial cell, redness of the cytoplasm and pycnosis of the cell nucleus, exfoliation of the synovial membrane epithelium in some areas. After Fenshipin’s treatment of a different dose group, there is a relief of inflammation of the synovial tissue of the joint, an increase in the formation of collagen fiber, exfoliation of part of the synovial cell, hyperplasia of the surface cartilage cells, its surface becomes smooth, the cartilage is in a state of recovery.

Experimental Example 2: Effect on the Delayed Hypersensitive Reaction (DTH) of the Ears of a Small Mouse Caused by 2,4,4-Dininthrochlorobenzene (2,4-DNFB)

50 small NIH mice were selected, including half female and half male, randomly divided into 5 groups. Sensitization is carried out with a 1% solution of acetone 2.4-DNFB at a dose of 0.025 ml for each mouse at an epilation site in the abdomen. Three days later, sensitization is enhanced by the same method. 5 days after sensitization, the right ear of a small mouse is smeared with 1% DNFB edible oil solution at a dose of 0.01 ml for each mouse for aggression, after 24 hours a small mouse is killed, the weight of the left and right ear is weighed on torsion scales, the difference between them (mg) represents the intensity of the DTH reaction of a small mouse. The experiments are carried out in different processes of immunization and reception of funds.

2.1 the effect of receiving funds in the whole process on DTH

The process of immunization and taking the drug is as follows:

table 2.1.
Fenshinin's Effect on the Delayed Hypersensitive NIH Mouse Response Caused by

Group dose (g / kg) term for receiving funds (day) the number of mice (piece) ear swelling percentage (%) percent inhibition (%) P value control 10 34.20 ± 3.77 Fan ship 27 0-5 10 26.24 ± 3.34 23.3 0.01 Fan ship 40 0-5 10 12.99 ± 4.96 62.0 <0.01 Fan ship 60 0-5 10 10.43 ± 7.53 69.5 <0.01 dexamethasone 0.003 0-5 10 13.93 ± 4.41 59.3 <0.01 control 10 42.43 ± 5.28 Fan ship 40 -2-0 10 31.50 ± 10.52 25.0 <0.01 Fan ship 40 -2-2 10 30.88 ± 7.92 27.2 0.01 Fan ship 40 -2-5 10 21.07 ± 4.62 * 50.3 0.01 Fan ship 40 5-6 10 32.00 ± 9.37 41.7 0.01 cyclofamide 0.05 -2-2 10 39.40 ± 10.78 8.1 > 0.05 cyclofamide 0.05 -2-0 10 37.47 ± 6.71 1 1.7 > 0.05 control 10 38.50 ± 4.67 cyclofamide 0.1 × 3 once and separately at 0, 2, 4 - day 10 23.00 ± 7.65 40.3 0.01 cyclofamide 0.25 -3d 10 41.84 ± 7.75 -8.7 <0.01 Fenshipin Cyclofamide 60 0-4 10 27.20 ± 10.20 29.4 + Fanshipin 0.25+ -3.0-4 10 38.07 ± 6.65 1.1 60 * compared with other groups P <0.05 or P <0.01

The results in Table 2.1 show that Fenshipin can significantly inhibit DTH of a small mouse caused by DNFB, the inhibition intensity is noticeably dose-dependent, with increasing doses, the inhibition also increases, at a dose of 60.9 g / kg, the inhibition of DTH is 69.5%.

2.2 The effect of different periods of receipt of the declared funds on the DTH of a small mouse

The immunization processes and the reception of funds and their results are presented in the lower column of table 2.1. The middle column shows 2 days before sensitization and on the same day of sensitization, 2 days before sensitization and 2 days after sensitization, 2 days before sensitization and 5 days after sensitization, before and after aggression, taking the drug can significantly inhibit the reaction , however, taking the drug throughout the process before and after sensitization, that is, two days before sensitization and 5 days after sensitization, shows a stronger inhibition. This indicates that the process and mechanism of Fenshipin's action on DTH can be associated both with cells that were earlier involved in inhibiting the DTH reaction, and with late effector cells and intermediate cells inhibiting the DTH reaction. This mechanism is different from the action of cyclofamide. 2 days before sensitization and on the same day of sensitization, or 2 days after sensitization, a small dose of cyclofamide will not affect the DTH reaction.

The lower column of Table 2.1 shows that a single dose of high-dose cyclofamide 3 days before sensitization does not only not inhibit the DTH reaction of the small mouse, but the reaction is also enhanced because Th cell function is relatively activated as a result of strong inhibition of Ts cells by cyclofamide. At this time, if cyclofamide is used in conjunction with Fenshipin, which has a marked inhibition effect on DTH, then the effect of Fanshipin inhibition can be negated. This indicates that Fenshipin differs from cyclofamide in the mechanism of action on DTH, since it has high activity in the inhibition of TH cells.

Experimental Example 3: Effect on Humoral Immunity

3.1 Influence on the antibody formation of hemolysin in a normal small mouse caused by chicken erythrocyte immunity (SEC)

190 small mice weighing 18-22 g, including half female and half male, were randomly divided into 19 groups, each mouse was immunized with an injection of 5% CRBC at a dose of 0.2 ml. 7 days after immunization, blood is taken by removal of the eyeball. After dilution of blood samples with saline, the effect of Fenshipin on the hemolysin antibody formation of small mice of different groups is determined. Fanshipin infusion is carried out at different times of immunization. The results are presented in tables 3.1, 3.2, 3.3.

table 3.1
Fenshipin's effect on small mouse mouse hemolysin antibody production

Group dose (g / kg) deadline for receiving funds the number of mice (piece) hemolysin value percent inhibition (%) P value control 10 169.0 ± 62.0 Fan ship eighteen 0-7 10 46.0 ± 15.6 72.8 <0.01 Fan ship 27 0-7 10 35.4 ± 12.0 79.1 <0.01 Fan ship 40 0-7 10 28.2 ± 5.9 83.3 <0.01 Fan ship 60 0-7 10 16.7 ± 3.0 90.1 <0.01 Tripterygium hypoglaucum (Levl.) Hutch 13.3 0-7 10 121.0 ± 88.0 ** 28.4 <0.015 cyclofamide 0.02 0-7 10 35.0 ± 12.0 79.3 <0.01 ** in comparison with Fenshipin with the same content (40 g / kg) Tripterygium hypoglaucum (Levl.) Hutch P <0.01

table 3.2
The effect of Fenshipin on the antibody formation of hemolysin in a small mouse

Group dose (g / kg) deadline for receiving funds the number of mice (piece) hemolysin value percent inhibition (%) P value control - - 10 124.70 ± 42.60 Fan ship 12 0-7 10 75.00 ± 53.10 39.9 <0.05 Fan ship eighteen 0-7 10 45.60 ± 22.70 63.4 <0.01 Fan ship 27 0-7 10 29.10 ± 22.10 76.8 <0.01 Fan ship 40 0-7 10 28.20 ± 5.30 77.4 <0.01 Tripterygium hypoglaucum (Levl.) Hutch 6.0 0-7 10 143.50 ± 67.90 ** > 0.05 cyclofamide 0.02 0-7 10 27.80 ± 6.60 77.9 <0.01 ** in comparison with Fenshipin of the same dose (18 g / kg) P <0.0

table 3.3
Fznshipin effect on small mouse mouse hemolysin antibody production

Group dose (g / kg) period of reception the number of mice (piece) hemolysin value percent inhibition (%) P value control - - 10 256.0 ± 26.0 Fan ship eighteen -7-7 10 198.0 ± 50.0 22.7 <0.01 Fan ship eighteen -3-7 10 156.0 ± 85.0 39.1 <0.01 Fan ship eighteen 0-7 10 98.0 ± 35.0 61.7 <0.01 cyclofamide 0.02 0-7 10 25.0 ± 4.0 90.2 <0.01

The results in the above three tables show that Fenshipin significantly inhibits the hemolysin antibody formation of small mice from different populations, and the effect increases with increasing dose. There is a good dose-response relationship, the minimum dose of inhibition is 12 g / kg, compared with Tripterygium hypoglaucum (Levl.) Hutch, which is the main composition of Fanshipin, has a more noticeable strong effect of inhibition on antibody formation. The results in Table 3.1 show that Fenshipin's activity is 2.25 times higher than Tripterygium hypoglaucum (Levl.) Hutch. The action of Tripterygium hypoglaucum (Levl.) Hutch at a dose of 13.5 g / kg is weaker than the action of Fenshipin containing Tripterygium hypoglaucum (Levl.) Hutch at a dose of 6 g / kg.

3.2 Effect on the immunological function of the humor of a small mouse with AA

Small NIH mice with a body weight of 20 ± 2 g were selected, intradermal injection with Freund's adjuvant into the toe of the right hind leg at a dose of 0.05 ml for each mouse was performed. After 3 weeks, a small mouse model AA is formed. After randomly dividing into 6 groups, separate funds are poured separately for 5 days. Simultaneously with taking the drug, the mouse is immunized with an injection of 10% sheep erythrocytes (CRBC) at a dose of 0.5 ml for each, after 5 days they are killed, and the spleen is taken. After washing with Hank's liquid, a suspension of lymphocytes is prepared by adjusting the cell concentration to 2 × 10 ⁷ / ml. Take 1 ml of lymphocyte suspension and place in a test tube, adding 1 ml of 0.2% SRBC and 1 ml of complement 1:30. It is placed in a water bath and incubated for 1 hour at 37 ° C, centrifuged at 2000 ppm for 5 minutes, the supernatant is taken and the optical density is measured with a 722 spectrophotometer at a wavelength of 415 nm. The value represents the amount of PFC.

The other part is taken from the blood of the above sensitized small mouse, the serum is separated, its antibody titer is determined by an agglutination test, and it is represented by the value Log2, the results are shown in table 3.4.

table 3.4
Fenshipin's influence on the immunological function of the humor of a small mouse that becomes ill

Group dose (g / kg) the number of mice (piece) PFC (OD) lgM (Log2) control group - 8 0.819 ± 0.013 # 6.875 ± 0.641 model group AA - 10 0.940 ± 0.019 ** 7.700 ± 0.599 * Fan ship 5 8 0.834 ± 0.012 ** # 6.875 ± 0.641 # Fan ship 10 8 0.834 ± 0.012 ** # 6.750 ± 0.886 # Fan ship twenty 8 0.830 ± 0.014 ** # 6.375 ± 0.518 ## Glucosidorum Tripterygll Totorum 0.012 10 0.835 ± 0.015 ** # 6.950 ± 0.597 #

Compared with the control group * P <0.05, ** P <0.01; compared with the reference group # P <0.05, ## P <0.01.

Table 3.4 shows that the PFC, IgM of a small mouse suffering from AA is significantly higher than that of a normal mouse. Fenshipin can significantly reduce the amount of antibody-forming cells (PFC) and the amount of antibody formation (IgM) of the spleen of a small mouse that develops AA.

Experimental example 4: effect on cutaneous passive anaphylactic reaction (PCA) of a large mouse

A large mouse is taken and an intramuscular injection of 10 mg / kg of ovalbumin is administered, at the same time, an injection of 2 × 10 ¹⁰ /0.2 ml into the abdominal cavity of Bacillus pertussis is carried out for immunization. After 2 weeks, kill a large mouse, take blood and separate the serum in the reserve.

Again, 60 large mice with a body weight of 150 ~ 200 g, including half female and half male, were randomly selected into 6 groups. In case of light anesthesia with ether, intramuscular injection of the above anti-valbumin serum is carried out (dilution degree 1: 5 and 1:10, d1, d2 separately) 0.1 ml at the epilation site on the mouse back, 2 points for injection are taken for each degree of dilution. After 48 hours, aggression is carried out by intravenous injection of 0.5% physiological solution of blue Evans in a dose of 1 ml containing 1 mg of ovalbumin. After 20 minutes, kill the large mouse with decapitation, turn the skin of the back of the mouse, based on the color depth and area of the blue spot, many people evaluate the category of blue spot by the degree of exudation of the dye. Then blue skin is cut into small pieces and soaked in 5 ml of 0.1% sodium sulfonate acetone solution (7: 3), centrifugation is carried out after 48 hours. Take the supernatant and determine the optical density at 590 nm, calculate the degree of PCA reaction of a large mouse and the percentage of inhibition. The results are presented in table 4.

table 4
Fenshipin's influence on the PCA of a large mouse

Group dose (g / kg) mark d1 d2 light coefficient d1 absorption d2 control - 5.60 ± 1.78 2.40 ± 2.46 0.191 ± 0.129 0.096 ± 0.106 Fan ship 12 7.50 ± 2.51 4.20 ± 2.49 0.402 ± 0.213 * 0.192 ± 0.175 Fan ship 24 7.10 ± 2.13 4.10 ± 1.79 0.310 ± 0.177 0.137 ± 0.099 Fan ship 48 6.00 ± 1.83 1.70 ± 1.95 0.121 ± 0.109 0.024 ± 0.026 * Tripterygium
hypoglaucum
(Levl.) Hutch 8 6.11 ± 1.27 2.56 ± 1.67 0.223 ± 0.122 0.074 ± 0.045 Ketotifen 0.1 2.78 ± 1.64 ** 0.67 ± 1.41 0.033 ± 0.024 ** 0.027 ± 0.019 * Compared with the control group * P <0.05, ** P <0.01

Table 4 shows that Fenshipin weakly inhibits the PCA of a large mouse, only with a large dose compared with the control group there is a noticeable difference.

Experimental Example 5: Effect on Cell Factor

5.1 Effect on small mouse TNFα, IL-2

60 small ICR mice with a body weight of 18 ~ 22 g, including half of the female and half of the male, are randomly divided into 6 groups, individually Fenshipin is poured into them in different doses or other means. The infusion is carried out once a day, continuously for 10 days, 24 hours after the last dose, they are killed. Under aseptic conditions, a macrophage or splenocyte is taken from the abdominal cavity of a small mouse, washed with Hank's solution 2 times, RPIM 1640 solution without serum 1 time, 2 × 10 ⁸ / ml cell suspension is prepared using a 5% FCS-RPMI 1640 solution, separately added LPS 10 ng / ml or ConAlong / ml. Cultivation is carried out at a temperature of 37 ° C under conditions of 5% CO ₂ 48 hours, TNFα or IL-2 is determined.

Definition of TNFα:

Take the bar coated with monoclonal antibody TNF-α of the small mouse. A 50 μl / hole culture supernatant is added to the bar. After 60 minutes of finding the bar at room temperature, labeled Biotin antibody is added, at 25 ° C for 2 hours, then enzyme-labeled streptavidin is added, after 30 minutes the substrate is added. After 30 minutes, a cliff solution is added, the OD value is determined at a wavelength of 450 nm. Based on the OD values, the TMF-α content (ng / ml) is calculated on a standard curve.

Definition of IL-2:

CTLL logarithmic cells whose growth is dependent on IL-2 are taken and a 1 × 10 ⁵ / ml cell suspension is prepared using a 5% FCS-RPMI 1640 solution. CTLL cell suspension is poured into a flat-bottomed cell culture plate with 96 openings 100 μl / hole and pour the supernatant for cultivation 100 μl / hole, copy each sample to 3, at the same time compare the standard sample rHIL-2 of different degrees of dilution with the nutrient liquid. Under conditions of 37 ° C, 5% CO _{2 is} cultivated 24 hours, 6 hours before the end of the cultivation, centrifuged, a supernatant of 110 μl / hole is isolated, MTT 10μl / hole is added, at 37 ° C after 3 hours the OD value at 570 nm and the OD value are determined at 630 nm. The final OD value of each sample lies in the OD range at 570 nm - OD at 630 nm.

table 5.1
Fenshipin's influence on TNF and

Group dose (g / kg) the number of mice (piece) TNF (pg / ml) 1L-2 (lU / ml) control - 10 87.80 ± 14.63 26.30 ± 4.22 12 10 62.14 ± 13.13 ** 16.00 ± 2.89 ** Fan ship 24 10 58.60 ± 9.63 ** 18.80 ± 2.86 ** 36 10 54.40 ± 10.88 ** 18.20 ± 2.86 ** Tripterygium hypoglaucum (Levl.) Hutch 8 10 58.25 ± 10.32 ** 16.00 ± 2.88 ** cyclofamide 0.02 10 42.20 ± 9.57 ** 10.10 ± 3.00 ** * P <0.05, ** P <0.01

The results in Table 5.1 show that Fenshipin significantly inhibits TNFα, at a dose of 12 g / kg the inhibition effect is very noticeable, the effect increases with increasing dose, however, the dose-response curve is shallow. For IL-2, Fenshipin also has a noticeable inhibition effect, but the dose-response is not obvious.

5.2 Effect on IL-1, IL-6

70 small NIH mice with a body weight of 18 ~ 22 g, including half of the female and half of the male, were randomly divided into 7 groups. Each one is injected with Fenshipin of a different dose or other means once a day, continuously 10 days, 24 hours after the last dose, they are killed, a macrophage or splenocyte is taken from the abdominal cavity and IL-1 and IL-6 are determined.

Definition of IL-1:

Under aseptic conditions, a macrophage or splenocyte is taken from the abdominal cavity, washed with Hank's solution 2 times, serum-free RPIM 1640 solution 1 time, 4 × 10 ⁶ / ml cell suspension is prepared using 5% FCS-RPMI 1640 solution. 1 ml of suspension is taken and infused in a centrifugal tube with a round bottom, under conditions of 37 ° C, 5% CO _{2 is} cultivated for 1 hour, non-adherent cells are isolated, 5% FCS-RPMI 1640 and LPS solutions (10 ng / ml) are added, at 37 ° C 5% CO ₂ cultivate 72 hours, repeatedly repeat freezing and thawing, keep at 4 ° C. Again, a small C57 mouse was taken, thymocytes were taken under aseptic conditions, a 1 × 10 ⁶ / ml cell suspension was prepared using a 5% FCS-RPMI 1640 solution.

Take 100 μl of a thymocyte cell suspension and 100 μl of supernatant, which underwent repeated freezing and thawing, and place them on a flat-bottomed cell culture bar with 96 holes, copy each sample by 3, at the same time compare a standard rHIL-1 sample of different dilution levels with nutrient liquid, then add ConA 2 ng / hole, at 37 ° C, 5% CO _{2 is} cultured for 72 hours, 14H before the end of cultivation, add 3H-TdR 0.1 μCi / hole, cells are harvested with a multi-stem cell collection device, the values are determined cpm

Definition of IL-6:

Under aseptic conditions, a small mouse splenocyte is taken, washed 2 times with Hank's solution, 1 time without serum RPIM 1640 solution, 2 × 10 ⁶ / ml cell suspension is prepared using 5% FCS-RPMI 1640 solution. 1 ml is taken and placed in a centrifugal tube with a round bottom, ConA (10 ng / ml) was added, at 37 ° C. 5% CO _{2 was} cultivated for 72 hours.

CTLL logarithmic growth cells whose growth is dependent on IL-6 are injected into a 1 × 10 ⁵ / ml cell suspension using a 5% FCS-RPMI 1640 solution.

Place the MH60 cell suspension into a flat-bottomed cell culture bar with 96 holes 100 μl / hole, and culture supernatant 25 μl / hole is poured, supplemented with 5% FCS-RPMI 1640 200 μl / hole solution, copy each sample to 3, at the same time compare the standard rHIL-sample 6 different degrees of dilution with nutrient fluid. Under conditions of 37 ° C, 5% CO _{2 is} cultivated for 72 hours, 6 hours before the end of the cultivation, centrifuged, a supernatant of 110 μl / hole is isolated, MTT 10μl / hole is added, at 37 ° C after 3 hours the OD value at 570 nm and the OD value are determined at 630 nm. The final OD value of each hole is determined in the range of OD at 570 nm - OD at 630 nm.

table 5.2
Effect on IL-1 and IL-6 of a small mouse

Group dose (g / kg) the number of mice (piece) IL-1 (ng / ml) IL-6 (IU / ml) control - 10 78.7 ± 7.1 94.6 ± 6.8 7.5 10 59.3 ± 4.9 ** 64.9 ± 4.8 ** Fan ship fifteen 10 53.3 ± 5.7 ** 60.5 ± 4.3 ** thirty 10 54.4 ± 4.8 ** 56.0 ± 4.6 ** 60 10 47.0 ± 16.6 ** 56.6 ± 6.1 ** Tripterygium hypoglaucum (Levl.) Hutch 5 10 57.6 ± 4.7 ** 65.7 ± 4.9 ** cyclofamide 0.02 9 44.5 ± 7.7 49.6 ± 6.7 **

The results in the table show that Fenshipin strongly inhibits the formation of macrophages in the production of IL-1 and the formation of abdominal splenocytes in the production of IL-6 in a small mouse, and the effect increases with increasing dose.

5.3 Effect on plasma NO of a large mouse suffering from arthritis from AA

60 large SD mice with a body weight of 160 ~ 200 g, including half of the female and half of the male, were randomly divided into 6 groups. Mice in the control group undergo an intradermal NS 0.5 ml injection in the right hind toe. The exemplary group, Fanshipin groups of large, medium and small doses, the Glucosidorum Tripterygll Totorum group, are injected intradermally with Freund's complete adjuvant (FCA) 0.5 ml of each mouse in the right hind toe. 18 days after the formation of arthritis from an auxiliary agent (AA), the large mouse begins to pour the agent into the stomach once a day, continuously continues for 5 days, give distillation water to the blank control group and the model group; individually, give Fanshipin a large, medium and small dose to groups of large, medium and small doses, give Glucosidorum Tripterygll Totorum tablets to the group of the positive control group. 1 hour after the last use of the drug, 2 ml of blood is taken from the abdominal aorta, blood plasma is separated and stored at -70 ° C for determination. The determination of NO is carried out in accordance with the explanatory note of the NO reactive box: take 0.1 ml of blood plasma and add 0.6 ml of reagent C and mix evenly, then add 0.4 ml of double distillation water and mix evenly, add 0.1 ml of reagent D and mix evenly, incubate on ice 60 minutes, centrifuged at 12,000 rpm for 2 minutes. Take 0.6 ml of the supernatant and add 0.4 ml of double distillation water and 0.1 ml of reagent A, incubate in ice water for 15 minutes, then add 0.1 ml of reagent B, place at room temperature for 1 hour, and determine the OD value by colorimetry at 545 nm. Based on the OD values of the samples, the NO content of the standard curve is calculated, the results are presented in table 5.3.

table 5.3
Fenshipin's effect on the NO plasma of a large mouse arthritic with an adjuvant

Group dose (g / kg) the number of mice (piece) NO content (μmol / L) y (y = lgx) control group - 8 13.55 ± 1.11 * 1.131 ± 0.032 model group AA - 9 17.56 ± 4.15 1.235 ± 0.097 Fan ship 12 7

0.985 ± 0.087 Fan ship 24 7

1.001 ± 0.067 Fan ship 48 7

1.026 ± 0.062 Glucosidorum Tripterygll Totorum 0.006 7 15.25 ± 3.48 1.173 ± 0.099 Compared with the reference group * P <0.05, ** P <0.01; compared to the Glucosidorum Tripterygll Totorum group

Table 5.3 shows that the blood plasma NO level of the large mouse of the model group is significantly higher than the blank control group. Fenshipin can significantly reduce the blood plasma level of NO in a large mouse that develops AA. Glucosidorum Tripterygll Totorum can also reduce the blood plasma level of NO in a large mouse that has arthritis, but its effect is noticeably weaker.

Experimental Example 6: Effect on T lymphocytes, NK, CD4, CD8 cells of a small mouse

6.1 Effect on the transformation of lymphocytes of a normal small mouse

80 small NIH mice are taken, including half of the female and half of the male, randomly divided into 8 groups, individually infused with different means, once a day, continuously continued for 10 days, 24 hours after the last dose they were killed, under aseptic conditions, a small mouse splenocyte is taken, washed with Hank's solution 2 times, RPIM 1640 solution without serum 1 time, 2 × 10 ⁶ / ml cell suspension is prepared using a 5% FCS-RPMI 1640 solution. The cell suspension is poured into a flat-bottomed culture bar cells with 96 holes 100μl / hole, each sample is copied by 3, the stimulus (ConA 2 ng / hole) is poured into two holes of them as a transformation hole, and the irritant is not added to the other hole as a contrast hole. Under conditions of 37 ° C, 5% CO _{2 was} cultured for 72 hours, 3H-TdR 0.1μ Ci / hole was added 14 hours before the end of cultivation. I collect cells with a multi-barrel cell collection device, determine the cpm value, calculate the average value of the copied holes. Directly compare cpm of different groups or irritation indicators, irritation indicators are calculated according to the following formula:

The results are presented in table 6.1.

table 6.1
Effect on ConAA-induced lymphocyte transformation in a small mouse

Group dose (g / kg) the number of mice (piece) cpm irritation indicator control - 10 20433 ± 3579 25.87 ± 3.06 7.5 10 13566 ± 1779 ** 27.29 ± 7.67 Fan ship fifteen 10 12708 ± 1692 ** 18.04 ± 3.76 thirty 10 12809 ± 2575 ** 16.17 ± 4.37 60 10 12090 ± 1706 ** 19.05 ± 3.80 Tripterygium 2.5 10 18038 ± 3359 17.11 ± 2.60 hypoglaucum (Levl.) Hutch 5 10 12081 ± 1039 ** 17.58 ± 4.37 cyclofamide 0.02 9 9922 ± 1145 ** 13.66 ± 2.28 Compared with the control group * P <0.05, ** P <0.01

Table 6.1 shows that Fenshipin can significantly inhibit the transformation of lymphocytes caused by ConA, and there is a certain dose-response relationship.

6.2 Effect on NK cells and CD ₄ , CD ₈ normal small mouse

The experiment is the same as 5.1, 24 hours after stopping the preparation, 2 × 10 ⁸ / ml cell suspension of a small mouse is prepared using 5% FCS-RPMI 1640 solution. CD ₄ , CD ₈ and their ratio and NK cells are determined.

Definition of CD ₄ and CD ₈ :

Take 50 μl suspension of small mouse splenocytes, add them to a glass slide coated with polylysine, and make a cell smear. T cells isolated by nylon guns are used as a positive control. After fixation with acetone, the cell smear is blocked with the serum of a normal small mouse, a biotin-labeled anti-CD ₄ , CD ₈ antibody is added, incubated for 2 hours at 37 ° C, enzyme-labeled streptavidin is added, stored for 10 minutes at room temperature, then the substrate is added, washed after 10 minutes, carried out complex hematoxylin staining for 2 minutes. After gradient dehydration with alcohol, the smear is blocked with gelatin glycerol, 200 cells are counted under a high magnification microscope.

NK cell definition:

Preparation of the EC cell: under aseptic conditions, splenocytes of a small mouse are taken, washed with Hank's solution 2 times, 1-time RPIM 1640 solution without serum, 2 × 10 ⁸ / ml cell suspension is prepared as an EC using 5% FCS-RPMI 1640 solution.

preparation of TS cells: take the cells that are sensitive to the NK cells of a small mouse located in the logarithmic growth period of Yack-1, prepare 4 × 10 ⁴ ml cell suspension as TS.

determination: pour 100μl EC and 100μl TC into a flat-bottomed cell culture bar with 96 holes, copy each sample to 3, simultaneously compare the EU and TC (EC control: EC100μl + 5% FCS RPMI 1640 100μl; TC control: TC100μl + 5% FCS RPMI 1640 100μl). Under conditions of 37 ° C, 5% CO _{2 is} cultivated 24 hours, 6 hours before the end of the cultivation, centrifuged, a supernatant of 110 μl / hole is isolated, MTT 10μl / hole is added, at 37 ° C after 3 hours the OD value at 570 nm and the OD value are determined at 630 nm. The OD value of each hole lies in the OD intervals at 570 nm - OD at 630 nm.

The results in Table 6.2 show that Fenshipin has a certain inhibition effect on CD ₄ and CD ₈ , and there is also a dose-response relationship, however, the dose-response curve is flat. For CD _{4, the} effective dose is 24 g / kg, and for a CD ₈ cell, only with a large dose of 36 g / kg the inhibition effect is noticeable. Accordingly, the ratio of CD ₄ / CD ₈ . Fanshipin imperceptibly affects. Cyclofamide has a strong inhibition effect on CD ₄ and CD ₈ , and the effect on CD ₈ is stronger, with the result that the ratio of CD ₄ / CD _{8 is} significantly increased.

For NK cells, Fenshipin also has a noticeable inhibition effect, however, the dose-dependent response is not obvious, however cyclofamide has a strong inhibition effect, there is a very significant difference between the action of cyclofamide at a dose of 20 mg / kg and the effects of Fenshipin at a dose of 12, 24, 36 g / kg .

6.3 Effect on the number and function of T lymphocytes of a small mouse suffering from AA

A small NIH mouse weighing 20 ± 2 g is taken, an intradermal injection of Freund's complete adjuvant into the toe of the right hind leg at a dose of 0.05 ml is carried out, after 3 weeks arthritis from AA is formed. For the negative control group, an intradermal injection of saline into the toe of the right hind leg at a dose of 0.05 ml is performed. The drug is poured once a day, continuously for 5 days. After 5 days, the peripheral blood of a small mouse of a different group is taken separately and a blood smear is made, lipase staining is performed. Under an immersion lens, the percentage of positive cells stained with lipase is observed (namely, the percentage of T cells in peripheral blood). After anesthesia, a small mouse is taken from the spleen and a unicellular suspension is prepared, washed once with PBS, the supernatant is isolated, 4 ml of the red blood cells are added, it is shaken completely for 2-3 minutes, after the red blood cells are completely dissolved, they are centrifuged, the supernatant is isolated and washed. After centrifugation, the supernatant was isolated, adjusting the cell concentration to 1 × 10 ⁶ / ml, separately diluted anti-CD ₄ , CD ₈ 50μl antibody was added to each tube, incubated for 1 hour at 4 ° C, and 2 times after washing with a fluorescent wash liquid 2 times ml of fixing solution. Filter the network with 400 cells per FCA tube, analyze through a Flow cytometer, the results are presented in table 6.3.

Table 6.3 shows that there is no noticeable difference between the different groups of the positive ANAE cell, however, the CD ₄ cells _{of a} small mouse that develops AA increase and the CD ₈ cells decrease significantly, the CD ₄ / CD ₈ ratio increases markedly, treatment with Fenshipin can restore the anomaly CD ₄ , CD ₈ , CD ₄ / CD ₈ to normal.

Experimental example 7: Effect on the phagocytic function of macrophages of the abdominal cavity of a small mouse

50 small NIH mice weighing 18 ~ 22 g, including half of the female and half of the male, are randomly divided into 5 groups, and drug fluid of the same volume of a different dose is poured separately, once a day, continuously for 7 days. After 1 hour after the last dose, 10% chicken erythrocytes are injected into the abdominal cavity, 0.2 ml each, after 4 hours they are killed, the abdominal fluid is taken, the number of macrophages phagocytosed by CRBC is observed and counted under a microscope, and the number of CRBC phagocytosed single macrophage, the results are presented in table 7.

table 7
Effect of Fenshipin on the phagocytosis function of the abdominal CRBC of a small mouse

Group dose (g / kg) the number of mice (piece) phagocytosis percentage (%) phagocytosis rate control - 10 25.75 ± 9.40 1.28 ± 0.20 Fan ship 27 10 33.20 ± 12.77 1.46 ± 0.36 Fan ship 40.5 10 35.20 ± 10.16 1.21 ± 0.20 Fan ship 60.9 10 37.78 ± 20.14 1.53 ± 0.32 dexamethasone 0.005 10 8.33 ± 10.13 * 1.10 ± 0.18 * P <0.05

The table shows that at a dose of 27, 40.5 and 60.9 g / kg Fenshipin slightly affects the phagocytic function of macrophages in the abdominal cavity of a small mouse.

Experimental example 8: Effect on the increased permeability of the capillaries of the abdominal cavity of a small mouse

Take 90 small NIH mice weighing 18 ~ 22 g, including half of the female and half of the male, randomly divided into 9 groups, individually injected drug fluid of the same volume of a different dose once, or once a day and continuously for 3 days . 1 hour after the last dose, 0.7% physiological solution of NAS is injected into the abdominal cavity, at the same time, 0.5% physiological solution of blue Evans is injected at a dose of 0.1 ml / 10 g of body weight, after 30 minutes the mouse is killed by prolapse of the cervical vertebrae, the abdominal cavity is cut off, washed the abdominal cavity several times with physiological saline 5 ml for each mouse, then the wash liquid is pumped out and drained. Saline is then added to a volume of 8 ml / mouse, after centrifugation at 3000 pnm, the supernatant is taken, the OD value is determined at 590 nm, the results are presented in table 8.

table 8
The effect of Fenshipin on the increased permeability of capillaries of the abdominal cavity of a small mouse caused by acetic acid

Group dose (r / kg) frequency of administration the number of mice (piece) amount of paint effusion (OD) P value control - - 10 0.29 ± 0.13 Fan ship 27 qd × 1 10 0.26 ± 0.14 > 0.05 Fan ship 40 qd × 1 10 0.25 ± 0.10 > 0.05 Fan ship 60 qd × 1 10 0.25 ± 0.09 > 0.05 control - - 10 0.28 ± 0.15 Fan ship 27 qd × 3 10 0.25 ± 0.12 > 0.05 Fan ship 40 qd × 3 10 0.18 ± 0.10 <0.05 Fan ship 60 qd × 3 10 0.15 ± 0.13 <0.05 dexamethasone 0.15 qd × 3 10 0.11 ± 0.07 <0.01

The results in table 8 show that for increased permeability of the capillaries of the abdominal cavity of a small mouse caused by acetic acid, Fenshipin imperceptibly acts after one administration of the drug, and after continuous use of the drug for 3 days it has a noticeable inhibition effect on it.

Experimental Example 9: Effect on exudation of pleurisy and aggregation of inflammatory cells of a small mouse caused by gelatin from ocular chondrus

After a random separation into different groups, they separately inject 0.5% physiological solution of blue Evans at 0.1 ml / 10 g of body weight into the coccygeal vein, and a small mouse is anesthetized with ether. Injection is done with a special needle with a 1% gelatin solution from ocular chondrus into the right chest cavity at a dose of 0.03 ml of each mouse. Separately, after 4 hours and 32 hours after inflammation, they kill by decapitation of small mice, cut the abdomen, expose the diaphragm. A syringe with a volume of 1 ml is injected twice into the abdomen with a washing liquid, the aggregate of which is 2 ml, then the washing liquid is collected in a test tube. Take 20 ml of the above eluate and add it to 400 ml of leukocyte-diluting fluid, under the microscope count the number of leukocytes. The residual fluid is centrifuged at 3000 pnm for 10 minutes, the supernatant is taken and the absorbance is determined at 600 nm, a thoracic lavage stock solution is used to check the zero point. The results are presented in table 9.

table 9
The effect of Fenshipin on the aggregation of pleuric cells of a small mouse caused by gelatin from ocular chondrus

Group dose (g / kg) white blood cell count (2 × 105) paint exudation (OD) 4 hour 32 hour 4 hour 32 hour control - 46.0 ± 6.9 16.0 ± 9.6 0.156 ± 0.066 0.109 ± 0.019 Fan ship 27 26.8 ± 4.5 * 14.2 ± 8.0 0.121 ± 0.062 0.116 ± 0.031 Fan ship 40.5 10.9 ± 4.0 ** 17.3 ± 4.6 0.100 ± 0.048 0.153 ± 0.032 Fan ship 60 8.0 ± 5.5 ** 6.6 ± 4.7 * 0.129 ± 0.066 0.092 ± 0.051 dexamethasone 0.05 12.7 ± 10.2 ** 4.4 ± 4.0 * 0.085 ± 0.045 0.063 ± 0.017 * P <0.05, ** P <0.01

Table 9 shows that Fenshipin can markedly inhibit the aggregation of leukocytes of the pleurisy of a small mouse; this effect is especially strong for early aggregation. At the moment of 4 hours, the regression equation y = 44.13-2.01x, r = -0.9625 is obtained, for late aggregation the effect is weak, only at a dose of 20 g / kg a noticeable effect occurs, and for the exudation of pleurisy it is not obvious.

Experimental example 10: Effect on the aggregation of leukocytes in the CMC capsule of a large mouse

Take 64 large SD mice weighing 150 ~ 180 g, including half of the female and half of the male, randomly divided into 8 groups, individually injected drug fluid of the same volume of a different dose once or once a day and continuously for 3 days. On the day of the experiment, 20 ml of a 1% CMC solution is injected into a balloon formed by pre-injecting 20 ml of air into the back of a large mouse on the previous day. After 3.5 hours and 7.5 hours, 0.1 ml was separately pumped out and poured into a 0.01% solution of diamond cresol blue PBS for staining, then the number of leukocytes in the CMC capsule liquid was examined under a microscope. The results are presented in table 10

Table 10
The effect of Fenshipin on the number of leukocytes in a capsule of a mouse carboxymethyl cellulose

Group dose (r / kg) quantity (piece) amount of WBC (× 107 / L) 3.5 hours 7.5 hours control - 8 9.7 ± 4.2 57.7 ± 17.3 Fan ship 27 × 1 8 8.5 ± 3.5 39.4 ± 16.5 Fan ship 40 × 1 8 8.7 ± 7.3 35.3 ± 23.2 Fan ship 60 × 1 8 6.6 ± 3.3 18.1 ± 8.6 ** control - 8 10.97 ± 6.7 35.6 ± 11.2 Fan ship 27 × 3 8 15.4 ± 9.7 38.6 ± 15.5 Fan ship 40 × 3 8 4.8 ± 3.4 ** 18.4 ± 12.2 ** Fan ship 60 × 3 8 3.0 ± 2.8 ** 11.0 ± 9.2 * cortisone 0.1 × 3 8 14.2 ± 8.0 41.7 ± 16.0 control - 8 10.9 ± 3.0 41.3 ± 6.9 Fan ship 18 × 7 8 6.2 ± 3.0 * 11.4 ± 6.4 * Fan ship 27 × 7 8 3.7 ± 1.7 ** 6.4 ± 3.1 ** Fan ship 40 × 7 8 2.5 ± 1.9 ** 5.9 ± 3.9 ** cortisone 2 mg × 1 8 1.5 ± 0.7 ** 3.0 ± 1.0 ** Compared with the control group ** P <0.01

Table 10 shows that Fenshipin can markedly inhibit leukocyte aggregation in the CMC capsule of a large mouse; there is an obvious dose-response relationship. With an increase in the period of taking the drug, the effect increases, continuous administration of the drug for 7 days at a dose of 18 g / kg can very noticeably inhibit leukocyte migration, injection of cortisone into the capsule also has a strong inhibitory effect.

Experimental Example 11: Effect on Ear Swelling of a Small Mouse Caused by Creton Oil

60 small NIH mice weighing 18-22 g, including half of the female and half of the male, were randomly divided into 6 groups, individually injected with liquid or mucus of a gummitragant of the same volume of a different dose once a day and continuously for 3 days. 1 hour after the last dose, two sides of the auricle of the left ear are spread evenly with 2% croton oil potion 0.02 ml of each mouse. After 4 hours, kill the mouse with prolapse of the cervical vertebrae, cut off the left and right ear, weigh the weight of the sleeper and the weight of the control ear, the difference between them, expressed in mg, is presented as the degree of swelling of the ear, the results are presented in table 11.

Table 11
Effect of Fznshipin on the swelling of the ear of a small mouse caused by croton oil

Group dose (g / kg) quantity (piece) degree of swelling of the ear (mg) percent inhibition (%) P value control - 10 44.38 ± 9.40 Fan ship 27 10 39.05 ± 12.33 12.00 > 0.05 Fan ship 40 10 36.65 ± 5.83 17.64 <0.05 Fan ship 60 10 34.91 ± 9.71 21.34 <0.05 dexamethasone 0.003 10 14.13 ± 5.75 68.16 <0.01

The results in table 11 show that Fenshipin significantly inhibits the swelling of the ear of a small mouse caused by cretonone oil, and there is a dose-dependent response, however, the dose response curve is gentle, at a dose of 13.5 g / kg the inhibition effect is very noticeable.

Experimental Example 12: Effect on Ace Twist of a Small Mouse Caused by Acetic Acid

60 small mice of the Kunming genus weighing 18-22 g, including half of the female and half of the male, are randomly divided into 6 groups, and drug fluid or mucus of a gummitragant of different doses is individually infused. 1 hour after taking the drug, an injection of 0.7% physiological solution of NAS in the abdominal cavity of 0.2 ml of each mouse is made. Put a small mouse in a glass pot and observe the latent period of the reaction of the body twist of different mice and the number of body twist for 20 minutes, the results are presented in table 12.

Table 12
Fenshipin's influence on the amount of body twist of a small mouse caused by acetic acid

Group dose (g / kg) quantity (piece) body twist latent period (minute) control - 10 34.6 ± 14.1 3.13 ± 0.80 Fan ship 27 10 28.2 ± 5.76 3.82 ± 0.85 Fan ship 40 10 31.0 ± 18.4 3.86 ± 2.00 Fan ship 60 10 20.7 ± 12.3 * 3.95 ± 1.42 Tripterygium hypoglaucum (Levl.) Hutch twenty 10 25.1 ± 11.9 3.60 ± 0.93 hydrochloric morphine 10 mg / kg 10 0.0 ± 0.0 0.00 ± 0.00

The results in Table 12 show that with a relatively large dose, Fenshipin can slow down the body twist reaction of a small mouse caused by acetic acid, and significantly reduce the number of body twist of a small mouse within 20 minutes. This indicates that Fenshipin has a certain analgesic effect.

Experimental Example 13: Effect on the Rheological Property of the Blood of a Large AA AA Mouse

A large SD mouse with a body weight of 180 ± 20 g is selected, an intradermal injection of Freund's complete adjuvant into the right hind toe of 0.05 ml for each mouse is made, and an arthritis sample from the adjuvant is formed. An intradermal injection of physiological saline into the right hind toe of 0.05 ml of each large mouse of the negative control group is made. 3 weeks after the formation of the samples, the mice were divided into a model group, large, medium and low dose groups, a negative control group and a positive control group, Glucosidurum Tripterygll Totorum tablets were given to the positive control group. Give funds by infusion once a day and continuously for 5 days. 1 hour after the last dose, 3 ml of blood is taken from the abdominal aorta and poured into an anticoagulative tube with 1% heparin, the viscosity of whole blood is determined with a conical-lamellar viscometer of the NXE-1 type at a shear rate of 230, 115, 46, 23, 11.5 and 5.75 S-1. Determine the viscosity of blood plasma with an adjustable isobaric capillary viscometer type WTP-B; hematocrit and erythrocyte aggregation are determined by hematocrit tube by centrifugation, hard red blood cell counts are obtained by counting the above results, the results are presented in table 13.

Table 13 shows that the rheological property of the blood of a large mouse suffering from AA is obviously changing, the viscosity of whole blood and blood plasma is increasing, the hematocrit will decrease, erythrocyte aggregation and tough indicators increase, Fenshipin treatment can significantly improve the above rheology.

The above experiments testified to the pharmacological action of Fenshipin, many important pharmacological actions of Fenshipin have a good dose-response relationship, which indicates that in clinical practice the dose can be adjusted to achieve the best treatment efficacy.

A study of the effectiveness of Fengshingin treatment in clinical practice was conducted in China, Japan and Australia. The results of observation according to international standards of diagnosis, treatment and treatment effectiveness of the corresponding types of the disease show that the effectiveness of the separate use of the Fengshipin capsule on RA is about 94%. Significant efficacy is about 60%, the Fenshipin capsule is capable of rapidly improving morning stiffness, swelling and other symptoms and the corresponding RA indices, the results are presented in tables 14 ~ 21.

Table 14
Comparison of the effectiveness of treatment of the treatment group with the control group Group number of examples relief (clinical
cure) significantly effective effectively not effective effectiveness of significant action (%) efficiency (%) medical 32 5 fourteen eleven 2 59.38 93.74 control thirty 3 10 12 5 43.33 83.33 Table 15
effect on IgG, IgA, IgM (X ± S) Group number of examples IgG before after IgA before after W before after normal 32 12.45 ± 1.48 2.37 ± 1.00 1.58 ± 0.59 medical 32 16.92 ± 3.49 14.17 ± 1.39 ** 3.65 ± 1.03 2.39 ± 1.18 ** 1.89 ± 0.88 1.48 ± 1.01 control thirty 17.03 ± 4.12 15.14 ± 2.21 ** 3.45 ± 1.86 2.32 ± 1.75 ** 2.03 ± 0.95 1.76 ± 1.28 Compared to pre-treatment ** P <0.0

Table 16
effect on C3, C4 (X ± S) Group number of examples (piece) C3 C4 before after before after normal 32 0.62 ± 0.13 0.14 ± 0.15 medical 32 1.88 ± 0.72 0.48 ± 0.12 1.25 ± 0.66 ** 0.26 ± 0.06 * control thirty 2.13 ± 0.64 0.40 ± 0.16 1.56 ± 0.62 ** 0.25 ± 0.07 ** Compared to pre-treatment * P <0.05, ** P <0.01 Table 17
effect on ESR, CRP (X ± S) Group number of examples (piece) ESR to after CRP up after normal 32 8.37 ± 5.26 4.12 ± 1.88 medical 32 66.58 ± 9.01 30.31 ± 6.53 ** 13.35 ± 6.67 8.86 ± 3.34 * control thirty 73.33 ± 9.09 14.21 ± 6.29 35.83 ± 11.61 ** 9.04 ± 3.15 ** Compared to pre-treatment * P <0.05, ** P <0.01

Table 18
hand strength comparison before and after treatment (X ± S) Group treatment group control group before after before after left hand strength 39.13 ± 20.24 (15) 24.00 ± 17.63 (21) (mmHg) 80.47 ± 34.61 ** (15) 55.15 ± 23.27 ** (21) right hand strength 35.85 ± 22.46 (15) 22.80 ± 12.32 (21) 85.32 ± 36.32 ** (15) 58.17 ± 20.59 ** (21) Compared to pre-treatment * P <0.05, ** P <0.01

Table 19
Effect on swelling and joint pain and rigor mortis (X ± S) thing treatment group control group before after before after joint swelling and pain stiffness (minute) morning 5.79 ± 0.52 3.14 ± 0.83 * 5.56 ± 2.15 3.92 ± 0.26 * 50.33 ± 6.47 20.24 ± 3.27 ** 48.75 ± 8.34 27.50 ± 3.78 ** Compared to pre-treatment * P <0.05, ** P <0.01 Table 20
Effect on turning RF into negativity Group number of examples RF negativity before after negative conversion percentage (%)
54.2
44.4 medical 32 24 eleven
10 control thirty eighteen

Along with achieving a noticeable treatment effect, Fenshipin can also reduce the serum S1L - 2R, STNF, SIL - 6R of patients' blood, the results are presented in table 21.

Table 21
impact on key indicators: SIL - 2R, STNF, SIL - 6R (X ± S) Group number of examples (piece) SlL-2R (u / ml) STNF R1 (ng / ml) SIL-6R (ng / ml) before after before after before after normal 32 299 ± 68 1.56 ± 0.48 72.05 ± 18.26 (n = 32) (n = 24) (n = 22) Fan ship fifteen 683 ± 189 2.87 ± 0.66 136.18 ± 28.57 381 ± 157 ** 1.75 ± 0.54 ** 90.15 ± 20.12 ** control 10 765 ± 203 2.63 ± 0.72 148.21 ± 30.31 412 ± 167 ** 2.38 ± 0.39 (n = 8) 99.02 ± 26.70 ** Compared to pre-treatment ** P <0.01

EXAMPLE 1

TREATMENT OF CHRONIC NEphritis RENAL GLOBULES FUNSHIPIN IN PRACTICE

60 patients with chronic nephritis were examined, the main manifestations: albuminuria, bloody urine. They are freely divided into two groups: an experimental group and a comparative group of 30 people. To the experimental group: 2 capsules of funshipin every time, three times a day, taken after meals. Comparative group: each time 20 mg of Leiguntentua, three times a day, treatment course of 8 weeks. The symptom and body changes of patients are recorded, urine is checked: dosing of urine protein for 24 hours (24h UPQ), white plasma protein (A), liver function (GPT), kidney function (BUN SCr), blood law (WBC). After 4 and 8 weeks of treatment, the above tests are repeated, the standard "principle of studying the practice of the new Chinese medicine" is reviewed, the effect is evaluated. As a result, in the experimental group of 30 people, 18 people have complete remission (60%), 9 people have private remission (30%), 2 people have influence (6.7%), 1 person without influence (3.3%), overall efficiency 96.7%. In the comparative group of 30 people. 15 people have complete remission (50%), 6 people have private remission (20%), 6 people have influence (20%), 2 people without influence (10%), overall efficiency is 90%. The significant efficacy of the two groups is compared (complete remission + private remission), P <0.01. In addition, the 24h indicator of UPQ (g) in the experimental group decreases faster and more than in the comparative group, before treatment, at the time of treatment for 4 weeks and at the time of 8 weeks, values 2.63 ± 0.72, 1.10 ± 0.56 (P <0.05), 0.42 = 0.28 (P <0.01); values in the comparative group 2.48 ± 0.92, 1.78 ± 0.64, 1.05 ± 0.66 (P <0.01). In the experimental group, in 3 people after taking swelling in the stomach appeared, after taking the medicine, the symptom disappears, in the comparative group in 4 people swelling in the stomach and nausea, in 3 people there is a slight destruction of liver function, the highest GPT is 68.5 U / L, in 12 sick women with regulations, there was a decrease and disorganization of regulation.

EXAMPLE 2

TREATMENT OF PATIENTS WITH SLE DISEASE SYSTEMATICALLY ACTIVE PERIOD (SLE).

60 patients are freely divided into two groups: the experimental group takes 2-3 capsidin capsules every time, three times a day, after meals; comparative group: taking 50 mg per day of prednisone (every two weeks to reduce by 10 mg, when the dosage is exactly 30 mg, every two weeks to reduce by 10 mg). Plus injections into a vein of 0.2 g of cyclophosphamide once every two days. Continuously treat for 3 months. As a result, in the comparative group 2 people were cured, 7 people have significant effectiveness, 14 people have influence, 7 people without influence. In the experimental group, 3 people were cured in practice, 8 people have significant effectiveness, 13 people have an effect, 6 people have no effect, patients of the two groups got a good effect. Active patient SLE (SLEDAI), blood sedimentation (ESR), urinary protein dosing C ₃ 24h and ds DNA antibody droplet size are significantly reduced in the two groups without a significant difference. In the comparative group, in 5 people, white cells decrease, in 2 people thrombocytopenia, in 5 people nausea, in 2 people hair loss, in 2 people an increase in liver function. In the experimental group receiving funchipine, small and slight malaise, only 2 people had stomach pain and bloating in the upper part of the stomach, in one person a slight fall of white cells.

Example 3

Woman, 40 years old, abdominal pain, diarrhea with blood, low temperature, weight loss, points in the abdomen. When checking with a radiometer using a barium agent, an internal mirror and examining the cause of the disease, a clone disease from 6 years was determined.

After treatment with hormones and depressants of the immune system, the situation of the disease is not stable, there is abdominal pain, 3-4 times diarrhea per day, high temperature (37.8 ° C), body weight 46 kg, significant anemia. After taking Farshipine 3-4 capsules once and 3 times a day, after 4 weeks the situation of the disease is stabilized, after 3 months of treatment the abdominal pain and diarrhea disappear, body temperature is normal, anemia decreases, weight increases (52 kg). According to the active indicator, clone disease (CDA1) is considered cured.

The following are specific examples of the preparation of pharmaceutical compositions according to the invention, which will provide the above effects.

Example 1

Goryanka large-flowered 2222 g

Tripterygium hypoglaucum (Levl.) Hutch 2222 g

Berries of Dereza 1111 g

Grape seeds 1111 g

They crumble Tripterygium hypoglaucum (Levl.) Hutch into small pieces, separately extracted three times in 13-, 10-, 10-fold waters, each time for 1 h; Goryanka is cut into large pieces, separately extracted three times in 15-, 10-, 10-fold waters, each time for 1 hour; crushed berries of dereza into a coarse material, soaked in 20-fold warm water at 80 ° C for 1 h; crushed Seeds of dodder in coarse powder, soaked in 31-fold warm water at 80 ° C for 1 h; separately broths and soaking liquids of four raw medicinal materials are filtered, then they are separately passed through a wide-necked resin adsorption column and eluted with 70% ethanol until a dark eluent flows out, which is collected before its color changes to a light color, the eluent is recycled until it is isolated alcohol. The resulting extracts are concentrated and dried.

Up to 200 grams of medicinal starch is added to the obtained medicinal extract powders, they are evenly mixed, then they are packaged in 1000 capsules. Each capsule made by the claimed method contains 0.2 g of a mixture of medicinal extracts, and the content of icariin C ₃₃ H ₄₀ O ₁₅ in each capsule less than 2.0 mg is not allowed. The usual oral dose is 3 capsules 3 times a day.

Example 2

Tripterygium hypoglaucum (Levl.) Hutch 2000 g

Goryanka large-flowered 2000 g

They crumble Tripterygium hypoglaucum (Levl.) Hutch into small pieces, separately extracted three times in 13-, 10-, 10-fold waters, each time for 1 h; Goryanka is cut into large pieces, separately extracted three times in 15-, 10-, 10-fold waters, each time for 1 hour; Separately filtered decoctions of two raw medicinal materials. The broths are passed separately through a wide-necked resin adsorption column and eluted with 70% ethanol until a dark eluent flows out, which is collected before its color changes to light, the eluent is recycled until alcohol is isolated. The resulting extracts are concentrated and dried.

The obtained medicinal powders of the extracts are combined and evenly mixed with medicinal starch, packaged in 1000 capsules. Each capsule prepared by the method according to the invention contains 0.2 g of a mixture of medicinal extracts, and the content of icariin C ₃₃ H ₄₀ O ₁₅ in each capsule less than 2.0 mg is not allowed. The usual oral dose is 3 capsules 3 times a day.

Example 3

Tripterygium hypoglaucum (Levl.) Hutch 2000 g

Goryanka large-flowered 2000 g

Dereza berries 1000 g

They crumble Tripterygium bypoglaucum (Levl.) Hutch into small pieces, separately extracted three times in 13-, 10-, 10-fold waters, each time lasting 1 hour; Goryanka is cut into large pieces, separately extracted three times in 15-, 10-, 10-fold waters, each time for 1 hour; crushed berries of dereza into coarse material, soaked in 20-fold warm waters of 80 ° C for 1 hour; The broths and soaked liquids of the three raw medicinal materials are filtered separately, then they are passed separately through a wide-necked resin adsorption column, eluted with 70% alcohol until the dark eluent flows out, which is collected before its color changes to light, the eluent is recycled until the alcohol is isolated. The resulting extracts are concentrated and dried.

The obtained medicinal powders of the extracts are combined and evenly mixed with medicinal starch, packaged in 1000 capsules.

Each capsule prepared by the method according to the invention contains 0.2 g of a mixture of medicinal extracts, and the content of icariin C ₃₃ H ₄₀ O ₁₅ in each capsule less than 2.0 mg is not allowed. The usual oral dose is 3 capsules 3 times a day

Example 4

Tripterygium hypoglaucum (Levl.) Hutch 2000 g

Goryanka large-flowered 2000 g

Milk seeds 1000 g

They crumble Tripterygium hypoglaucum (Levl.) Hutch into small pieces, separately extracted three times in 13-, 10-, 10-fold waters, each time lasting for 1 hour; Goryanka is cut into large pieces, separately extracted three times in 15-, 10-, 10-fold waters, each time lasts 1 hour; chopped Seeds of dodder on coarse powder, heat soaked in 31-fold water 80 for 1 hour; the boiling and soaking liquids of three medicinal materials are separately filtered, then a wide-necked resin adsorption column is passed separately, then it is eluted with 70% alcohol until the dark eluent flows out, which is collected before its color changes to light, the eluent is recycled until alcohol is recovered. The resulting extracts are concentrated and dried.

The obtained four medicinal powder extracts are combined and evenly mixed with medicinal starch, packaged in 1000 capsules.

Each capsule prepared by the method according to the invention contains 0.2 g of a mixture of medicinal extracts, and the content of icariin C ₃₃ H ₄₀ O ₁₅ in each capsule less than 2.0 mg is not allowed. The usual oral dose is 3 capsules 3 times a day

Example 5

Tripterygium hypoglaucum (Levl.) Hutch 2000 g

1000 seeds

They crumble Tripterygium hypoglaucum (Levl.) Hutch into small pieces, separately extracted three times in 13-, 10-, 10-fold waters, each time 1 hour. Crushed Seeds of dodder on coarse powder, soaked in 31-fold warm water at 80 ° C for 1 hour; Separately, the broth and soaked liquids of two medicinal materials are filtered, then they are passed separately through a wide-necked resin adsorption column, then eluted with 70% alcohol until the dark eluent flows out, which is collected before its color changes to light, the eluent is recycled until the alcohol is isolated. The resulting extracts are concentrated and dried.

The obtained two medicinal powder extracts are combined and evenly mixed with medicinal starch, packaged in 1000 capsules.

The daily dosage of the capsule prepared by the described method according to the invention is equal to the amount of medicinal raw materials - 30 g / day.

Example 6

Tripterygium hypoglaucum (Levl.) Hutch 2000 g

Dereza berries 1000 g

They crumble Tripterygium hypoglaucum (Levl.) Hutch into small pieces, separately extracted three times in 13-, 10-, 10-fold waters, each time for 1 hour; crushed berries of dereza into coarse material, soaked in 20-fold warm waters of 80 ° C for 1 hour; The broth and the soaked liquid of medicinal materials are separately filtered, then they are passed separately through a wide-necked resin adsorption column, then they are eluted with 70% alcohol until the dark eluent flows out, which is collected before its color changes to light, the eluent is recycled until the alcohol is isolated. The resulting extracts are concentrated and dried.

The obtained medicinal powders of the extracts are combined and evenly mixed with medicinal starch, packaged in 1000 capsules.

The daily dosage of the capsule made by this invention is equal to the amount of medicinal raw materials - 30 g / day.

Claims (8)

1. Pharmaceutical composition for the treatment of rheumatism, prepared from the raw medicinal material Tripterygium hypoglaucum (Levl.) Hutch and at least one of the raw medicinal materials selected from the group: Goryanka large-flowered (Epimedium brevicomum Maxim.), Berries of dereza (Lycium barbarum L. ), Seeds of dodder (Cuscuta chinensis Lam., Cuscuta australis R.Br.), or mixtures thereof, taken in the following weight ratio: Tripterygium hypoglaucum (Levl.) Hutch 1-4 Goryanka, large-flowered (Epimedium brevicomum Maxim.) 1-4 Dereza berries (Lycium barbarum L.) 1-4 Seeds of dodder (Cuscuta chinensis Lam., Cuscuta australis R. Br.) 1-4 2. The pharmaceutical composition according to claim 1, characterized in that it is prepared from the named raw medicinal materials taken in the following weight ratio: Tripterygium hypoglaucum (Levl.) Hutch 2 Goryanka, large-flowered (Epimedium brevicomum Maxim.) 2 Dereza berries (Lycium barbarum L.) one Seeds of dodder (Cuscuta chinensis Lam., Cuscuta australis R. Br.) one 3. The method of preparing the pharmaceutical composition according to any one of claims 1 and 2, comprising the following operations: weighing the raw medicinal materials and grinding Tripterygium hypoglaucum (Levl.) Hutch and the named Goryanka krupnotsvetkovaya, separate boiling them in water 3 times, soaking the named Berries of dereza, Seed caught with water at a temperature of 80-95 ° C 1-3 times, separate transmission of the resulting broths and obtained liquids through a wide-necked resin adsorption column, washing with water of the resin column until the effluent is clarified, eluya 30-99.3% ethyl alcohol before the dark liquid flows out, collecting the eluent before its color changes from dark to light, removing the alcohol liquid from the column with water, mixing the eluate with the alcohol liquid, while the total weight of the eluate is 1- 8 times heavier than the weight of the named raw medicinal products, recycling the eluent of each of the raw medicinal material to extract alcohol, concentrating to a specific gravity of 1.10, drying extracts of each raw medicinal material individually or in combination, mixing them evenly or mixing in proportion to obtain a dosage form acceptable in clinical practice. 4. The method of preparing the pharmaceutical composition according to any one of claims 1 and 2, characterized in that Tripterygium hypoglaucum (Levl.) Hutch is crushed into small pieces, extracted three times separately in 13-, 10-, 10-fold waters for one hour , Goryanka, large-flowered, is crumbled into segments, extracted separately three times in 15-, 10-, 10-fold waters for one hour; Dereza berries are crushed into coarse material and soaked in 20-fold waters at a temperature of 80-95 ° С for one hours, Dodder seeds are crushed into coarse powder, soaked in 31-fold waters at 80 ° C for one hour, the extracts or soaking liquids of the four raw medicinal materials are filtered separately, then passed through a wide-necked resin adsorption column, eluted with 70% ethanol until the dark eluent flows out, which is collected before its color changes from dark to light, the eluent from these raw materials is recycled to extract alcohol, concentrated, dried, the obtained medicinal powders of the extracts are mixed uniformly or in proportion with obtaining a dosage form acceptable in clinical practice. 5. The method of preparing the pharmaceutical composition according to claim 3, characterized in that any dosage forms acceptable in clinical practice are obtained, such as a hard capsule, soft capsule, tablet, granule, injection. 6. The method of preparing the pharmaceutical composition according to claim 4, characterized in that any dosage forms acceptable in clinical practice are obtained, such as a hard capsule, soft capsule, tablet, granule, injection. 7. The use of the pharmaceutical composition according to any one of claims 1 and 2 for the preparation of agents for the treatment of rheumatism or rheumatoid arthritis. 8. The use of the pharmaceutical composition according to any one of claims 1 and 2 for the preparation of agents for the treatment of chronic nephritis.