CN1682748A - Fengshian traditional Chinese medicine preparation and its preparation and application - Google Patents

Abstract

The Chinese medicine preparation, Fengshi'an, for treating rheumatism has the effective component 3-O-alpha-L-pyranorhamnose (1->2)-beta-D-xylopyranose-oleanolic acid-28-O-alpha-L-pyranorhamnose(1->4) beta-D-pyranoglucose (1->6) beta-D-glucopyranoside, named flaccid anemone saponin W3, and with molecular expression of C59H96O25. Flaccid anemone saponin W3 is prepared with coarse flaccid anemone rhizome powder, and through reflux extraction, concentration, adsorption and elution in macroposrous resin, adsorption and elution in dry silicon gel column and liquid separation. Flaccid anemone saponin W3 is further prepared into Chinese medicine preparation with flaccid anemone saponin W3 content over 90 wt%, and the Chinese medicine preparation is used in treating rheumatic diseases with high curative effect and less toxic side effect.

Description

Fengshian traditional Chinese medicine preparation and its preparation and application

technical field

The invention relates to a traditional Chinese medicine preparation for rheumatic diseases and its preparation and application.

Background technique

Rheumatic diseases are common autoimmune diseases that can affect multiple organs and tissues. Clinical morbidity is high. Because its pathogenesis is not completely clear, there is no specific treatment at present (Lu Junjian, Chinese and Western Medicine Diagnosis and Treatment of Connective Tissue Diseases, People's Medical Publishing House, 1992: 348). Clinically, comprehensive treatment such as corticosteroid hormones and cytotoxic drugs is often used, but this often causes adverse consequences such as non-specific immunosuppression of the body.

The dried rhizome of Anemone flaccida Fr. Schmidt, a plant of the genus Anemone of the family Ranunculaceae. It is recorded in "Guizhou Folk Medicine" and "Zhejiang Tianmu Mountain Drug Chronicle" that it is warm in nature and slightly bitter in taste. (Dictionary of Traditional Chinese Medicine, Shanghai: Shanghai Science and Technology Press, 2002: 802)

The total effective parts obtained from Diwu made Diwu extract and Diwu Fengshi'an Capsules were approved for Phase I and II clinical trials of the National Traditional Chinese Medicine Category 5 new drug on September 2, 2004, and have now entered the clinical trial stage. It is clinically used to treat rheumatoid arthritis (RA) with remarkable curative effect.

Contents of the invention

The object of the present invention is to provide a rheumatism preparation and its application made from the active ingredient W ₃ extracted, separated and purified from Diwu, the molecular formula being C ₅₉ H ₉₆ O ₂₅ .

The Fengshian traditional Chinese medicine preparation of the present invention has an active ingredient of: 3-O-α-L-rhamnopyranose (1→2)β-D-xylopyranose-oleanolic acid-28-O-α -L-rhamnopyranose (1→4)-β-D-glucopyranose (1→6)-β-D-glucopyranoside, [named as diguanin W ₃ , as described below Saponin W ₃ refers to this compound], the molecular formula is C ₅₉ H ₉₆ O ₂₅ .

The content of diguanin _W3 in the Fengshian traditional Chinese medicine preparation of the present invention is more than or equal to 90wt%.

The preparation method of Fengshi'an traditional Chinese medicine preparation of the present invention, its processing step is:

1. Take Diwu coarse powder, add 8 to 12 times of water to reflux and extract for 3 to 4 times, each time for 1 to 3 hours, and combine the extracts;

2. Concentrate the extract obtained in step 1 to dryness under reduced pressure, add 50% to 80% ethanol, filter out the precipitate, and obtain the alcohol extract;

3. Concentrate the alcohol extract obtained in step 2 to dryness under reduced pressure, add water to dissolve, filter, absorb on D101 macroporous resin on the filtrate, and successively elute with water, 40%-80% ethanol, and 80%-95% ethanol; Collect 40%-80% ethanol eluate, recover ethanol and evaporate to dryness in a water bath to obtain diglucoside;

4th, put the diglucoside obtained in step 3 on silica gel column chromatography, elute with chloroform:methanol:water=20:10:1 mixture, roughly divide into 5 sections, detect by high performance liquid chromatography, collect rich The second section of diguanin W ₃ was subjected to silica gel column chromatography, eluted with a mixture of chloroform: methanol: water = 7: 4; 1, detected by high performance liquid chromatography, and collected rich in diguanin W ₃ The part of the compound was obtained by preparative high-efficiency liquid phase separation and purification, and the content of guanin _W3 was more than or equal to 90wt%. Preparative high-efficiency liquid phase separation conditions: stationary phase: octadecylsilane bonded silica gel; mobile phase: a mixture of methanol: water with a volume ratio of 6:4 to 7:3; flow rate: 2.0 to 10ml/min; column temperature : 25℃～40℃; detection wavelength: 205～250nm.

Fifth, prepare the compound diguanin W ₃ obtained in step 4 into a preparation. Its formulations include powders, pills, drops and tablets.

The Fengshian traditional Chinese medicine preparation of the invention is used for treating rheumatic diseases. When treating rheumatic diseases, the Fengshi'an traditional Chinese medicine preparation regulates immunity by aiming at the specific immune regulation state of the body, avoids non-specific immunosuppression and drug toxicity, and has the advantages of significant curative effect and less toxic and side effects.

In the present invention, unless otherwise specified, the percentage concentration of ethanol refers to the percentage concentration by volume, and the percentage content of the compound refers to the percentage content by mass.

Description of drawings

Fig. 1 process flow diagram for the preparation of diguanin W ₃ bulk drug

Detailed ways

Example 1

Preparation of API _W3

See attached picture 1

Take Diwu coarse powder, add 8 to 12 times of water to reflux and extract 3 to 4 times, each time for 1 to 3 hours, combine the extracts, concentrate the extracts under reduced pressure to dryness, add 50% to 80% ethanol, filter out the precipitate, The alcohol extract was obtained, concentrated to dryness under reduced pressure, dissolved in water, filtered, adsorbed on D101 macroporous resin on the filtrate, and eluted with water, 40%-80% ethanol, 80%-95% ethanol in sequence; collected 40%-80% Ethanol eluate, recovered ethanol and evaporated to dryness in a water bath to obtain diguanoside; dry column chromatography on silica gel for the obtained diguanoside, and eluted with a mixture of chloroform:methanol:water=20:10:1 volume ratio , roughly divided into 5 sections, high performance liquid chromatography (TLC) detection, collecting the second section part rich in diguanin W3, and then going to silica gel column chromatography, with chloroform: methanol: water = 7: 4: 1 volume ratio The mixture was eluted and identified by TLC. The fraction rich in diguanin W3 was collected and purified by preparative high performance liquid phase separation to obtain compound W ₃ . Preparative high-efficiency liquid phase separation conditions: stationary phase: octadecylsilane bonded silica gel; mobile phase: a mixture of methanol: water with a volume ratio of 6:4 to 7:3; flow rate: 2.0 to 10ml/min; column temperature : 25℃～40℃; detection wavelength: 205～250nm.

Example 2

Rat long-term toxicity test:

To investigate the long-term toxicity of Diwu extract 864, 288, 96mg/kg doses of continuous intragastric administration for 6 months. Observe the general condition of the animals, body weight changes, hematology, blood biochemistry, general anatomy of organs, organ coefficients and histopathological changes. Results None of the animals died. There was no statistical difference in the general situation of the animal test of each dose group at the end of the 3-month test period and the recovery period, body weight change, hematology, blood biochemistry, general anatomy of the whole body organs, and organ coefficients compared with the negative control group. At the end of the test, the histopathological examination of each dose group indicated that the proportion of hyperplastic lobules in the thyroid follicular lobules was different, among which the hyperplastic lobules accounted for more than 50% of the thyroid follicular lobules in the high-dose group, and 30-30% in the middle-dose group. 50%, low dose group or distilled water control group accounted for 10 to 30%. Hypertrophic lobules appeared in 100% of the animals in each dose group and distilled water group, and there was no significant difference in the X ² test, but the severity increased with the increase of the dose. Hyperplastic thyroid follicles generally reflect insufficient iodine intake in animals. In this experiment, the proportion of proliferative thyroid follicles in the low-dose group or the distilled water control group was consistent, suggesting that the insufficient intake of iodine in animals was caused by insufficient dietary iodine content; while the high and medium dose groups aggravated the insufficient intake of iodine in animals; The metabolism (growth curve, organ coefficient, etc.) is good, so it is considered that the high proportion of hyperplastic thyroid follicles does not belong to the range of irreversible toxic damage of Diwu extract. The long-term toxicity test of rats showed that it is safe to administer Diwu extract 96mg/kg orally to rats for 6 consecutive months.

Example 3.

Beagle dog long-term toxicity test:

Investigate the long-term toxicity of the extract of Diwu 128mg/kg, 256mg/kg and 512mg/kg for 9 months. Observe the general condition of the animals, body weight change, fecal and urine routine, electrocardiogram, hematology, blood biochemistry, general anatomy of the whole body organs, organ coefficient and histopathological changes. As a result, none of the animals died. There was no statistical difference in the indicators of the high, medium and low dose groups in the animal test for 3 months, 6 months, 9 months and the recovery period compared with the blank control group. The long-term toxicity test results show that it is safe to feed and administer Diwu extract to Beagle dogs at 128mg/kg, 256mg/kg and 512mg/kg for 9 consecutive months.

Example 4.

Effect of Radix Diwu Extract on Adjuvant Arthritis in Rats

1. Observe the effect of Diwu extract on primary lesions

Take 50 Wistar rats with a body weight of about 180 g, half male and half male, and randomly divide them into 5 groups according to body weight, and measure the normal volume (ml) of the left hind paw. Each mouse was intradermally injected with Freuneds complete adjuvant [using liquid paraffin and lanolin (2:1) to shake at 70°C, autoclave, and then add 6 mg of BCG per ml to make 0.1 ml of inflammation. The administration started from the day before the injection of the adjuvant. Observe the effect of the drug on the primary lesion.

Diwu extract 3 dose groups, the doses were 288, 144, 72mg/kg; prednisolone acetate tablet group 20mg/kg; distilled water group 20ml/kg. Both were administered by intragastric administration, once a day for 10 consecutive days. The left hind paw volume of each mouse was measured every day, and the results were compared with those of the distilled water group (t test) in Table 1.

2. Observe the preventive effect of Diwu extract on secondary lesions

Another 50 rats about 180 g in weight, half male and half male, were randomly divided into 5 groups. The grouping situation and dosage are the same as (1). On the 7th day of adjuvant injection, administration was started, and for 7 consecutive days, the contralateral (right side) paw swelling, body weight change, the incidence of ear, forelimb and tail lesions, the number or severity of nodules were observed, and each Measure body weight every 3 days. The results are listed in Table 2 by t-test (exact probability method) compared with the distilled water group.

3. Observe the therapeutic effect of Diwu extract on secondary lesions

Another 50 rats with secondary lesions were taken and administered on the 19th day after adjuvant injection. Observe the swelling degree of the plantar on the opposite side (right side) of the adjuvant injection, and the change in body weight, and observe until the 30th day after continuous administration. Body weight was measured every 3 days. The results are listed in Table 3 compared with the distilled water group (t test).

The results show that: after the inflammation of each dose group of Diwu extract, the degree of swelling of the soles of the feet is significantly different from that of the distilled water group at the same time (P<0.05, P<0.01, P<0.001), suggesting that the drug It has the effect of anti-adjuvant arthritis primary lesions.

Table 1 Effect of Diwu extract on primary lesions in rats with adjuvant arthritis (X±S, n=10) group Dose (mg/kg) Left hind foot volume (ml) normal After administration (days) 1 2 3 4 high dose group 288 0.76±0.03 ^* 0.99±0.07 ^*** 0.98±0.07 ^*** 0.96±0.05 ^**** 0.87±0.06 ^**** Middle dose group 144 0.75±0.02 ^* 1.00±0.07 ^*** 0.99±0.03 ^**** 0.98±0.03 ^**** 0.92±0.04 ^*** low dose group 72 0.74±0.02 ^* 1.03±0.05 ^** 1.00±0.05 ^*** 0.97±0.06 ^*** 0.89±0.05 ^**** prednisone acetate group 20 0.74±0.02 ^* 0.96±0.05 ^**** 0.95±0.06 ^**** 0.92±0.09 ^**** 0.86±0.08 ^**** Distilled water group 20ml 0.74±0.02 1.09±0.06 1.09±0.07 1.08±0.07 1.00±0.06 5 6 7 8 9 10 0.82±0.05 ^**** 0.80±0.04 ^**** 0.80±0.04 ^*** 0.83±0.03 ^**** 0.82±0.03 ^**** 0.79±0.02 ^**** 0.86±0.04 ^*** 0.80±0.02 ^**** 0.81±0.02 ^*** 0.84±0.03 ^**** 0.82±0.04 ^**** 0.78±0.02 ^**** 0.83±0.06 ^**** 0.80±0.03 ^**** 0.79±0.04 ^*** 0.85±0.03 ^**** 0.82±0.03 ^**** 0.79±0.02 ^**** 0.82±0.05 ^**** 0.80±0.04 ^**** 0.78±0.02 ^**** 0.86±0.03 ^*** 0.82±0.03 ^**** 0.79±0.02 ^**** 0.93±0.05 0.89±0.06 0.87±0.06 0.93±0.05 0.93±0.05 0.90±0.05

Compared with the distilled water group (t test) *P>0.05, **P<0.05, ***P<0.01, ****P<0.001.

Table 2 Preventive effect of Diwu extract on secondary lesions (X±S, n=10) group Dose (mg/kg) Normal right foot plantar volume (ml) The contralateral (right hind foot) volume of adjuvant injection at different times after inflammation (ml) 7 days 8 days 9 days 10 days high dose group 288 0.73±0.02 ^* 0.73±0.02 ^* 0.73±0.02 ^* 0.74±0.03 ^* 0.75±0.03 ^** Middle dose group 144 0.74±0.03 ^* 0.75±0.03 ^* 0.75±0.02 ^* 0.76±0.03 ^* 0.77±0.02 ^* low dose group 72 0.73±0.02 ^* 0.74±0.02 ^* 0.75±0.02 ^* 0.77±0.04 ^* 0.78±0.04 ^* prednisone acetate group 20 0.73±0.02 ^* 0.73±0.02 ^* 0.73±0.03 ^* 0.74±0.02 ^* 0.75±0.02 ^** Distilled water group 20ml 0.73±0.02 0.73±0.02 ^* 0.74±0.02 ^* 0.76±0.03 0.79±0.04 group Dose (mg/kg) The contralateral (right hind foot) volume of adjuvant injection at different times after inflammation (ml) 11 days 12 days 13 days high dose group 288 0.75±0.03 ^*** 0.75±0.03 ^*** 0.75±0.03 ^**** Middle dose group 144 0.78±0.02 ^** 0.78±0.02 ^* 0.78±0.02 ^** low dose group 72 0.80±0.05 ^* 0.81±0.05 ^* 0.81±0.05 ^* prednisone acetate group 20 0.75±0.03 ^*** 0.75±0.03 ^*** 0.76±0.03 ^*** Distilled water group 20ml 0.81±0.04 0.82±0.06 0.83±0.06

Compared with the distilled water group (t test) *P>0.05, **P<0.05, ***P<0.01, ****P<0.001.

Table 3 Therapeutic effect of Diwu extract on secondary lesions in rats with adjuvant arthritis

(Volume of the right rear foot, X±S, n=10) group Dose (mg/kg) Normal right hind foot volume (ml) Volume of right hind paw at different times after inflammation (ml) 19 days 20 days 21 days 22 days high dose group 288 0.75±0.02 ^* 0.96±0.09 ^* 0.88±0.04 ^*** 0.86±0.04 ^**** 0.85±0.04 ^**** Middle dose group 144 0.74±0.02 ^* 0.94±0.06 ^* 0.92±0.05 ^** 0.90±0.05 ^**** 0.89±0.05 ^*** low dose group 72 0.74±0.02 ^* 0.94±0.06 ^* 0.93±0.06 ^* 0.93±0.05 ^*** 0.91±0.05 ^** prednisone acetate group 20 0.74±0.02 ^* 0.95±0.07 ^* 0.94±0.04 ^* 0.92±0.05 ^*** 0.91±0.05 ^** Distilled water group 20ml 0.74±0.03 0.95±0.06 0.98±0.07 1.01±0.07 1.00±0.09 twenty three twenty four 25 26 27 28 0.84±0.04 ^**** 0.84±0.03 ^**** 0.83±0.02 ^**** 0.82±0.03 ^**** 0.80±0.02 ^**** 0.78±0.02 ^**** 0.88±0.05 ^**** 0.86±0.05 ^**** 0.85±0.04 ^**** 0.84±0.04 ^**** 0.82±0.04 ^**** 0.79±0.04 ^**** 0.89±0.05 ^**** 0.88±0.05 ^*** 0.86±0.05 ^**** 0.84±0.04 ^**** 0.82±0.04 ^**** 0.80±0.04 ^**** 0.89±0.04 ^**** 0.87±0.04 ^**** 0.84±0.04 ^**** 0.81±0.04 ^**** 0.79±0.03 ^**** 0.76±0.03 ^**** 1.00±0.06 1.01±0.10 0.99±0.07 0.97±0.05 0.97±0.06 0.95±0.05 29 30 0.76±0.02 ^**** 0.75±0.02 ^**** 0.78±0.03 ^**** 0.75±0.03 ^**** 0.79±0.03 ^**** 0.74±0.02 ^**** 0.75±0.02 ^**** 0.74±0.02 ^**** 0.94±0.05 0.93±0.04

Compared with the distilled water group (t test) *P>0.05, **P<0.05, ***P<0.01, ****P<0.001.

Claims (4)

1, treating rheumatism Chinese medicine preparation, it is characterized in that forming effective ingredient is: 3-O-α-L-pyrans rhamnose (1 → 2) β-D-xylopyranose-oleanolic acid-28-O-α-L-pyrans rhamnose (1 → 4)-β-D-Glucopyranose. (1 → 6)-β-D-pyranglucoside, called after Rhizoma Anemones flaccidae saponin W ₃, molecular formula is C ₅₉H ₉₆O ₂₅

2, treating rheumatism Chinese medicine preparation as claimed in claim 1 is characterized in that Rhizoma Anemones flaccidae saponin W in the said preparation ₃Content 〉=90wt%.

3, the preparation method of the described Chinese medicine preparation of claim 1 is characterized in that processing step is:

1st, get the Rhizoma Anemones flaccidae coarse powder, add 8～12 times of water reflux, extract, 3～4 times, each 1～3h, merge extractive liquid;

2nd, the extracting solution of step 1 gained is evaporated to dried, adds 50%～80% ethanol, the filtering precipitation, alcohol extract;

3rd, the alcohol extract of step 2 gained is evaporated to dried, is dissolved in water, filters, D101 macroporous resin adsorption on the filtrate, and water, 40%～80% ethanol, 80%～95% ethanol elution successively; Collect 40%～80% ethanol elution thing, reclaim ethanol and water bath method, get Rhizoma Anemones flaccidae total glucosides;

4th, with silica gel column chromatography on the Rhizoma Anemones flaccidae total glucosides of step 3 gained, with chloroform: methanol: water=20: 10: 1 mixed liquor eluting, rough segmentation becomes 5 sections, and Gao Xiao Ye Xiang Se Spectrum examines knowledge, collects to be rich in the Rhizoma Anemones flaccidae saponin W ₃The 2nd section part, go up silica gel column chromatography again, with chloroform: methanol: water=7: 4: 1 mixed liquor eluting, Gao Xiao Ye Xiang Se Spectrum examines knowledge, collects to be rich in the Rhizoma Anemones flaccidae saponin W ₃Part, get chemical compound Rhizoma Anemones flaccidae saponin W with preparation type high performance liquid chromatogram separation and purification ₃Content 〉=90wt%;

5th, with the chemical compound Rhizoma Anemones flaccidae saponin W of step 4 gained ₃Be prepared into preparation.

4, the described Chinese medicine preparation of claim 1 is used, and it is characterized in that being used for the treatment of rheumatism.