WO2003084945A1

WO2003084945A1 - An antispastic, analgetic pharmaceutical composition and the preparation method thereof as well as the quality control technique therefor

Info

Publication number: WO2003084945A1
Application number: PCT/CN2003/000255
Authority: WO
Inventors: Benxiang Wang; Shengwu Chen; Lianzhu Zhang; Hairi Li; Donghua Yang
Original assignee: Jilin Tianyao Science And Technology Co. Ltd.
Priority date: 2002-04-10
Filing date: 2003-04-10
Publication date: 2003-10-16
Also published as: CN1241574C; AU2003236118A1; CN1449761A

Abstract

The present invention discloses an antispastic, analgetic pharmaceutical composition and the preparation method thereof, also discloses a quality control technique therefor. The composition consists of 80-130 part by weight glycyrrhetinic acid and 120-70 part by weight paeoniflorin. Glycyrrhetinic acid and paeoniflorin extracted from licorice and radices paeoniae alba respectively, are added into pharmaceutical acceptable carrier/or excipients and formed into clinical acceptable dosage forms, such as tablets, capsules, pills, granulas, dropping pills, oral liquid injections, aerosol, suppository. The present invention also provides a quality control technique for ingredients identification and content determination of the composition. The composition according to the invention can be dosed easily, and, has a good curative effect and no toxic and side-effects, ie, has an excellent antispastic and analgetic effect.

Description

Antispasmodic and analgesic pharmaceutical composition and

Preparation method and quality control method

Technical field:

The invention relates to a combination medicine, in particular to a combination medicine for antispasmodic and analgesic, its preparation method and quality control method.

Background technique:

Shaoyao Gancao Decoction is a well-known prescription in the field of traditional Chinese medicine. Traditional Chinese medicine is mainly used to treat abdominal smooth muscle cramps such as menstrual pain, uterine cramps and stomach pain.

Although Shaoyao Gancao Decoction has unique curative effect, its dosage form is still traditional decoction. Obviously, it cannot be produced industrially, but the users themselves cook it. Because the whole material is used for medicine, the active ingredients of the drug are unclear, and no effective or even harmful impurities are mixed in it. Therefore, the pharmacological analysis of Chinese herbal medicines, the isolation of medicinal active substances, and the production of modern medicines without impurities, small size, easy circulation and convenient taking through large-scale production are the development trends of Chinese medicine.

In order to explore the drug activity of Shaoyao Gancao Decoction, we administer Shaoyao Gancao Decoction to rats, then take the serum of the rats and test the serum of the animals with HPLC and HPLC-MS. Glycyrrhet inic acid (or glycyrrhet icacid), glycyrrhizic acid (or glycyrrhizin), paeoni f lorin, and paeonimetabol inel (PM-1) were further tested to prove that the above compounds Glycyrrhetinic acid and glycyrrhetic acid are effective ingredients for antispasmodic and anti-inflammatory, and glycyrrhetic acid is an inhaled blood metabolite of the oral compound glycyrrhizic acid. The drug concentration was significantly higher than that of glycyrrhizic acid. For this purpose, glycyrrhetic acid can be used as an antispasmodic component in a combination medicine. The above experiments also proved that paeoniflorin and paeoniflorin metabolites are effective ingredients for analgesia and sedation. Although paeoniflorin metabolites have stronger analgesic effects than paeoniflorin, it is difficult to mass preparation. Moreover, paeoniflorin metabolites are products of inhaled blood after paeoniflorin is metabolized by enteric bacteria, so paeoniflorin can be used as an analgesic component in medicine.

Summary of the invention:

An object of the present invention is to disclose a new drug combination with antispasmodic and analgesic effects. Another object of the present invention is to disclose a method for preparing a new pharmaceutical composition having antispasmodic and analgesic effects; and the object of the present invention is to disclose a new method for quality control of a pharmaceutical composition.

The composition and formulation of the drug substance of the pharmaceutical composition of the present invention are as follows (by parts by weight): 200-110 parts by weight of glycyrrhizic acid 140-60 parts by weight of total glucosides of paeony, preferably 170-140 parts by weight of glycyrrhizic acid 110-70 Parts by weight of the pharmaceutical composition of the present invention may also be composed of the following drug substances, which are formulated as follows (by parts by weight):

Glycyrrhetinic acid 180-130 parts by weight Paeoniflorin 120-70 parts by weight

Preferably: 165-145 parts by weight of glycyrrhetic acid 110-80 parts by weight of paeoniflorum Most preferably: 153 parts by weight of glycyrrhetic acid 97 parts by weight of paeoniflorin

The medicament of the present invention can also be added to pharmaceutically acceptable carriers and / or excipients to make clinically acceptable dosage forms, such as tablets, capsules, pills, granules, suspensions, drip pills, oral liquid preparations, injections, gas Aerosols and suppositories.

The preparation method of the pharmaceutical composition of the present invention is:

1. Glycyrrhetinic acid preparation: Glycyrrhiza uralensis powder is boiled with water for 2 to 4 times, and the medicinal solution is filtered, and concentrated sulfuric acid is added so that no precipitate can be precipitated. Place, filter, brown precipitate, and wash with water to obtain crude glycyrrhizic acid; use acetone Extract 2-4 times under reflux to obtain an acetone extract, let cool, and add 15-25% K0H ethanol solution under mild stirring until weakly alkaline, leave to crystallize, filter the crystals of tripotassium glycyrrhizinate, dry, heat with water acetic acid Dissolve, let cool, precipitate crystals, and filter to obtain monopotassium glycyrrhizinate, add 4-7% sulfuric acid, heat for 8-12 hours, suction filter, wash with water to neutrality, and dry to obtain crude white glycyrrhetic acid, and then Dissolve in hot chloroform, filter while hot, cool the chloroform solution, pass through an alumina chromatography column, and elute with chloroform to obtain glycyrrhetic acid, which is hot-soluble in ethanol, and pour into 1/2 volume of boiling water while hot. Crystals are obtained and filtered to obtain medicinal glycyrrhetic acid crystals;

2. Paeoniflorin extraction: take Paeonia lactiflora with 3-8 times the amount of 30%-60% ethanol for hot reflux or cold dip extraction 2-3 times, and concentrate the ethanol extract to a relative density of 40-60 ° C 1.1, Then add 90-96% ethanol to an alcohol concentration of 70%-90%, let it stand overnight, filter, add 1%-2% (M / V) activated carbon to the ethanol filtrate, stir, filter, ethanol filtrate Concentrated until no alcohol taste, the above-mentioned extracts were purified on two macroporous resins D101 and D201, washed with water until the Molish reaction was negative, and then eluted with 10%-30% ethanol, collected 10%-30% The ethanol eluate was concentrated under reduced pressure and dried to give pale yellow paeoniflorin.

The quality control method of the pharmaceutical preparation of the composition includes identification and / or content determination. Identification methods include one and / or several of the following methods:

a. Take an appropriate amount of the preparation of the composition, dissolve it with methanol, and make a solution containing about 1 mg per lral as a test solution; take another glycyrrhetic acid reference substance and make a solution containing about 1 mg per ml with methanol as a control. Test solution according to thin layer chromatography (Appendix VI B of the Chinese Pharmacopoeia 2000 Edition), suck 10 μΙ of each of the two solutions above, and place them on the same silica gel GF ₂₅₄ thin-layer plate with sodium carboxymethyl cellulose as the binder. Above, using 3-6: 1-3: 0. 1-0. 5 n-hexane-ethyl acetate-glacial acetic acid as a developing agent, unfolded, taken out, dried, and examined under a 200-300nm ultraviolet light; In the chromatography, the same dark spots should be displayed at the positions corresponding to the reference color language;

b. Take the paeoniflorin reference substance with methanol to make a solution containing about 1mg per 1ml as the reference solution; follow the thin layer chromatography (Chinese Pharmacopoeia 2000 edition, Appendix VI B) test, draw the above reference solution and identify 1 Each test solution under this item was 10 μΙ, and each was spotted on the same silica gel G thin-layer plate with carboxymethyl cellulose sodium as a binder, and 7-10: 1-3 chloroform-methanol was used as a developing agent, and the samples were taken out and taken out. , Dry, spray with 5% vanillin acid solution, heat at 95-120 ° C until the spots are clear; in the test product chromatography, the spots of the same color should be displayed at the corresponding position with the reference product chromatography;

Content determination includes one and / or several of the following methods:

a. Glycyrrhetinic acid, determined according to high performance liquid chromatography (Appendix VI D of the Chinese Pharmacopoeia 2000 Edition); chromatographic conditions and system suitability tests, using octadecylsilane bonded silica gel as a filler; 85-100 : 8-14 methanol-water (H 3 7 adjusted by phosphoric acid) is the mobile phase; the detection wavelength is 250nm and the column temperature is room temperature; the theoretical number of plates calculated from the glycyrrhetic acid peak should not be less than 2000; the preparation of the reference solution, Lg ^: ， Take an appropriate amount of glycyrrhetic acid reference substance, accurately weigh, add methanol to make a solution containing 25 g of glycyrrhetic acid per 1 ml, as a reference solution; for the preparation of the test solution, take the composition preparation 0. lg ^: ， Precise weighing, put in 10Offll measuring flask; Take 10-25 minutes, let it cool, add methanol to the mark, shake well, let it stand, filter through a microporous membrane, and take the filtrate to obtain; Assay method: Inject the above reference solution and test solution separately. 10μΙ Omg; Injected into a high-performance liquid chromatography, measuring the peak area, calculated by external standard method, that is, obtained; per unit amount containing glycyrrhetic acid should not be less than 70. Omg;

b. Total glucosides of paeony, measured according to high performance liquid chromatography (Appendix VI D of the Chinese Pharmacopoeia 2000 Edition); chromatographic conditions and system suitability tests, using octadecylsilane bonded silica as a filler; 25- 35 : 65-80 methanol-water is the mobile phase, the detection wavelength is 230nm; the number of theoretical plates should be not less than 2000 according to the peony peak; A solution containing 25 g of paeoniflorin per 1 ml is used as the paeoniflorin reference solution; the preparation of the test solution is the preparation of the test solution under the same content determination item 1; the test method is to combine the above reference solution with the test product 0mg _o Each solution was injected ΙΟμΙ, injected into a high performance liquid chromatograph, the peak area was measured, and calculated by the external standard method;

The unit amount refers to an amount equivalent to one capsule preparation.

The composition of the present invention has a small volume, good curative effect, no toxic and side effects, convenient taking, and good smooth muscle antispasmodic and analgesic effects.

The following experimental examples further illustrate the present invention.

Experimental example 1

In this experiment, 2 hours after the rats were given gastric peony and licorice soup, the blood was anesthetized and centrifuged to remove blood. The serum was extracted with ether and n-butanol, and the absorbed components were analyzed by HPLC-MS. The activity of each isolated fraction and monomer compound was analyzed, and the conclusion: the effective component of Shaoyao Gancao Decoction was paeoniflorin and its metabolite PM-I, and the effective component of antispasmodic effect was glycyrrhetic acid.

1. Preparation of paeonia licorice (paeonia: licorice = 1: 1) decoction and decoction of supernatant and sediment (residue-1): decoction 3 times with 3 times (W / V) water for 1 hour each time. After concentrating to an appropriate volume, add 3 times the volume of 95% ethanol, stir and let stand overnight, filter to obtain the supernatant and residue-1. Recover the ethanol from the supernatant and concentrate to 2.5 g crude drug / ml for later use. 5g 生药 / ml。 Pharmacological experiments, the residue was formulated with water to 2.5 g raw drugs / ml. Supernatant and residue doses are 40, 20, 10 ml / kg, intragastric administration once a day for 2 days, and do analgesia test 1 hour after the last administration.

2. Preparation and activity detection of serum fractions of rats administered with peony and licorice decoction: 200 rats were divided into two groups, of which 20 rats were used as controls, 30 ml / kg of saline was administered to the stomach, and the remaining 180 rats Gavage peony licorice decoction decoction (peony: licorice = 1: 1, decoction concentration 2.5 g crude drug / ml) 30 ml / kg. 10% chloral hydrate (4 ml / kg) intraperitoneally 2 hours after administration ) Anesthetize the rats, collect blood from the abdomen and collect serum (60 ml in the control group and 540 ml in the administration group), extract the serum 3 times with ether and n-butanol, recover the solvent, combine the above extracts, and prepare a high-performance liquid phase ( Model: Ag i lent type 1100, column type: Zorbax C-18 column, mobile phase: acetonitrile: acidic water = 10% → 90%: 90-10%, gradient elution). Sections 1-6 (Figure 3). After pharmacological identification, the effective part of the analgesic and antispasmodic effect is determined. Then, each active ingredient is further analyzed by HPLC-MS and pharmacological activity test until the chemical structure of the active ingredient is determined.

3. Detection of pharmacological effects of analgesics and antispasmodics: Take 1/2 of each separated part (equivalent to the amount of each in 1/2 (ie 270ml) original serum), and dissolve it into 13.5 ml with 10% Tween 80 aqueous solution. (Equivalent to 20 times the original serum concentration), for the analgesic and antispasmodic pharmacological activity test.

Analgesic experiments according to the literature method ^] , take the solution of the above isolated site α-6), and give the mice tail vein injection doses of 5 and 10 ml / kg (after intravenous injection, make the drug serum concentration reach its original serum respectively. 1 and 2 times), 5 minutes after dosing, after intraperitoneal injection of 1% acetic acid 10 ml / kg, record the number of writhing times for 10 minutes immediately; antispasmodic experiments, using isolated mouse uterus to test the solution of each sample according to literature methods Spasm effect. Take female mice weighing about 28g and inject estradiol at a rate of 0.4ml / subcutaneously once a day for 3 consecutive days. The mice will be killed by cutting their heads, the uterus will be removed, and the uterus will be washed with Typhoon's solution. Suspend the uterus in a 50ml bath and equilibrate for 30 minutes to test the sample. Take each of the experimental samples prepared from each of the serum fractions and serum residues above, and test each sample for 2 concentrations, that is, the original drug in the serum Medium concentration and half concentration. The volume of the actual test sample added to the serum is 2.5 and 1.25ml. Before adding the serum, the same volume of Tyrode's solution is released from the bath. The antispasmodic effect of each sample on spasm of the mouse uterus induced by pituitary hormone was recorded. 4. Serum residue (residue 2) preparation of test samples: take serum residue and add 27 ml of 10% Tween-80 aqueous solution, stir to dissolve, centrifuge 1000 g for 10 minutes, take the supernatant, and determine with 10% Tween-80 aqueous solution. The content is up to 27ml (that is, the concentration is 20 times of the original serum). For pharmacological activity test. In the analgesic and antispasmodic experiment, the dosage of the medicinal solution is the same as that of the serum sample.

Results

I. Pilot study of the optimal absorption time of known chemical constituents of Shaoyao Gancao Decoction. Twenty rats were evenly divided into two groups, one group was administered with saline 30 ml / kg as a control, and the other group was administered with Shaoyao and Licorice soup. Decoction (peony: licorice = 1: 1, decoction concentration is 2.5 g crude drug / ml) 30 ml / kg. At 1, 1, 4, and 8 hours after administration, rats in the control group and the administration group were given Two rats each were anesthetized with intraperitoneal injection of 10% chloral hydrate (4 ml / kg), placed from the abdominal aorta ^, placed in a 37 ° C water bath, and treated with blood ^^ to separate the serum. It was extracted with ether and n-butanol three times. After recovering the solvent, both were dissolved in methanol and combined. The extracts were subjected to high-performance liquid chromatography (Agi lent 1000) chromatography, column type: Zorbax C18, mobile phase: acetonitrile: Acidic water (pH 3.5) = 10% ~ 90%: 90% → 10%, gradient elution. The contents of paeoniflorin, glycyrrhizic acid and glycyrrhetic acid in the serum were measured, and the results showed that the content of paeoniflorin, glycyrrhizic acid and glycyrrhetic acid in the serum basically reached a high level 2 hours after the administration. Therefore, in the following study, when the rats were given gastric peony licorice soup to absorb the active ingredients into blood, the blood collection time was set to 2 hours after the administration.

Analgesic results.

1. Effects of different fractions from serum of rats treated with peony glycosides decoction on writhing response in mice induced by acetic acid.

The preparation of the pharmacological activity test samples of the serum isolated fractions and serum residues of the drug-treated rats is as described above. The intravenous injection volume of each group is 10ml / kg, and the actual dosage is shown in Table 2. 5 minutes after the administration After intraperitoneal injection of 1% acetic acid at 10 ml / kg, the number of twists for 10 minutes was recorded immediately. The results are shown in Table 1. Table 1. Effects of different fractions of rat serum administered with peony glycosides decoction on writhing response in mice induced by acetic acid

Γ ± S, n = 5.

Group (ml (mg) / kg) Number of twists / 10 minutes P

1. Saline 10 22. 8 Soil 6. 1

2. Morphine hydrochloride 10 (mg) 0 <0.001

3. Part 1 10 20. 6 Taxi 2. 9> 0. 05

5 22. 0 Taxi 4. 6> 0. 05

4. Part 2 10 13. 0 soil 1. 9 <0. 01

5 15. 2 Taxi 3. 6 <0. 05

5. Part 3 10 10. 0 soil 3. 4 <0. 01

5 12. 9 soil 2. 3 <0. 01

6. Section 4 10 21. 4 Taxi 3. 8> 0. 05

5 22. 8 soil 3. 8> 0. 05

7. Section 5 10 21. 8 Taxi 4.1 1> 0. 05

5 25. 0 taxis 3. 5> 0. 05

8. Section 6 10 19. 6 Soil 1. 5> 0. 05

5 22. 4 Taxi 4. 2> 0. 05

9. Serum residue 10 23. 0 ± 4. 6> 0. 05

5 21. 8 soil 6. 1> 0. 05 As can be seen from Table 1, serum fractions 2 and 3 have analgesic effects.

The serum part 2 was separated and identified by HPLC-MS. The original part 2 was single-absorbed. The retention time of the HPLC chromatogram peak was also consistent with the standard of paeoniflorin (Figure 1-1). The ion peak m / z is 481.3 (Figure 1-2), which is consistent with paeoniflorin, so part 2 is initially identified as paeoniflorin.

Take the remaining 1/2 part 3 and separate it with high-performance liquid phase to obtain three separation parts such as 3-a, 3-b and 3-c (see Figure 2-1). Take the above separated parts (equivalent to 270ml in 1/2 of original serum), after dispersing the solvent, dissolve it into 13.5 ml (corresponding to 20 times of original serum) with 10% soil temperature-80 aqueous solution. The following pharmacological experiments. Continue to observe the effect of the above three separated parts on the writhing response of mice induced by acetic acid. The experimental procedure is the same as the aforementioned analgesic experiment. Pharmacological experiments have proven that the separated part 3-C has obvious analgesic action. (Table: 2 ).

Table 2.Effects of isolated fractions from rat serum part 2 on mouse writhing response induced by acetic acid. X + S, n = 5.

Group ml (mg) / kg Number of twists / 1Q minutes P

1. saline 10 19.4 ± 3.7

2. Morphine hydrochloride 1 (mg) 0 <0.001

3. Paeoniflorin (Part 2) 150 (mg / kg, ο) 12.8 + 3.6 <0.05

75 (mg / kg, ο) 14.4 soil 2.3 <0.05

4. Serum fraction 3-a 10 19.2 ± 5.4> 0.05

5 19.0 soil 3.6> 0.05

5. Serum fraction 3-b 10 16.2 + 5.5> 0.05

5 17.6 soil 3.6> 0.05

6. Serum fraction 3-c 10 12.8 soil 4.8 <0.05

5 14.0 ± 1.4 <0.05

Serum fraction 3-C was detected by HPLC-MS and was absorbed by paeoniflorin into the blood metabolite PM-I (paeonimetabolin I) because of the retention time of some 3-c peaks in HPLC and molecular ions given by HPLC-MS. The peak m / z amounts are all the same as PM-1, so it is preliminarily considered that part 3-c is the same compound as PM-1, and part 3-b may be glycyrrhizic acid (Figure 2-1, 2, 3)

3. Detection of antispasmodic effect of each separated part of serum of rats

According to the literature method ^] The antispasmodic effect of each sample was detected with isolated mouse uterus. Female mice weighing about 28g were taken and injected subcutaneously with 0.4ml / estradiol once a day for 3 consecutive days. The head was sacrificed, the uterus was taken out, and the uterus was rinsed with Typhoon's solution. The uterus was suspended in a bath with a volume of 50 ml. After equilibration for 30 minutes, it was used to detect the sample. Each of the above-mentioned serum fractions and serum residues were prepared as experimental samples. each sample detection 2 concentration, i.e. half the original concentration and the concentration of drug in the serum. the actual volume of ^2.5 and the addition of 1.25 ml. the volume of the supplementary bath made 50 ml. each sample recorded on vasopressin-induced The spasmolytic effect of uterine spasm in mice. Crusting showed that the isolated part 6 had a spasmolytic effect, and other samples had no obvious spasmolytic effect (Figure 3-1, 3-2). Part-6 was further obtained by high-performance liquid phase separation. Part 6-1— 6-5 and 5 parts (Figure 4-1).

Further antispasmodic experiments proved that part 6-d was the effective component of antispasmodic action (Figure 5). After HPLC-MS analysis, its retention time on HPLC chromatography and molecular ionization given by mass spectrometry The sub-peak m / z is consistent with glycyrrhetic acid (Figure 6). Therefore, some ^6- d are initially determined to be glycyrrhetic acid.

Experimental example 2. Effects on spontaneous activities in mice

Ninety mice were divided into 9 groups, each group was divided into male and female, and the drug was administered once according to the dosage and administration route shown in Table 1. The first group was the gastric control group, and the gastric 0.2% CMC aqueous suspension 10 ml / kg. The sixth group is a control group for injection administration, and the saline is injected subcutaneously at 10 ml / kg. The mice in the 1-5 group are administered for 1 hour after the administration, and the mice in the 6-9 group are administered for 30 minutes after the administration. In the observation device, the number of spontaneous activities of every eight mice within 5 minutes was recorded. The results are shown in Table 3. It can be seen from Table 3 that both the glutinous capsules and ganzhi injection significantly inhibit the spontaneous activity of mice. ―

Table 3. Effects of Gancao Capsule and Gancao Injection on Spontaneous Activity in Mice (i ± S) n = 10 Group Dose (mg / kg) Number of Mouse Activities Inhibition Rate (%)

(5min)

1. Control 0.2% CMC po 144.8 ± 22.5

2.Stability 5 po 56.8 ± 23.1 "· 60.7

3.Glycan capsule 300 po 45.2 ± 9.9 * '* 68.8

4. 150 po 53.5 ± 20.8 "63.1

5. 75 po 73.5 + 22.5 * 49.2

6. Saline 10ml / kg im 132.7 ± 36.7

7. Gan Zhi injection 60 sc 50.4 ± 12.9 · "62.0

8. 30 sc 64.3 ± 16.5 "51.5

9. 15 sc 71.4 ± 13.6. 44.2 Note: Compared with the negative control, P <0.05, ** P <0.01, *** ρ <0.001 Experiment Example 3 Torsion response and abdominal exudation induced by intraperitoneal injection of acetic acid in mice Effects of sexual inflammatory response:

In this experiment, the mice were grouped and administered in the same manner as in the experimental 1.1-5 group. Chlorine was administered 1 hour after administration, and the 6-9 group was administered 30 minutes after administration. The mice were injected with Evan's blue (2%, 5ml / kg), 2 minutes after the injection of Evans blue, the mice were injected intraperitoneally with 1% 'acetic acid 10ml / kg. The number of twists in each mouse within 10 minutes after the injection of acetic acid was recorded, and then the mice _: Sacrifice by pushing, laparotomy with saline 4 ml / mouse flush the abdominal cavity to exudate the dye. With photoelectric colorimeter (620 nm) measurement to determine the amount of dye bleeding. The results are shown in Table 4.

Table 4. Effects of Glycoside and Glycoside injection on writhing response and peritoneal exudative inflammation in mice induced by acetic acid.

(X soil S), n = 10.

Intraperitoneal penetration number of twisted body inhibition rate group dose (mg / kg)

Amount of dye μ g / ffll lOmin (%)

1. Control 0.2% CMC po 56 ± 8 15.6 ± 2, 9

2.Aspirin 400 po 33 ± 11 "4.7 ± 2.2". 69.9

3. Ganzhi Capsules 300 o 26 ± 8 "· 3.1 + 0. Γ 80.1

4. 150 po 28 soil 8 · '· 5.3 ± 1.9 "66.0

5. 75 po 33 ± 3 * '6.1 ± 1.6. 60.9

6.Control saline 10ml / kg im 57 ± 11 14.1 ± 2.4

7. Gansu injection 60 sc 27 soil 5 ". 3.7 ± 1.6 · 70.8

8. 30 sc 32 soil 3 "5.0 ± 1.2 ·· 64.5

9. 15 sc 35 ± 4. 6.0 ± 1.5 · 57.5 Note: Compared with the respective control group, * p <0.05, ** p <0.01, *** p <0.001. Experimental example 4 Glycopene gelatin spasmolysis test 1 Effect on Small Intestinal Smooth Muscle Activity of Isolated Rats

Materials and Methods:

Animals: Wistar rats, weighing 250 ± 20g, both female and male. Drugs: Paeoniflorin, Glycyrrhetinic Acid, Glycyrrhizin Injection with different concentrations were prepared by the Plant Chemistry Laboratory of Jilin Institute of Natural Medicine; acetylcholine chloride (lg / branch, Beijing, Beijing Donghuan United Chemical Plant); atropine sulfate injection ( Specification: 0.5mg / mL, Kaifeng Pharmaceutical Factory, Henan Province). Apparatus: Powerlab / 8s data recording analyzer and related accessories (products of Australia Ede Company); YSD-4G multi-purpose instrument for pharmacological and physiological experiments and its constant temperature water bath device (product of Anhui Bengbu Radio 2 Factory). Methods: Rats were sacrificed after fasting for 48 hours. The abdominal cavity was opened. A section of the intestine was cut in the upper part of the small intestine and placed in culture medium I with Typhoon's solution. The mesentery was gently separated along the intestinal wall and rinsed with Typhoon «tube After that, cut into several small pieces of about 2cm for later use. Mediation by a mercury contact thermometer with 'YSD- 4G type multiple pharmacological physiological experiments spectrometer coupled to control the temperature of the constant temperature water bath, to be constant at ³ 7 ± 0.5. C, the volume of Typhoon solution in the Maxwell bath tube is about 50ml, The liquid level of the C. solution is basically the same as the liquid level of the water bath. The air flow is controlled at about 50 bubbles / minute. A small intestine is selected. One end is fastened and fixed on the hook of the ventilation tube with the other end. Water-filled capillary glass tubes are connected. When the isolated bowel is contracted, the water column in the capillary glass tube rises. The level of liquid level rise is related to the contractile strength of the intestinal canal. Stabilize for 20 minutes, adjust the liquid level in the capillary to the "0" position, and then add 0.001% acetylcholine chloride 0.1 ml to the Myrtle bath tube, causing intestinal spasm and tension. In addition to the most obvious intestinal contraction, it was administered in three ways: ① adding 1 mL of Typhoon's solution; ② adding 1 mL of azapine; ③ subsequently adding different concentrations of glycyrrhetic acid, paeoniflorin, and gan injection. Observe the antispasmodic effect of the drug. Complete one step, replace the new intestine and nutrient solution, and stabilize for 20 minutes before proceeding to the next step.

Experimental results: Glycyrrhetinic acid and glycyrrhizin injection have different degrees of inhibition on rat intestinal spasm induced by acetylcholine choline chloride, and the antispasmodic effect of glycyrrhizin injection is more obvious. However, the effect of paeoniflorin is not obvious (Table 5)

Table 5. Effects of Glycyrrhetinic Acid, Paeoniflorin, and Glycyrrhizin Injection on Isolated Intestinal Constriction in Rats. (X ± S), n = 3

Group Intestinal contraction amplitude Inhibition rate

Drug concentration in the bath (g / ml) See%

Acetylcholine chloride reduction 4 25 ± 3.0

Atropine 10 6 ± 1.0 76

Glycyrrhetinic acid 40 8 ± 2.0 68

20 12 ± 2.0 52

10 16 ± 1.7 36

5 24 ± 2.0 4

Acetylcholine chloride 4 20 ± 1.0

Atropine 10 7 ± 1.0 65

Glycyrrhetinic acid 40 19 ± 3.0 5

20 20 ± 2.0 0

10 20 ± 2.0 0

Acetylcholine chloride 4 24 ± 2

Atropine 10 6 ± 2 75

Glycyrrhetinic acid 40 6 ± 2 75

20 8 ± 2 67

10 12 ± 2 58

5 20 + 1 17

2.5 23 ± 1 4- Experimental example 5 Study on the ratio of glycyrrhetic acid and total glucosides of paeony

Glycyrrhetinic acid and total glucosides of paeony make up a modern Chinese medicine compound new medicine. The dosage of the two components is determined through the following orthogonal design:

Table 6-1 Factors of orthogonal test

-Total glucosides of paeony (mg) Glycyrrhetinic acid (mg) Factor A B

1 1750 1108

2 842 532

3 345 222 Table 6-2 Orthogonal test results of Glycerin capsule (dose 300mg / kg) No. A B Α χ Β Empty column

1 1 1 3 2. 62.3

2 2 1 1 1 39. 6

3 3 1 2 3 43. 7

4 1 2 2 1 54. 2

5 2 2 3 3 30. 9

6 3 2 1 2 43. 1

7 1 3 1 3 52, 4

8 2 3 2 2 33. 6

9 3 3 3 1 62. 4 168.9 145.6 135.1 156.2 κ ₂ 104.1 128.2 131.5 139

3 149.2 127

56.3 48.5 45.0 52.1 2 34.7 42.7 43.8 46.3

_-3 49.7 49.5 51.9 42.3

R 21.6 6 inch. 8 8.1 9. 8

O C

Inch

Table 6-3 Analysis of variance table

Deviation mean square and degrees of freedom mean square oo F P

S _A 735.7 5.12〉 0. 05

S _B 79.82 2 39.91 0.56> 0.05

s empty 143.6 2 71.8

Corpse 0.05 (2, 2) = 19.0

Table 6-4 Analysis of variance table

Deviation from mean square and degrees of freedom mean square P P

Error 336.06 6 56.01

/ ¾. ₀₅ (2, 6) = 5. 14

The analysis of variance showed that factor A was the main influencing factor, that is, the amount of total glucosides of paeony had a great effect on the analgesic effect, factor B was the secondary influencing factor, and the optimal ratio was A, but combined with the test results, There is a certain synergy, prescription 7 is A, but it is not as good as prescription 1 and prescription 9 for analgesic effect. Based on the results of the analgesic and antispasmodic test, prescription 1 is selected.

The following embodiments can achieve the effects of the present invention.

Example 1 Preparation of Glycyrrhetinic Acid and Paeoniflorin

1. Glycyrrhetinic acid preparation: Glycyrrhiza uralensis powder is boiled 3 times with water, the liquid medicine is filtered, and concentrated sulfur is added The acid is no longer precipitated, placed, filtered, brown precipitate, washed and dried to obtain crude glycyrrhizic acid. Extract 3 times with refluxing acetone to obtain acetone extract, let cool, and add 20% KOH ethanol solution to weakly basic under stirring, leave to crystallize, filter crystals (tripotassium glycyrrhizinate), dry, add glacial acetic acid Hot melt, let cool, precipitate crystals, and filter to obtain monopotassium glycyrrhizinate, add 5% sulfuric acid, heat for 10 hours, suction filter, wash with water to neutrality, and dry to obtain crude white glycyrrhetic acid, then dissolve it in heat In chloroform, filter while hot, cool the chloroform solution, pass through an alumina chromatography column, and elute with chloroform to obtain glycyrrhetic acid, which is hot-melt in ethanol, and pour into 1/2 volume of boiling water while standing, and obtain crystals. Filtration yields medicinal glycyrrhetic acid crystals.

2. Paeoniflorin extraction: take Paeonia lactiflora materials for 5 times the amount of 45% ethanol for hot reflux or cold dip extraction once, concentrate the ethanol extract to a relative density of 1.1 (50 ° C), and add 95% ethanol to the alcohol concentration 80%, leave it overnight, filter, add 1.5% (M / V) activated carbon to the ethanol filtrate, stir, filter, and concentrate the ethanol filtrate until it has no alcohol. The above extracts are obtained in D101 and D201. Purify on the porous resin, first wash with water until the Molllious reaction is negative, and then elute with 10%-30% ethanol. Collect the 20% ethanol eluate, concentrate under reduced pressure, and dry to obtain pale yellow paeoniflorin.

Example 2 Preparation of Glycyrrhizin

Paeoniflorin (90% purity) 153 g

Glycyrrhetinic acid (purity 95%) 97g

Starch amount

Made into 1000 tablets (tablets)

Preparation method: Paeoniflorin and glycyrrhetic acid are thoroughly mixed with an appropriate amount of starch and filled into capsules. Each capsule contains 0.25g of medicine.

Indications: Abdomen: Abdominal smooth muscle cramps (menstrual pain, uterine cramps, gastrointestinal cramps due to gastric ulcers) and headaches.

Usage and Dosage: 1-2 capsules of Yueyue once for pain.

Example 3 Preparation of Gansu tablets

Paeoniflorin (90% purity) 148 g

Glycyrrhetinic acid (purity 95%) 102g Starch amount

Made into 1000 tablets

Preparation method: Paeoniflorin and glycyrrhetic acid are thoroughly mixed with an appropriate amount of starch, and granulated and compressed. each

1 tablet contains 0.25g of medicine.

Indications: Abdomen: Abdominal smooth machine spasm pain (menstrual pain, uterine spasm pain, gastrointestinal spasm pain caused by gastric ulcer) and headache.

Dosage and dosage: Take 1-2 tablets orally at a time for pain.

Example 4 Preparation of Ganzhi Granules

Paeoniflorin (90% purity) 160 g

Glycyrrhetinic acid (purity 95%) 90g

An appropriate amount of starch was made into granules of 2000 g, and the indications were the same as in Example 3. Usage and dosage: When painful, JI Liang 2g.

Example 5 Preparation of Ganzhi dripping pills Paeoniflorin (purity 90%) 33g

Glycyrrhetinic acid (purity 95%) 17g

1: 1.5 Add matrix PEG6000, mix well, drip into methyl silicone oil to make drop pills, a total of 5000 pills, each pill weighs 25mg (containing medicine 10mg), the indications are the same as in Example 3, usage and dosage: 1 in pain 15-30 pills orally.

Example 6 Gansu injection

Paeoniflorin (90% purity) 30g

Glycyrrhetinic acid (purity 9 · 5%) 20g

1, 2-propanediol it * _

1000ml 2ml ampoules

Preparation method: Dissolve peony licorice and glycyrrhetic acid in an appropriate amount of propylene glycol, filter, separate, and sterilize. Each contains 50mg of drug.

Indications: Abdominal smooth machine spasm pain (menstrual pain, uterine spasm pain, gastrointestinal spasm pain due to gastric ulcers, and headache. Usage and dosage: When using, dilute one Ganzhi injection in 500ml physiological saline and inject it intravenously. Every time a 1 ^2.

Example 7 Quality Control Method of Ganzhi Capsule

Identification: a. Take an appropriate amount of the content of this product, add methanol to dissolve, make a solution containing about 1mg per lml, as the test solution; take another glycyrrhetic acid reference substance to make a solution containing about 1mg per lml, As a reference solution; According to the thin-layer chromatography (Chinese Pharmacopoeia 2000 Edition Appendix VI B) test, draw the two solutions above 10μΙ, respectively, and point them on the same silica gel GF ₂₅₄ with carboxymethylcellulose sodium as the binder. On the layer plate, use 4: 1, 7: 0.2 n-hexane-ethyl acetate-glacial acetic acid as the developing agent, expand, remove, dry, and inspect under a 254nm ultraviolet light. The same dark spots should be displayed at the corresponding positions in the color language; b. Take the peony fragrant reference product and make a solution containing about 1 mg per ml with methanol as the reference solution; follow the thin-layer color consultation method (Chinese Pharmacopoeia 2000 Edition A Appendix VI B) test, suck the reference solution and the test solution under the identification 1 each 10μl, respectively point on the same silica gel G thin layer plate with carboxymethyl cellulose sodium as the binder, take 8 : 2 chloroform-methanol as a developing agent, Out, dried, 'sprayed with 5% vanilla meal sulfuric acid solution was heated to a clear spot at 105 ° C; Chromatography of the test, the color corresponding to the reference position, the spots should be the same color;

Content determination: a. Glycyrrhetinic acid, determined according to high performance liquid chromatography (Appendix VI D of the Chinese Pharmacopoeia 2000 Edition); color ridge condition and system suitability test, using octadecylsilane bonded silica as a filler ; 90: 10 methanol-water (pH 3. 7 adjusted by phosphoric acid) as mobile phase; detection wavelength 250nm, column temperature is room temperature; theoretical plate number should be not less than 2000 calculated from glycyrrhetic acid peak; preparation of reference solution, take Lg Glycyrrhetinic acid reference substance, accurately weighed, add methanol to make a solution containing 25 pg of glycyrrhetic acid per 1 ml, as a reference solution; for the preparation of test solution, take the content of this product 0. lg, precision weighing In a ¾ 100ml volumetric flask, ultrasonic extraction with a power of 100W and a frequency of 50 z for 15 min, let cool, add methanol to the mark, shake well, and let it stand, filter through a 0.45 μm microporous filter, and take the filtrate to obtain; In the determination method, inject 10 μl of the reference solution and the test solution respectively, and inject them into a high-performance liquid colorimeter, determine the peak area, and calculate using the external standard method. Each capsule contains essential glycyrrhetic acid to 70. Omg; b. Total glucosides of paeony, determined according to high performance liquid chromatography (Appendix VI D of the Chinese Pharmacopoeia 2000 Edition); chromatographic conditions and system suitability tests, using octadecylsilane bonded silica as a filler 30: 70 methanol-water as mobile phase, detection wavelength 230nm; theoretical plate number should be not less than 2000 calculated from paeoniflorin peak; preparation of reference solution, take appropriate amount of paeoniflorin reference, accurately weigh and add methanol A solution containing 25pg of paeoniflorin per 1ml is used as the paeoniflorin reference solution; the preparation of the test solution, the preparation of the test solution under the same content determination item 1; the test method, the above reference solution and the test solution 0mg。 Inject 10μΙ, inject high-performance liquid chromatography "i universal instrument, determine the peak area, calculated by external standard method, that is; each capsule of paeoniflorin should not be less than 48.0mg.

Claims

Rights request

1. A pharmaceutical composition with antispasmodic and analgesic properties, characterized in that the pharmaceutical composition is made of the following raw materials:

Glycyrrhizic acid 200-110 parts by weight Paeoniflorin total glycosides 140-60 parts by weight.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is made of the following raw materials:

170-140 parts by weight of glycyrrhizic acid 110-70 parts by weight of total glycosides of paeonia lactiflora.

3. A pharmaceutical composition with antispasmodic and analgesic properties, characterized in that the pharmaceutical composition is made of the following raw materials:

Glycyrrhetinic acid 180-130 parts by weight Paeoniflorin 120-70 parts by weight.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is made of the following raw materials:

Glycyrrhetinic acid 165-145 parts by weight Paeoniflorin 110-80 parts by weight.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is made of the following drug substance:

153 parts by weight of glycyrrhetic acid 97 parts by weight of paeoniflorin.

6. The pharmaceutical composition according to claim 1, 2, 3, 4 or 5, characterized in that the pharmaceutical composition can also be added with excipients to make tablets, oral liquid preparations, capsules, granules, Injectable preparation.

7. The pharmaceutical composition according to claim 6, characterized in that the method for preparing each composition is:

Glycyrrhetinic acid preparation: Glycyrrhiza uralensis powder is boiled with water for 2-4 times, the solution is filtered, concentrated sulfuric acid is added to prevent precipitation, and it is placed, filtered, brown precipitated, washed and dried to obtain crude glycyrrhizic acid; reflux extraction with acetone 2 -4 times, get acetone extract, let cool, add 15-25% K0H ethanol solution to weakly alkaline, leave to stand, crystallize, filter the crystals of tripotassium glycyrrhizinate, dry, heat with glacial acetic acid, Allow to cool, precipitate crystals, and filter to obtain monopotassium glycyrrhizinate, add 4-7% sulfuric acid, heat for 8-12 hours, pump> Ϊ, wash with water until neutral, and dry to obtain white licorice Crude hypoacid, then dissolve it in hot chloroform, filter while hot, cool the chloroform solution, pass through an alumina chromatography column, and elute with chloroform to obtain glycyrrhetic acid, hot melt ethanol, and pour into 1 / while hot 2 volumes of boiling water, left to obtain crystals, and filtered to obtain medicinal glycyrrhetic acid crystals;

Extraction of Paeoniflorin: Take 3 to 8 times the amount of 30% -60% ethanol and extract it with hot reflux or cold dip for 2-3 times. Concentrate the ethanol extract to 40-60 ° C relative density 1.1, then use 90- Add 96% ethanol to an alcohol concentration of 70% -90%, leave it overnight, filter, add 1%-2% activated carbon to the ethanol filtrate, stir, filter, and concentrate the ethanol filtrate until it has no alcohol taste. The product was purified on two kinds of macroporous resins D101 and D201, washed with water until the Molish reaction became negative, and then eluted with 10% -30% ethanol. The 10% -30% ethanol eluate was collected, concentrated under reduced pressure, and dried to obtain Light yellow paeoniflorin.

The pharmaceutical composition according to claim 7, characterized in that the method for preparing each composition is:

Glycyrrhetinic acid preparation: Glycyrrhiza uralensis powder is boiled 3 times with water, and the liquid medicine is filtered, and concentrated sulfuric acid is added so that no precipitate can be precipitated. Place, filter, brown precipitate, wash and dry to obtain crude glycyrrhizic acid. Extract 3 times under reflux with acetone to obtain an acetone extract, let cool, and add 20% KOH ethanol solution to weakly basic under stirring, leave to crystallize, filter the crystals, dry, add water acetic acid to heat dissolve, let cool, and precipitate crystals After filtration, the monopotassium glycyrrhizinate salt was added, 5% sulfuric acid was added, and the mixture was heated for 10 hours, filtered with suction, washed with water to neutrality, and dried to obtain a crude white glycyrrhetic acid, which was then dissolved in hot chloroform and filtered while hot. The chloroform solution was allowed to cool, passed through an alumina chromatography column, and eluted with chloroform to obtain glycyrrhetic acid, which was hot-dissolved in ethanol, and poured into 1/2 volume of boiling water while hot, and left to crystallize. Acid crystallization

Paeoniflorin extraction: take the Paeonia lactiflora medicinal material for 5 times the amount of 45% ethanol under hot reflux or cold dip extraction twice, concentrate the ethanol extract to 50 ° C relative density 1.1, and then add 95% ethanol to 80% alcohol concentration, Let it stand overnight, filter, add 1.5% activated carbon to the ethanol filtrate, stir, filter, and concentrate the ethanol filtrate until it has no alcohol taste. The above extracts are purified on two macroporous resins D101 and D201, and washed with water until The Moll ious reaction was negative, and then eluted with 10%-30% ethanol. The 20% ethanol eluate was collected, concentrated under reduced pressure, and dried to obtain pale yellow paeoniflorin.

9. The method for quality control of a pharmaceutical composition formulation according to claim 3, 4 or 5, characterized in that the identification method in the method includes one or more of the following identifications:

a. Take an appropriate amount of the composition preparation, dissolve it with methanol, and make a solution containing about 1 mg per 1 ml as a test solution; take another glycyrrhetic acid reference substance and make a solution containing about 1 mg per 1 ml with methanol as Control solution: According to the thin layer chromatography test, pipette the two solutions mentioned above 10 μl each, and point them on the same silica gel GF ₂₅₄ thin-layer plate with sodium carboxymethyl cellulose as a binder, with 3-6: 1-3 : 0. 1-0. 5 n-hexane-ethyl acetate-water acetic acid as a developing agent, unfolded, taken out, air-dried, inspected under a 200-300nm ultraviolet light; for the chromatogram of the test product, corresponding to the reference color In the position, the same dark spots should be displayed;

b. Take the paeoniflorin reference substance and make a solution containing about 1mg per 1ml as the reference substance solution; according to the thin layer chromatography test, pipette the above reference substance solution and the test solution under the identification 1 each 10μΙ Click on the same silica gel G sheet with sodium carboxymethylcellulose as the binder, use 7-10: 1-3 chloroform-methanol as the developing agent, unfold, remove, dry, and spray with 5% vanilla The acid sulfuric acid solution is heated at 95-120 ° C until the spots are clear. In the test product color, the spots of the same color should be displayed at the corresponding positions in the chromatogram of the reference substance.

10. The method for quality control of a pharmaceutical composition formulation according to claim 9, characterized in that the identification method of the capsules includes one or more of the following identifications:

a. Take an appropriate amount of the content of this product, add methanol to dissolve, make a solution containing about 1mg per lml, as a test solution; take a glycyrrhetic acid reference substance, and use methanol to make a solution containing about 1mg per lffll, as a control. Product solution; According to the thin layer color test, pipette the above two solutions 10 μl each, and point them on the same silica gel GF ₂₅₄ thin-layer plate with sodium carboxymethyl cellulose as a binder, and take 4: 1. 7: 0. . 2 n-hexane-ethyl acetate-glacial acetic acid as the developing agent, unfold, remove, dry, view under 254nm UV light; for the chromatography of the test product, at the corresponding position of the chromatography of the reference substance, it should show the same Dark spots

b. Take the paeoniflorin reference substance and make a solution containing about 1mg per ml with methanol as the reference solution; according to the thin-layer chromatography test, pipette the reference solution and the test solution under the identification 1 each 10 μl, respectively Point on the same silica gel G sheet with sodium carboxymethylcellulose as the binder, use 8: 2 chloroform-methanol as the developing agent, unfold, remove, dry, spray with The 5% vanillin sulfuric acid solution is heated at 105 ° C until the spots are clear. In the chromatogram of the test product, the spots of the same color should be displayed at the positions corresponding to the reference color.

11. The method for quality control of a pharmaceutical composition preparation according to claim 3, 4, or 5, characterized in that the content determination method in the method includes one or more of the following methods: a. Glycyrrhetinic acid, according to High performance liquid chromatography; Color language conditions and system applicability test, using octadecylsilane bonded silica as filler; 85-100: 8-14 methanol-water as mobile phase; detection wavelength 250nm, column temperature is room temperature; number of theoretical plates of glycyrrhetinic acid peak not lower than 2000; preparation of the reference solution, take glycyrrhetinic acid reference standard amount, accurately weighed, add methanol per lml containing glycyrrhetinic acid 2 ⁵ g of The solution was used as a reference solution; for the preparation of the test solution, 0.1 g of the composition preparation was accurately weighed, placed in a 100 ml measuring flask, ultrasonically extracted for 10-25 min, allowed to cool, added methanol to the mark, and shaken, Allow to stand, filter through a microporous membrane, and take the filtrate to obtain; Assay method: Inject 10μΙ of the reference solution and test solution respectively, and inject it into a high-performance liquid color spectrometer to determine the peak area. External standard method Calculated to get licorice per unit Acid not less than 70. Omg;

b. Total glucosides of Paeonia lactiflora, measured according to high performance liquid chromatography, test of color and conditions and system suitability, using octadecane bonded silica gel as filler; 25-35: 65-80 methanol-water as mobile phase, The detection wavelength is 230nm. The number of theoretical plates should not be less than 2000 calculated from the paeoniflorin peak. For the preparation of the reference solution, take an appropriate amount of the paeoniflorin reference and accurately weigh it. Add methanol to make a solution containing 25u ₈ of paeoniflorin per ml. As the paeoniflorin reference solution; the preparation of the test solution, the preparation of the test solution under the same content determination item 1; the determination method, the above reference solution and the test solution were injected 10μΙ, and injected into the HPLC color Omg。 I Pu Yi, measured peak area, calculated by external standard method, that is, each unit amount containing paeoniflorin should not be less than 48.0mg.

12. The method for quality control of a pharmaceutical composition formulation according to claim 11, characterized in that the method for determining the content of a capsule liniment includes one or more of the following methods:

a. Glycyrrhetinic acid, measured according to high performance liquid chromatography, color and condition and system suitability test, using octadecylsilane bonded silica gel as filler; 90: 10 methanol-water as mobile phase,-phosphoric acid to adjust pH ^3.7; detection wavelength 250 nm, column temperature was room temperature; number of theoretical plates of glycyrrhetinic acid peak not lower than 2000; preparation of the reference solution, take glycyrrhetinic acid reference standard amount, Lg, add methanol to make a solution containing 25 g of glycyrrhetic acid per lml, as a reference solution; for the preparation of the test solution, take the content of this product 0. lg, accurately weigh, place in a 100ml measuring bottle , Power 100W, 50KHz ultrasonic extraction for 15min, let cool, add methanol to the mark, shake well, let stand, filter with 0.45μm. Microporous filter, take the filtrate to obtain; assay method, the above reference Omg; The solution and the test solution were injected into ΙΟμΙ, injected into a high-performance liquid colorimeter, and the peak area was determined. The external standard method was used to obtain that the content of glycyrrhetic acid in each capsule was not less than 70. Omg;

b. Paeonia lactiflora, measured according to high performance liquid chromatography, test of color and conditions and system suitability, using octadecylsilane bonded silica gel as filler; 30: 70 methanol-water as mobile phase, detection wavelength 230nm The number of theoretical plates should be not less than 2000 according to the paeoniflorin peak. For the preparation of the reference solution, take an appropriate amount of the paeoniflorin reference and accurately weigh it. Add methanol to make a solution containing 25 g of paeoniflorin per ml. Preparation of reference solution; preparation of test solution, preparation of test solution under the same content determination item 1; determination method, injecting the above reference solution and test solution into 10μΙ and injecting them into a high performance liquid chromatography 0mg。 Determination of peak area, calculated by external standard method, that is, each capsule containing paeoniflorin should not be less than 48.0mg.

13. The method for quality control of a pharmaceutical composition formulation according to claim 3, 4 or 5, comprising the following steps:

Identification: a. Take an appropriate amount of the preparation of the composition, dissolve it in methanol, and make a solution containing about 1 mg per 1 ml as a test solution; take another glycyrrhetic acid reference substance and make a solution containing about 1 mg per 1 ml with methanol. As a reference solution solution: According to the thin layer chromatography test, pipette the above two solutions 10 μl each, and point them respectively on the same ^^ GF ₂₅₄ -thin layer plate with sodium carboxymethyl cellulose as a binder, and take 3-6: 1-3: 0. 1-0. 5 n-hexane-ethyl acetate-water acetic acid as a developing agent, unfolded, taken out, dried, and examined under a 200-300nm ultraviolet light; for the chromatography of the test product, At the corresponding position of color "i", the same dark spots should be displayed;

b. Take the paeoniflorin reference substance and make a solution containing about 1mg per ml with methanol as the reference substance solution. According to the thin-layer chromatography test, pipette the reference substance solution and the test solution under the identification 1 into 10 μl, respectively. Point on the same silica gel G thin plate with sodium carboxymethylcellulose as the binder, use 7-10: 1-3 chloroform-methanol as a developing agent, unfold, remove, and dry, Spray with 5% vanillin casein solution and heat at 95-120 ° C until the spots are clear. In the chromatogram of the test product, the spots of the same color should be displayed at the positions corresponding to the reference color;

Content determination: a. Glycyrrhetinic acid, determined according to high performance liquid chromatography; Chromatographic conditions and system suitability tests, using octadecylsilane bonded silica as a filler; 85-100: 8-14 methanol-water as Mobile phase; detection wavelength is 250nm, column temperature is room temperature; theoretical plate number should be not less than 2000 calculated from glycyrrhetic acid peak; preparation of reference solution, take appropriate amount of glycyrrhetic acid reference, accurately weigh, add methanol to make each A solution containing 25 pg of glycyrrhetic acid in lral was used as a reference solution. For the preparation of the test solution, 0.1 g of the composition preparation was accurately weighed, placed in a 100 ml measuring bottle, ultrasonically extracted for 10-25 min, and allowed to cool. Add methanol to the scale, shake well, let it stand, filter through a microporous membrane, and take the filtrate to obtain; Assay method: Inject the above reference solution and test solution separately into 10μΙ, and inject it into a high performance liquid chromatography ， Measurement of peak area, calculated by external standard method, that is, the content of glycyrrhetic acid per unit amount should not be less than 70. Omg; b. Total glucosides of paeony, measured according to high performance liquid chromatography, color language conditions and system suitability test, Filled with octadecane bonded silica gel 25-35: 65-80 methanol-water as mobile phase, detection wavelength 230nm; theoretical plate number should be not less than 2000 calculated from paeoniflorin peak; preparation of reference solution, take appropriate amount of paeoniflorin reference substance, accurately weigh , add methanol solution containing each lral paeoniflorin 25μ ₈ as a reference solution paeoniflorin; preparation of test solution supply, preparing a test solution with a determination; assay, above the reference 0mg。 The solution and the test solution solution were injected ΙΟμΙ, injected into a high-performance liquid color error analyzer, measured the peak area, calculated by external standard method, that is, each unit containing no less than 48.0 mg paeoniflorin.

14. The method for quality control of a pharmaceutical composition tincture according to claim 13, comprising the following steps:-identifying: a. Taking an appropriate amount of the content of the product, dissolving it in methanol, and making a solution containing about 1 mg per 1 ml as Test solution; Glycyrrhetinic acid reference substance was also prepared with methanol to make a solution containing about 1 mg per 1 ml as the reference solution solution. According to the thin-layer chromatography test, each of the two solutions mentioned above was sucked 10 μΙ, respectively, and the carboxylic acid Silica gel GF ₂₅₄ sheet with sodium methylcellulose as a binder, 4: 1. 7: 0.2 n-hexane-ethyl acetate-glacial acetic acid as a developing agent, unfolded, taken out, air-dried, 254nra ultraviolet light View under lamp; in test chromatography The same dark spots should appear at the corresponding positions of the language;

b. Take the paeoniflorin reference substance and make a solution containing about 1mg. per lml as the reference solution; according to the thin-layer chromatography test, pipette the reference solution and the test solution under the identification 1 each 10μ1 Point on the same silica gel G thin plate with sodium carboxymethylcellulose as the binder, use 8: 2 chloroform-methanol as the developing agent, unfold, remove, dry, spray with a 5% vanilla sulfuric acid solution. Heat at 105 ° C until the spots are clear. In the chromatogram of the test product, the spots of the same color should be displayed at the positions corresponding to the reference color;

Determination of content: a. Glycyrrhetinic acid, determined according to high performance liquid chromatography, color test conditions and system applicability test, using octadecane hydrazone bonded silica gel as filler; 90: 10 methanol-water as mobile phase, phosphoric acid adjustment pH 3. 7; The detection wavelength is 250nm and the column temperature is room temperature. The number of theoretical plates should not be less than 2000 calculated based on the glycyrrhetic acid peak. For the preparation of the reference solution, take an appropriate amount of the glycyrrhetic acid reference and accurately weigh it. A solution of 25 pg of glycyrrhetic acid in 1 ml was used as a reference solution. For the preparation of the test solution, the content of this product was 0.1 g, accurately weighed, placed in a 100 ml measuring bottle with a power of 100 W and a frequency of 50 KHz. Extract for 15min, allow to cool, add methanol to the mark, shake well, let it stand, filter through a 0.45μm microporous filter, and take the filtrate to obtain; Assay method, inject the above reference solution and test solution separately Omg; ΙΟμΙ, injected into a high performance liquid chromatography, measured the peak area, calculated by external standard method, that is, each capsule containing glycyrrhetic acid should not be less than 70. Omg;

b. Total glucosides of Paeonia lactiflorum, measured according to high performance liquid chromatography, colorimetry conditions and system suitability tests, using octadecylsilane bonded silica gel as filler; 30: 70 methanol-water as mobile phase, detection wavelength 230nm; number of theoretical plates calculated paeoniflorin peak should not be less than 2000; preparation of the reference solution, paeoniflorin reference substance, accurately weighed, add methanol solution of paeoniflorin 25μ ₈ lml each containing, as peony Glycoside reference solution; preparation of test solution, preparation of test solution under the same content determination item 1; determination, i, 'Inject the above reference solution and test solution respectively 10μΙ, and inject high-performance liquid color Omg。 Phosphor, measuring the peak area, calculated by external standard method, that each capsule of paeoniflorin containing no less than 48.0 mg. '