CN104997922A - Refined traditional Chinese medicine composition extract with hemostasis and miscarriage prevention effect and application thereof - Google Patents

Abstract

The invention discloses a refined extract of a traditional Chinese medicine composition with the effect of stopping bleeding and preventing miscarriage. The n-butanol part is composed of 1:3 by weight. The present invention compares and screens through a large number of experiments, and combines multiple parts to obtain a refined extract with an optimal weight ratio. The results of pharmacological experiments show that the refined extract with the optimal weight ratio screened by the present invention has good hemostasis, anti-fetal and other effects, better clinical curative effect, and smaller dosage.

Description

Refined extract of traditional Chinese medicine composition with hemostatic and miscarriage effects and application thereof

technical field

The invention relates to a traditional Chinese medicine composition, in particular to a refined extract of the traditional Chinese medicine composition with the effect of stopping bleeding and preventing miscarriage.

Background technique

"Golden Chamber Yaolue Heart Code" says: "Women are dripping with menstrual water, and those who have bleeding before and after childbirth are all deficient in the Ren channel, and Yin Qi cannot be kept. Chinese medicine has the advantages of nourishing blood, promoting blood circulation, regulating menstruation, hemostasis and miscarriage. Unique curative effect, but the existing traditional Chinese medicine compound composition is generally used in a large amount, which does not remove the roughness and preserve the essence according to the clinical symptoms, which brings some inconvenience to the patient's clinical use, and the inactive ingredients have certain adverse effects on the body.

Therefore, on the basis of traditional Chinese medicine, the present invention screens out the combination of refined parts with the best curative effect through modern separation and extraction methods combined with pharmacological experiments, which can improve clinical curative effect and reduce dosage, which is of great significance.

Contents of the invention

Purpose of the invention: The purpose of this invention is to carry out in-depth research and screening of traditional Chinese medicine compound prescriptions, combine modern extraction and separation methods and pharmacological experiments, screen out the best refined combination parts, and provide a refined extraction of Chinese medicine compositions with hemostatic and anti-fetal effects thing.

Technical solution: In order to achieve the above object, the present invention adopts the following technical solution:

The refined extract of the traditional Chinese medicine composition with the effect of hemostasis and miscarriage, which is composed of the ethyl acetate part and the n-butanol part of the traditional Chinese medicine composition with a weight ratio of 1:3;

The ethyl acetate part and the n-butanol part of the Chinese medicine composition are prepared by the following method:

Weigh donkey-hide gelatin, chuanxiong, licorice, wormwood, angelica, white peony root and rehmannia glutinosa with a weight ratio of 2:2:2:3:3:4:4, add water to decoct, and decoct to obtain an extract under reduced pressure , the extract is mixed with diatomaceous earth while it is hot, then placed in a vacuum drying oven under reduced pressure, and dried to obtain a medicinal powder, and then the dried medicinal powder is extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and each solvent extract is vacuum-dried under reduced pressure , to obtain petroleum ether part extract, ethyl acetate part extract and n-butanol part extract.

As a preferred solution, the refined extract of the above-mentioned traditional Chinese medicine composition with hemostatic and tocolytic effects is boiled and extracted 1 to 3 times with 6 to 12 times the volume of water of the weight of the medicinal materials, and each time is 1 to 3 hours.

As a preferred option, the above-mentioned refined extract of the traditional Chinese medicine composition with hemostasis and anti-tocolysis effect, the mass volume ratio of the medicinal powder to petroleum ether, ethyl acetate and n-butanol is 1:6-12, and the extraction method is reflux extraction, Continuous reflux extraction or ultrasonic extraction.

Application of the refined extract of the traditional Chinese medicine composition with hemostatic and tocolytic effect in the preparation of hemostatic and tocotropic medicine.

A traditional Chinese medicine preparation with hemostatic and tocolytic effect, which is prepared into granules, tablets, capsules or pills from the refined extract of the traditional Chinese medicine composition with hemostatic and tocolytic effect according to the present invention and a pharmaceutically acceptable carrier.

Beneficial effects: the present invention obtains the refined extract with the best weight ratio through a large number of experiments, comparative screening, and combination of multiple parts. The results of pharmacological experiments show that the refined extract with the optimal weight ratio screened by the present invention has good hemostasis and anti-fetal effects, better clinical curative effect and smaller dosage.

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art will readily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims .

Example 1 Screening test

1.1 Medicinal materials

Angelica, Rhizoma Chuanxiong, Radix Paeoniae Alba, Shengdi, Artemisia argyi, and Licorice were all purchased from Anhui Fengyuan Tongling Traditional Chinese Medicine Pieces Co., Ltd.

1.2 Instruments

LG-PABER-1 semi-automatic coagulation analyzer (Beijing Shidi Scientific Instrument Co., Ltd.); MKIII type microplate reader (Thermo fisher); Anke-LXJ-11B centrifuge (Shanghai Anshou Scientific Instrument Factory); SHANGPINGYP601N type 1/ 10 electronic balance (Shanghai Precision Scientific Instrument Co., Ltd.); ultraviolet/visible spectrophotometer (PerkinElmer).

1.3 Reagent

Mifepristone Tablets (Zhejiang Xianju Pharmaceutical Co., Ltd.); Misoprostol (PIRAMAL HEALTHCARE UKLIMITED); Gongxuening Capsules (Yunnan Baiyao Group Co., Ltd.); Sodium Citrate (Sinopharm Chemical Reagent Co., Ltd. , batch number: F20110506); chloral hydrate (Kermel, batch number: B1420001); sodium carboxymethylcellulose (Shanghai Jiuyi,); NaCl (Xilong Chemical Co., Ltd.); Reagent Co., Ltd.); Petroleum ether (60-90°C), ethyl acetate, and n-butanol were all domestic analytically pure. Kit: prothrombin time (PT, lot number: STG20101-49), thrombin time (TT, lot number: STG20301-35A), activated partial thromboplastin time (APTT, lot number: ST20201-41), plasma fibrinogen ( _FIB , batch number: _STY20401-42 ) were purchased from Beijing Shidi Scientific Instrument Company; , estradiol (E2, batch number: 1022XG06), and progesterone (P, batch number: 1020VH11) were purchased from Beijing North Institute of Biotechnology.

1.4 Animals

80 female SD rats, SPF grade, weighing 220-250g, 40 male SD rats, SPF grade, weighing 250-300g, provided by Zhejiang Experimental Animal Center, license number: SCXK (Zhejiang) 2014-0001.

1. Method

2.1 Preparation of the test solution

Take by weighing 2880g of Chinese medicine compound composition (wherein donkey-hide gelatin: Chuanxiong: licorice: Artemisia argyi: Angelica: white peony root: Rehmannia glutinosa weight ratio is respectively 2:2:2:3:3:4:4), add medicinal material weight 10 times volume The decoction was decocted twice, each time for 2 hours, and the decoction was concentrated under reduced pressure to obtain an extract, and the extract was mixed with diatomaceous earth while hot (diatomaceous earth: extract 1:1.5) and then vacuum-dried under reduced pressure oven at 55°C. Then use petroleum ether, ethyl acetate and n-butanol to reflux extract the dried medicinal powder for 3 times in turn, and vacuum-dry each solvent extract to dryness to obtain petroleum ether extract, ethyl acetate extract, and n-butanol extract. The extracts were 6.2g (0.22% in yield), 40.2g (1.40% in yield) and 268.8g (9.3% in yield) respectively.

According to the weight ratio of ethyl acetate part and n-butanol part of 1:3, 1:5, 1:7, 1:9, 1:11, take appropriate amount of ethyl acetate part extract and n-butanol part extract , add an appropriate amount of 0.5% CMC-Na solution to prepare each test solution whose concentration (calculated according to the amount of crude drug) is 1.20 g mL ^-1 , and additionally take an appropriate amount of Gongxuening capsule and add 0.5% CMC-Na solution to prepare Concentration is 15.60mg·mL ^-1 Gongxuening solution, and each test solution is stored in a refrigerator at 4°C for later use.

2.2 Animal grouping and administration

Female SD rats were randomly divided into 10 groups according to the random number table method, 10 rats in each group, namely: blank group, model group, Gongxuening group, ethyl acetate group, n-butanol group, and ethyl acetate group : n-butanol part 1:3 group (referred to as 1:3 group), ethyl acetate part: n-butanol part 1:5 group (referred to as 1:5 group), ethyl acetate part: n-butanol part 1:7 group (1:7 group for short), ethyl acetate part: n-butanol part 1:9 group (1:9 group for short), ethyl acetate part: n-butanol part 1:11 group (1:11 group for short) . Each administration group was converted into an animal administration dose according to twice the clinical administration dose, that is, the Gongxuening group was 0.156kg ^-1 ·d ^-1 , and the rest of the administration groups were 12g·kg ^-1 ·d ^-1 ( Calculated according to crude drug amount). Each administration group was intragastrically administered corresponding drugs at the dosage of 1ml·100g ^-1 on pregnancy day 8, and the blank group and model group were intragastrically administered the same dose of 0.5% CMC-Na solution once a day for 7 consecutive days.

2.3 Model replication

At 17:00, the rats were caged at a ratio of 2:1 for males and females, and a vaginal smear was performed at 8:00 the next day to find that the sperm was pregnancy d1. On d7, mifepristone 8.3mg·kg ^-1 (8:00) and misoprostol 100μg·kg ^-1 (18:00) were intragastrically administered to reproduce the incomplete abortion model of early pregnancy rats.

2.4 Index detection

On the seventh day, after intragastric administration of misoprostol, a quantitative cotton ball (cotton ball weighing 85-90 mg) was inserted into the vagina (the quantitative cotton ball was half-wrapped with plastic film to prevent blood leakage and urine reflux), respectively. At 8 o'clock and 18 o'clock the next day, the quantitative cotton balls were taken out and new cotton balls were placed. The taken out cotton balls were placed in a ziplock bag and stored in a refrigerator at 4°C (for the measurement of uterine bleeding). The uterine bleeding time (Uterine Bleeding Time, UBT) of group animals was calculated, and the amount of uterine bleeding (Uterine Bleeding Quantity, UBQ) was calculated.

On the 14th day, 40 minutes after the administration of each administration group, the animals were anesthetized with 10% chloral hydrate, blood was collected from the common carotid artery, anticoagulated with 3.8% sodium citrate (V:V 1:9), and the whole blood was treated with 3000rr Centrifuge at min ^-1 for 10 min, take the upper platelet-poor plasma (PPP) and use a semi-automatic coagulation analyzer to measure the four coagulation items according to the instructions of the four coagulation items, and take another 2ml of whole blood (without anticoagulant) and let it stand for 30 min. ^-1 Centrifuge for 10 min, take the upper serum and use a 96-well plate to measure the contents of TXB ₂ , 6-keto-PGF _la , E2, P, in the serum according to the instructions of the ELISA kit.

2.5 Statistical methods

SPSS19.0 statistical software was used to conduct double-sample t-test and analysis of variance on the experimental data, and all experimental data were used Indicates that P<0.05 or P<0.01 is statistically significant.

3 Experimental results

3.1 Effects on coagulation function of uterine bleeding model rats

As shown in the experimental results in Table 1, compared with the blank group, the TT, PT, and APTT of the rats in the model group were significantly prolonged, and the FIB was significantly reduced (P<0.01); compared with the model group, the Gongxuening group and the 1:3 group could significantly shorten the TT , PT, APTT, significantly increased FIB (P<0.01, P<0.05); 1:5 group, 1:7 group can significantly shorten TT, PT, APTT (P<0.01, P<0.05); 1:9 group It can significantly shorten APTT (P<0.05); 1:11 group can significantly shorten TT (P<0.01), and the weight ratio of ethyl acetate part to n-butanol part is 1:3, which has the best effect. And the experimental results show that when the ethyl acetate part and the n-butanol part are in a weight ratio of 1:3, it has a more superior effect of shortening TT, PT and APTT than a single ethyl acetate part or n-butanol part, indicating that ethyl acetate After the ester part and n-butanol part are combined at a ratio of 1:3 by weight, a good synergistic effect has been achieved.

Table 1 Effects of each administration group on TT, PT, APTT, and FIB of uterine bleeding model rats

Compared with the model group, ^** P<0.01, ^* P<0.05

3.2 Effects on UBT and UBQ in Uterine Hemorrhage Model Rats

The experimental results in Table 2 show that compared with the model group, the Gongxuening group, 1:3 group, 1:5 group, 1:7 group, 1:9 group, and 1:11 group can significantly shorten UBT and reduce UBQ (P<0.01, P<0.05), wherein the weight ratio of ethyl acetate part to n-butanol part is 1:3, which has the best effect. And the experimental results show that when the ethyl acetate part and the n-butanol part are 1:3 by weight, it has a more superior effect of shortening UBT and reducing UBQ than a single ethyl acetate part or n-butanol part, indicating that ethyl acetate After the ester part and n-butanol part are combined at a ratio of 1:3 by weight, a good synergistic effect has been achieved.

Table 2 Effects of each administration group on UBT and UBQ in uterine bleeding model rats

group Dose/g·kg ^-1 UBT/hours UBQ/mL blank group - 0.00±0.00 0.00±0.00 model group - 148.00±38.45 0.43±0.04 Gongxuening Group 0.156 96.00±15.18 ^* 0.32±0.04 ^** Ethyl acetate fraction group 12 101.00±27.02 ^* 0.38±0.03 ^* n-butanol site group 12 106.29±43.50 0.40±0.03 1:3 groups 12 96.00±10.73 ^** 0.33±0.04 ^** 1:5 groups 12 104.73±10.85 ^** 0.36±0.06 ^* 1:7 groups 12 114.00±11.11 ^* 0.36±0.02 ^** 1:9 groups 12 108.00±9.07 ^* 0.41±0.09 1:11 group 12 108.00±0.00 ^* 0.36±0.07 ^*

Compared with the model group, ^** P<0.01, ^* P<0.05

3.3 Effects on TXB2 and 6-keto-PGF _la in uterine bleeding model rats

The experimental results in Table 3 show that, compared with the blank group, the model group rats TXB2 significantly decreased, 6-keto-PGF _la significantly increased, and TXB2/6-keto-PGF _la significantly decreased (P<0.01); In comparison, the Gongxuening group, 1:3 group, 1:5 group, 1:7 group, 1:9 group, and 1:11 group could significantly increase TXB2, decrease 6-keto-PGF _la , and regulate TXB2/6 - Balance of keto-PGF _la (P<0.01, P<0.05). Among them, the weight ratio of ethyl acetate part to n-butanol part is 1:3, which has the best effect. And the experimental results show that when the ethyl acetate site and the n-butanol site are in a weight ratio of 1:3, compared with a single ethyl acetate site or n-butanol site, there is more superiority in raising TXB2 and reducing 6-keto-PGF _la , the effect of regulating the balance of TXB2/6-keto-PGF _la , indicating that the ethyl acetate part and the n-butanol part are combined at a ratio of 1:3 by weight, and a good synergistic effect has been achieved.

Table 3 Effects of each administration group on TXB2 and 6-keto-PGFla in uterine bleeding model rats

Compared with the model group, ^** P<0.01, ^* P<0.05

3.4 Effects on E2 and P in uterine bleeding model rats

The experimental results in Table 4 show that compared with the blank group, the E2 and P of the rats in the model group decreased significantly (P<0.01); compared with the model group, the Gongxuening group, 1:3 group, and 1:5 group could significantly increase High E2, P (P<0.01, P<0.05); 1:7 group can significantly increase P (P<0.05); 1:11 group can significantly increase E2 (P<0.05). Among them, the weight ratio of ethyl acetate part to n-butanol part is 1:3, which has the best effect. And the experimental results show that when the ethyl acetate part and the n-butanol part are in a weight ratio of 1:3, it has a more superior effect of increasing E2 and P than a single ethyl acetate part or n-butanol part, indicating that ethyl acetate After the ester part and n-butanol part are combined at a ratio of 1:3 by weight, a good synergistic effect has been achieved.

Table 4 Effects of each administration group on E2 and P in uterine bleeding model rats

group Dose/g·kg ^-1 E2/pg·ml ^-1 P/ng·ml ^-1 blank group - 365.43±17.65 6.33±0.30 model group - 299.21±38.31 ^## 5.66±0.40 ^## Gongxuening Group 0.156 336.94±14.55 ^* 6.31±0.26 ^** Ethyl acetate fraction group 12 350.61±19.10 6.10±0.21 ^* n-butanol site group 12 323.46±30.98 6.01±0.34 1:3 groups 12 362.97±20.20 ^* 6.54±0.43 ^** 1:5 groups 12 339.04±16.57 ^* 6.12±0.34 ^* 1:7 groups 12 319.97±13.83 6.07±0.22 ^* 1:9 groups 12 325.32±17.44 5.99±0.20 1:11 group 12 336.99±13.66 ^* 5.78±0.45

Compared with the model group, ^** P<0.01, ^* P<0.05

The results of the screening and comparison experiments from the above Table 1 to Table 4 show that when the ethyl acetate part provided by the present invention is combined with the n-butanol part in a weight ratio of 1:3, it has a higher ratio than other weight ratios and a single ethyl acetate part. Or the n-butanol part has more superior effects such as hemostasis and miscarriage protection. That is, after the ethyl acetate part and the n-butanol part are combined at a weight ratio of 1:3, they can play a very good synergistic effect, and can be developed into a hemostatic and tocolytic drug with better curative effect.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (5)

1. there is the refining extract of the Chinese medicine composition of arresting bleeding and miscarriage prevention effect, it is characterized in that: it is that the ethyl acetate extract of the Chinese medicine composition of 1:3 and n-butanol portion form by weight ratio;

The ethyl acetate extract of described Chinese medicine composition and n-butanol portion are prepared by following method:

Take Colla Corii Asini that weight ratio is 2:2:2:3:3:4:4, Rhizoma Chuanxiong, Radix Glycyrrhizae, Folium Artemisiae Argyi, Radix Angelicae Sinensis, the Radix Paeoniae Alba and Radix Rehmanniae medical material, decoct with water, obtain decocting liquid concentrating under reduced pressure and obtain extractum, extractum mixes the rearmounted reduced vacuum drying baker of sample with kieselguhr while hot, dry to obtain medicated powder, then extract dry medicated powder with petroleum ether, ethyl acetate and n-butyl alcohol successively, each solvent extractable matter reduced vacuum is dry, obtains petroleum ether part extractum, ethyl acetate extract extractum and n-butanol portion extractum.

2. the refining extract with the Chinese medicine composition of arresting bleeding and miscarriage prevention effect according to claim 1, is characterized in that: the water adding up medical material weight 6 ~ 12 times of volumes, decocts extraction 1 ~ 3 time, each 1 ~ 3 hour.

3. the refining extract with the Chinese medicine composition of arresting bleeding and miscarriage prevention effect according to claim 1, it is characterized in that: the mass volume ratio of medicated powder and petroleum ether, ethyl acetate and n-butyl alcohol is 1:6 ~ 12, extracting method is reflux, extract, continuous circumfluence extraction or supersound extraction.

4. the refining extract with the Chinese medicine composition of arresting bleeding and miscarriage prevention effect described in any one of claims 1 to 3 is preparing the application in arresting bleeding and miscarriage prevention medicine.

5. one kind has the Chinese medicine preparation of arresting bleeding and miscarriage prevention effect, it is characterized in that: be prepared into granule by the refining extract with the Chinese medicine composition of arresting bleeding and miscarriage prevention effect described in any one of claims 1 to 3 and pharmaceutically acceptable carrier, tablet, capsule or pill.