CN1899367B - Herba sinensis extract, its preparation method, application and compound preparation - Google Patents

Abstract

The invention discloses a herba sinensis extract, a preparation method of the extract, a use of the extract in preparing medicine for treating uterine fibroids or mammary gland hyperplasia, and a compound preparation containing the extract. The extract and compound preparation of the present invention have significant curative effect on treating uterine fibroids or mammary gland hyperplasia, and long-term use has no obvious toxic and side effects. The main components of the extract of the present invention are 8-acetyl harpaside and harpaside.

Description

Herba sinensis extract, its preparation method, application and compound preparation

technical field

The present invention relates to the extract of Herba sinensis, more specifically, the invention relates to the extract of Herba sinensis for treating uterine fibroids or hyperplasia of mammary glands.

Background technique

Jingucao preparations mainly include: Jingucao Tablets (Ministerial Standard, WS3-B-1446-93) and Capsules (Ministry Standard, WS3-B-3704-98), which have the functions of clearing heat and detoxifying, eliminating phlegm and relieving cough. The medicinal properties of Jingucao recorded in traditional Chinese medicine are bitter, sweet, and cold; "Supplements to Materia Medica" records that it is sweet, flat, and non-toxic; "Supplements to Compendium" records that it is cold in nature and bitter in taste; through". It has the effects of clearing the lung and relieving cough, invigorating the gallbladder and reducing jaundice, cooling the liver and dispelling wind, softening hard masses and dispelling stagnation. Cure mainly bronchitis, hematemesis, epistaxis, red dysentery, gonorrhea, sore throat, furuncle, carbuncle, traumatic injury.

Existing herbal preparations (tablets and capsules) clear heat and detoxify, relieve cough, eliminate phlegm, relieve asthma, and are used for acute and chronic bronchitis and lung abscess. Among these products, the extract obtained by ethanol extraction is mixed with the whole herb and coarsely crushed to make tablets and capsules, without any qualitative and quantitative indicators to control its quality. The quality of the existing products is unstable, the dosage is large, the solubility is poor, the bioavailability is low, there is no specific quality control standard, and there are many defects in the process technology and quality control.

Jingucao tablet is a species recorded in the 77th edition of the Chinese Pharmacopoeia. It is the whole plant of Ajugadecumbens Thunb. and T FUJITA. Phytochemistry, 1987, 26(8): 2303 records, the main chemical components in Herba sinensis are iridoids, diterpenoids, flavonoids and phenylethanol glycosides. Among them, the iridoids include 8-acetylharpagide (8-O-acetylharpagide), harpagide (Harpagide), leptoside (Reptoside), and echinoside A, B, C, D (decumboside A, B, C, D) and decumbesterone, etc.

To sum up, so far, there are no relevant reports or publications about the effective fractions of the extracts of Herba sinensis, and there are no reports or publications about the use of Herba sinensis for the treatment of uterine fibroids and hyperplasia of mammary glands.

Uterine fibroids and mammary gland hyperplasia are common and frequently-occurring diseases that threaten women's health, and are also difficult diseases that are being studied by medical circles at home and abroad. Modern studies have confirmed that the pathogenesis of these two diseases is related to the long-term mental tension. Today, with the accelerated pace of life, its incidence is on the rise. So far, both Chinese and Western medicine have no effective treatment drugs. Surgical hysterectomy is the ultimate treatment method, and hysterectomy will bring new physical and mental pain to patients, especially for women in childbearing period. Therefore, the development of therapeutic drugs with definite curative effect and small side effects for such diseases will surely benefit patients.

Contents of the invention

Based on the narration in the background art above, there has been an urgent need for a long time to develop a drug that has a significant curative effect on uterine fibroids or mammary gland hyperplasia and has less toxic and side effects. After many years of research and a large number of tests, the inventor found that the extract obtained by using Herba sinensis as raw material and using a specific extraction method can effectively treat uterine fibroids or hyperplasia of mammary glands, and has very little toxic and side effects.

The technical problem to be solved by the present invention is to provide an extract of Herba sinensis for treating uterine fibroids or mammary gland hyperplasia. Another technical problem to be solved by the present invention is to provide the preparation method of this extract. Another technical problem to be solved by the present invention is to provide the use of the extract for the preparation of medicaments for treating uterine fibroids or breast hyperplasia. Another technical problem to be solved by the present invention is to provide a compound preparation containing the extract.

Herba sinensis extract of the present invention is prepared by one of the following methods:

(1). Using the Herba sinensis coarse powder as raw material, use water or ethanol with a concentration lower than 80%, heat extraction at 30-90°C, preferably at 45-70°C, or perform percolation, and concentrate the extract to a specific gravity of 1.0 -1.3, preferably to 1.12-1.16, filter, treat with macroporous adsorption resin, wash with water to remove impurities, elute the resin with 30%-95% ethanol solution until the active ingredients are completely washed out, recover the solvent, and dry to obtain the above Extract;

Or (2). Using the Herba sinensis coarse powder as a raw material, extracting with 40%-95% ethanol with a concentration, the extract is concentrated to a specific gravity of 1.0-1.3, preferably 1.12-1.16, and then water or 5%-30% ethanol The solution was redissolved, the eluate was filtered, the solvent was recovered, and dried to obtain the above extract.

The effective part group of the extract of the present invention is 8-acetylharpagide (8-O-acetylharpagide) and harpagide (Harpagide), and their content in the extract is 50-99%. Harpagide and 8-acetylharpagide The ratio of glycosides is in the range of about 1:2-4.

The compound preparation of the present invention contains the above extract and at least one traditional Chinese medicine of promoting blood circulation to remove (broken) blood stasis, promoting blood circulation and regulating menstruation, clearing heat and dampness, clearing heat and detoxification, clearing heat and cooling blood, regulating qi and relieving pain, nourishing blood and calming liver-yang. Among them, the traditional Chinese medicine for promoting blood circulation (breaking) blood stasis is selected from at least one of Chuanxiong, frankincense, myrrh, turmeric, and turmeric; The traditional Chinese medicine is selected from at least one of Prunella vulgaris, Sophora flavescens, Scutellaria baicalensis, and Gentiana; the traditional Chinese medicine for clearing away heat and detoxification is selected from Patrinia, Guanzhong, Houttuynia cordata, wild chrysanthemum, Dijincao, lobelia, boxing ginseng, At least one of Hedyotis diffusa, Indigo Daisy, Shanzigu, Loulu, and Eucalyptus eucalyptus; at least one of heat-clearing and blood-cooling traditional Chinese medicines selected from Shengdi, Scrophulariaceae, Comfrey, Cortex Moutan, and Radix Paeoniae Rubra; Qi-regulating Analgesic traditional Chinese medicine is selected from at least one of Cyperus cyperi, bergamot, rose, sandalwood, black medicine, and litchi core; From oysters.

The compound preparation of the present invention can be prepared by the conventional process of preparing compound preparations in the field of pharmacy, for example, the extract of the present invention and various pharmaceutical excipients (celluloses, starches, dextrins, stearin) commonly used in the field of medicine are prepared. Magnesium acid, lactose, micropowder silica gel, etc.) are mixed, and the preparation is formed; or the extract of the present invention, the above-mentioned promoting blood circulation (breaking) blood stasis, promoting blood circulation and regulating menstruation, clearing away heat and dampness, clearing away heat and detoxification, clearing away heat and cooling blood, regulating qi and relieving pain, enriching blood and calming down The drug powder or its extract of at least one kind of liver-yang traditional Chinese medicine is mixed with the above-mentioned various pharmaceutical excipients, and the preparation is formed; At least one of them is extracted after being mixed, and then the extract is mixed with the above-mentioned various pharmaceutical excipients to shape the preparation.

The extract of the present invention and its compound preparation can be made into various conventional dosage forms in the field of medicine, such as tablets, pills, capsules, gels, powder sprays, aerosols, suspensions, emulsions, syrups Various common dosage forms such as , sterile injection, etc., can also be immediate-release or long-acting or localized drug delivery or drug-release preparations of the above-mentioned various dosage forms. It is obtained by colonic enzymatic degradation and controlled-release encapsulation technology to make micro-particles or nanoparticles.

The above-mentioned extract or compound preparation of the present invention can be administered through various commonly used routes, including oral, mucosal, transdermal, subcutaneous, intravenous and intramuscular injection administration, according to the body weight and symptoms of the subject, Doses are generally administered in the range of 10-500 mg (extract)/day.

The extract and its compound preparation of the present invention have been proved by a large number of pharmacodynamic and toxicological tests by the inventors to have significant curative effect on treating uterine fibroids or mammary gland hyperplasia, and long-term use has no obvious toxic and side effects. In order to facilitate a clearer understanding of the present invention, in the following part of the specification, the inventor further describes the present invention through specific embodiments, but this specific embodiment is not exhaustive and does not limit the protection scope of the present invention. constituting a restriction.

Detailed ways

Example 1-4

Prepare the extracts of Examples 1-4 according to any of the following extraction methods, and the specific process parameters used are shown in Table 1:

(1) Use Herba sinensis coarse powder as raw material, heat extraction with water or ethanol, or percolate, concentrate the extract, filter, treat with macroporous adsorption resin, wash with water to remove impurities, and elute the resin with ethanol solution until effective The components are completely washed out, the solvent is recovered, and dried to obtain the above extract;

Or (2) extracting with ethanol, concentrating the extract, then redissolving with water or ethanol solution, filtering the eluate, recovering the solvent, and drying to obtain the above extract.

Table 1 Process parameters of Herba sinensis extract

Physicochemical parameters and experimental basis of effective parts in the extract of the present invention

According to experimental analysis, the effective fraction group of the extract of the present invention is 8-acetylharpagide (8-O-acetylharpagide) and harpagide (Harpagide), and their content in the extract is 50-99%. - The ratio of acetyl harpaside is in the range of about 1:2-4.

Harpagide has the structure of the following general formula I, which was first discovered and reported in [Biological analysis of Harpagophytum procumbens D.C.II.Pharmacological analysis of the effects of harpagoside, harpagide and harpagogenine on the -isolated ig guinea] ileum, J PharmBelg., 1981 Sep-Oct; 36 (5): 321-4]; 8-acetyl harpaside has the structure of the following general formula II, which was first discovered and reported in [ Vasoconstrictor activity of 8-O-acetylharpagide from Ajuga reptans., J.Nat.Prod.1992; Aug;55(8):1145-8].

General formula I: General formula II:

Structural analysis of harpaside:

The maximum absorption wavelength is 197nm, indicating that the molecular structure does not have a double bond; IR gives the following signals 3381→-OH, 1655→C=C, 1237, 1075→-COC-, 947→cis-CH=CH-; ¹ H -NMR (DMSO-d6) shows: one angle methyl signal δ1.23 (3H, s), five sugar proton signals δ3.21-3.89, one sugar terminal hydrogen signal δ4.56 (1H, d, J= 7.8Hz), two alkene hydrogen signals δ4.94 (1H, d, J=6.3Hz) and 6.30 (1H, d, J=6.3Hz); ¹³ C-NMR (DMSO-d6) showed a total of 16 carbon signals , except for the six carbon signals δ99.4(d), 74.5(d), 78.1(d), 71.7(d), 77.5(d), and 62.8(t) on glucose, there are still 10 carbon signals, suggesting It is a monoterpenoid compound; ¹³ C-NMR shows two alkene carbon signals δ142.6(d) and 108.5(d), one acetal carbon δ93.2(d), the compound is an iridoid glycoside. Compared with compound 1, the information of compound 2 is basically the same except for one less acetoxy group; FAB-MS also verified this point: the molecular weight of compound 2 is 364, which is exactly 42 different from the molecular weight of compound 1 which is 406 (-COCH ₃ ); therefore, compound 2 was speculated to be harpagide. ¹ H-NMR and ¹³ C-NMR data were assigned according to DEPT, HMQC and HMBC spectra.

Structural identification of harpaside:

UVλ: 197nm; IR v _max ^KBr : 3381, 2930, 1655, 1379, 1237, 1075, 947cm ^-1 ; FAB-MS (m/z): 387[M+Na] ⁺ , 347[M-OH], 274 , 207, 167 (100%), 145, 115; ¹ H-NMR (300Hz, DMSO) δ: 1.23 (3H, s, H-10), 1.78 (1H, dd, J=3.9, 14.1Hz, 7α- H), 1.90 (1H, dd, J=4.5, 13.5Hz, 7β-H), 2.53 (1H, brs, H-9), 3.21 (1H, d, J=7.8Hz, H-2'), 3.26 (1H, m, H-4'), 3.29 (1H, m, H-3'), 3.40 (1H, m, H-5'), 3.63 (1H, d, J=5.4Hz, H-6) , 3.68(1H, m, H1-6'), 3.89(1H, dd, J=1.8, 10.2Hz, _H2-6 '), 4.56(1H, d, J=7.8Hz, H-1'), 4.94(1H, d, J=6.3Hz, H-4), 5.73(1H, d, J=1.2Hz, H-1), 6.30(1H, d, J=6.3Hz, H-3); ¹³ C - See table for NMR data.

Structural analysis of 8-acetylharpaside:

The maximum absorption wavelength is 197nm, indicating that there is no conjugated double bond in the molecular structure; IR shows the following functional groups: 3440→-OH, 1711→C=O, 1652→C=C, 1238, 1075→-COC-, 960→cis -CH=CH-; ¹ H-NMR (DMSO-d ₆ ) shows: one angle methyl signal δ1.35 (3H, s), one acetoxy signal δ1.92 (3H, s), six sugar protons Signal δ2.98-3.68, a sugar terminal hydrogen signal δ4.38 (1H, d, J = 7.8Hz), two cis-ene hydrogen signals δ4.87 (1H, d, J = 6.3Hz) and 6.35 ( 1H, d, J=6.3Hz); ¹³ C-NMR (DMSO) showed a total of 16 carbon signals, except one acetoxy signal 22.0(q), 170.1(s) and six carbon signals on glucose δ97.3 (d), 73.0(d), 77.1(d), 70.2(d), 75.7(d), and 61.6(t), there are still 9 carbon signals, suggesting that they are monoterpenoids; ¹³ C-NMR shows two An ene carbon signal δ141.2, 107.4 and an acetal carbon signal 92.4(d), indicating that it is an iridoid compound. Comparing the ¹³ C-NMR data with the literature value of 8-acetylharpagide ^[4] , the two are basically consistent, therefore, the compound is determined to be 8-acetylharpagide. ¹ H-NMR and ¹³ C-NMR data were assigned according to DEPT, HMQC and HMBC spectra.

Structural identification of 8-acetylharpaside:

UVλ: 197nm; IR v _max ^KBr : 3440, 2912, 1711, 1652, 1375, 1238, 1075, 960; FAB-MS (m/z): 429[M+Na] ⁺ , 407[M+H] ⁺ , 369, 329 (100%), 311, 277, 241, 209; ¹ H-NMR (DMSO-d6) δ: 1.35 (3H, s, 10-CH ₃ ), 1.92 (3H, s, -COCH ₃ ), 1.77 (1H, dd, J = 4.5, 14.7Hz, 7α-H), 2.05 (1H, dd, J = 3.9, 12.9Hz, 7β-H), 2.64 (1H, brs, H-9), 2.98 (1H , d, J=6Hz, H-2'), 3.06(1H, m, H-5'), 3.10(1H, m, H-4'), 3.14(1H, m, H-3'), 3.46 (1H, d, J=1.2Hz, H ₁ -6'), 3.56 (1H, brs, H-6), 3.68 (1H, dd, J=6.6Hz, H ₂ -6'), 4.38 (1H, d, J=7.8Hz, H-1'), 4.87(1H, d, J=6.3Hz, H-4), 5.86(1H, d, J=1.2Hz, H-1), 6.35(1H,d , J=6.3Hz, H-3).

Determination of the content of the extract

Method: Determination according to high performance liquid chromatography (Appendix VID of Chinese Pharmacopoeia 2000 edition). Chromatographic conditions and system suitability test, octadecylsilane bonded silica gel was used as filler; acetonitrile-water (10:90) was used as mobile phase; detection wavelength was 197nm; column temperature was 30°C.

Preparation of reference substance solution: Accurately weigh an appropriate amount of harpaside and acetylharpaside reference substance, dissolve in methanol, and prepare 0.088mg/ml and 0.124mg/ml reference substance solutions respectively.

Preparation of the test solution: take 0.1 g of the extract prepared according to the above-mentioned method and accurately weigh it, put it in a conical flask, add 100 ml of 20% ethanol accurately, seal it tightly, weigh it, sonicate it for 10 minutes, let it cool, and weigh it. Make up the lost weight with 20% ethanol, shake well, and filter with a microporous membrane (0.45 μm).

Determination method: Accurately draw 5 μl of the reference substance solution and 5 μl of the test solution respectively, inject it into a liquid chromatograph, measure it, and obtain it.

Results: The determinations of 10 batches of samples are all in the range of 50-70%. The measurement results of the recovery rate are as follows.

Table 2 Acetylharpaside recovery rate assay result

The recovery determination result of table 3 harpaside

Referring to the accompanying drawings of this description, what is represented in the figure is the high-efficiency liquid phase diagram of the determination of the content of the herba sinensis extract of the present invention.

Example 5-12

Preparation of Compound Extract of Chinese Medicine Containing Grass Japonica

According to any compatibility combination in Examples 5-12, the Chinese medicine compound extract compatible with the extract of the herba sinensis can be mixed and applied after preparation using the conventional process for preparing compound preparations in the field of pharmacy, or it can also be mixed with the herba sinensis and the extract of Example 5. - At least one drug in 12 is mixed and extracted, and then the extract is mixed with the above-mentioned various pharmaceutical excipients, and the preparation is formed. The specific compatible drugs used are shown in Table 2:

            Table 4 Drugs used in combination with the extracts of Phyllostachys sinensis

Example 13

The pharmacodynamic test data of the effective part of the extract of Herba sinensis on the model of mammary gland hyperplasia in rats

The mammary gland hyperplasia model in rats is marked by the following pathological changes in the mammary gland tissue of the rats in the model group: the number of mammary gland lobules increases, the number of acini in the lobules increases, the lobules show diffuse enlargement, and even merge with each other, and the shape of the lobules is irregular , deformation, or loss of lobular structure, unclear outline, but still maintain the characteristics of lobular hyperplasia. In some animals, the lobular ductal epithelium proliferated significantly, and the basement membrane of some vesicles was obviously thickened, and there was hyaline fibrous secretion or serous exudation in the vesicles, and the connective tissue in individual lobules proliferated slightly, and there were a large number of lymphocytes around the lobule infiltration.

The curative effect judging standard is based on the semi-quantitative grading standard of the above-mentioned pathological tissue changes, using pharmacological statistical software for statistics, and the significant difference compared with the model group shall prevail.

1. Test material

1.1 Drugs: Herba sinensis extract: each gram contains 700mg of acetylharpaside. Rupixiao: Liaoning Hengren Pharmaceutical Co., Ltd., batch number: 02042; estradiol benzoate injection: 1mg/ml, produced by Shanghai Ninth Pharmaceutical Factory, batch number: 001102. Progesterone injection: 10mg/ml, produced by Shanghai Ninth Pharmaceutical Factory, batch number 020115. Serum follicle-stimulating hormone (hFSH), estradiol (E2), and serum luteinizing hormone (LH) radioimmunoassay kits: all were purchased from Beijing Furui Bioengineering Company, batch number: 0109.

1.2 Animals: SD rats, female, 200-250 grams, purchased from Weitong Lihua Experimental Animal Co., Ltd., certificate number: SCXK (Beijing) 2002-0003.

1.3 Instruments: FG-630 microcomputer multi-channel gamma measuring instrument, manufactured by Beijing 262 Factory; SAKUPA automatic dehydrator, made in Japan; Leitz rotary microtome, made in Germany; SAKURA RSH-100 automatic staining machine, made in Japan; Nikon optical microscope, Made in Japan; DLYMPUS BH-2 biological automatic photomicrograph system, made in Japan.

2. Method

The SD rats were randomly divided into 6 groups according to body weight, 10 in each group. Except for the blank control group, the other animals were intramuscularly injected with 0.5 mg/Kg of estradiol benzoate, once a day, for 25 consecutive days. Intramuscular injection of progesterone 4mg/Kg, once a day, for 5 consecutive days. Simultaneously, 40, 20, and 10 mg/Kg of Herba sinensis extract was administered by intragastric administration for 30 days, and the positive control drug Rupixiao 2.4 g/Kg was continuously administered by intragastric administration for 30 days (equivalent to 30 times the clinical dose). On the 31st day, the animal was anesthetized with 3% pentobarbital, blood was collected from the abdominal aorta, centrifuged, and the serum was taken to measure estradiol (E2), follicle-stimulating hormone (hFSH), and luteinizing hormone (LH); The mammary glands were inspected visually; the 2nd and 3rd pair of breasts were weighed with an 8mm punch, fixed in 10% formalin, sectioned in paraffin, stained with HE, and examined with a light microscope; the ovaries and uterus were weighed. Differences between groups were compared using t test.

3. Results

3.1 Effects on Rat Mammary Gland Hyperplasia Model

3.1.1 The effect on the general shape of the breast

Visual observation showed that the 2nd and 3rd pair of breasts of the rats in the model group were obviously red and swollen, and the nipples were enlarged. Increase, increase is not obvious.

3.1.2 Effect on breast weight

The results showed that the weights of the 2nd and 3rd pair of breasts of rats in the model group were significantly increased, which was significantly different from that of the control group; compared with the model group, the weights of the breasts of the rats in the positive drug Rupixiao group and the administration groups were significantly reduced (See Table 1).

3.1.3 Effects on breast tissue morphology

Observed under the light microscope, the mammary glands of the rats in the normal control group had no obvious hyperplasia, and the structure was normal. In the model group, the number of mammary gland lobules increased, the number of acini in the lobules increased, the lobules showed diffuse enlargement, and even merged with each other. . In some animals, the lobular ductal epithelium proliferated significantly, and the basement membrane of some vesicles was obviously thickened, and there was hyaline fibrous secretion or serous exudation in the vesicles, and the connective tissue in individual lobules proliferated slightly, and there were a large number of lymphocytes around the lobule infiltration. Compared with the model group, the mammary gland lesions in each administration group and the positive drug control group were improved to varying degrees, with increased acinus, thickened glandular epithelium, exudation in the glandular duct, and inflammatory infiltration around the acinus. Compared with the model group, it was significantly relieved, and the effects of large, medium and small dose groups on breast tissue showed a dose-effect relationship with the increase of the dose.

3.1.4 Effect on uterine coefficient

The results showed that the uterine weight and uterine coefficient of rats in the model group increased, and the difference was significant compared with the control group; compared with the model group, the uterine weight of the rats in the positive drug and high-dose administration group decreased, and the uterine coefficient decreased, and the difference was significant. The uterine coefficient of the rats in the middle and small dose groups has a tendency to decrease, but there is no significant difference (see Table 5).

3.2 Effects on Hormone Levels

The results showed that the estradiol (E2) and serum luteinizing hormone (LH) of the rats in the model group were significantly increased, and the difference was significant compared with the blank control group; compared with the model group, the positive drugs and administration groups could reduce the E2 Compared with the model group, the difference was significant (P<0.05); there was a tendency to affect follicle-stimulating hormone (hFSH) and luteinizing hormone (LH), but there was no significant difference (P>0.05) (see Table 4) .

4. Summary

The extracts of the Herbs sinensis were given by intragastric administration at doses of 40, 20, and 10 mg (extract)/Kg, and Rupixiao granule 2.4 g/Kg (equivalent to 30 times the clinical dose) was used as the positive control drug, which can resist estrogen and progesterone The induced rat experimental mammary gland hyperplasia model can reduce the degree of mammary gland hyperplasia and breast weight, reduce the level of estradiol, and reduce the uterine coefficient, which is significantly different from the model group.

Table 1. Effects on Rat Udder Weight (X±S; n=10)

Note: Compared with the blank control group: ^# P<0.05;^##P<0.01; compared with the model group: ^* P<0.05; ^** P<0.01 (the same below).

Table 2. Effects on Rat Mammary Tissue Morphology (X±S; n=10)

Note: 3 consecutive fields of view under a 10×10 magnification lens;

The number of acini is the mean of the one with the largest number of acini in each field of view.

Table 3. Effect on the degree of mammary gland hyperplasia in rats (X ± S; n=10)

Note: Grading standards for mammary gland hyperplasia:

+++Severe: Inflammatory cell infiltration and epithelial hyperplasia are more diffuse, and some have connective tissue.

++Moderate: inflammatory cell infiltration and epithelial hyperplasia are limited, involving multiple lobules.

+ Mild: Inflammatory cell infiltration and epithelial hyperplasia are scattered in flakes.

-Normal: no obvious hyperplasia and inflammatory cell infiltration.

Table 4. Effects on Hormone Levels (X±S; n=8)

Table 5. Effects on rat uterine coefficients (X±S; n=10)

Example 14

The pharmacodynamic test data of the effective part in the extract of the herba sinensis on the animal model of uterine fibroids

The animal uterine fibroid model is marked by the increase in uterine weight, estrogen level, uterine smooth muscle thickness and uterine tissue following pathological changes in the model group: the smooth muscle layer at the root of the uterine horns shows obvious uneven thickening , the arrangement of smooth muscle cells is disordered, showing that the smooth muscle cells are mainly cord-like, the arrangement direction is a vortex, the nuclei are long rod-shaped, and some smooth muscle cells are round or polygonal. Red degeneration, and mild inflammatory cell infiltration, mainly lymphocytes, with a small amount of neutrophils.

The curative effect judging standard is based on the changes of the above indicators and the semi-quantitative grading standard of the pathological tissue, and the pharmacological statistics software is used for statistics, and the significant difference compared with the model group shall prevail.

1. Materials

1.1 Animals Guinea pig: female, body weight 300-350g; Rat: SD species, female, body weight 200-250g, SPF grade, purchased from Weitong Lihua Experimental Animal Co., Ltd., certificate number: SCXK (Beijing) 2002-0003 . Rabbit: female, New Zealand species, 2-2.5Kg, all purchased from Beijing Tongli Breeding Factory (Jing Dong Xu Zi 2000 No. 010, Total No. 55). Mice: Kunming species, half male and half female, weighing 18-20 g, purchased from the Breeding Plant of the Institute of Zoology, Chinese Academy of Medical Sciences (certificate number: SCXK 11-00-0006).

1.2 Drugs and Reagents Grass sinensis extract: Each gram contains 700mg of acetylharpaside. Guizhi Fuling Pills: produced by Chengdu Jinding Pharmaceutical Co., Ltd., batch number: 0020627. Estradiol Benzoate Injection: 1mg/ml, produced by Shanghai Ninth Pharmaceutical Factory, batch number: 991102. Progesterone injection: 10mg/ml, produced by Shanghai Ninth Pharmaceutical Factory, batch number 980201. Serum follicle-stimulating hormone (hFSH), estradiol (E2), and serum luteinizing hormone (LH) radioimmunoassay kits: all were purchased from Beijing Furui Bioengineering Company, batch number: 0009.

1.3 Instrument BIOPACSY STEM eight-channel physiological recorder: Made in the United States. FG-630 microcomputer multi-channel gamma measuring instrument: manufactured by Beijing 262 Factory.

2. Methods and results

2.1 Effects on experimental uterine leiomyoma in guinea pigs

60 adult non-pregnant guinea pigs were selected and randomly divided into normal control group, model control group, positive drug control group, large, medium and low dose groups of Herba sinensis extract, 10 in each group. Intramuscular injection of estradiol benzoate, 0.2mg/mouse, 3 times/week in the model group and each administration group, after continuous injection for 3 months, intramuscular injection of 1.5mg/mouse of progesterone, 2 times/week, 1 consecutive month moon. At the same time as the modeling, each dose group of the chingucao was administered orally every day, and the model group and the normal control group were given the same dose of water every day. After 4 months, all animals were administered with 0.5h, anesthetized by intraperitoneal injection of 3% pentobarbital (0.1ml/100g), weighed, blood was taken from abdominal aorta, centrifuged, serum was taken to measure estradiol (E2), Serum follicle-stimulating hormone (hFSH) and serum luteinizing hormone (LH); the general shape of the uterus was observed by laparotomy, and the uterus was cut along the uterine-vesical junction, weighed, and the uterine coefficient was calculated (uterine weight/body weight); Take the transverse diameter of the uterine root above the cervix and the angled uterus; fix it in 20% neutral formalin, cut 0.5 cm from the same part above the angled uterus, and take 3 pieces from each side, embedding in paraffin, staining with HE, and observing with light microscope For uterine histological morphology, 5 parts were selected for each slice, and the smooth muscle thickness was measured with a micrometer under a 10×10 microscope, and the average value was taken, and the differences among groups were compared by t test.

2.1.1 Effect on uterine weight The uterine weight of the animals in the model group increased significantly compared with the control group. Compared with the model group, each administration group lost weight, and the difference was significant (P<0.05). See Table 1.

2.1.2 Effects on the transverse diameter of the uterus The transverse diameters of the sub-angle and root of the uterus in the model group were thicker than those of the control group, and the difference was significant. See Table 1.

2.1.3 Effects on the thickness of uterine smooth muscle Taking the number of small divisions of the eyepiece micrometer as the thickness unit, the thickness of the uterine smooth muscle in the model group was significantly thicker than that in the normal control group (P<0.001), and the administration groups were significantly thinner than the model group , significantly different from the model group (P<0.001). See Table 2.

Table 1. Effects on uterine weight and uterine transverse diameter of guinea pigs with uterine fibroids (X±S; n=10)

Note: Compared with the model group *P<0.05; **P<0.01 (the same below)

Table 2. Effect on uterine smooth muscle thickness of guinea pigs with uterine fibroids (X±S; n=10)

2.1.4 Effects on hormone levels The levels of E2 and LH in the serum of animals in the model group were significantly increased, and the levels of E2 could be reduced in various degrees in each group after administration, and the difference was significant compared with the model group (P<0.05); Affect the trend, but no significant difference (P>0.05). See Table 2 for details.

Table 2. Effects on hormone levels in guinea pigs with uterine fibroids (X±S; n=10)

2.1.5 Morphological changes of uterine tissue Under the microscope, the uterine mucosa and glands of the animals in the normal control group belonged to the secretory phase, no obvious thickening of the smooth muscle layer, no hemorrhage and infiltration of inflammatory cells in the interstitium. The smooth muscle layer at the root of the horned uterus in the model group was obviously unevenly thickened, and the smooth muscle cells were arranged in a disorderly manner, showing that the smooth muscle cords were mainly in the form of a vortex, and the nucleus was long rod-shaped, and some smooth muscle cells were round or polygonal. The interstitium of the uterine muscle of the animal had obvious hemorrhage, red degeneration of the myometrium, and mild infiltration of inflammatory cells, mainly lymphocytes, with a small amount of neutrophils. The uterine smooth muscle layer of the animals in each administration group was thinner to varying degrees than that of the model group, and the arrangement was regular. There was no inflammatory infiltration in the uterus of the animals in the large and medium dose groups, and no obvious bleeding in the interstitium of the uterine muscle. There is mild inflammatory infiltration.

2.2 Effects on experimental uterine leiomyoma in rats

60 adult non-pregnant rats were selected and randomly divided into normal control group, model group, positive control group, large, medium and low dose groups of Herba sinensis extract, 10 in each group. Intramuscular injection of estradiol benzoate 0.5mg/Kg, 3 times/week, for 3 months in the model group and each administration group, followed by intramuscular injection of progesterone 5mg/Kg, 2 times/week, for 1 month in a row. At the same time as the modeling, each dose group of the chingucao was administered orally every day, and the model group and the normal control group were given the same dose of water every day. After 4 months, all animals were given 0.5h after administration, anesthetized by intraperitoneal injection of 3% pentobarbital (0.1ml/100g), weighed, blood was taken from the abdominal aorta, centrifuged, and serum was taken to measure estradiol (E2); Observe the general shape of the uterus by laparotomy, cut the uterus along the uterine-vesical junction, weigh it, and calculate the uterine coefficient (uterine weight/body weight); measure the transverse diameter of the uterine root above the cervix and the angled uterus; put it in 20% neutral formalin Fixed, the same part of the uterus above the angle of the uterus was cross-sectioned 0.5cm, and 3 pieces were taken from each side, embedded in paraffin, stained with HE, and the shape of the uterus was observed under a light microscope. The smooth muscle thickness was measured with a micrometer, and the average value was taken, and the difference between groups was compared by t test.

2.2.1 Effects on body weight, uterus and ovary weight The weight of the animals in the model group decreased, the weight of the uterus increased, and the weight of the ovary decreased, which was significantly different from that in the control group. The body weight and ovary weight of the animals in each drug administration group increased to varying degrees, and the uterine weight decreased to varying degrees, and the difference was significant compared with the model group. See Table 3 for details.

Table 3. Effects on the body weight of uterine fibroid rats and uterus and ovary coefficients (n=10, X±S)

2.2.2 Effects on immune organs Thymus atrophy, weight decreased, and spleen weight increased in the model group animals, which was significantly different from that in the normal control group. The thymus and body weight of the animals in each administration group increased to varying degrees, and the spleen weight decreased to varying degrees, and the difference was significant compared with the model group. See Table 4.

Table 4. Effects on immune organs of rats with uterine fibroids (n=10, X±S)

2.2.3 The effect on the thickness of the uterine root The vertical diameter, transverse diameter and lower diameter of the uterine root of the model group were thicker than those of the control group, and there was a significant difference compared with the normal control group. The diameters were smaller than the model group, and the difference was significant compared with the model group (P<0.05). See Table 5.

Table 5. Effects on the thickness of the uterine roots of rats with uterine fibroids (n=10, X±S)

2.2.4 Effects on hormone levels The estradiol (E2) of the animals in the model group was significantly increased, and the levels of E2 could be reduced in various degrees in each group after administration, and the difference was significant compared with the model group (P<0.05). See Table 6.

Table 6. Effects on estrogen levels in rats with uterine fibroids (X±S; n=10)

2.2.5 Morphological changes of uterine tissue Under the microscope, the myometrium of the animals in the normal control group was composed of smooth muscle, and the muscle fascicles were layered with connective tissue. The layering of the muscle layer was not obvious, no thickening, bleeding and inflammation were seen cell infiltration. In the model group, the uterine smooth muscle was significantly thickened, the nuclei were long spindle-shaped, densely arranged, the nuclei were mostly long rod-shaped, the two ends were blunt, the chromatin was fine, the cytoplasm was pink, and there were a small amount of fibrovascular tissue and light The degree of inflammatory cell infiltration is dominated by neutral segmental nuclei. The degree of thickening of uterine smooth muscle and infiltration of inflammatory cells in each group of animals with drug administration was reduced to varying degrees compared with the model group. See Table 7, 8.

Table 7. Effects on the thickness of uterine smooth muscle in rats with uterine fibroids (X±S; n=10)

Note: (measured under a 16×16 magnification lens)

Table 8. Effects on the degree of uterine tissue lesions (n=10)

Note: rank sum test: ^* P<0.05 compared with the model group; ^** P<0.01

Grading standards:

"-": no thickening of myometrium, no inflammatory infiltration, normal structure.

"+": Myometrial thickening is not obvious, and inflammatory infiltration is mild.

"++": The myometrium is slightly thickened, and the infiltration of inflammatory cells is obvious, mainly with neutral lobulated nuclei.

"+++": The myometrium is obviously thickened, and the infiltration of inflammatory cells is heavy.

"++++": The myometrium is obviously thickened, the arrangement of muscle cells is disordered, and diffuse inflammatory cell infiltration.

2.3 Effects on rabbit uterus in vivo

Rabbits were anesthetized by injecting pentobarbital sodium 30mg/Kgb.w through the ear vein, fixed in the supine position, the lower abdomen was sheared, and a midline incision was made in the lower abdomen with a length of about 4-5cm. Open the abdominal cavity, put an air bag into the uterus from the junction of the cervix and vagina, the air bag is connected with a silicone tube, the air in the air bag is exhausted, and the air bag is filled with water, the silicone tube is connected with the pressure transducer. A multichannel physiological recorder monitors uterine activity. After the operation, when the uterine activity is constant (about 30 minutes after the operation), record a period of normal contraction curve, then administer the drug through the duodenum, record the uterine activity curve at 30, 60 and 90 minutes after the drug, and calculate it with BIOPAC System Acknowledgment3.5 calculation software Uterine contraction amplitude, contraction frequency and contraction area under the curve.

Statistical method: Subtract the corresponding values before the administration of the animal from the uterine contraction amplitude recorded at 30, 60, and 90 minutes after administration, the number of contractions at 4 minutes, and the area under the contraction curve per minute, respectively, to obtain the values before and after administration. The difference, that is, the change value of uterine contraction amplitude, the change value of contraction frequency and the change value of the area under the curve. All the data are expressed as mean ± standard deviation, and the difference between each administration group and the model control group is compared by t-test.

Results: Large doses of chingucao can increase 30min-90min after administration; small doses can increase the contraction range of rabbit uterus in vivo 30min-60min after administration, and the two doses have no effect on the number of contractions and the area under the curve. See Table 9.

Table 9. The effect of Herba sinensis extract on uterine smooth muscle of rabbits in vivo (X±S; n=10)

3. Conclusion

Herba sinensis extract has obvious inhibitory effect on the experimental uterine fibroid model of rats and guinea pigs induced by estrogen and progesterone. Reduce the thickness of smooth muscle; reduce the level of estrogen (estradiol); inhibit the hyperemia and swelling of animal uterus and pathological changes of uterine tissue; increase the contraction range of rabbit uterus in vivo; inhibit cotton ball granuloma in rats and ear swelling in mice ; Inhibit the pain response of mice induced by oxytocin, chemical stimulation and heat stimulation, and the difference is significant compared with the model group.

Example 15

The safety test of the extract of Herba sinensis

1. Acute toxicity test

Under the condition of the maximum administration capacity and concentration, most of the animals showed decreased activity after intragastric administration of 25g extract/kg (equivalent to 750g crude drug/kg) of Herba chinensis, and then gradually returned to normal. Animals did not die. After about 24 hours after administration, the behavior and activities basically returned to normal. During the following 14-day observation period, the weight gain was normal, and no obvious abnormal symptoms were seen.

Under the conditions of the maximum administration capacity and the maximum administration concentration, after intragastric administration of 10 g of Herba sinensis extract/kg (equivalent to 300 g of crude drug/kg) in rats, most of the animals showed a decrease in spontaneous activity, often lying still, Then it gradually returned to normal. Animals did not die. After about 24 hours after administration, the behavior and activities basically returned to normal. During the subsequent observation period, the weight gain was normal, and no obvious abnormal symptoms were seen.

2. Long-term toxicity test

Abstract: Large, medium and small doses of 1200mg/kg, 220mg/kg and 40mg/kg of the extract of Herba sinensis were given to rats by intragastric administration for 25 weeks. The results showed that there were no obvious abnormalities in the general state, hematology, blood biochemical indicators and major organ tissues of the rats in each administration group.

Test purposes

Observe the possible toxic and side effects and the severity of the animals' bodies after feeding the extracts of the herba sinensis to SD female rats continuously for 25 weeks, so as to determine the safe dosage range of this product and provide the basis for human clinical safety drug use.

experiment material

Tested drug: Herba sinensis extract: each gram of extract contains 3.6 grams of crude drug, provided by the preparation room of the Institute of Traditional Chinese Medicine, China Academy of Traditional Chinese Medicine. Add distilled water before use to make the corresponding concentration.

Kit: total cholesterol kit: produced by Zhongsheng Beikong Biotechnology Co., Ltd., and the remaining 9 blood biochemical index determination kits are all produced by Beijing Beihua Kangtai Clinical Reagent Co., Ltd. Urine test strips (8N all items) are produced by the First Pharmaceutical Factory in Suzhou, China.

Animals: SD rats, female, weighing 95+5 grams, purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd., certificate number: SCXK (Beijing) 2002-0003.

Instruments: MEK6318K automatic blood cell recorder, produced by Hitachi; HUMALYZER2000 biochemical analyzer, produced in Germany.

Animal feed: Rat feed, produced by Jiujiangkou Feed Factory, Chaoyang District, Beijing [Jing Dong Xu Zi (2000) No. 007, Total No. 052]. Main ingredients: crude ash 8.0%, salt 0.54%, crude protein 26.7%, crude fat 6.8%, crude fiber 3.7%, calcium 1.4%, total phosphorus 0.92%.

Animal feeding conditions: Grade III animal feeding facilities, room temperature: 22-24°C, relative humidity 45-60%.

experiment method

Grouping of animals and dosage

Five animals per cage were fed with full-price nutritious feed and free to drink water. Before the experiment, they were adaptively fed for one week and then randomly divided into 4 groups with 40 animals in each group. The high-dose group was 1200mg (extract)/kg, the medium-dose group was 220mg (extract)/kg, and the small-dose group was 40mg (extract)/kg, the administration volume was 1ml/100g, and the normal control group was fed with the same volume of 0.5 %CMC-Na. Continuous intragastric administration for 25 weeks, 6 days a week. The body weight and food intake were weighed every Monday, and the dosage was adjusted according to body weight.

Test items and methods

Observe the general state of the animals every day, that is, the mental state, behavioral activities, coat color and urine and stool. At 12 weeks and 25 weeks after administration, 10 and 20 animals in each group were taken respectively, and blood was taken from the orbit to measure hemoglobin (HB), red blood cell count (RBC), white blood cell count (WBC) and classification, platelet (PLT) and other hematology Index, measure coagulation time; femoral artery blood is taken and centrifuged to get serum, and automatic biochemical analyzer is used to measure aspart aminotransferase (GOP), alanine aminotransferase (GPT), alkaline phosphatase (ALP), urea nitrogen (BUN ), muscle liver (Crea), total protein (TP), albumin (ALb), blood glucose (Glu), total bilirubin (T-Bili), total cholesterol (T-CHO) and other biochemical indicators; Liver, spleen, lung, kidney, adrenal gland, brain (cerebrum, cerebellum), thyroid gland, thymus, pancreas, pituitary gland, optic nerve, stomach, duodenum, colon, jejunum, ileum, uterus, ovary, bladder, sternum, mammary gland Weight, calculate the organ coefficient; do the histopathological examination of the organs. The test results were statistically processed, and the inter-group T test was performed to compare the differences between the groups. The remaining 10 animals in each group underwent the above-mentioned inspection after 5 weeks after drug withdrawal, and the observation indexes were the same as above.

test results

Effects on general state and body weight of animals

General state of animals: after administration for 12 weeks, 25 weeks and drug withdrawal for 5 weeks, the rats in each group were active, their mental state was good, their hair color was shiny, their stools were normal, and there was no obvious abnormality in urine routine examination (see Table 5). No other obvious abnormalities were found, and no animals in each group died.

Animal body weight and food intake: After administration for 12 weeks, 25 weeks and drug withdrawal for 5 weeks, the body weight and food intake of rats in each group were normal, and there was no significant difference compared with the blank control group (see Table 1, 2).

Effects on hematological indicators in rats

The measurement result of hematology shows, after 12 weeks, 25 weeks and 5 weeks of drug withdrawal in rats, after 5 weeks of large, Chinese and medicine, compared with the blank control group, the general state and hematology of rats in each administration group were significantly improved. , blood biochemical indicators and major organ tissues have no significant effects related to drugs. Compared with the control group, there was no statistically significant difference in the measured indicators of the P3 dose group (see Table 3-1, 3-2, 6).

Effects on blood biochemical indicators

The measurement result of blood biochemical index shows, after rat administration 25 weeks, the measured index of three dosage groups all fluctuates in the normal range, compares no statistically significant difference with matched group (see table 4-1, 4-2 ).

Effects on the Coefficients of Major Viscera in Rats

After 12 weeks, 25 weeks and 5 weeks of drug withdrawal, the rats were dissected, and the viscera of the rats in each group were generally observed with the naked eye without obvious adhesion, degeneration and other abnormalities. There is no statistically significant difference (see Table 7-1, 7-2).

Effects on major organ histology

The results of pathological examination showed that after 12 weeks, 25 weeks of administration of this product and 5 weeks of drug withdrawal, no obvious pathological damage related to the test substance was found in the major organ tissues of the rats in each administration group, see the pathological examination report for details and photos.

in conclusion

Herba sinensis extract 1200mg/kg, 310mg/kg, 80mg/kg, continuous intragastric administration to rats for 12 weeks, 25 weeks and 5 weeks after stopping the drug has a great influence on the general state, hematology, blood biochemical indicators and Histological examination of major organs showed no drug-related pathological changes.

Table 1. Effect of administration on body weight of female rats for 25 weeks (X±SD; n=40; g)

Table 2. Effect of administration on food intake of female rats for 25 weeks (X±SD; n=40; g/100g)

Table 3-1, the influence of 25 weeks of administration on blood routine indexes of female mice (X±SD; n=20)

Table 3-2, the effect of 25 weeks of administration on blood routine indicators of female mice (X±SD; n=20)

Table 4-1. Effects of 25-week administration on serum biochemical indicators in female mice (X±SD; n=20)

Table 4-2. Effects of 25-week administration on serum biochemical indicators in female mice (X±SD; n=20)

Note: Compared with the control group, ^* P<0.05, ^** P<0.01

Table 5, 25 weeks of administration on the impact of rat urine routine (n=20)

Note: *was weakly positive; # X±SD

The influence of table 6 on coagulation time (X±SD; n=20; sec)

Table 7-1, 25 weeks of administration to the organ coefficient of female rats (X±SD; n=20; g/100g)

Table 7-2, 25 weeks of administration to the organ coefficient of female rats (X±SD; n=20; g/100g)

Claims (7)

1. for the treatment of uterine fibroids or mammary gland hyperplasia extract, it is characterized in that the extract is prepared by the following method: Use the coarse powder of the sinensis as the raw material, heat and extract with water or ethanol with a concentration lower than 80% at 30-90°C, or perform percolation, concentrate the extract to a specific gravity of 1.0-1.3, filter, and treat with macroporous adsorption resin , after washing with water to remove impurities, elute the resin with 30%-95% ethanol solution until the active ingredients are completely washed out, recover the solvent, and dry to obtain the above extract 2. The extract according to claim 1, wherein the heating extraction temperature is 45-70° C., and the extract is concentrated to a specific gravity of 1.12-1.16. 3. The extract according to claim 1, which contains 50-99% of 8-acetylharpaside and harpaside. 4. The extract according to claim 1, which contains harpagside and 8-acetyl harpagside in a ratio of 1:2-4. 5. the preparation method of the extract of one of claims 1-4, it is characterized in that the method is: Use the coarse powder of the sinensis as the raw material, heat and extract with water or ethanol with a concentration lower than 80% at 30-90°C, or perform percolation, concentrate the extract to a specific gravity of 1.0-1.3, filter, and treat with macroporous adsorption resin , after washing with water to remove impurities, elute the resin with 30%-95% ethanol solution until the active ingredients are completely washed out, recover the solvent, and dry to obtain the above extract. 6. The method according to claim 5, wherein the heating extraction temperature is 45-70° C., and the extract is concentrated to a specific gravity of 1.12-1.16. 7. The use of the herba sinensis extract according to any one of claims 1-4, characterized in that it is used for the preparation of medicines for treating uterine fibroids or hyperplasia of mammary glands.