COMPOSITION, PREPARATION METHOD THEREOF, AND APPLICATION THEREOF IN THE PREVENTION AND TREATMENT OF MAMMARY GLAND DISEASE

Abstract

The present disclosure relates to the technical field of traditional Chinese medicine technology, especially to a composition, a preparation method thereof and an application in the prevention and treatment of mammary gland diseases. The present disclosure provides a composition consisting of Rehmanniae Radix Praeparata, Lycii Fructus, Polygonati Rhizoma, Radix Codonopsis Pilosulae, Paeoniae Radix Alba, Cyperi Rhizoma (processed with vinegar) and Grape Seed. The composition has functions of improving hyperplasia of mammary gland and/or anti-oxidation. Compared with each control group, the composition provided by the present disclosure has the optimal effects, and the effect has statistical differences, indicating that the components in the composition provided by the present disclosure are reasonably arranged and can play a good synergistic effect. Furthermore, the preparation is safe and has no side effects.

Claims

1. A composition, consisting of the following components in parts by weight TABLE-US-00017 Rehmanniae Radix Praeparata 10 parts-200 parts; Lycii Fructus 8 parts-100 parts; Polygonati Rhizoma 8 parts-100 parts; Radix Codonopsis Pilosulae 4 parts-50 parts; Paeoniae Radix Alba 4 parts-50 parts; Cyperi Rhizoma (processed with vinegar) 4 parts-50 parts; Grape Seed 1 part-30 parts.

2. The composition according to claim 1, consisting of the following components in parts by weight TABLE-US-00018 Rehmanniae Radix Praeparata 30 parts-150 parts; Lycii Fructus 40 parts-80 parts; Polygonati Rhizoma 40 parts-80 parts; Radix Codonopsis Pilosulae 10 parts-50 parts; Paeoniae Radix Alba 10 parts-50 parts; Cyperi Rhizoma (processed with vinegar) 10 parts-50 parts; Grape Seed 1 part-20 parts.

3. The composition according to claim 1, consisting of the following components in parts by weight TABLE-US-00019 Rehmanniae Radix Praeparata 90 parts; Lycii Fructus 60 parts; Polygonati Rhizoma 60 parts; Radix Codonopsis Pilosulae 30 parts; Paeoniae Radix Alba 30 parts; Cyperi Rhizoma (processed with vinegar) 30 parts; Grape Seed 7 parts.

4. An extract made from the composition according to claim 1.

5. A method for preparing the extract according to claim 4, comprising extracting Cyperi Rhizoma (processed with vinegar) and Paeoniae Radix Alba with water, concentrating to powder by spraying drying, to give component A; extracting a combination of Rehmanniae Radix Praeparata, Polygonati Rhizoma, Lycii Fructus and Radix Codonopsis Pilosulae by water extraction and alcohol deposit to obtain a precipitation, and drying the precipitation to powder, to give component B; extracting Grape Seed with an alcohol, column separating, gathering effluent fraction of ethanol, and drying the effluent fraction to powder, to give a Grape Seed Extract; and mixing component A, component B and the Grape Seed Extract to give the extract.

6. The method according to claim 5, wherein in the process of water extraction, the weight of water is 5-20 times based on a total weight of the medicinal materials, the extraction temperature is 80-100 C., the duration is 0.5-4 h, and the extraction is carried out for 2 times.

7. The method according to claim 5, wherein the alcohol precipitation is carried out by using an ethanol aqueous solution having a volume fraction of 80%-95%, until the finally volume fraction of ethanol is 75% or more.

8. A method for regulating endocrine levels, regulating expression levels of estrogen receptor and/or progesterone receptor, improving hyperplasia of mammary gland, or resisting oxidation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition according to claim 1.

9. The method according to claim 8, wherein endocrine hormone is estradiol, progesterone, luteinizing hormone, follicle stimulating hormone and/or prolactin.

10. The method according to claim 8, wherein improving hyperplasia of mammary gland comprises improving diffuse hyperplasia of mammary gland, improving breast pain and/or improving breast tenderness.

11. The method according to claim 8, wherein the resisting oxidation comprises lowering LPO and/or MDA levels in serum, improving total anti-oxidative capacity, improving GSH-PX enzyme activity, and improving SOD enzyme activity.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0050] FIG. 1 shows a total evaluation of breast pain (T2B) after administering with the compositions; * represents the baseline of the group, and compared with the first, the second and the third month, the difference is significant (p<0.05); obvious improvement (TOP 2)=very obvious improvement (5-6 points)+obvious improvement (3-4 points).

[0051] FIG. 2 shows a total evaluation of improvement of breast pain after administering with compositions (N=122,%), very obvious improvement=5-6 points; obvious improvement=3-4 points; a little obvious improvement=1-2 points; and no improvement=0 point.

[0052] FIG. 3 shows average number of days of breast pain during the administration period of the composition (N=122, mean); * represents the baseline of the group, and compared with the first, the second, and the third month, the difference is significant (p<0.05).

[0053] FIG. 4 shows the improvement of the number of days of breast pain before and after the administration of the composition (the value in the figure is the difference from that in 0 week).

[0054] FIG. 5 shows the evaluated average value of symptoms and signs before and after the administration of the composition; * represents that compared with the initial values, the difference is significant (p<0.05).

[0055] FIG. 6 shows the improvements of symptoms and signs (N=122, %).

[0056] FIG. 7 shows the proportion of people whose symptoms and signs are improved (N=122, %).

[0057] FIG. 8 shows optical microscope observation of the mammary gland tissue of each group, 100; wherein, FIG. 8-a shows optical microscope observation of the mammary gland tissue of normal control group, 100; FIG. 8-b shows optical microscope observation of the mammary gland tissue of model control group, 100; FIG. 8-c shows optical microscope observation of the mammary gland tissue of composition 1 group, 100; FIG. 8-d shows optical microscope observation of the mammary gland tissue of composition 2 group, 100; FIG. 8-e shows optical microscope observation of the mammary gland tissue of composition 3 group, 100; FIG. 8-f shows optical microscope observation of the mammary gland tissue of composition 4 group, 100; FIG. 8-g shows optical microscope observation of the mammary gland tissue of composition 5 group, 100; FIG. 8-h shows optical microscope observation of the mammary gland tissue of control sample 1 group, 100; FIG. 8-i shows optical microscope observation of the mammary gland tissue of control sample 2 group, 100; FIG. 8-j shows optical microscope observation of the mammary gland tissue of Rupixiao group, 100.

DETAILED DESCRIPTION

[0058] The present disclosure provides a composition, a preparation method thereof and an application in the prevention and treatment of mammary gland diseases. Those skilled in the art can learn from the contents of this document and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art, and are considered to be included in the present disclosure. The methods and applications of the present disclosure have been described in the preferred embodiments. It will be apparent to those skilled in the art that the methods and applications herein may be modified or changed and combined to implement and apply the techniques of the present invention without departing from the content, spirit and scope of the disclosure.

[0059] In an embodiment of the present disclosure, provided is a composition, consisting of the following components in parts by weight

TABLE-US-00008 Rehmanniae Radix Praeparata 10 parts-200 parts; Lycii Fructus 8 parts-100 parts; Polygonati Rhizoma 8 parts-100 parts; Radix Codonopsis Pilosulae 4 parts-50 parts; Paeoniae Radix Alba 4 parts-50 parts; Cyperi Rhizoma (processed with vinegar) 4 parts-50 parts; Grape Seed 1 part-30 parts.

[0060] In a preferred embodiment of the present disclosure, the composition consists of the following components in parts by weight

TABLE-US-00009 Rehmanniae Radix Praeparata 30 parts-150 parts; Lycii Fructus 40 parts-80 parts; Polygonati Rhizoma 40 parts-80 parts; Radix Codonopsis Pilosulae 10 parts-50 parts; Paeoniae Radix Alba 10 parts-50 parts; Cyperi Rhizoma (processed with vinegar) 10 parts-50 parts; Grape Seed 1 part-20 parts.

[0061] In a more preferred embodiment of the present disclosure, the composition consists of the following components in parts by weight

TABLE-US-00010 Rehmanniae Radix Praeparata 90 parts; Lycii Fructus 60 parts; Polygonati Rhizoma 60 parts; Radix Codonopsis Pilosulae 30 parts; Paeoniae Radix Alba 30 parts; Cyperi Rhizoma (processed with vinegar) 30 parts; Grape Seed 7 parts.

[0062] In an embodiment of the present disclosure, an extract made from the composition is provided.

[0063] In an embodiment of the present disclosure, a method for preparing the extract is provided, comprising

[0064] extracting Cyperi Rhizoma (processed with vinegar) and Paeoniae Radix Alba with water, concentrating to powder by spraying drying, to give component A;

[0065] extracting a combination of Rehmanniae Radix Praeparata, Polygonati Rhizoma, Lycii Fructus and Radix Codonopsis Pilosulae by water extraction and alcohol deposit to obtain a precipitation, and drying the precipitation to powder, to give component B;

[0066] extracting Grape Seed with an alcohol, column separating, gathering effluent fraction of ethanol, and drying the effluent fraction to powder, to give a Grape Seed Extract;

[0067] and mixing component A, component B and the Grape Seed Extract to give the extract.

[0068] In a preferred embodiment of the present disclosure, in the process of water extraction, the weight of water is 5-20 times based on a total weight of the medicinal materials, the extraction temperature is 80-100 C., the duration is 0.5-4 h, and the extraction is carried out for 2 times.

[0069] In a preferred embodiment of the present disclosure, the alcohol precipitation is carried out by using an ethanol aqueous solution having a volume fraction of 80%-95%, until the finally volume fraction of ethanol is 75% or more.

[0070] In an embodiment of the present disclosure, a method for regulating endocrine levels in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of the composition of the present disclosure.

[0071] In a preferred embodiment of the present disclosure, endocrine hormone is estradiol, progesterone, luteinizing hormone, follicle stimulating hormone and/or prolactin.

[0072] In an embodiment of the present disclosure, a method for regulating expression levels of estrogen receptor and/or progesterone receptor in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of the composition of the present disclosure.

[0073] In an embodiment of the present disclosure, a method for improving hyperplasia of mammary gland in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of the composition of the present disclosure.

[0074] In a preferred embodiment of the present disclosure, improving hyperplasia of mammary gland comprises improving diffuse hyperplasia of mammary gland, improving breast pain and/or improving breast tenderness.

[0075] In an embodiment of the present disclosure, a method for resisting oxidation is provided, comprising using the composition of the present disclosure.

[0076] In a preferred embodiment of the present disclosure, the resisting oxidation comprises lowering LPO and/or MDA levels in serum, improving total anti-oxidative capacity, improving GSH-PX enzyme activity, and improving SOD enzyme activity.

[0077] In an embodiment of the present disclosure, a product for improving hyperplasia of mammary gland and/or resisting oxidation is provided, comprising the composition of the present disclosure.

[0078] The materials used in the present disclosure are all normal products, all of which are commercially available.

[0079] The present disclosure will be further illustrated hereinafter in conjunction with the embodiments.

Example

[0080] Formulation of the composition in each group is shown in Table 1.

TABLE-US-00011 TABLE 1 Formulations of the compositions (parts by weight) Cyperi Rhizoma Rehmanniae Radix (processed Radix Lycii Polygonati Codonopsis Paeoniae with Grape Praeparata Fructus Rhizoma Pilosulae Radix Alba vinegar) Seed Composition 90 60 60 30 30 30 7 1 Composition 10 100 8 50 4 50 1 2 Composition 200 8 100 4 50 4 30 3 Composition 30 80 40 50 10 50 1 4 Composition 150 40 80 10 50 10 20 5 Control 90 60 60 30 Example 1 Control 90 60 60 30 30 30 Example 2 Control 30 30 7 Example 3

[0081] Preparation Process

[0082] The Cyperi Rhizoma (processed with vinegar) and Paeoniae Radix Alba were mixed, drinking water was added in 8 times of the amount of the total material input, and the mixture was extracted at a heating temperature of 100 C. for 2.0 h. In a second extraction, drinking water was added in 7 times of the amount of the total material input, and the mixture was extracted at a heating temperature of 100 C. for 1.0 h. Filtration was conducted. The two extract solutions were combined and concentrated at a temperature of 65 to 85 C. and a vacuum degree of 0.04 to 0.07 Mpa, until the Solid Content was concentrated to 205%, following which the material was removed in hot to use. The Rehmanniae Radix Praeparata, Polygonati Rhizoma, Lycii Fructus and Radix Codonopsis Pilosulae were mixed, drinking water was added in 10 times of the amount of the total material input, and the mixture was extracted at a heating temperature of 100 C. for 2.0 h. In a second extraction, drinking water was added in 8 times of the amount of the total material input, and the mixture was extracted at a heating temperature of 100 C. for 1.0 h. Filtration was conducted. The two extract solutions were combined and concentrated at a temperature of 65 to 85 C. and a vacuum degree of 0.04 to 0.07 Mpa, until the Solid Content was concentrated to 205%, following which the material was removed in hot to use. Ethanol of over 85% was added, wherein the concentrated solution was added firstly and then ethanol was slowly added with stirring, until the concentration of ethanol was 75%. The resultant was stood overnight (over 12 h).

Efficacy Study

1. Inhibitory Effects on Hyperplasia of Mammary Gland

[0083] 1.1 Institution that Carried Out the Experiment

[0084] Guangdong Provincial Hospital of Traditional Chinese Medicine, Engineering Technology Research Institute

[0085] 1.2 Object of the Experiment

[0086] Inhibiting effects of the traditional Chinese medicine composition on hyperplasia of mammary gland in rats were observed and evaluated.

[0087] 1.3 Experimental Materials

[0088] 1.3.1 Medicinal Materials

[0089] The traditional Chinese medicine composition is provided by Infinitus (China) Co., Ltd. The blank control sample is distilled water. Positive control sample is Rupixiao tablet, Liaoning Herbapex Pharmaceutical Group Co Ltd. Estradiol benzoate injection.

[0090] 1.3.2 Reagents

[0091] Estradiol benzoate injection, Shanghai General Pharmaceutical Co., Ltd., Batch No:1207241.

[0092] Progesterone injection, Shanghai General Pharmaceutical Co., Ltd., Batch No:13003146.

[0093] Enzyme Immunoassay Kit for Rat Estradiol (ELISA kit for Rat E.sub.2), Shanghai Westang Biological Technology Co., Ltd., Batch No:1307251.

[0094] Enzyme Immunoassay Kit for Rat Prolactin (ELISA Kit for Rat PRL), Shanghai Westang Biological Technology Co., Ltd., Batch No:1308083.

[0095] Enzyme Immunoassay Kit for Rat Progesterone (ELISA Kit for Rat P), Shanghai Westang Biological Technology Co., Ltd., Batch No:1307251.

[0096] Enzyme Immunoassay Kit for Rat Follicle Stimulating Hormone (ELISA Kit for Rat FSH), Shanghai Westang Biological Technology Co., Ltd., Batch No:1306251.

[0097] Enzyme Immunoassay Kit for Rat Luteinizing Hormone (ELISA Kit for Rat LH), Shanghai Westang Biological Technology Co., Ltd., Batch No:1306252.

[0098] 0.9% sodium chloride injection, Sichuan Kelun Pharmaceutical Co., Ltd., Batch No: M12071027.

[0099] 1.3.3 Dosage Definition

[0100] Blank control group, traditional Chinese medicine formulation group 1 (752.32 mg.Math.kg.sup.1), traditional Chinese medicine formulation group 2 (752.32 mg.Math.kg.sup.1), traditional Chinese medicine formulation group 3 (752.32 mg.Math.kg.sup.1), traditional Chinese medicine formulation group 4 (752.32 mg.Math.kg.sup.1), traditional Chinese medicine formulation group 5 (752.32 mg.Math.kg.sup.1), control group 1 (752.32 mg.Math.kg.sup.1), control group 2 (752.32 mg.Math.kg.sup.1).

[0101] 1.4 Experimental Method and Test Indicators

[0102] Modeling method: except for the normal control group, rats in the other groups were intramuscularly injected with 0.5 mg.Math.kg.sup.1 of estradiol benzoate once a day for 25 d. Subsequently, the rats were intramuscularly injected with 4 mg.Math.kg.sup.1 of progesterone once a day for 5 d. The rats in the normal control group were not injected with the hormone, but only intramuscularly injected with 0.2 ml normal saline per rat once a day for 30 d. Each administered group was intragastrically administered according to the dosage, and the normal control group and the model control group were intragastrically administered with an equal volume of distilled water. There were 10 rats in each group, administered once a day for 30 d. 1 h after the last administration, the diameters and heights of the second pair of nipples were measured with a vernier caliper, and the appearance was observed. Blood was collected from the venous plexus of orbit of the animals, and serum was separated. Contents of estradiol E.sub.2, progesterone P, Prolactin PRL, luteinizing hormone LH, follicle-stimulating hormone FSH in the serum of the animals were determined. Partial depilation treatment was carried out using 8% sodium sulfide. After the animals were sacrificed, the second pair of nipples in chests of the rats was integrally peeled and taken off. The nipples were fixed with 10% formaldehyde solution, subjected to paraffin embedded section, and the second pair of mammary tissue was dyed with hematoxylin-eosin (HE) and observed with light microscope (FIG. 8).

[0103] 1.5 Calculation of Q Value of Drug Combination (Jin Zhengjun Method)

[0104] Q value of drug combination was calculated using a modified formula: Q=E.sub.A+B/(E.sub.A+E.sub.BE.sub.AE.sub.B) according to the method of literature. E.sub.A+.sub.B indicates the data of drug combination, E.sub.A indicates the data of a single use of A, E.sub.B indicates the data of a single use of B, and the calculation is performed using the inhibition rate or the increase rate data. A Q value of less than 0.85 indicates that the two drugs have an antagonistic effect after combination; a Q value of greater than 0.85 and less than 1.15 indicates that the two drugs have an additive effect after combination; a Q value of greater than 1.15 indicates that the two drugs have a synergistic effect after combination.

[0105] 1.6 Experimental Results

[0106] 1.6.1 Influence on Diameters and Heights of the Second Pair of Nipples of the Rats Having Hyperplasia of Mammary Gland

[0107] 1 h after the last administration, the diameters and heights of the second pair of nipples were measured with a vernier caliper, and the appearance was observed.

TABLE-US-00012 TABLE 2 Influence on the diameters and heights of the second pair of nipples of the rats having hyperplasia of mammary gland (x s, n = 10) Diameter of Inhibition Height of nipple Inhibition Group Dosage nipple (mm) rate (mm) rate Normal / 1.89 0.09 1.61 0.07 control group Model / 2.60 0.10** 2.47 0.08** control group Composition 752.32 2.19 0.07.sup.## 16.00% 1.98 0.06.sup.## 20.10% 1 mg .Math. kg.sup.1 Composition 752.32 2.35 0.08.sup.## 9.50% 2.18 0.08.sup.## 12.10% 2 mg .Math. kg.sup.1 Composition 752.32 2.26 0.08.sup.## 13.10% 2.19 0.05.sup.## 11.50% 3 mg .Math. kg.sup.1 Composition 752.32 2.27 0.07.sup.## 12.70% 2.13 0.06.sup.## 13.70% 4 mg .Math. kg.sup.1 Composition 752.32 2.26 0.07.sup.## 13.10% 2.18 0.06.sup.## 11.80% 5 mg .Math. kg.sup.1 Control 752.32 2.47 0.11.sup. 5.2% 2.35 0.06.sup. 5.00% Sample 1 mg .Math. kg.sup.1 Control 752.32 2.50 0.08.sup. 3.8% 2.37 0.08.sup. 4.4% Sample 2 mg .Math. kg.sup.1 Control 752.32 2.46 0.10.sup. 5.5% 2.44 0.11.sup. 1.5% Sample 3 mg .Math. kg.sup.1 Rupixiao 960.00 2.28 0.06.sup.## 12.50% 2.15 0.09.sup.## 12.90% tablet group mg .Math. kg.sup.1 Comments: Compared with the normal control group, *p < 0.05, and **p < 0.01. Compared with the model control group, #p < 0.05, and ##p < 0.01. Compared with composition group1, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 2, .sup.p < 0.05, and.sup.p < 0.01. Compared with composition group 3, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 4, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 5, .sup.p < 0.05, and .sup.p < 0.01.

[0108] Results: The degree of hyperplasia of mammary gland of human was irregular, and was usually localized mass; whereas the hyperplasia of mammary gland of rats was usually diffusive, showing an increase in the overall size of the breast, an increase in the height and the diameter of the nipples. In the results of the present experiment, compared with the normal control group, the diameters and heights of the second pair of nipples of the rats in the model control group significantly increased (p<0.01). Compared with the model control group, the diameters and heights of the second pair of nipples of the rats in the formulation groups 1, 2, 3, 4, 5 and the Rupixiao tablet group significantly decreased (p<0.01). The diameters and heights of the second pair of nipples of the rats in the formulation groups 2, 3, 4, 5 were all larger than those of the formulation group 1 (p<0.01 or p<0.05). The diameters and heights of the second pair of nipples of the rats in the control sample group 1 were all larger than those of the formulation groups 1, 2, 3, 4, 5 (p<0.01). The diameters and heights of the second pair of nipples of the rats in the control sample group 2 were all larger than those of the formulation groups 1, 2, 3, 4, 5 (p<0.01). The diameters and heights of the second pair of nipples of the rats in the control sample group 3 were all larger than those of the formulation groups 1, 2, 3, 4, 5 (p<0.01 or p<0.05). E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample1+E.sub.control sample3E.sub.control sample1*E.sub.control sample3)=0.160/(0.052+0.0550.052*0.055)=1.536. 1.536>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing the diameter of nipple. E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample1+E.sub.control sample3E.sub.control sample1*E.sub.control sample3)=0.201/(0.050+0.0150.050*0.015)=3.152, 3.152>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing the height of nipple.

[0109] 1.6.2 Influence on Contents of Estradiol E.sub.2 and Progesterone P in Serum of the Rats Having Hyperplasia of Mammary Gland

[0110] Blood was collected from the venous plexus of orbit of the animals, and the serum was separated. Contents of estradiol E.sub.2 and progesterone P in serum of the animals were determined.

TABLE-US-00013 TABLE 3 Influence on contents of estradiol E.sub.2 and progesterone P in serum of the rats having hyperplasia of mammary gland ( x s, n = 10) Estradiol Inhibition Progesterone Inhibition Group Dosage E.sub.2 (pg/ml) rate P (pg/ml) rate Normal / 59.95 13.79 64.92 18.39 control group Model / 491.30 83.82** 225.32 32.96** control group Composition 752.32 315.76 54.04.sup.## 35.73% 164.17 20.65.sup.## 27.14% 1 mg .Math. kg.sup.1 Composition 752.32 396.81 70.11.sup.## 19.23% 172.87 19.24.sup.## 23.28% 2 mg .Math. kg.sup.1 Composition 752.32 343.76 51.46.sup.## 30.03% 175.39 23.04.sup.## 22.16% 3 mg .Math. kg.sup.1 Composition 752.32 386.65 53.3.sup.## 21.30% 174.14 24.93.sup.## 22.71% 4 mg .Math. kg.sup.1 Composition 752.32 374.15 49.2.sup.## 23.84% 173.04 32.53.sup.## 23.20% 5 mg .Math. kg.sup.1 Control 752.32 448.79 62.79.sup. 8.65% 201.13 24.56.sup. 10.74% Sample 1 mg .Math. kg.sup.1 Control 752.32 463.33 51.12.sup. 5.69% 207.64 30.29.sup. 7.85% Sample 2 mg .Math. kg.sup.1 Control 752.32 470.30 46.22.sup. 4.27% 205.30 25.77.sup. 8.89% Sample 3 mg .Math. kg.sup.1 Rupixiao 960.00 324.39 74.32.sup.## 33.97% 184.79 27.75.sup.## 17.99% tablet group mg .Math. kg.sup.1 Comments: Compared with the normal control group, *p < 0.05, and **p < 0.01. Compared with the model control group, #p < 0.05, and ##p < 0.01. Compared with composition group 1, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 2, .sup.p < 0.05, and.sup.p < 0.01. Compared with composition group 3, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 4, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 5, .sup.p < 0.05, and .sup.p < 0.01.

[0111] Results: Compared with the normal control group, the contents of estradiol E.sub.2 and progesterone P of the rats in the model control group significantly increased (p<0.01). Compared with the model control group, the contents of estradiol E.sub.2 and progesterone P of the rats in composition groups 1, 2, 3, 4, 5 significantly decreased (p<0.01). The content of E.sub.2 in the composition groups 2, 4, 5 was higher than that in composition group 1 (p<0.01 or p<0.05). The contents of estradiol E.sub.2 and progesterone P in control sample groups 1, 2, 3 were higher than those in composition groups 1, 2, 3, 4, 5 (p<0.01 or p<0.05). E.sub.composition1 Q value=E.sub.composition1/(E.sub.control sample1+E.sub.control sample3E.sub.control sample1E.sub.control sample3)=0.357/(0.087+0.0430.0870.043)=2.846. 2.846>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing E.sub.2. E.sub.composition1 Q value=E.sub.composition1/(E.sub.control sample1+E.sub.control sample3E.sub.control sample1E.sub.control sample3)=0.271/(0.107+0.0880.1070.088)=1.454. 1.454>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing P

[0112] 1.6.3 Influence on Contents of Prolactin PRL, Luteinizing Hormone LH, Follicle Stimulating Hormone FSH in Serum of the Rats Having Hyperplasia of Mammary Gland

[0113] At the end of the experiment, blood was collected from the venous plexus of orbit of the animals, and the serum was separated. Contents of prolactin PRL, luteinizing hormone LH, follicle stimulating hormone FSH in serum of the animals were determined.

TABLE-US-00014 TABLE 4 Influence on contents of PRL, LH, FSH in serum of the rats haying hyperplasia of mammary gland ( x s, n = 10) Follicle Prolactin Luteinizing stimulating PRL Inhibition hormone Inhibition hormone Inhibition Group Dosage (pg/ml) rate LH (pg/ml) rate FSH (pg/ml) rate Normal / 1.25 0.12 2.48 0.34 6.50 1.65 control group Model / 8.13 1.13** 3.83 0.42** 19.07 4.40** control group Composition 752.32 3.92 1.96.sup.## 51.80% 2.63 0.30.sup.## 31.20% 13.49 2.25.sup.## 29.30% 1 mg .Math. kg.sup.1 Composition 752.32 5.75 1.67.sup.## 29.20% 3.10 0.28.sup.## 19.20% 14.57 2.72.sup.## 23.60% 2 mg .Math. kg.sup.1 Composition 752.32 4.27 1.91.sup.## 47.50% 2.84 0.32.sup.## 26.00% 14.30 3.47.sup.## 25.00% 3 mg .Math. kg.sup.1 Composition 752.32 3.88 1.05.sup.## 52.30% 3.14 0.29.sup.## 18.00% 14.67 3.05.sup.## 23.10% 4 mg .Math. kg.sup.1 Composition 752.32 4.65 1.11.sup.## 42.80% 3.04 0.59.sup.## 20.80% 14.45 3.81.sup.## 24.20% 5 mg .Math. kg.sup.1 Control 752.32 7.57 1.86.sup. 6.80% 3.69 0.42.sup. 3.7% 17.82 2.10.sup. 6.60% Sample 1 mg .Math. kg.sup.1 Control 752.32 7.49 1.12.sup. 7.90% 3.61 0.43.sup. 5.7% 17.78 3.06.sup. 6.80% Sample 2 mg .Math. kg.sup.1 Control 752.32 7.90 1.10.sup. 2.80% 3.63 0.56.sup. 5.2% 17.62 2.23.sup. 7.60% Sample 3 mg .Math. kg.sup.1 Rupixiao 960.00 4.36 1.11 .sup.## 46.40% 2.77 0.40.sup.## 27.60% 14.85 3.17.sup.## 22.10% tablet group mg .Math. kg.sup.1 Comments: Compared with the normal control group, *p < 0.05, and **p < 0.01. Compared with the model control group, #p < 0.05, and ##p < 0.01. Compared with composition group 1, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 2, .sup.p < 0.05, and.sup.p < 0.01. Compared with composition group 3, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 4, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 5, .sup.p < 0.05, and .sup.p < 0.01.

[0114] Results: Compared with the normal control group, the contents of prolactin PRL, luteinizing hormone LH and follicle stimulating hormone FSH of the rats in the model control group significantly increased (p<0.01). Compared with the model control group, the contents of prolactin PRL, luteinizing hormone LH and follicle stimulating hormone FSH of the rats in the composition groups 1, 2, 3, 4, 5 and the Rupixiao tablet group significantly decreased (p<0.01). The content of PRL of composition group 2 was higher than that of composition group 1 (p<0.05). The content of LH of composition groups 2, 4, 5 was higher than that of composition 1 (p<0.01 or p<0.05). The contents of prolactin PRL, luteinizing hormone LH, and follicle stimulating hormone FSH of control sample groups 1, 2, 3 were higher than those of composition groups 1, 2, 3, 4, 5 (p<0.01 or p<0.05). E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample 1+E.sub.control sample 3E.sub.control sample 1E.sub.control sample 3)=0.518/(0.068+0.0280.0680.028)=5.460, 5.460>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing PRL. E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample 1+E.sub.control sample 3E.sub.control sample 1E.sub.control sample 3)=0.312/(0.037+0.0520.0370.052)=3.595, 3.595>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing LH. E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample 1+E.sub.control sample 3E.sub.control sample 1E.sub.control sample 3)=0.293/(0.066+0.0760.0660.076)=2.139, 2.139>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing FSH.

[0115] 1.6.4 Pathology Morphologic Observation of the Second Pair of Nipples of the Rats Having Hyperplasia of Mammary Gland

[0116] At the end of the experiment, the second pair of nipples in chests of the rats was taken off. The nipples were fixed with 10% formaldehyde solution, subjected to paraffin embedded section, and the second pair of mammary tissue was dyed with hematoxylin-eosin (HE) and observed with light microscope. The results were shown in FIG. 8.

[0117] Results: In a general observation, the morphology of nipples in the normal control group was the same as that at the beginning of the experiment. The position of the nipples was not visible through the hair. After the hair was separated, the nipples were millet-like, which were clung to the skin, pale, soft, and a few nipples were slightly swelled (about 1 mm). The morphological changes of the rats in the model group having hyperplasia of mammary gland were characterized by erected, firmer, hyperemic, and height-increased nipples (2-3 mm). The nipple morphology of rats in the Chinese medicine formulation group in each dose showed that the nipples were erected, and the hyperemia significantly decreased. When observing under a microscope, there was no obvious hyperplasia in lobules of mammary gland and acinus in the normal control group. The number of acinus was 4-5, and the lumen of acinar did not expand. The number of lobules of mammary gland of rats in the model group significantly increased, and the number of acinus significantly increased. As for some of the mammary glands, lumen of the acinus expanded, having secreta inside. As for some of the mammary glands, epithelial cells of the ducts were significantly proliferated, locally showing papillary or multi-layer hyperplasia, dilatation of lumen, which had exfoliated epithelial cells and secreta inside, accompanying with interstitial fibrous tissue hyperplasia. The number of lobules of mammary gland of rats in the Chinese medicine formulation group in each dose significantly reduced, the number of acinus significantly decreased, and hyperplasia of the epithelial cells of the mammary gland duct significantly reduced. The number of lobules of mammary gland in control sample groups 1, 2 and 3 significantly increased, the number of acinus significantly increased, and hyperplasia of the epithelial cells of the mammary gland duct significantly increased.

[0118] 1.7 Experimental Results

[0119] In the present disclosure, the present formulation in each dose and the Rupixiao tablet had significantly inhibitory effects on hyperplasia of mammary gland of the rats, and the drug combination in high dose group had the best overall effect.

2. Research on Anti-Oxidation Effect

[0120] 2.1 Object of the Experiment

[0121] Anti-oxidation effects of the traditional Chinese medicine composition were observed and evaluated.

[0122] 2.2 Experimental Materials

[0123] 2.2.1 Medicinal Materials

[0124] The traditional Chinese medicine composition is provided by Infinitus (China) Co., Ltd. The blank control sample is distilled water. Positive control sample is Pingxiao capsule, national medicine permission number Z61021330, Xi'an C.P. Pharmaceutical Co. Ltd.

[0125] 2.2.2 Reagents

[0126] D-(+)-Galactose, Sinopharm Chemical Reagent Co., Ltd., Batch No: 20130917.

[0127] Total Antioxidant Capacity (T-AOC) kit, Nanjing Jiancheng Bioengineering Institute, Batch No.: 20131211.

[0128] Glutathione-peroxidase (GSH-PX) kit, Nanjing Jiancheng Bioengineering Institute, Batch No.: 20140220.

[0129] Superoxide dismutase (SOD) kit measured by WST-1 method, Nanjing Jiancheng Bioengineering Institute, Batch No.: 20140416.

[0130] Lipid Peroxide (LPO) kit, Nanjing Jiancheng Bioengineering Institute, Batch No.: 20140228.

[0131] Malondialdehyde (MDA) kit, Nanjing Jiancheng Bioengineering Institute, Batch No.: 20140225.

[0132] 2.2.3 Dosage Definition

[0133] Blank control group, traditional Chinese medicine formulation group 1 (752.32 mg.Math.kg.sup.1), traditional Chinese medicine formulation group 2 (752.32 mg.Math.kg.sup.1), traditional Chinese medicine formulation group 3 (752.32 mg.Math.kg.sup.1), traditional Chinese medicine formulation group 4 (752.32 mg.Math.kg.sup.1), traditional Chinese medicine formulation group 5 (752.32 mg.Math.kg.sup.1), control group 1 (752.32 mg.Math.kg.sup.1), control group 2 (752.32 mg.Math.kg.sup.1).

[0134] 2.3 Experimental Method and Test Indicators

[0135] 80 SPF grade female SD rats were taken, which were randomly divided into 10 rats as the normal control group, and the other 70 rats as the model group. The rats in model groups were injected intraperitoneally with 125 mg/kgBW of D-galactose at an injection amount of 0.2 mL/100 g once a day for 6 weeks. The 10 rats in the normal control group were injected intraperitoneally with the same volume of normal saline. 6 weeks after modeling, blood was collected to measure the MDA. The rats were grouped according to the levels of MDA, which were respectively model control group, Chinese medicine formulation group, control sample group and Pingxiao capsule group, with 10 rats in each group.

[0136] Each administration group was intragastrically administered with different concentrations of test samples. The normal control group and the model control group were intragastrically injected with an equal volume of distilled water once a day for 30 d. While administering the test samples, the model control group and each administration group were continuously intraperitoneally injected with an equal amount of D-galactose. At the end of the experiment, the animals were sacrificed, and blood was collected to determine the total anti-oxidative capacity (T-AOC) of serum and contents of glutathione-peroxidase (GSH-PX), lipid peroxide (LPO), malondialdehyde (MDA) and superoxide dismutase (SOD).

[0137] 2.4 Calculation of Q Value of Drug Combination (Jin Zhengjun Method)

[0138] Q value of drug combination was calculated using a modified formula: Q=E.sub.A+B/(E.sub.A+E.sub.BE.sub.AE.sub.B) according to the method of literature. E.sub.A+B indicates the data of drug combination, E.sub.A indicates the data of a single use of A, E.sub.B indicates the data of a single use of B, and the calculation is performed using the inhibition rate or the increase rate data. A Q value of less than 0.85 indicates that the two drugs have an antagonistic effect after combination; a Q value of greater than 0.85 and less than 1.15 indicates that the two drugs have an additive effect after combination; a Q value of greater than 1.15 indicates that the two drugs have a synergistic effect after combination.

[0139] Experimental Results

[0140] 2.5.1 Influences on T-AOC and GSH-PX of D-Galactose-Induced Aging Rats

[0141] At the end of the experiment, the animals were sacrificed, and blood was collected to determine the total anti-oxidative capacity (T-AOC) and glutathione-peroxidase (GSH-PX) of serum.

TABLE-US-00015 TABLE 5 Influences on T-AOC and GSH-PX of D-galactose-induced aging rats ( x s, n = 10) Increase Increase Group Dosage T-AOC rate GSH-PX rate Normal / 11.48 3.88 368.87 145.03 control group Model / 3.39 2.05** 218.56 38.38** control group Composition 752.32 8.86 3.34.sup.## 161.30% 320.56 40.54.sup.## 46.70% 1 mg .Math. kg.sup.1 Composition 752.32 6.89 2.48.sup.## 103.30% 302.22 33.48.sup.## 38.30% 2 mg .Math. kg.sup.1 Composition 752.32 7.27 3.09.sup.## 114.60% 322.89 36.68.sup.## 47.70% 3 mg .Math. kg.sup.1 Composition 752.32 8.18 2.91.sup.## 141.40% 314.33 33.59.sup.## 43.80% 4 mg .Math. kg.sup.1 Composition 752.32 7.08 3.53.sup.# 108.90% 302.19 26.45.sup.## 38.30% 5 mg .Math. kg.sup.1 Control 752.32 4.52 1.59.sup. 33.30% 276.35 24.18.sup. 26.40% Sample 1 mg .Math. kg.sup.1 Control 752.32 4.26 1.48.sup. 25.80% 260.77 33.63.sup. 19.30% Sample 2 mg .Math. kg.sup.1 Control 752.32 4.59 1.64.sup. 35.40% 261.30 33.57.sup. 19.60% Sample 3 mg .Math. kg.sup.1 Pingxiao 960.00 7.50 2.42.sup.## 121.10% 325.09 28.67.sup.## 48.70% capsule mg .Math. kg.sup.1 group Comments: Compared with the normal control group, *p < 0.05, and **p < 0.01. Compared with the model control group, #p < 0.05, and ##p < 0.01. Compared with composition group 1, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 2, .sup.p < 0.05, and.sup.p < 0.01. Compared with composition group 3, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 4, .sup.p < 0.05, and .sup.p < 0.01. Compared with composition group 5, .sup.p < 0.05, and .sup.p < 0.01.

[0142] Results: Compared with the normal control group, the T-AOC of rats in the model control group significantly decreased (p<0.01) and the GSH-PX activity significantly decreased (p<0.05). Compared with the model control group, the content of T-AOC of rats in the composition groups 1, 2, 3, 4, 5 significantly increased, and the GSH-PX activity significantly increased, showing a significant difference (p<0.01 or p<0.05). The content of T-AOC of rats in the control sample groups 1, 2 and 3 was significantly lower than that of the composition groups 1, 2, 3, 4 and 5, and the GSH-PX activity was significantly lower than that of the composition groups 1, 2, 3, 4 and 5 (p<0.01 or p<0.05). E .sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample 1+E.sub.control sample 3E.sub.control sample 1 E.sub.control sample 3)=1.613/(0.333+0.3540.3330.354)=2.006. 2.006>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on increasing T-AOC. E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample 1+E.sub.control sample 3E.sub.control sample 1 E.sub.control sample 3)=0.467/(0.264+0.1960.2640.196)=0.912. 0.85<0.912<1.15, indicating that the combination of control sample 1 and control sample 3 has an additive effect, working together to increase the level of GSH.

[0143] 2.5.2 Influence on Contents of SOD, LPO and MDA of D-Galactose-Induced Aging Rats

[0144] At the end of the experiment, blood was collected to determine the contents of lipid peroxide (LPO) and malondialdehyde (MDA), and superoxide dismutase (SOD) in serum.

TABLE-US-00016 TABLE 6 Influence on contents of SOD, LPO and MDA of D-galactose-induced aging rats ( x s, n = 10) Increase Inhibition Inhibition Group Dosage SOD rate LPO rate MDA rate Normal / 103.10 19.26 5.51 0.70 2.68 1.20 control group Model / 75.09 14.82** 11.34 3.69** 12.83 0.85** control group Composition 752.32 110.37 14.82.sup.## 47.00% 6.09 1.10.sup.## 46.30% 5.57 1.22.sup.## 56.60% 1 mg .Math. kg.sup.1 Composition 752.32 107.73 20.72.sup.## 43.50% 6.38 1.15.sup.## 43.70% 6.03 0.99.sup.## 53.00% 2 mg .Math. kg.sup.1 Composition 752.32 108.18 21.88.sup.## 44.10% 6.82 0.91.sup.## 39.90% 6.30 1.20.sup.## 50.90% 3 mg .Math. kg.sup.1 Composition 752.32 106.94 23.09.sup.## 42.40% 7.56 1.22.sup.## 33.40% 6.70 0.82.sup.## 47.70% 4 mg .Math. kg.sup.1 Composition 752.32 109.56 16.60.sup.## 45.90% 7.12 1.08.sup.## 37.20% 7.44 0.97.sup.## 42.00% 5 mg .Math. kg.sup.1 Control 752.32 90.50 11.85.sup. 20.50% 10.27 1.41.sup. 9.40% 10.00 1.68.sup. 22.00% Sample 1 mg .Math. kg.sup.1 Control 752.32 90.43 13.13.sup. 20.40% 9.72 1.45.sup. 14.30% 10.33 1.82.sup. 19.50% Sample 2 mg .Math. kg.sup.1 Control 752.32 89.90 14.48.sup. 19.70% 9.86 1.31.sup. 13.00% 9.84 2.02.sup. 23.30% Sample 3 mg .Math. kg.sup.1 Pingxiao 960.00 102.48 7.33.sup.## 36.50% 8.76 1.30.sup.# 22.70% 8.24 1.12.sup.## 35.70% capsule mg .Math. kg.sup.1 group Comments: Compared with the normal control group, *p < 0.05, and **p < 0.01. Compared with the model control group, #p < 0.05, and ##p < 0.01. Compared with the composition group 1, .sup.p < 0.05, and .sup.p < 0.01. Compared with the composition group 2, .sup.p < 0.05, and.sup.p < 0.01. Compared with the composition group 3, .sup.p < 0.05, and .sup.p < 0.01. Compared with the composition group 4, .sup.p < 0.05, and .sup.p < 0.01. Compared with the composition group 5, .sup.p < 0.05, and .sup.p < 0.01.

[0145] Results: Compared with the normal control group, the SOD activity of rats in the model control group significantly decreased (p<0.05), and content of LPO and MDA significantly increased (p<0.01). Compared with the model control group, the SOD activity of rats in the composition groups 1, 2, 3, 4 and 5 significantly increased (p<0.01), and the content of LPO and MDA significantly decreased (p<0.01). The content of LPO and MDA of rats in the composition groups 4 and 5 was lower than that of the composition group 1 (p<0.01 or p<0.05). The SOD activity of the control sample groups 1, 2 and 3 was significantly lower than that of the composition groups 1, 2, 3, 4 and 5 (p<0.01 or p<0.05), and the content of LPO and MDA was significantly higher than that of the composition groups 1, 2, 3, 4 and 5 (p<0.01). E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample 1+E.sub.control sample 3E.sub.control sample 1E.sub.control sample 3)=0.470/(0.205+0.1970.2050.197)=1.061, 0.85<1.06<1.15, indicating that the combination of control sample 1 and control sample 3 has an additive effect, working together to increase the level of SOD. E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample 1+E.sub.control sample 3E.sub.control sample 1E.sub.control sample 3)=0.463/(0.094+0.1300.0940.130)=2.179, 2.179>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing the LPO. E.sub.composition 1 Q value=E.sub.composition 1/(E.sub.control sample 1+E.sub.control sample 3E.sub.control sample 1 E.sub.control sample 3)=0.566/(0.220+0.2330.2200.233)=1.408, 1.408>1.15, indicating that the combination of control sample 1 and control sample 3 has a synergistic effect on decreasing the MDA.

3. Statistics of Clinical Application

[0146] The sample of composition 1 was used as the test sample.

[0147] 3.1 Selected conditions: samples of subjects having slight mammary gland pain and moderate mammary gland pain were suitable samples, and samples that had developed into lesion were removed. The actual subjects were 122.

[0148] 3.2 Reference was made according to the standard adopted by the 8th meeting of the Breast Diseases Professional Committee of the Traditional Chinese Surgery Branch of Chinese Medicine Association in 2002. Doctors assessed symptoms and signs using hand diagnose according to the degree of breast pain, degree of breast tenderness, size of lump, texture of lump and distribution range of lump.

[0149] 3.3 Pain was evaluated with reference to the visual analogue scale (VAS). The method was widely used in clinical practice. The same method was used before and after clinical treatment to make a relatively objective score, and to make a relatively objective evaluation on the effect of pain treatment. VAS gives its breast pain scores according to the internationally recognized self-pain assessment based on the number representing the pain level on the scale, in which 0-10 points indicates painless to severe pain. The results are shown in FIGS. 1-2.

[0150] 3.4 According to the self-perceived pain score of the subjects, after administration of the products for three months, 56.56% of the subjects had a significant improvement in pain, 38.52% of the subjects had a slight improvement, and only 4.92% of the subjects had no improvement in the pain scores, fully indicating that the traditional Chinese medicine composition had a significant effect on improving breast pain.

[0151] 3.5 According to FIGS. 3-4, after cured the products for one month, compared with the baseline period, the number of days of breast pain in the subjects administrated with the traditional Chinese medicine composition significantly reduced (p<0.05). As the administration time increased, the number of days of breast pain in the subject decreased. Three months after administration of the traditional Chinese medicine composition, the number of days of breast pain in the subjects decreased by 1.65 days.

[0152] 3.6 Reference was made according to the standard adopted by the 8.sup.th meeting of the Breast Diseases Professional Committee of the Traditional Chinese Surgery Branch of Chinese Medicine Association in 2002. Improvement rates of before and after the treatment were calculated: improvement rate=(total score before treatmenttotal score after treatment)/total score before treatment100%. Cured: improvement rate>90%, obviously effective: improvement rate 70%-89%; effective: improvement rate 30%-69%; and not effective: improvement rate<30%.

Total effective rate=cured rate+obviously effective rate+effective rate.

[0153] FIGS. 5-6 showed the improvements in the subjects after administration of the traditional Chinese medicine composition. From the symptoms and signs obtained by hand diagnose of specialists, after administration of the composition for three months, the average value of breast symptom and signs of the subject decreased from 6.69 points to 3.12 points. From the aspect of improvement in total effective rate, the total effective rate reached 64.75% after three months with a statistical difference (p<0.05).

Comment: * represented that compared with the initial vale, the difference was significant (p<0.05).

[0154] 3.7 As it shown in FIG. 7, after administration of the product for 1 month, degree of breast tenderness of the subject obviously improved (40.2%), and the other indicators improved in some degree. As the administration time increased, each symptom and sign in the subjects gradually improved. After administration of the traditional Chinese medicine composition for three months, degree of breast pain, degree of breast tenderness, size of the lump, texture of the lump and the distribution range of the lump in the subject obviously improved (p<0.05). Especially, degree of breast pain (51.5%) and degree of breast tenderness (51.6%) improved the most obviously.

[0155] The above are only preferred embodiments of the present disclosure. It should be noted that a number of modifications and refinements may be made by one ordinary skilled in the art without departing from the principles of the disclosure, and such modifications and refinements are also considered to be within the scope of protection of the disclosure.