Traditional Chinese medicine composition and traditional Chinese medicine preparation for treating hyperplasia of mammary glands and precancerous lesions and preparation method of traditional Chinese medicine preparation

Abstract

The invention discloses a traditional Chinese medicine composition for treating hyperplasia of mammary glands and precancerous lesions, a traditional Chinese medicine preparation and a preparation method of the traditional Chinese medicine composition. Comprising 8-12 parts of vinegar-processed radix bupleuri, 12-18 parts of radix paeoniae alba, 12-18 parts of selfheal, 9.5-14.5 parts of thunberg fritillary bulb, 8-12 parts of vinegar-processed curcuma zedoary, 8-12 parts of radix curcumae, 12-18 parts of bighead atractylodes rhizome, 12-18 parts of poria cocos, 12-18 parts of raw oyster shell, 12-18 parts of hedyotis diffusa, 6.4-9.6 parts of schisandra chinensis, 9.5-14.5 parts of glossy privet fruit, 8-12 parts of vinegar-processed turtle shell, 8-12 parts of astragalus membranaceus and 4.8-7.2 parts of honey-fried licorice root. The traditional Chinese medicine composition has the effects of soothing the liver, strengthening the spleen, reducing phlegm, resolving masses, activating blood and eliminating diseases, and can be used for treating hormone-dependent proliferative diseases such as hyperplasia of mammary glands and atypical hyperplasia Multi-target intervention is achieved, the curative effect is remarkable, side effects are small, and wide market and application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to a traditional Chinese medicine composition for treating breast diseases, and in particular to a traditional Chinese medicine composition for treating breast hyperplasia and precancerous lesions, a traditional Chinese medicine preparation and a preparation method thereof. Background Art

[0002] Breast cancer is the most common and most fatal cancer type in women. Precancerous lesions primarily refer to atypical hyperplasia and ductal carcinoma in situ (DCIS) lesions, which are still confined to the basement membrane. Timely intervention during this "window period" can effectively prevent the development of invasive cancer. Traditional Chinese Medicine (TCM) believes that precancerous lesions are often caused by emotional imbalance, which leads to liver dysfunction and spleen dysfunction. This leads to stagnation of qi and the formation of phlegm, which in turn transforms into blood stasis. Ultimately, the phlegm and blood stasis form a complex, forming a "liver depression, phlegm coagulation, and blood stasis syndrome." Traditional Chinese medicines that soothe the liver, promote qi circulation, and activate blood circulation can effectively inhibit the progression of precancerous lesions.

[0003] At present, surgery and endocrine therapy are the main treatments for precancerous lesions of breast cancer. However, surgery is prone to overtreatment, while endocrine therapy can cause systemic side effects such as hot flashes, thrombosis, and endometrial thickening, resulting in low patient compliance. Existing Chinese herbal combination drugs are good at soothing the liver, resolving phlegm, or promoting blood circulation, but lack the effects of anti-fibrosis and reversing precancerous lesions. It is difficult to systematically regulate this pathological change, and the preparation is not standardized enough. Therefore, there is currently no multi-target compound that covers the "liver depression-phlegm stasis-vital deficiency" pathogenesis chain at home and abroad. There is a lack of low-toxic, high-efficiency, and easily accepted solutions and drugs for the progression of precancerous lesions of breast cancer. Summary of the Invention

[0004] The first technical problem to be solved by the present invention is to provide a traditional Chinese medicine composition for treating breast hyperplasia and precancerous lesions. The traditional Chinese medicine composition has the effects of soothing the liver and strengthening the spleen, resolving phlegm and dispersing nodules, and promoting blood circulation and eliminating symptoms. It can be used to treat hormone-dependent proliferative diseases such as breast hyperplasia and atypical breast hyperplasia (AH).

[0005] The second technical problem to be solved by the present invention is to provide a method for preparing a traditional Chinese medicine composition for treating breast hyperplasia and precancerous lesions.

[0006] The third technical problem to be solved by the present invention is to provide a traditional Chinese medicine preparation for treating breast hyperplasia and precancerous lesions.

[0007] The technical solution adopted by the present invention to solve the first technical problem is a traditional Chinese medicine composition for treating breast hyperplasia and precancerous lesions, wherein the raw material components thereof include, by weight:

[0008] 8-12 parts of vinegar bupleurum, 12-18 parts of white peony root, 12-18 parts of selfheal, 9.5-14.5 parts of thunbergia bulb, 8-12 parts of vinegar zedoaria, 8-12 parts of turmeric, 12-18 parts of atractylodes, 12-18 parts of poria, 12-18 parts of raw oyster, 12-18 parts of hedyotis diffusa, 6.4-9.6 parts of schisandra chinensis, 9.5-14.5 parts of glossy privet fruit, 8-12 parts of vinegar turtle shell, 8-12 parts of astragalus, and 4.8-7.2 parts of roasted liquorice.

[0009] Preferably, the raw material components include:

[0010] 10 parts of vinegar bupleurum, 15 parts of white peony root, 15 parts of selfheal, 12 parts of thunbergia bulbs, 10 parts of vinegar zedoaria, 10 parts of turmeric, 30 parts of raw oyster, 15 parts of herba hedyotis diffusae, 10 parts of vinegar turtle shell, 10 parts of astragalus, 15 parts of atractylodes, 15 parts of poria, 12 parts of glossy privet fruit, 8 parts of schisandra chinensis, 6 parts of roasted liquorice.

[0011] Preferably, for patients with severe liver depression, the raw material components also include: 8-12 parts of citron, 8-12 parts of cyperus rotundus, 4.8-7.2 parts of immature bitter orange, and 4.8-7.2 parts of green peel.

[0012] Preferably, for patients with severe phlegm-dampness, the raw material components further include: 8-12 parts of Alisma orientalis, 8-12 parts of Atractylodes lancea, 8-12 parts of Magnolia officinalis, and 4.8-7.2 parts of Amomum villosum.

[0013] Preferably, the ratio of Bupleurum Root: White Peony Root: Prunella Vulgaris = 1:1.4-1.6:1.4-1.6.

[0014] Vinegar Bupleurum: Pungent and bitter in flavor, slightly cold in nature; enters the Liver, Gallbladder, and Lung meridians. It dispels heat, relieves liver qi stagnation, and elevates Yang Qi. It is commonly used to treat flank distension, breast swelling, and emotional distress caused by liver qi stagnation.

[0015] White Peony Root: Bitter and sour in flavor, slightly cold in nature; enters the Liver and Spleen meridians. Nourishes blood and regulates menstruation, astringes Yin and stops sweating, softens the liver and relieves pain; primarily used to treat hypochondriac pain due to blood deficiency, irregular menstruation, and hyperactivity of liver yang.

[0016] Prunella Vulgaris: Pungent, bitter, and cold in nature; enters the Liver and Gallbladder Meridians. It clears the liver and purges heat, improves eyesight, and reduces swelling. It is used for scrofula, breast ulcers, and liver-fire headaches.

[0017] Fritillaria thunbergii: Bitter in flavor, cold in nature; enters the Lung and Heart meridians. Clears heat, resolves phlegm, relieves cough, detoxifies, disperses stagnation, and eliminates carbuncle; particularly effective in treating breast abscesses, breast lumps, and phlegm-fire stagnation.

[0018] Curcuma zedoaria (Curcuma longa) is pungent, bitter, and warm in nature. It enters the Liver and Spleen meridians. It promotes blood circulation and qi circulation, eliminates stagnation, and relieves pain. It is commonly used for treating lumps, amenorrhea, dysmenorrhea, and chest pain.

[0019] Curcuma: Pungent and bitter in flavor, cold in nature; enters the Liver, Gallbladder, Heart, and Lung meridians. It promotes blood circulation and relieves pain, promotes Qi and relieves depression, clears the heart and cools the blood, promotes bile secretion and reduces jaundice. It also treats symptoms of Qi stagnation and blood stasis, such as breast tenderness and irregular menstruation.

[0020] Atractylodes macrocephala: Sweet and bitter in flavor, warm in nature; enters the spleen and stomach meridians. It tonifies Qi and strengthens the spleen, dispels dampness and promotes diuresis, stops sweating and stabilizes pregnancy; used for diarrhea caused by spleen deficiency, edema caused by dampness, and sinking of Qi in the middle meridians.

[0021] Poria: Sweet, bland, neutral in nature; enters the Heart, Lung, Spleen, and Kidney meridians. It promotes diuresis and eliminates dampness, strengthens the spleen, and calms the mind; treats edema, phlegm and fluid retention, dizziness, palpitations, and insomnia due to heart and spleen deficiency.

[0022] Raw oysters: A mildly cold flavor, they enter the liver, gallbladder, and kidney meridians. They suppress yang and replenish yin, calm the mind, soften and disperse lumps, and astringe. They are commonly used for scrofula, phlegm nodules, bone steaming, hot flashes, and acid reflux.

[0023] Hedyotis diffusa: Slightly bitter and sweet, with a cold nature; it enters the stomach, large intestine, and small intestine meridians. It clears heat and detoxifies, promotes dampness and relieves stranguria; it is widely used as an adjunct treatment for carbuncles, ulcers, intestinal carbuncles, and various tumors.

[0024] Schisandra chinensis: Sour, sweet, and warm in nature; enters the Lung, Heart, and Kidney meridians. It has astringent properties, replenishes Qi and promotes fluid production, nourishes the kidneys, and calms the mind. It is suitable for treating chronic cough, asthma, nocturnal emission, spermatorrhea, and palpitations and insomnia.

[0025] Ligustrum lucidum fruit: sweet, bitter, and cooling in nature; enters the liver and kidney meridians. It nourishes the liver and kidneys, improves eyesight, and darkens hair; it also has modern benefits such as antioxidant, lipid-regulating, and inhibiting breast hyperplasia.

[0026] Vinegar-soaked Turtle Shell: A mildly cold herb with a mild flavor; it enters the Liver and Kidney meridians. It nourishes Yin and suppresses Yang, reduces fever and eliminates steam, softens and disperses lumps; it has been used throughout history for amenorrhea, masses, scrofula, and fever due to Yin deficiency.

[0027] Astragalus: Sweet in flavor, slightly warm in nature; enters the spleen and lung meridians. It tonifies Qi and raises Yang, strengthens the defense system and consolidates the exterior, promotes diuresis and reduces swelling, and removes toxins and promotes tissue regeneration. It is a key herb for strengthening the body's health and boosting immunity.

[0028] Roasted Licorice: Sweet in flavor, neutral in nature; enters the Heart, Lung, Spleen, and Stomach meridians. It tonifies the spleen and replenishes Qi, clears heat and detoxifies, eliminates phlegm and relieves cough, relieves urgency and pain, and harmonizes various herbs; it is often used as an adjuvant to enhance synergy and moderate harsh properties of other herbs.

[0029] The present invention uses vinegar bupleurum and white peony root as the main drugs. Women are born with the liver as their innate nature. Bupleurum is pungent and dispersing, bitter and purgative, soothes the liver and relieves depression, and regulates the qi mechanism. It is a key drug for treating milk meridian qi stagnation. White peony root is sour and sweet to soften the liver, nourish blood and restrain yin, and with the dispersing effect of bupleurum, it allows liver qi to flow smoothly without consuming yin blood. The auxiliary drug Curcuma "breaks blood and promotes qi circulation, dissipates lumps and masses", and together with Curcuma, enters the liver to promote qi circulation and break blood stasis, transform qi and promote blood circulation to relieve stagnation; Prunella vulgaris and Fritillaria thunbergii are bitter and cold to clear the liver and resolve phlegm, soften and disperse lumps to eliminate phlegm nodules; raw oyster and vinegar turtle shell soften cold and hard masses, suppress yang and disperse lumps, and help break up phlegm and blood stasis. Atractylodes macrocephala and Poria cocos are used to strengthen the spleen and dry dampness, cultivate the earth and generate gold to prevent phlegm from accumulating; Hedyotis diffusa clears heat and disperses lumps, Ligustrum lucidum nourishes the liver and kidneys, Astragalus invigorates qi and strengthens the body, and Schisandra chinensis is sour and astringes yin, all of which protect the root and strengthen the essence, cultivate the body and eliminate evil. Roasted liquorice is used to harmonize the various drugs. It can soothe the liver and strengthen the spleen, resolve phlegm and disperse nodules, activate blood circulation and eliminate symptoms, which is in line with the core pathogenesis of "liver depression, phlegm coagulation and blood stasis" in breast cancer prelesions, so that the qi can flow smoothly, phlegm and blood stasis can be resolved, and the positive energy can be consolidated, so as to achieve the effects of softening and dispersing nodules, harmonizing yin and yang, and reversing the progression of phlegm and blood stasis.

[0030] The Chinese medicine composition of the present invention has the following effects:

[0031] It soothes the liver and strengthens the spleen, resolves phlegm and dissipates stagnation, and activates blood circulation to eliminate symptoms. It is used for the syndrome of liver depression, phlegm coagulation, and blood stasis in precancerous lesions of breast cancer. Symptoms include a breast lump, pathologically confirmed as atypical hyperplasia, adenosis, sclerosing adenosis, ductal epithelial hyperplasia (papillary, solid, or cribriform hyperplasia), or ductal carcinoma in situ, accompanied by chest tightness and flank distension, depression and irritability, insomnia, restlessness, bitter taste in the mouth, or yellow urine, a dark red tongue with a thin yellow coating, and a wiry and slippery pulse.

[0032] The present invention solves the second technical problem by adopting a technical solution that is a method for preparing a traditional Chinese medicine composition for treating breast hyperplasia and precancerous lesions, comprising the following steps:

[0033] S1, Prunella vulgaris and Curcuma aromatica were extracted twice with 60% ethanol under reflux and the filtrates were combined;

[0034] S2. Decoction of vinegar bupleurum root, white peony root, thunbergia thunbergii, vinegar zedoaria, atractylodes macrocephala, poria, raw oyster, herba hedyotis diffusae, schisandra chinensis, glossy privet fruit, vinegar turtle shell, astragalus root, and roasted liquorice root twice, and concentrate to a concentrate with a relative density of 1.10-1.30;

[0035] S3. Mix the filtrate obtained in step S1 and the concentrated solution obtained in step S2.

[0036] Preferably, the vinegared bupleurum root, vinegared zedoary turmeric and vinegared turtle shell are prepared by the following method: bupleurum root, zedoary turmeric and turtle shell are soaked in 6% rice vinegar for 48 hours and then dried at 60°C.

[0037] Preferably, in step S2, the relative density is 1.20.

[0038] The technical solution adopted by the present invention to solve the third technical problem is a traditional Chinese medicine preparation for treating breast hyperplasia and precancerous lesions, which includes: an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the above-mentioned traditional Chinese medicine composition.

[0039] The present invention is not limited to the type of preparation, and may be a decoction, paste, granules, tablets, capsules, gels, etc.; therefore, the pharmaceutically acceptable carrier is also not limited, as long as it can be formed into a preparation of the corresponding form with the active ingredient. For example, the pharmaceutically acceptable carrier can be selected from one or a combination of dextrin, starch, cellulose, starch or cellulose derivatives, etc.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] The Chinese medicine composition provided by the present invention can inhibit ERα expression, reverse AH lesions, and is suitable for treating various types of breast hyperplasia. It can also alleviate the burden of breast hyperplasia drugs on the liver and reduce the possibility of liver toxicity.

[0042] The Chinese medicine composition of the present invention is based on Rutong Ruanjian Tablets and the empirical formula Chaishao Rupi Decoction. By removing hepatotoxic drugs and improving the drug composition, it can exert the effects of soothing the liver and strengthening the spleen, resolving phlegm and dispersing stagnation, and promoting blood circulation and eliminating symptoms, showing a good therapeutic effect on precancerous lesions of breast cancer. This Chinese medicine combination has the efficacy of synergistically regulating hormones, resisting fibrosis, and reversing precancerous lesions. It has multi-target intervention, significant efficacy, and minimal side effects, and has broad market and application prospects. It can be developed into the first evidence-based Chinese medicine preparation for reversing precancerous lesions of breast cancer, filling the treatment gap between hormone therapy and surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 These are microscope photos (100×) of HE-stained mammary gland tissues of experimental rats in each group of Example 1.

[0044] Figure 2 This is a bar chart of the final pathological diagnosis of rats in each experimental group in Example 1.

[0045] Figure 3 This is a bar chart of the TCM syndrome scores of rats in each experimental group in Example 1.

[0046] Figure 4 Microscopic photographs and bar graphs of microvascular density of rats in each experimental group in Example 1;

[0047] Among them, A is a microscope photo of rat microvessel density results of CD31 immunohistochemistry, and B is a bar graph of statistical analysis results.

[0048] Figure 5 This is a bar graph showing the inhibition rate of HRAS-MCF10A cells after intervention with the Chinese medicine composition in Example 2;

[0049] Among them, A, B, and C are bar charts of cell inhibition rates at 24h, 48h, and 72h intervention, respectively.

[0050] Figure 6 The figure shows the cell colony formation and the bar chart of the statistical analysis results in Example 2;

[0051] Among them, A is a picture of cell clone formation, and B is a bar graph of statistical analysis results. DETAILED DESCRIPTION

[0052] The specific implementation of the present invention is further described in detail below with reference to the accompanying drawings and specific examples.

[0053] The following examples are used to illustrate specific embodiments of the present invention, but are not used to limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0054] Unless otherwise specified, the experimental reagents and materials used in the examples of the present invention can be obtained through conventional commercial channels; unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.

[0055] Example 1 Experimental study on the therapeutic effect of the Chinese medicine composition of the present invention on pre-breast cancer lesions in rats

[0056] 1. Materials

[0057] 1.1 Experimental Animals

[0058] 40 SPF SD rats, 8-9 weeks old, weighing (200±20) g.

[0059] 1.2 Experimental drugs and reagents

[0060] The Chinese medicine composition of this embodiment (i.e., Chaishao Xiaozheng Recipe) comprises: 10g of vinegared bupleurum, 15g of white peony root, 15g of selfheal, 12g of thunbergia bulb, 10g of vinegared zedoaria, 10g of turmeric, 15g of atractylodes macrocephala, 15g of poria, 30g of raw oyster, 15g of hedyotis diffusa, 8g of schisandra chinensis, 12g of glossy privet fruit, 10g of vinegared turtle shell, 10g of astragalus, and 6g of roasted liquorice (dosage can be ±20%, wherein the ratio of bupleurum: white peony root: selfheal is 1:1.5:1.5). It was provided by the First Affiliated Hospital of Hunan University of Chinese Medicine. All raw medicinal materials were soaked in 10 times water for 30 minutes and then decocted for 1 hour. The medicinal materials were filtered out and decocted in 5 times water for 1 hour. The combined medicinal liquid was concentrated to 100mL. Tamoxifen (purchased from Yangtze River Pharmaceutical Group Co., Ltd.) was dissolved in sterile saline and stored at 4°C. 7,12-Dimethylbenz(a)anthracene (DMBA) was purchased from Sigma-Aldrich, USA, dissolved in sesame oil and used immediately after preparation. CD31 was purchased from Xiamen Tongling Biological Co., Ltd.

[0061] 1.3 Instruments

[0062] Pathological tissue bleaching and drying machine; paraffin embedding machine, rotary paraffin slicer, upright microscope.

[0063] 2 Methods

[0064] 2.1 Animal modeling

[0065] After one week of adaptive feeding, 60 SD rats were randomly divided into a blank group (10 rats) and a modeling group (30 rats). The modeling group rats were gavaged with 100 mg / kg of DMBA solution at week 0 and week 4, and were fitted with a neck shackle starting at week 5 and continuing until week 9 to establish a liver depression and blood stasis syndrome model. Modeling success was assessed by observing mammary gland pathological morphology and symptom scores.

[0066] 2.2 Animal grouping and drug administration

[0067] After the model was successfully established, the rats were randomly divided into a model group, a Chaishao Xiaozheng Decoction group, and a tamoxifen group. The equivalent dose for rats, calculated based on body surface area, was calculated: the Chaishao Xiaozheng Decoction group received a daily gavage dose of 17.4 g / kg of the crude drug; the tamoxifen group received a daily gavage dose of 3.2 mg / kg. Both the blank and model groups received an equal volume of normal saline daily for 30 consecutive days.

[0068] 2.3 Index detection

[0069] Calculate symptom scores before and after medication administration. The following symptoms were scored as 0, 1, or 2 points, respectively, for "absent," "mild," or "significant": sluggish reaction, slow movement, squinting eyes, eye discharge, dreary fur, dry, small, and scanty stools, brownish-yellow tail or scaly patches, thin body, and dark purple tongue with ecchymosis. Symptom improvement index = [(sum of symptom scores before medication administration - sum of symptom scores after medication administration) / sum of symptom scores before medication administration] × 100%.

[0070] After the experiment, the rats were euthanized and the second pair of breasts were removed and fixed in 4% formaldehyde solution. HE staining and immunohistochemical detection of CD31 were performed, and the microvessel density was calculated.

[0071] 2.4 Statistical analysis

[0072] Graphpad Prism 9.4 software was used for data processing. Normally distributed quantitative data were expressed as (x ± s); those not normally distributed were expressed as medians and interquartile ranges. If normal distribution and homogeneity of variance were achieved, one-way analysis of variance was used for comparisons between multiple groups, and the Tukey method was used for further pairwise comparisons. In cases of heterogeneity of variance, the Krugal-Wallis H test was used. P < 0.05 indicated statistical significance.

[0073] 3 Results

[0074] 3.1 HE staining of mammary gland tissue of rats in each group

[0075] Depend on Figure 1 It can be seen that in the blank group, the alveoli and terminal ducts proliferated, the cells were arranged neatly, and there was no dysplasia; in the model group, the epithelial layers increased, the cell size and morphology showed differences, the ductal lumen was filled with proliferating cells, there was pushing and overlapping between cells, the chromatin increased, and it showed atypical hyperplasia changes; the degree of cell proliferation in the ductal lumen in the Chaishao Xiaozheng Decoction group and the tamoxifen group was less than that in the model group.

[0076] 3.2 Final pathological diagnosis of rats in each group

[0077] The pathological diagnosis of each group at the end of the intervention was shown in Figure 2 The results show that pathological differences were significant between the intervention groups (P < 0.05); there were significant differences between each group and the model control group (P < 0.05); and there were no significant differences between the tamoxifen group and the Chaishao Xiaozheng Decoction group (P > 0.05). This suggests that both tamoxifen and Chaishao Xiaozheng Decoction can inhibit the progression of precancerous lesions to a certain extent, with no significant difference in their inhibitory effects.

[0078] 3.3 TCM syndrome scores of rats in each group

[0079] The rats in the model group often showed symptoms and signs such as slow reaction, withered fur, scales, thin body, dark purple tongue and ecchymosis. Figure 3 As shown in the data, compared with the model group, the TCM syndrome score of the Chaishao Xiaozheng Decoction group was significantly improved (P<0.05), while there was no significant change in the tamoxifen group (P>0.05).

[0080] 3.4 Microvessel density of rats in each group

[0081] Depend on Figure 4 Compared with the blank group, the model group had significantly increased vascular proliferation. The MVD values in the tamoxifen and Chaishao Xiaozheng Decoction groups were significantly lower than those in the model group (P<0.05), but there was no statistically significant difference between the two groups (P>0.05). This indicates that both tamoxifen and Chaishao Xiaozheng Decoction can significantly inhibit vascular proliferation in rats with pre-breast cancer lesions, and their effects are comparable.

[0082] 3.5 Changes in liver function of rats in each group

[0083] As shown in Table 1, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the model group and tamoxifen group were significantly higher than those in the blank group (P < 0.05). There were no significant differences in ALT and AST between the Chaishao Xiaozheng Decoction group and the blank group, suggesting that Chaishao Xiaozheng Decoction did not damage the liver function of rats.

[0084] Table 1 Changes in liver function of rats in each group

[0085]

[0086] Note: Compared with the blank group, * indicates P < 0.05.

[0087] 4 Conclusion

[0088] The Chinese medicinal composition Chaishao Xiaozheng Decoction can inhibit angiogenesis in rats with pre-breast cancer lesions, inhibit the progression of canceration, and has no damage to the liver and kidney functions of the rats.

[0089] Example 2 Experimental study on the intervention effect of the serum containing the Chinese medicine composition of the present invention on breast cancer precursor lesion cells

[0090] 1. CCK8 test to investigate the effect of the serum containing the Chinese medicine composition of the present invention on the proliferation of breast cancer prelesion cells

[0091] Human mammary epithelial cells transfected with HRAS lentivirus were used to establish a breast cancer pre-lesion model (HRAS-MCF10A). The cells were treated with 0.5%, 1%, 2.5%, 5%, 10%, 20%, 40% and 60% (v / v) drug-containing serum for 24h, 48h and 72h, respectively, and the absorbance (A 450 nm) and convert the inhibition rate. Figure 5 As shown, after 24 hours of treatment, cell viability in the 40% and above concentration groups decreased significantly. After 48 hours of treatment, the growth inhibition rates in the 0.5%-60% groups were 15.93%, 24.72%, 41.35%, 49.07%, 33.40%, 10.10%, 16.53%, and 27.55%, respectively, showing a clear dose-time dependence. After 72 hours, the inhibition rate reached 100% for cells above 5%. These results demonstrate that the Chinese herbal composition of the present invention effectively inhibits the proliferation of precancerous lesion cells.

[0092] 2. Investigate the effect of Chinese medicine combination-containing serum on the proliferation and invasion of breast cancer precursor lesion cells through clone formation experiment

[0093] HRAS-MCF10A cells were seeded in 6-well plates (500 cells / well) and treated with 5% (v / v) drug-containing serum or tamoxifen for 48 h, followed by replacement with normal culture medium. MCF10A cells were used as a control. After 14 days of continuous culture, the cells were fixed with methanol and stained with crystal violet, and the number of clones was calculated. Figure 6 As shown, the number of clones in the model group increased significantly, and the cell clones were significantly reduced after intervention with tamoxifen and Chinese medicine combination-containing serum, and the inhibitory effects of the two were comparable.

[0094] Example 3 Clinical Observation on the Treatment of Liver Depression and Phlegm Coagulation Type Hyperplasia of the Mammary Glands with the Chinese Medicine Composition of the Present Invention

[0095] 1 Clinical Data

[0096] 1.1 General Information

[0097] Patients who visited the Breast Clinic of the First Affiliated Hospital of Hunan University of Chinese Medicine between October 1, 2015, and December 31, 2018, were randomly divided into a treatment group and a control group, with 50 patients in each group. In the treatment group, patients aged 20 to 54 years (mean, 34.23±9.60) years, with a disease duration of 6 months to 9.5 years (mean, 3.53±0.47) years. In the control group, patients aged 19 to 55 years (mean, 35.42±9.13) years, with a disease duration of 5.5 months to 10 years (mean, 3.69±0.61) years. No statistically significant differences in age, disease duration, and other data were found between the two groups (P>0.05), indicating comparability.

[0098] 1.2 Case selection criteria

[0099] Inclusion criteria: (1) those who meet the above diagnostic criteria and TCM syndrome differentiation criteria; (2) women aged 18 to premenopausal; (3) those who have not used other therapeutic drugs within 14 days; (4) those who voluntarily participate in the observation.

[0100] Exclusion criteria: (1) postmenopausal women or women under 18 years old; (2) those who have recently used other therapeutic drugs; (3) those with premenarchal gynecomastia, gynecomastia, or other benign or malignant breast lumps; (4) those with chest pain or breast discharge caused by other reasons; (5) those with other types of breast problems; (6) pregnant and lactating women; (7) patients with other serious systemic diseases; (8) patients with diabetes; (9) patients with mental and psychological diseases.

[0101] 2 Treatment methods

[0102] The treatment group received a modified version of the traditional Chinese medicine composition of the present invention. The composition includes: 10g of Bupleurum Root, 15g of White Peony Root, 15g of Prunella Vulgaris, 10g of Fritillaria thunbergii, 10g of Atractylodes macrocephala, and 10g of Poria. Also included are 10g of Citrus Palmatum, 10g of Cyperus Rhizoma, 6g of Immature Citrus Aurantii, and 6g of Citrus Aurantii. For patients with severe phlegm-dampness, 10g of Alisma Orientalis, 10g of Atractylodes Rhizoma, 10g of Magnolia Bark, and 6g of Amomum Villosum were added. One dose per day was administered, decocting the decoction to obtain 300mL of the juice, which was taken orally once in the morning and evening.

[0103] The control group was treated with Rupixiao Tablets (produced by Liaoning Shangyao Haohushi Pharmaceutical Group Co., Ltd.), 4 tablets each time, 3 times a day.

[0104] Both groups took a 12-week course of treatment. During the treatment period, all patients maintained a positive and stable mood, appropriately controlled their intake of fatty foods, and promptly treated gynecological diseases such as irregular menstruation.

[0105] 3. Observation of therapeutic effects

[0106] 3.1 Observation indicators (1) Observe the overall efficacy and TCM syndrome efficacy of the two groups; (2) Observe the relief of breast lumps and pain. According to the clinical manifestations, the main symptom of breast lumps is measured in centimeters, and the maximum diameter of the lump is taken. The main symptom of breast pain is scored as 0, 2, 4, and 6 points as none, mild, moderate, and severe, respectively. The accompanying symptoms of chest and flank distension, irritability, abdominal pain during menstruation, and scanty and dark menstrual flow are scored as 0, 1, 2, and 3 points as none, mild, moderate, and severe, respectively.

[0107] 3.2 Efficacy criteria (1) Overall efficacy criteria: Cured: Lump and pain disappear; Markedly effective: Lump disappears ≥1 / 2 and pain disappears; Effective: Lump disappears <1 / 2 and pain is relieved; Ineffective: Lump and pain remain unchanged. (2) TCM syndrome efficacy criteria: Cured: Main symptoms and signs basically disappear, and the total score decreases ≥90% compared with before treatment; Markedly effective: Main symptoms and signs mostly disappear, and the total score decreases <90% but ≥70% compared with before treatment; Effective: Main symptoms and signs partially disappear, and the total score decreases <70% but ≥30% compared with before treatment; Ineffective: Main symptoms and signs do not improve significantly or worsen, and the total score decreases <30% compared with before treatment.

[0108] Statistical analysis was performed using SPSS 17.0. Enumeration data were analyzed using the chi-square test, ranked data were analyzed using the Ridit test, and measurement data were analyzed using the t-test for homogeneity of variance and normal distribution. P < 0.05 indicated statistical significance.

[0109] 4 Results

[0110] 4.1 Comparison of comprehensive efficacy between the two groups

[0111] The total effective rate was 94% in the treatment group and 78% in the control group. The difference between the two groups was statistically significant. (See Table 2)

[0112] Table 2 Comparison of comprehensive efficacy between the two groups (cases)

[0113]

[0114] Note: Compared with the control group, a means P < 0.05.

[0115] 4.2 Comparison of TCM syndrome efficacy between the two groups

[0116] The total effective rate of TCM syndrome treatment was 96% in the treatment group and 74% in the control group. The difference between the two groups was statistically significant. (See Table 3)

[0117] Table 3 Comparison of TCM syndrome efficacy between the two groups (examples)

[0118]

[0119] Note: Compared with the control group, a means P < 0.05.

[0120] 4.3 Comparison of breast mass reduction between the two groups

[0121] There was statistically significant difference in breast mass reduction between the two groups before and after treatment, as well as between the two groups after treatment (see Table 4).

[0122] Table 4 Comparison of breast mass reduction between the two groups (x±s, cm)

[0123]

[0124] Note: Compared with the group before treatment, a means P < 0.01; compared with the control group, b means P < 0.05.

[0125] 3.7 Comparison of breast pain relief between the two groups In terms of pain relief, there were statistically significant differences in the comparisons within the groups before and after treatment, and between the groups after treatment (see Table 5).

[0126] Table 5 Comparison of breast pain relief between the two groups (x±s, points)

[0127]

[0128] Note: Compared with the group before treatment, a means P < 0.01; compared with the control group, b means P < 0.05.

[0129] 5 Conclusion

[0130] The Chinese medicinal composition of the present invention has satisfactory therapeutic effect in treating liver depression and phlegm coagulation type hyperplasia of mammary glands.

[0131] Example 4 Clinical Observation on the Effect of the Chinese Medicine Preparation of the Present Invention on Hormone Levels and Tumor Size in Patients with Hyperplasia of the Mammary Glands

[0132] 1 Clinical Data

[0133] 1.1 General Information

[0134] All cases were recruited from outpatient clinics of the Department of Traditional Chinese Medicine Surgery, First Affiliated Hospital of Hunan University of Chinese Medicine, between January 2022 and January 2023. They were randomly divided into two groups: an observation group of 41 patients with a mean age of (32.2±8.12) years and a mean disease duration of (3.8±3.6) years; 32 patients were married and 9 were unmarried. A control group of 41 patients with a mean age of (31.9±7.22) years and a mean disease duration of (3.6±4.1) years; 33 patients were married and 8 were unmarried. There was no statistically significant difference in general information between the two groups (P>0.05), indicating that the two groups were comparable.

[0135] 1.2 Case selection criteria

[0136] All patients met the Western medical diagnostic criteria for mammary hyperplasia, as outlined in the "Diagnosis, Syndrome Differentiation, and Therapeutic Efficacy Evaluation Criteria for Mammary Hyperplasia," revised in 2002 by the Mammary Disease Professional Committee of the Surgery Branch of the Chinese Association of Traditional Chinese Medicine. The TCM syndrome differentiation criteria were based on the syndrome classification criteria for TCM diseases and syndromes, the TCM industry standard of the People's Republic of China, issued by the State Administration of Traditional Chinese Medicine. Liver depression and phlegm stagnation type: more common in young and middle-aged women, with symptoms including breast lumps that wax and wane with mood swings, accompanied by chest tightness and flank distension, depression and irritability, insomnia, frequent dreams, restlessness, bitter taste in the mouth, a thin yellow tongue coating, and a wiry and slippery pulse. Patients had regular menstruation, a course of more than three months, and had not taken any relevant medications in the past two weeks. Patients with other underlying systemic diseases, suspected breast cancer, or breast fibroids, as well as those taking birth control pills or other sex hormone medications, were excluded.

[0137] 1.3 Efficacy criteria

[0138] The two groups were compared before and after treatment for TCM syndrome efficacy, ultrasound size of the largest mass, and serum estrogen and progesterone levels. TCM syndrome efficacy criteria: clinical cure (efficacy index ≥ 90%); marked efficacy (efficacy index 70%-89%); effective (efficacy index 30%-69%); and ineffective (efficacy index <30%). Efficacy index = (pre-treatment syndrome score - post-treatment syndrome score) / pre-treatment syndrome score × 100%. TCM symptom grading and quantitative criteria were developed in accordance with the "Modern Chinese Medicine Mammology" standard.

[0139] 2 Treatment methods

[0140] The observation group took the Chaishao Xiaozheng granules of the present invention orally (active ingredients: vinegar Bupleurum 10g, white peony root 15g, selfheal 15g, thunbergia thunbergii 12g, vinegar Curcuma 10g, Curcuma 10g, Atractylodes 15g, Poria 15g, raw oyster 30g, Hedyotis diffusa 15g, Schisandra 8g, Ligustrum lucidum 12g, vinegar turtle shell 10g, Astragalus 10g, and preheated Licorice 6g) (granule preparation commissioned by Changsha Xinlin Pharmaceutical Co., Ltd. for trial production), twice a day, and stopped taking it during menstruation; the control group took Hongjin Xiaojie concentrated pills (composed of golden buckwheat, five-spice blood vine, Dahongpao, bupleurum, Panax notoginseng, Cyperus rotundus, eight-angle lotus, woodlic insect, black ant, and Caulis Aconiti Lateralis Preparata, produced by Yunnan Chuxiong Yunzhong Pharmaceutical Co., Ltd., 0.2g×60 pills, national medicine standard Z20080315, batch number 091104) 10 pills, 3 times a day, and stopped taking it during menstruation. Both groups took the medicine for 3 months.

[0141] 3. Observation of therapeutic effects

[0142] 3.1 Comparison of TCM syndrome efficacy between the two groups of patients

[0143] As shown in Table 6, the cure and marked improvement rates of the two groups were compared, and the observation group was superior to the control group (P<0.05).

[0144] Table 6 Comparison of TCM syndrome efficacy between the two groups [cases (%)]

[0145]

[0146] 3.2 Comparison of serum estradiol (E2) and progesterone (P) levels between the two groups before and after treatment

[0147] As shown in Tables 7 and 8, the serum E2 levels of the two groups of patients were significantly decreased after treatment, and the serum P levels were significantly increased compared with those before treatment (P<0.05). Compared with the two groups after treatment, the therapeutic effect of the observation group was significantly better (P<0.05).

[0148] Table 7 Comparison of serum E2 levels before and after treatment in the two groups (pmol / L, x±s)

[0149]

[0150] Note: Compared with the group before treatment, * indicates P < 0.05; compared with the control group after treatment, △ indicates P < 0.05.

[0151] Table 8 Comparison of P content in the two groups before and after treatment (nmol / L, x±s)

[0152]

[0153] Note: Compared with the group before treatment, * indicates P < 0.05; compared with the control group after treatment, △ indicates P < 0.05.

[0154] 3.3 Comparison of breast tumor size between the two groups before and after treatment

[0155] After treatment, the breast lumps in both groups of patients were significantly smaller than those before treatment (P<0.05). Compared with the two groups after treatment, the improvement in the observation group was more obvious (P<0.05), see Table 9.

[0156] Table 9 Comparison of breast mass size under B-ultrasound before and after treatment in the two groups of patients (cm, x ± s)

[0157]

[0158] Note: Compared with the group before treatment, * indicates P < 0.05; compared with the control group after treatment, △ indicates P < 0.05

[0159] 4 Conclusion

[0160] The Chinese medicinal preparation Chaishao Xiaozheng granules of the present invention has satisfactory efficacy in treating breast hyperplasia diseases, can regulate sex hormone levels and reduce lumps under B-ultrasound.

[0161] Finally, the above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for treating breast hyperplasia and precancerous lesions, characterized in that: By weight, the raw material components include: 8-12 parts of vinegar bupleurum, 12-18 parts of white peony root, 12-18 parts of selfheal, 9.5-14.5 parts of thunbergia bulb, 8-12 parts of vinegar zedoaria, 8-12 parts of turmeric, 12-18 parts of atractylodes, 12-18 parts of poria, 12-18 parts of raw oyster, 12-18 parts of hedyotis diffusa, 6.4-9.6 parts of schisandra chinensis, 9.5-14.5 parts of glossy privet fruit, 8-12 parts of vinegar turtle shell, 8-12 parts of astragalus, and 4.8-7.2 parts of roasted liquorice.

2. The Chinese medicine composition according to claim 1, characterized in that The raw material components include: 10 parts of vinegar bupleurum, 15 parts of white peony root, 15 parts of selfheal, 12 parts of thunbergia bulbs, 10 parts of vinegar zedoaria, 10 parts of turmeric, 30 parts of raw oyster, 15 parts of herba hedyotis diffusae, 10 parts of vinegar turtle shell, 10 parts of astragalus, 15 parts of atractylodes, 15 parts of poria, 12 parts of glossy privet fruit, 8 parts of schisandra chinensis, 6 parts of roasted liquorice.

3. The Chinese medicine composition according to claim 1 or 2, characterized in that The raw material components further include: 8-12 parts of bergamot, 8-12 parts of cyperus rotundus, 4.8-7.2 parts of immature bitter orange, and 4.8-7.2 parts of green peel.

4. The Chinese medicine composition according to claim 1 or 2, characterized in that The raw material components further include: 8-12 parts of Rhizoma Alismatis, 8-12 parts of Rhizoma Atractylodis Macrocephalae, 8-12 parts of Cortex Magnoliae Officinalis, and 4.8-7.2 parts of Fructus Amomi.

5. The Chinese medicine composition according to any one of claims 1 to 4, characterized in that in, Bupleurum: White Peony Root: Prunella Vulgaris = 1:1.4-1.6:1.4-1.

6.

6. A method for preparing a Chinese medicine composition for treating breast hyperplasia and precancerous lesions according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, Prunella vulgaris, and Curcuma aromatica were extracted twice with 60% ethanol under reflux, and the filtrates were combined; S2. Decoction of vinegar bupleurum root, white peony root, thunbergia thunbergii, vinegar zedoaria, atractylodes macrocephala, poria, raw oyster, herba hedyotis diffusae, schisandra chinensis, glossy privet fruit, vinegar turtle shell, astragalus root, and roasted liquorice root twice, and concentrate to a concentrate with a relative density of 1.10-1.30; S3. Mix the filtrate obtained in step S1 and the concentrated solution obtained in step S2.

7. The preparation method according to claim 6, characterized in that Vinegar-coated bupleurum root, vinegar-coated zedoary turmeric and vinegar-coated turtle shell are prepared by the following method: bupleurum root, zedoary turmeric and turtle shell are soaked in 6% rice vinegar for 48 hours and then dried at 60°C.

8. The preparation method according to claim 6, characterized in that In step S2, the relative density is 1.

20.

9. A Chinese medicine preparation for treating breast hyperplasia and precancerous lesions, characterized in that: include: An active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the traditional Chinese medicine composition according to any one of claims 1 to 5.

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