Pharmaceutical composition containing selfheal polysaccharide, triterpene and volatile oil and application thereof
Technical Field
The invention relates to a pharmaceutical composition, in particular to a pharmaceutical composition containing selfheal polysaccharide, triterpene and volatile oil.
Background
The genus Prunella is L5 species of perennial herb of Labiatae, and is widely distributed in temperate regions and tropical mountainous regions of Eurasia, northwest of Africa and North America. China is mainly distributed in the river basin of Huaihe river and the middle and lower reaches of Yangtze river, the wild resources of Jiangsu, Anhui, Zhejiang and Henan are most abundant, and the compounds mainly comprise triterpenes and saponins thereof, phenolic acids, sterols and glycosides thereof, flavones, organic acids, volatile oil, saccharides and the like.
The current treatment methods of mammary gland mainly comprise surgical excision, chemotherapy, radiotherapy, endocrine treatment and molecular targeted treatment, but all have great toxic and side effects and influence the life quality of patients. The traditional Chinese medicine for treating breast cancer has unique advantages, selfheal is applied to treatment of hyperplasia of mammary glands since ancient times, but researches and applications of selfheal are mostly focused on extracts and monomer components at present, and active ingredients are not combined and used for treating breast cancer.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a pharmaceutical composition containing selfheal polysaccharide, triterpene and volatile oil and application thereof, aiming at realizing remarkable synergistic effect through compatibility of selfheal monomer components.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a pharmaceutical composition (PPTV, or PTV for short, wherein both PPTV and PTV in the figures of the present specification refer to the same substance) comprising a Polysaccharide (Polysaccharide), a Triterpene (Triterpene) and a Volatile oil (volalite oil) of prunella vulgaris.
The prunella vulgaris polysaccharide, the triterpenes and the volatile oil are effective components which can be extracted by a conventional extraction means in the field, and the content of the effective components in corresponding extracts is not less than 6 percent, and the extraction method is not limited additionally, and only the following optional extraction methods are provided:
(1) selfheal polysaccharide: crushing selfheal, weighing 500g of water, heating and refluxing for 4 times in an amount which is 10 times that of the water, combining extracting solutions, centrifuging at 4800r/min to remove impurities, concentrating under reduced pressure to 2L, adding 95% ethanol to enable the final concentration of the ethanol to reach 70%, carrying out ethanol precipitation in a refrigerator at 4 ℃ for 24h, carrying out centrifugal separation (4800r/min, 10min), discarding supernate, sequentially washing obtained precipitates with absolute ethyl alcohol, acetone and absolute ethyl ether twice respectively, and carrying out vacuum drying at 55 ℃ to obtain crude selfheal polysaccharide. The extraction rate is about 7.5% -9.5%.
(2) Prunellae Spica triterpene: pulverizing Prunellae Spica, heating and reflux-extracting with 500g water 10 times, mixing extractive solutions, centrifuging at 4800r/min to remove impurities, and concentrating under reduced pressure to 800 ml. Separating the concentrated solution by macroporous resin, eluting by 75% ethanol, collecting the eluent, concentrating under reduced pressure to obtain extract, freeze-drying, storing at-20 deg.C for later use, and extracting at 6% -7%.
(3) Selfheal volatile oil: pulverizing Prunellae Spica with pulverizer, and adding 200g into supercritical fluid (CO)2) An extraction device, wherein the extraction temperature is 35 ℃, and the extraction pressure is 15-18 MPa; the temperature of the separation kettle 1 is 35-40 ℃, and the separation pressure is 7-9 MPa; the temperature of the separation kettle is 2 ℃ to 30 ℃, the separation pressure is 5-7MPa, the extraction time is 3h, and an entrainer is not used. Using the same extraction conditions, recharging and repeatedly extracting once, combining the two extracted volatile oils, and storing in a refrigerator at-20 ℃ for later use, wherein the extraction rate is about 4-6%.
The invention discovers through experimental research that the selfheal polysaccharide, the triterpenes and the volatile oil can obviously improve the antiproliferative activity and the antitumor activity after being used together, can effectively induce and promote the apoptosis of breast cancer cells, and can enhance the immunologic function of an organism. The compatibility can enhance the antitumor activity of the single medicine, so the compatibility can also effectively reduce the dosage of the medicine and reduce the adverse reaction of the medicine.
Preferably, the compatibility proportion of the selfheal polysaccharide, the triterpenoids and the volatile oil is 3-5: 1-3: 1-2.
More preferably, the compatibility proportion of the selfheal polysaccharide, the triterpene and the volatile oil is 3:2: 1.3. The drug was added to the feed and the administration was started one week before the model creation, 4T1 cells were injected subcutaneously into the right fat pad of BALB/c mice (6-8 weeks old) to establish a mouse breast cancer model, and tumor growth and mouse body weight were recorded every 3 days. After sacrifice of the neck, tumor tissues, spleen and thymus were harvested and organ indices were calculated. The mouse body mass was not significantly changed compared to the normal group; the growth rate of the mouse tumor is obviously reduced, which shows that PPTV can inhibit the proliferation rate of the tumor-bearing mouse tumor. Thymus indexes of the normal group, the model group and the treatment group are similar; after the treatment of the medicine, the spleen of the mouse is obviously enlarged, which shows that PPTV can enhance the immune system of the mouse, delay the development process of tumor through the autoimmune system, balance the immune microenvironment in the organism and fundamentally change the growth and living environment of tumor cells. In addition, an important characteristic in the process of generating the tumor is cell apoptosis and proliferation imbalance, therefore, the recovery of the proportion of induced cell apoptosis and proliferation is a good treatment strategy for treating the tumor.
Therefore, the invention provides the application of the pharmaceutical composition in preparing a medicine for inducing breast cancer cell apoptosis.
The invention also provides application of the pharmaceutical composition in preparing a medicine for promoting breast cancer cell apoptosis.
The invention also provides application of the pharmaceutical composition in preparing anti-cell proliferation medicines.
The invention also provides application of the pharmaceutical composition in preparing antitumor drugs. The tumor is preferably breast cancer.
The invention also provides application of the pharmaceutical composition in preparing a medicine for improving immunity.
The invention can obviously enhance the anti-tumor activity when used alone after the three components are compatible (PPTV), and presents time-dose dependence; and (3) proving that: PPTV has good anti-tumor activity. The medicine is used for treating a mouse breast cancer model, has good treatment effect, obviously inhibits the growth speed of tumor, and can obviously reduce tumor metastasis.
It should be noted that the above-mentioned preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The invention has the beneficial effects that:
the invention provides a pharmaceutical composition (PPTV) containing selfheal polysaccharide, triterpene and volatile oil, the PPTV can be used for treating breast cancer, a new application method of the selfheal is developed, a new choice is provided for medicaments for treating the breast cancer, the PPTV can inhibit the growth of tumors, and can induce the apoptosis of tumor cells; the composition enhances the immune system of an organism, delays the development process of tumors through the autoimmune system, balances the immune microenvironment in the organism, fundamentally changes the growth and living environment of tumor cells, has great potential in the aspect of tumor resistance after the compatibility of active parts of the selfheal, can be used as a new potential choice for treating breast cancer, and is worthy of further research and exploration.
Drawings
FIG. 1 shows the effect of Prunella vulgaris polysaccharides, triterpenes, volatile oils and PPTV treated cells for 48h on breast cancer cells in Experimental example 1.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1A pharmaceutical composition
1. Raw materials:
selfheal polysaccharide: crushing selfheal, weighing 500g of water, heating and refluxing for 4 times in an amount which is 10 times that of the water, combining extracting solutions, centrifuging at 4800r/min to remove impurities, concentrating under reduced pressure to 2L, adding 95% ethanol to enable the final concentration of the ethanol to reach 70%, carrying out ethanol precipitation in a refrigerator at 4 ℃ for 24h, carrying out centrifugal separation (4800r/min, 10min), discarding supernate, sequentially washing obtained precipitates with absolute ethyl alcohol, acetone and absolute ethyl ether twice respectively, and carrying out vacuum drying at 55 ℃ to obtain crude selfheal polysaccharide. The extraction rate is about 7.5% -9.5%.
Prunellae Spica triterpene: pulverizing Prunellae Spica, heating and reflux-extracting with 500g water 10 times, mixing extractive solutions, centrifuging at 4800r/min to remove impurities, and concentrating under reduced pressure to 800 ml. Separating the concentrated solution by macroporous resin, eluting with 75% ethanol, collecting the eluate, concentrating under reduced pressure to obtain extract, freeze drying with extraction rate of 6% -7%, and storing at-20 deg.C for use.
Selfheal volatile oil: pulverizing Prunellae Spica with pulverizer, and adding 200g into supercritical fluid (CO)2) An extraction device, wherein the extraction temperature is 35 ℃, and the extraction pressure is 15-18 MPa; the temperature of the separation kettle 1 is 35-40 ℃, and the separation pressure is 7-9 MPa; the temperature of the separation kettle is 2 ℃ to 30 ℃, the separation pressure is 5-7MPa, the extraction time is 3h, and an entrainer is not used. Using the same extraction conditions, recharging and repeatedly extracting once, combining the two extracted volatile oils, and storing in a refrigerator at-20 ℃ for later use, wherein the extraction rate is about 4-6%.
2. The preparation method comprises the following steps:
the extraction rates of the prunella vulgaris polysaccharide, the triterpenoids and the volatile oil are respectively 9.3%, 6.2% and 4.3%, and the three components are mixed according to the proportion, namely 3:2:1.3 combining to obtain the medicine. Specifically, the method comprises the following steps: weighing polysaccharide, triterpene and volatile oil in proportion, and dissolving polysaccharide and triterpene in pure water; adding trace amount of tween-80 into the volatile oil to assist dissolution (the final concentration of tween-80 is not more than 0.1%); mixing the three components evenly to obtain an experimental sample.
Example 2
The difference between the embodiment and the embodiment 1 is that the contents of the effective components of the prunella vulgaris polysaccharide, the triterpenes and the volatile oil are calculated according to the extraction rate, and the medicine is obtained according to the advantages and disadvantages with the effective content ratio of 5:1: 2.
Example 3
The difference between the embodiment and the embodiment 1 is that the contents of the effective components of the prunella vulgaris polysaccharide, the triterpenes and the volatile oil are calculated according to the extraction rate, and the medicine is obtained according to the advantages and disadvantages with the effective content ratio of 3:3: 1.
Experimental example 1
The antiproliferative activity of the drug is detected by MTT method.
Taking MCF-7 cells (1 × 10) in logarithmic growth phase5One/well) were seeded in 96-well plates and incubated for 24h until the cells attached. Adding concentration gradient Prunellae Spica polysaccharide, Prunellae Spica triterpene, Prunellae Spica volatile oil and the pharmaceutical composition of example 1, treating cells for 48 hr, removing the culture medium containing medicine, adding 100ul 0.5mg/ml MTT into each well, incubating at 37 deg.C for 4 hr, discarding the culture medium, and adding DMSO to dissolve crystal. And detecting the light absorption value A at 490nm of the microplate reader, repeating the experiment for 3 times, and calculating the cell survival rate.
Cell viability was ═ 100% (experimental a-blank a)/(control a-blank a).
The test results are shown in FIG. 1. The results show that: after the selfheal polysaccharide, the triterpene and the volatile oil are compatible, the anti-tumor activity is obviously enhanced, and the dosage of the medicament can be reduced.
Experimental example 2
Babl/c mice, female, SPF grade, body mass 18-22g, provided by the Silikedada laboratory animal center, Hunan province, animal license number SCXK Xiang 2014-. Adaptive feeding for 1 week, and starting preventive administration one week before molding. Mu.l (1.0X 10)6Individual cells) 4T1 cells were injected subcutaneously into the right fat pad of BALB/c mice (6-8 weeks old), and the mice were randomly divided into a control group, a positive group, a high dose (4g/kg) administration group and a low dose (2g/kg) administration group (10 mice per group), and administration was continued for 25 days. Tumor volume was measured on day 7 after molding, and thereafter tumor volume and mouse body weight were measured every 3 days. Tumor volume (mm) in mice3) When mice were sacrificed by cervical dislocation, tumor and lung tissues as well as spleen and thymus were taken and spleen and thymus indices were calculated as 0.52 xl × W2.
Organ index is the organ weight g/body weight g × 10.
Experimental example 3
HE staining examined pathological changes: the tumor tissue is picked up, the tumor tissue is fixed in 4 percent paraformaldehyde for 24 hours, the fixed tissue block of the target part is trimmed and leveled, running water washing is carried out for about 12 hours, and then the tissue soaked with wax is embedded in an embedding machine. And after cooling and solidification, taking the wax block out of the embedding frame and trimming the wax block. The slices were taken in a paraffin microtome and were 4 μm thick. Hematoxylin-eosin (H & E) staining, histopathological examination of tumor tissue sections. The pathological structural changes of the tumor tissue were observed using a microscope to take 200 × images.
Experimental example 4
Detecting in situ apoptosis with TUNEL, fixing tumor tissue in 4% paraformaldehyde for 24h, embedding paraffin, slicing, deparaffinizing with gradient alcohol, adding distilled water for hydration, digesting with 20 μ g/ml proteinase K at 37 deg.C for 30min, washing with PBS for 3 times, adding 50 μ l TUNEL staining solution dropwise, incubating at 37 deg.C for 30min, washing with PBS for 3 times, adding 50 μ l alkaline phosphatase coupled anti-fluorescent antibody (CoverAP), incubating at 37 deg.C for 30 min; after 3 times of PBS washing, 50 mu l/piece of substrate DAB (diamino diphenyl ammonia) is added, color development is carried out for 10min, and after dehydration, resin sealing is carried out, and a picture is taken by observing under a microscope.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.