CN107496502B

CN107496502B - Application of prunella vulgaris volatile oil in preparation of medicine for treating hysteromyoma

Info

Publication number: CN107496502B
Application number: CN201710684360.1A
Authority: CN
Inventors: 林丽美; 廖端芳; 杨超; 吴萍
Original assignee: Hunan University of Chinese Medicine
Current assignee: Hunan Jialin Stem Cell Technology Co.,Ltd.
Priority date: 2017-08-11
Filing date: 2017-08-11
Publication date: 2020-07-07
Anticipated expiration: 2037-08-11
Also published as: CN107496502A

Abstract

The invention relates to a traditional Chinese medicine extract, and particularly discloses a new application of selfheal volatile oil in treating hysteromyoma. The main body is as follows: the application of the prunella vulgaris volatile oil in preparing the medicines for treating the hysteromyoma and the medicines for inhibiting the proliferation of the hysteromyoma cells. The invention also provides a medicament for treating hysteromyoma and a medicament for inhibiting hysteromyoma cell proliferation, wherein the medicaments contain the selfheal volatile oil. The invention develops new application of the selfheal volatile oil and provides a new choice for the medicine for treating hysteromyoma.

Description

Application of prunella vulgaris volatile oil in preparation of medicine for treating hysteromyoma

Technical Field

The invention relates to a traditional Chinese medicine extract, in particular to application of selfheal volatile oil in treating hysteromyoma.

Background

Prunella vulgaris L, which is known as withered after summer. The Prunella plants have upright stems with four edges, opposite single leaves, lip-shaped corolla, strong stamen, 2 carpels, 4 small nuts, a flowering period of 4-6 months and a fruit period of 7-10 months, and mainly grow on sparse forests, barren mountains, ridges and roadside roads. The self-heal has been applied in China for thousands of years, half-dry clusters are generally used as medicines in summer, and the self-heal is recorded in the book of the earliest existing pharmaceutical monograph 'Shennong herbal Jing' in China: prunellae Spica is bitter and pungent in flavor and cold in nature, and has the symptoms of scrofula, murine fistula, head trauma, laceration, goiter, beriberi, foot swelling, damp arthralgia and weight loss. One sunset, one jingdong. Raw Sichuan valley ". (Sun Feng Yi, Shennong Ben Cao Jing, inner Mongolia: inner Mongolian people's publishing house, 2006.) thereafter, the Chinese ancient herbal has collected the selfheal, and the effect of single drug administration is significant, for example, Taiping Shenghui Fang, Ben Cao gang mu and Ben Cao gang mu Shi Yi. Since the 1963 edition of pharmacopeia, the ancient pharmacopeia of China has collected selfheal, and the medicinal part is definitely half-dried or mature fruit cluster (the Chinese pharmacopeia, the Chinese pharmaceutical science and technology publisher, 2015.). Moreover, the selfheal is also one of the first medical and edible traditional Chinese medicines published by Ministry of health, and is safe and reliable.

The traditional effects of selfheal are clearing heat, improving eyesight, reducing swelling and dissipating stagnation. Aiming at the traditional efficacy, a series of researches are carried out, and the traditional Chinese medicine composition has the efficacies of resisting virus, resisting bacteria, resisting inflammation, resisting oxidation, resisting tumor, reducing blood pressure, reducing blood sugar and the like (Liuyue and the like, the research progress of the chemical components and the biological activity of the selfheal, J, Shenyang pharmaceutical university report, 2003, 20(1): 55-59.).

The main components of the prunella spike volatile oil are aliphatic compounds such as 1, 6-cyclodecanone diene, hexadecanoic acid, hexadecane and the like (Tianjing and the like, the structural identification of the prunella spike saponin A [ J ] in the pharmaceutical science report, 2000, 35(1): 29-31.). It was found that, from Prunella vulgaris, 40 volatile oil Compounds, mainly aliphatic aldehydes and alcohols, aromatic aldehydes, and small amounts of sesquiterpenes and monoterpene hydrocarbons were identified by GC-MS analysis, in which (E, E) -2, 4-heptadienal and phenylacetaldehyde were the main components and the contents were 17.8% and 13.2%, respectively (Jerkovic I, et al. phytochemical composition of the scientific of the cellular grandiflora [ J ]. Chemistry of Natural Compounds, 2013, 49(2): 371.). The method comprises the steps of extracting ursolic acid from prunella vulgaris clusters by using a supercritical carbon dioxide fluid extraction technology, and determining the content of the ursolic acid in an extract by using a high performance liquid chromatography (Jiansheng rock, Penchu peach, Zhao Liang Zhong, and the like, research on the supercritical extraction of the ursolic acid from the prunella vulgaris clusters [ J ]. Shaoyang academy of academic (Nature science edition), 2010, 7(1): 48-52.).

The spica prunellae volatile oil compounds enable plants to have strong aromatic odor and have the effects of sterilization, disinfection, analgesia, antivirus and the like (Yuandong plum, Yangyuan silk, Yan Shicai, the study on the chemical components of the pistacia chinensis bunge leaf volatile oil [ J ] the northwest plant bulletin, 2005, 25(12): 2539-2542.). Ursolic Acid and oleanolic Acid In volatile oil active ingredients of Prunella vulgaris have effects of inhibiting tumor cell proliferation and inducing Apoptosis, thereby achieving anti-tumor effect (Wang X, et al. Ursolic Acid inhibition and Apoptosis of Cancer Cells In Vitro and In Vivo [ J ]. Journal of Biomedicine & Biotechnology, 2011, 2011(1): 419343; Shuyu M H, Kao T C, Yen G C.Oleanolic Acid and Ursolic Acid index Apoptosis In H7 Humanopatocellular Carcinoma Cells through Mitochia drial-dependen Pathway and downlink regulation of Down [ J. Journal of Agricultural Chemistry & chemical 6110): 2010 (58). Besides ursolic acid and oleanolic acid, the prunella vulgaris volatile oil also has other triterpenoids and saponin compounds thereof with obvious anti-tumor effect: the total triterpenes and phenolic acids after the selfheal purification can obviously inhibit the growth of SPC-A-1 cells and tumors and show that the lung cancer resistance is realized by the synergistic effect of a plurality of compounds (Feng L, et al. combination of active components industries of lung cancer [ J ] Molecules, 2010, 15(11): 7893) 7906.).

The study on the prunella vulgaris volatile oil mainly focuses on the study on the aspects of antibiosis, anti-inflammation and the like, but the study on the aspect of tumor resistance is not deep enough; although the treatment of the hysteromyoma has the means of medicament treatment, operation treatment, interventional therapy and the like, the side effects of the means are great, and the female reproductive health is greatly influenced. Therefore, it is highly desirable to provide a safe and effective drug for treating uterine fibroids.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a new application of selfheal volatile oil in the aspect of treating hysteromyoma.

The invention finds the inhibition effect of the selfheal volatile oil on the proliferation of human hysteromyoma cells and the effect of treating the hysteromyoma; and identifying the chemical components of the selfheal volatile oil.

The prunella spike volatile oil is volatile oil substances extracted according to a conventional method in the field.

Preferably, a super zero boundary fluid (CO) is used₂) The extraction is carried out, and specifically comprises the following steps: pulverizing Prunellae Spica, weighing appropriate amount of pulverized Prunellae Spica (such as 200g), and placing in supercritical fluid (CO)₂) An extraction device, wherein the extraction temperature is 35 ℃, and the extraction pressure is 15 MPa; the temperature of the separation kettle 1 is 35 ℃, and the separation pressure is 7 MPa; the temperature of the separation kettle is 2 ℃ and 30 ℃, the separation pressure is 5MPa, the extraction time is 2h, and no entrainer is used. Repeating the extraction once under the same extraction conditions, and combining the two extractionsAnd (5) putting the volatile oil into a refrigerator at the temperature of-20 ℃ for storage for later use. Using a super zero boundary fluid (CO)₂) The extraction has the advantages of no interference of solvent and ensured purity of the medicine.

The invention discovers that the selfheal volatile oil has obvious inhibition effect on proliferation of the uterine fibroid cells through experimental research; promoting apoptosis of human hysteromyoma cells; has effect in inhibiting cell cycle of human hysteromyoma; has therapeutic effect on uterus myoma of SD rat.

Moreover, the invention also determines the chemical components of the selfheal volatile oil through experimental research.

The invention adopts GC-MS to carry out component analysis on the grass volatile oil:

the gas chromatography conditions were HP-5 elastic quartz capillary column, specification 30m × 250 μm × 0.25.25 μm, Agilent Inc., USA, initial temperature 100 deg.C, holding for 2.0 min, then rising to 270 deg.C at 5.00 deg.C/min, holding for 10min, carrier gas being helium with 99.99% purity, column flow 1.00mL/min, sample introduction 1.0 μ L, sample inlet temperature 270 deg.C, and quantification by area normalization.

Mass spectrum conditions: the ionization source is EI, the ionization energy is 70eV, the interface temperature is 280 ℃, and the ion source temperature is 200 ℃; the scanning range is 35-550 m/z. The standard spectral library is NISTOS and NISTOSs spectral library.

The total ion flow diagram and the mass spectrogram of each component of the sample volatile oil are obtained by gas chromatography-mass spectrometry, and each chemical component is analyzed and determined by NISTOS and NISTOSs spectral library retrieval and artificial spectrum solution, which is detailed in Table 1.

TABLE 1 Prunellae Spica volatile oil chemical composition

The chemical component analysis of the selfheal volatile oil shows that: the volatile oil contains squalene (28.03%), linoleic acid (9.96%), linolenic acid (9.95%), stearic acid (6.26%) and oleic acid (5.51%).

Based on the invention, the technical scheme of the invention is as follows:

in a first aspect, the invention provides an application of selfheal volatile oil in preparing a medicament for treating hysteromyoma.

And the application of the prunella vulgaris volatile oil in preparing the medicine for inhibiting the proliferation of the hysteromyoma cells.

In a second aspect, the invention also provides a medicament for treating hysteromyoma and a medicament for inhibiting the proliferation of hysteromyoma cells, wherein the medicaments contain the selfheal volatile oil disclosed by the invention.

The invention has the beneficial effects that:

the invention discovers the new application of the selfheal volatile oil in the aspect of treating the hysteromyoma for the first time, and provides a new choice for the medicine for treating the hysteromyoma.

Drawings

FIG. 1 is a curve showing the inhibition of Prunella vulgaris volatile oil on uterine leiomyoma cells in example 1 of the present invention.

FIG. 2 shows the inhibition rate of the volatile oil of Prunella vulgaris in example 1 on the proliferation of uterine leiomyoma cells.

FIG. 3 shows the result of apoptosis test after 24h of the Prunella vulgaris volatile oil with different concentrations on uterine leiomyoma cells in example 1.

FIG. 4 shows the result of apoptosis test after 48h of the Prunella vulgaris volatile oil with different concentrations on uterine leiomyoma cells in example 1.

FIG. 5 shows the result of apoptosis test after 72 hours of the Prunella vulgaris volatile oil with different concentrations acts on uterine leiomyoma cells in example 1 of the present invention.

FIG. 6 shows the cell cycle test results of 24h after the Prunella vulgaris volatile oil with different concentrations acts on uterine leiomyoma cells in example 1 of the invention.

FIG. 7 shows the cell cycle test results of 48h after the Prunella vulgaris volatile oil with different concentrations acts on uterine leiomyoma cells in example 1 of the invention.

FIG. 8 is a cell cycle test result of 72 hours after the Prunella vulgaris volatile oil with different concentrations acts on uterine leiomyoma cells in example 1 of the invention.

FIG. 9 is a graph showing HE staining of SD rat with Prunella vulgaris volatile oil of different concentrations after gastric lavage for 14 days in 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 1

The selfheal adopted by the invention is purchased in the GAP base of the selfheal of Anhui; high glucose medium DMEM (HyClone, 500mL/AB 216498); MTT (Sigma, 250 mg/M2128); foetal bone Serum (Gibco, 500 mL/20030116); trypsin Solution 0.25% (1X) (HyClone, 100 mL/J150018); PhosphateBuffered salt (1X) (HyClone, 500mL/AB 218110); annexin V-FITC/PI apoptosis kit (Union, 1mL/AP 101); PI Solution (Union organism, 1 mL/ZF-50-0001).

The invention sets seven concentration gradients (2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL, 6.0mg/mL, 7.0mg/mL and 8.0mg/mL) for the selfheal volatile oil, sets five compound holes for each concentration, measures the cell activity (OD) of uterine smooth muscle cells by adopting an MTT method after measuring the effect of the selfheal volatile oil, obtains a cytotoxicity trend line by taking the drug concentration as a horizontal coordinate and the absorbance as a vertical coordinate, and calculates the IC trend line of the selfheal volatile oil for the uterine smooth muscle cells by using the spss18.0₅₀. The results are shown in FIG. 1.

As can be seen from FIG. 1, the OD value of the uterine smooth muscle cells gradually increased with the increase of the concentration of the Prunella vulgaris volatile oilGradually decreases, when the concentration of the selfheal volatile oil reaches 6.0mg/ml, the OD value of the uterine smooth muscle cells tends to be linear and close to 0, which shows that the toxicity to the uterine smooth muscle cells is larger when the concentration of the selfheal volatile oil exceeds 6.0mg/ml, and the IC of the selfheal volatile oil on the human uterine smooth muscle cells is measured by using SPSS18.0₅₀＝4.58mg/mL。

The invention sets eight concentration gradients (1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL, 6.0mg/mL, 7.0mg/mL, 8.0mg/mL) for the selfheal volatile oil, sets five compound holes for each concentration, measures the cell activity (OD) of uterine smooth muscle cells by adopting an MTT method after 24 hours, 48 hours and 72 hours of the selfheal volatile oil action, obtains a cytotoxicity trend line (figure 2) by taking the drug concentration as an abscissa and the absorbance as an ordinate, and calculates the IC (integrated Circuit) of the selfheal volatile oil for the uterine leiomyoma cells by using a sps 18.0₅₀。

TABLE 2 inhibition of proliferation of uterine leiomyoma cells by volatile oil of Prunella Spica

The cell proliferation inhibition rate was 100% (1-drug group a (490 nm)/blank control group a (490nm) × 100%.

The IC of the selfheal volatile oil which acts on the human uterine leiomyoma cells for 24 hours is measured by the experimental data of tumor inhibition rate and the SPSS18.0₅₀2.61 mg/mL; IC acting for 48h₅₀2.48 mg/mL; function for 72h IC₅₀1.94 mg/mL. Prunellae Spica volatile oil IC with toxicity on uterine leiomyoma cells₅₀The concentration is far lower than that of human uterine smooth muscle cell IC₅₀(4.58mg/mL), which shows that the toxicity of the selfheal volatile oil concentration causing the death of the uterine leiomyoma cells to normal uterine smooth muscle cells is very low, and the inhibition rate of the uterine leiomyoma cells is increased along with the increase of the concentration of the selfheal volatile oil.

The invention sets the concentration gradient (0, 0.25 × IC) of the selfheal volatile oil₅₀、0.5×IC₅₀、0.75×IC₅₀、1.0×IC₅₀) After respectively acting on uterine leiomyoma cells for 24h, 48h and 72h, collecting the cellsThe apoptosis was measured by flow cytometry using FITC and PI double staining, and the results are shown in FIGS. 3 to 5.

As can be seen from figure 3, the prunella spike volatile oil has apoptosis induction effect on hysteromyoma cells after 24 hours of action, and the apoptosis rate is 0.25 × IC₅₀＝15.39％、0.5×IC₅₀＝21.41％、0.75×IC₅₀＝44.18％、1.0×IC₅₀57.40%, the apoptosis effect is obvious.

As can be seen from figure 4, the prunella vulgaris volatile oil has apoptosis induction effect on hysteromyoma cells for 48h, and the apoptosis rate is 0.25 × IC₅₀＝18.08％、0.5×IC₅₀＝27.29％、0.75×IC₅₀＝37.76％、1.0×IC₅₀64.06%, the apoptosis effect is obvious.

As can be seen from figure 5, the prunella vulgaris volatile oil has apoptosis inducing effect after 72h of action on hysteromyoma cells, and the apoptosis rate is 0.25 × IC₅₀＝20.98％、0.5×IC₅₀＝38.97％、0.75×IC₅₀＝50.75％、1.0×IC₅₀78.39%, the apoptosis effect is obvious.

The invention sets the concentration gradient (0, 0.25 × IC) of the selfheal volatile oil₅₀、0.5×IC₅₀、0.75×IC₅₀、1.0×IC₅₀) After 24h, 48h and 72h of action on the uterine leiomyoma cells, the cells are collected, and the cell cycle is determined by a flow cytometer by adopting a PI single staining method, and the results are shown in figures 6-8.

After the prunella vulgaris volatile oil is acted on uterine leiomyoma cells for 24 hours, 48 hours and 72 hours, the proportion of the uterine leiomyoma cells in G0-G1 phases is obviously increased along with the increase of the concentration, which indicates that the prunella vulgaris volatile oil can block the cells in the G0-G1 phases, so that the cells can not enter the G2 phases for DNA synthesis, and finally the in-vitro proliferation of tumor cells is inhibited.

In conclusion, the prunella vulgaris volatile oil can inhibit the proliferation of the human uterine leiomyoma cell cycle by blocking the cell cycle of the human uterine leiomyoma in G0-G1; the anti-uterine leiomyoma effect is exerted by inducing apoptosis of human uterine leiomyoma cells.

The invention adopts SD rat hysteromyoma model, sets the high, medium and low concentrations of selfheal volatile oil to be 4.58mg/mL, 2.29mg/mL and 1.15mg/mL respectively, performs gastric lavage treatment on SD model rat for 14 days at 2.0 mL/mouse/day, performs HE staining after the treatment course and compares with the model group, and the result is shown in figure 9.

According to HE staining, the high and low concentrations of the volatile oil of the selfheal have obvious proliferation inhibiting effect on hysteromyoma, reduce myoma cells and have certain treatment effect on hysteromyoma.

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.

Claims

1. The application of the prunella vulgaris volatile oil in preparing the medicine for treating hysteromyoma is characterized in that the preparation method of the prunella vulgaris volatile oil comprises the following steps: pulverizing Prunellae Spica, weighing the pulverized Prunellae Spica, and adding supercritical fluid CO₂An extraction device, wherein the extraction temperature is 33-37 ℃, and the extraction pressure is 14-18 MPa; the temperature of the separation kettle 1 is 30-33 ℃, and the separation pressure is 9-11 MPa; separating at 26-30 deg.C under 5-7MPa for 2 hr, repeatedly extracting under the same extraction conditions, and mixing the two extractive volatile oils.

2. The application of the prunella vulgaris volatile oil in preparing the medicine for inhibiting the proliferation of hysteromyoma cells is characterized in that the preparation method of the prunella vulgaris volatile oil comprises the following steps: pulverizing Prunellae Spica, weighing the pulverized Prunellae Spica, and adding supercritical fluid CO₂An extraction device, wherein the extraction temperature is 33-37 ℃, and the extraction pressure is 14-18 MPa; the temperature of the separation kettle 1 is 30-33 ℃, and the separation pressure is 9-11 MPa; separating at 26-30 deg.C under 5-7MPa for 2 hr, repeatedly extracting under the same extraction conditions, and mixing the two extractive volatile oils.

3. A medicine for treating hysteromyoma is characterized byThe prunella spike volatile oil is prepared by the following steps: pulverizing Prunellae Spica, weighing the pulverized Prunellae Spica, and adding supercritical fluid CO₂An extraction device, wherein the extraction temperature is 33-37 ℃, and the extraction pressure is 14-18 MPa; the temperature of the separation kettle 1 is 30-33 ℃, and the separation pressure is 9-11 MPa; separating at 26-30 deg.C under 5-7MPa for 2 hr, repeatedly extracting under the same extraction conditions, and mixing the two extractive volatile oils.

4. The medicine for inhibiting the proliferation of hysteromyoma cells is characterized by containing selfheal volatile oil, and the preparation method of the selfheal volatile oil comprises the following steps: pulverizing Prunellae Spica, weighing the pulverized Prunellae Spica, and adding supercritical fluid CO₂An extraction device, wherein the extraction temperature is 33-37 ℃, and the extraction pressure is 14-18 MPa; the temperature of the separation kettle 1 is 30-33 ℃, and the separation pressure is 9-11 MPa; separating at 26-30 deg.C under 5-7MPa for 2 hr, repeatedly extracting under the same extraction conditions, and mixing the two extractive volatile oils.