CN114288333B - Rosemary compound essential oil for inhibiting skin melanoma cell proliferation and application thereof - Google Patents
Rosemary compound essential oil for inhibiting skin melanoma cell proliferation and application thereof Download PDFInfo
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Abstract
The invention provides rosemary compound essential oil for inhibiting skin melanoma cell proliferation and application thereof in preparing a medicament for inhibiting skin melanoma cell proliferation. The rosemary compound essential oil for inhibiting skin melanoma cell proliferation provided by the invention comprises the following raw materials in parts by weight: 25-30 parts of alpha-pinene type rosemary essential oil, 10-15 parts of verbenone type rosemary essential oil and 5-8 parts of 1, 8-cineole type rosemary essential oil. IC for melanoma cells B16-F10 by comparing different chemical types of essential oils 50 And carrying out component proportion of essential oils of different chemical types, and finally screening out a compound essential oil formula with the strongest inhibition effect on B16-F10 proliferation; further, the influence of different concentrations of the rosemary compound essential oil on cell cycle change is explored through experiments, and the remarkable inhibition effect of the rosemary compound essential oil on melanoma cells is proved.
Description
Technical Field
The invention belongs to the field of pharmacology of natural plant extracts, and particularly relates to a new application of rosemary compound essential oil in inhibiting melanoma cell proliferation.
Background
Melanoma (melanoma) is a highly malignant tumor derived from malignant melanocytes, and has become one of the most rapidly growing malignant tumors worldwide. Research shows that Chinese skin malignant tumor, especially malignant melanoma, has a trend of increasing obviously in recent 20 years. At present, essential oil is used as a non-invasive treatment method with less risk, which can potentially improve the life quality of cancer patients and relieve the symptoms of chemotherapy of the cancer patients, and simultaneously relieve side effects and improve the prognosis condition. The rosemary essential oil has good activities of oxidation resistance, inflammation resistance and the like, has a certain antiproliferative effect on breast cancer, lung cancer, colon cancer, bladder cancer and cervical cancer cells, and can promote the apoptosis of liver cancer cells.
At present, rosemary essential oil has few and not deep enough anticancer researches, and the cell viability is detected by an MTT method after essential oil is treated for 24 hours or 48 hours at different concentrations and the IC is calculated 50 . Through an MTT method, the rosemary essential oil is found to have an antiproliferative effect on human breast cancer cells MDA-MB-231, lung adenocarcinoma cells A549, human non-small cell lung cancer cells H1299, human breast cancer cells MCF-7, cervical cancer HeLa, human T lymphoma cells Jurkat, human colon cancer cells HT-29 and human bladder cancer cells T24, can enhance the expression of bax in liver cancer cells HepG2, reduce the expression of bcl-2, induce apoptosis of the cells, and cause different influences on tumor cells due to the difference of essential oil components. However, the inhibition effect of rosemary volatile essential oil on melanoma cells is not reported at present, and only the influence of a nonvolatile component rosmarinic acid and the like on skin melanoma cell generation is existed. In addition, the essential oil related to the reported rosemary essential oil cancer inhibition technology has the defects of low utilization rate, single chemical type, poor effect and the like, and is prepared by adopting different chemical typesThe prepared compound essential oil is rare.
Disclosure of Invention
Aiming at the problems, the invention provides application of rosemary compound essential oil in a medicine for inhibiting skin melanoma cell proliferation and a preparation method thereof. The method mainly comprises the following steps: (1) extracting rosemary essential oil; (2) analyzing essential oil components; (3) preparing compound essential oil; (4) analyzing the components of the compound essential oil; (5) the concentration range used was determined in conjunction with toxicity experiments for melanoma cells B16-F10 and HaCaT.
The invention aims to provide a method for screening rosemary essential oil formula and concentration with stronger anticancer activity by treating different chemical rosemary essential oil aiming at melanoma cells, taking normal skin cell HaCaT as a control and analyzing cell cycle change to prove the effect of the rosemary essential oil on inhibiting the proliferation of the melanoma cells, simultaneously preparing different chemical rosemary essential oil into rosemary compound essential oil, treating the melanoma cells again, taking normal skin cell HaCaT as a control and analyzing the cell cycle change.
Firstly, the invention provides application of rosemary compound essential oil in a medicine for inhibiting skin melanoma cell proliferation and a preparation method thereof.
The technical scheme adopted by the invention is as follows:
1. the rosemary compound essential oil comprises the following raw materials in parts by weight: 25-30 parts of alpha-pinene type rosemary essential oil, 10-15 parts of verbenone type rosemary essential oil and 5-8 parts of 1, 8-cineole type rosemary essential oil.
The rosemary essential oil is obtained by extracting the overground part (including rosemary branches, leaves and flowers) of rosemary by a steam distillation method, wherein the chemical alpha-pinene type essential oil, the verbenone type essential oil and the 1, 8-cineole type essential oil comprise the following main components:
the main components of the alpha-pinene type rosemary essential oil are 28.32 percent of alpha-pinene, 20.48 percent of 1, 8-cineole, 14.21 percent of verbenone and 5.22 percent of borneol;
the verbenone type rosemary essential oil mainly comprises 24.46 percent of verbenone, 10.48 percent of 1, 8-cineole, 7.12 percent of alpha-terpineol and 5.88 percent of borneol;
the 1, 8-cineole type rosemary essential oil mainly comprises 25.46% of 1, 8-cineole, 13% of verbenone, 11.96% of alpha-pinene and 11.38% of camphor.
The main components of the rosemary compound essential oil are 18.56-20.59% of alpha-pinene, 16.70-20.26% of verbenone, 17.70-19.06% of 1, 8-cineole and 5.54-5.99% of borneol.
In another technical scheme of the invention, after the raw materials are mixed according to the mass part, the compound essential oil is subjected to nano crushing, homogenizing, dispersing and sieving to form nano particles with the particle size of 10-30 nm.
Respectively dissolving 3 kinds of chemical essential oil and the prepared compound essential oil in dimethyl sulfoxide (DMSO), uniformly swirling at the speed of 250-500 r/min by using a swirl mixer, filtering by using a sterile organic system filter membrane, diluting by using a DMEM culture medium, and preparing into a cell culture medium with the concentration of 0.01% -0.15% according to the volume ratio. The culture medium was used to treat melanoma B16-F10 cells for 24 hours, and then the cell viability was measured by the CCK-8 method, and the growth and morphology of the cells were observed by an inverted microscope. Meanwhile, PI staining and 75% ethanol fixation are carried out, flow analysis is carried out, the influence on the growth cycle of melanoma cells is observed, and the effect of the rosemary compound essential oil is optimal.
By referring to relevant documents in many ways, reports of rosemary essential oil on the aspect of inhibiting the proliferation of skin melanoma cells are not found. The invention has the advantages that the rosemary essential oil has obvious effect on inhibiting the proliferation of skin melanoma cells. Particularly, after different chemical essential oils of rosemary are combined in parts by weight, the prepared compound essential oil has more remarkable effect. The compound essential oil has obvious inhibition effect on the proliferation of skin melanoma cells B16-F10 in a cell culture medium with the concentration of 0.01 percent. At a concentration of 0.07%, the inhibitory effect reaches IC 50 Significantly lower than other chemical forms. At a concentration of 0.16%, inhibition of normal HaCaT cells was also observed. Compared with other chemical rosemary essential oil, the compound essential oil obviously reduces the inhibitory concentration on melanoma cells B16-F10, and has IC (integrated Circuit) toxic to normal cells by HaCaT 50 And in contrast, the application concentration range of the rosemary essential oil is enlarged. Meanwhile, by observing the growth condition and the morphology of cells, the compound essential oil is found to inhibit the cell DNA synthesis to cause the cycle block of G1/G0, thereby achieving the effect of inhibiting the proliferation of B16-F10. The invention lays a foundation for the development of new drugs for treating skin melanoma, though the invention provides the inhibition effect of the essential oil on melanoma cells B16-F10 cultured in vitro.
Drawings
FIG. 1 shows the mass spectrum of essential oil components of rosemary compound essential oil
FIG. 2 shows the effect of rosemary essential oil on the activity of melanoma cells B16-F10. ns indicates p > 0.05, no statistical difference compared to control; * Represents that p is more than 0.01 and less than 0.05, and the difference is obvious compared with the control; * Indicates p < 0.01, and the difference was very significant compared to the control.
FIG. 3 shows the morphological and quantitative effects of rosemary essential oil on melanoma cells B16-F10.
FIG. 4 shows the effect of the essential oil of the compound rosemary on the growth cycle of melanoma cells B16-F10 in example. ns indicates p > 0.05, no statistical difference compared to control; * Represents that p is more than 0.01 and less than 0.05, and the difference is obvious compared with the control; * Indicates p < 0.01, with very significant differences compared to the control.
FIG. 5 shows the effect of rosemary essential oil on the growth cycle of melanoma cells B16-F10 in example 4. ns indicates p > 0.05, no statistical difference compared to control; * Represents that p is more than 0.01 and less than 0.05, and has obvious difference compared with a control; * Indicates p < 0.01, with very significant differences compared to the control.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Experimental materials: 3 chemical forms of rosemary essential oil;
an instrument device: clevenger type distillation device, gas chromatography-mass spectrometer, super clean bench, autoclave, high-speed refrigerated centrifuge, cell incubator, electron microscope, fluorescence inverted microscope, multifunctional microplate reader, flow cytometer, etc.;
the test method comprises the following steps: drying 3 chemical rosemary materials in the shade, extracting essential oil by a steam distillation method, and performing gas chromatography-mass spectrometry combined analysis on the essential oil to obtain chemical components; mixing 25-30 parts of alpha-pinene type, 10-15 parts of verbenone type and 5-8 parts of 1, 8-cineole type in parts by mass; the compound essential oil is subjected to nano crushing, homogenizing, dispersing and sieving to form nano particles with the particle size of 10-30 nm.
The prepared compound essential oil and 3 chemical rosemary essential oils are respectively set to be 0.01%, 0.05%, 0.10%, 0.50%, 1.00%, 2.50% and 5.00% in concentration (v/v), the cell viability is measured after mouse melanoma cells B16-F10 and skin normal cells HaCaT are treated for 24 hours, and the IC of 4 essential oils on the melanoma cells and the skin normal cells HaCaT is calculated 50 (ii) a Will IC 50 Melanoma cells were treated with the lowest rosemary essential oil and the periodicity of the tumor cells was measured.
The cell culture conditions were: the temperature was 25. + -. 1 ℃ and the carbon dioxide concentration was 5%.
EXAMPLE 1 ingredients of Rosemary oil
Collecting 25-40cm branches of herba Rosmarini officinalis in Beijing in late 8 months of 2020, drying in shade for one month, and steam distilling the mixed material of leaves and stems with Clevenger type device to extract essential oil.
The volatile components of 3 rosemary essential oils were analyzed by GC-MS. Separating sample components by gas chromatography-mass spectrometer and simultaneously detecting n-alkane C 7 -C 40 The retention time of the sample component is substituted into the Kovats retention index formula to calculate the actual value of the component temperature programmed retention index. By searching NIST14 standard mass spectrum image library, using MS software to search the spectrum library, identifying the structure and name of the component according to the similarity of retention index and mass spectrum peak image, and determining the relative percentage content of each component by adopting an area normalization method. Analysis (table 1) revealed that the 3 essential oil components exhibited different trends in type and content: the main components of the alpha-pinene type rosemary essential oil are 28.32 percent of alpha-pinene, 20.48 percent of 1, 8-cineole, 14.21 percent of verbenone and 5.22 percent of borneol; horseThe main components of the verbenone type rosemary essential oil are 24.46 percent of verbenone, 10.48 percent of 1, 8-cineole, 7.12 percent of alpha-terpineol and 5.88 percent of borneol; the 1, 8-cineole type rosemary essential oil mainly comprises 25.46% of 1, 8-cineole, 13% of verbenone, 11.96% of alpha-pinene and 11.38% of camphor.
The preparation method comprises the steps of preparing 3 chemical types of rosemary essential oil according to 28 parts of alpha-pinene type, 15 parts of verbenone type and 7 parts of 1, 8-cineole type to form compound essential oil, analyzing and identifying the components of the compound essential oil, determining the relative percentage content of each component by adopting an area normalization method (table 1), wherein the component mass spectrogram of the compound essential oil is shown in an attached figure 1. Wherein the main components are 19.22 percent of alpha-pinene, 18.25 percent of 1, 8-cineole, 18.20 percent of verbenone and 5.58 percent of borneol.
TABLE 1 Rosemary 4 Rosemary oils component types and relative content (%)
And (3) annotation: RI (Ri) a Represented by the passage of n-alkane C on an HP-5MS capillary column 7- C 40 Measuring retention time and calculating a temperature programmed retention index; RI (Ri) b Is the retention index reported for the component in the relevant literature and NIST14 standards database; "-" indicates that the component was not detected; "t" means the relative content of the ingredients is less than 0.10%.
EXAMPLE 2 IC of Rosemary oil on melanoma cells B16-F10 and human epidermal keratinocyte HaCaT 50
Melanoma B16-F10 cells were treated with 3 kinds of chemical type essential oils such as alpha-pinene type and the complex essential oil prepared in example 1 at concentrations (v/v) of 0.01%, 0.05%, 0.10%, 0.50%, 1.00%, 2.50%, 5.00% by volume for 24 hours, and cell viability was measured by the CCK-8 method, and IC of 4 kinds of essential oils was calculated 50 。IC 50 A smaller size means a higher ability to inhibit B16-F10 proliferation. The results show that according to IC 50 From small to large: the compound essential oil is < verbena ketene type < 1, 8-cineole type < alpha-pinene type, wherein the compound essential oil type essential oil IC 50 The lowest, best effect, is 0.07 plus or minus 0.01%; secondly, verbenone type IC 50 0.10 plus or minus 0.003%;1, 8-cineole type IC 50 0.23 plus or minus 0.02 percent; IC (integrated circuit) 50 The highest was the alpha-pinene form, reaching 0.29. + -. 0.02% (Table 2). IC by detecting rosemary essential oil in human normal epidermal keratinocyte HaCaT 50 The toxicity of the compound essential oil type and verbenone type essential oil on B16-F10 is found to be significantly greater than that of HaCaT, wherein the compound essential oil on IC of B16-F10 50 Is 0.44 times of HaCaT, and is obviously lower than other 3 chemical essential oils (toxicity IC of the essential oil to the HaCaT) 50 And B16-F10 IC 50 The greater the difference, the greater the concentration range of essential oils used). In addition, IC of verbenone type essential oil on B16-F10 50 Is 0.77 times of HaCaT, and the IC of the rest 2 chemical type essential oils on HaCaT 50 Are all significantly greater than for B16-F10 and are not recommended.
TABLE 2 IC of rosemary essential oils for B16-F10 and HaCaT 50 (v/v，％)
Example 3 toxicity test and Effect on growth cycle of Rosemary Complex essential oil on melanoma cells B16-F10
The rosemary complex essential oil prepared in example 1 was selected, and after treating B16-F10 cells at 5 concentrations (v/v) of 0.010%, 0.025%, 0.050%, 0.075%, and 0.10% for 24 hours, cell viability was measured by the CCK-8 method, and cell growth and morphology were observed by an inverted microscope. The results show (figure 2) that the cytotoxicity of the compound essential oil on B16-F10 is concentration-dependent, the higher the concentration, the more the cytotoxicity is, the more serious the growth condition and the morphological effect on melanoma cells are (figure 3), and the treatment of 0.075% reduces the cell viability to 45.90%.
B16-F10 cells are treated by rosemary compound essential oil for 24 hours, then flow analysis is carried out after PI staining and 75% ethanol fixation. As shown in FIG. 4, the blank group contained 62.57% of cells at G1/G0, 34.22% of cells at S phase and 3.23% of cells at G2/M phase; under the treatment of 0.010% and 0.025% essential oils, the G2/M phase is 0, which respectively reduces S phase cells by 10.10% and 3.25%, and increases G1/G0 phase by 13.33% and 6.48%, which indicates that the two concentrations inhibit DNA synthesis of B16-F10, resulting in G1/G0 phase block; the treatments at 0.050% and 0.075% increased the G1/G0 phase of B16-F10 to 85.27% and 93.39%, respectively, and decreased the S phase to 12.82% and 5.43%, respectively, further indicating that the complex essential oil treatment significantly inhibited B16-F10 proliferation by inhibiting DNA synthesis, causing cycle arrest of G1/G0.
Example 4 Rosemary Compound essential oil and its use in inhibiting skin melanoma cell proliferation
The 3 rosemary essential oils extracted in the example 1 are prepared into compound essential oil according to 30 parts of alpha-pinene type, 12 parts of verbenone type and 8 parts of 1, 8-cineole type, the compound essential oil is subjected to essential oil component analysis and identification, and the relative percentage content of each component is determined by adopting an area normalization method. The main components of the medicine are 20.59 percent of alpha-pinene, 16.70 percent of verbenone, 18.46 percent of 1, 8-cineole and 5.54 percent of borneol.
The prepared complex essential oil was treated to a concentration (v/v) of 0.01%, 0.05%, 0.10%, 0.50%, 1.00%, 2.50%, 5.00% for 24 hours, respectively, to melanoma B16-F10 cells, and then cell viability was measured by the CCK-8 method, and IC was calculated 50 By detecting the IC of the compound essential oil on human normal epidermal keratinocyte HaCaT 50 . As a result, the IC of the compound type essential oil on B16-F10 and HaCaT is found 50 0.07 plus or minus 0.02 percent and 0.17 plus or minus 0.01 percent respectively. IC for B16-F10 50 IC significantly higher than that of human normal epidermal keratinocytes 50 And the expected result is met.
Selecting the prepared rosemary compound essential oil, setting 5 concentrations (v/v) of 0.010%, 0.025%, 0.050%, 0.075% and 0.10% to treat B16-F10 cells for 24 hours, detecting the cell viability by a CCK-8 method, and observing the growth condition and the morphology of the cells by an inverted microscope. The results show that the compound essential oil is concentration-dependent on the cytotoxicity of B16-F10, and the higher the concentration is, the greater the cytotoxicity is, the more serious the influence on the growth condition and the morphology of melanoma cells is; treatment at 0.075% reduced cell viability to 46.55%.
B16-F10 cells were treated with rosemary complex essential oil for 24 hours, then subjected to PI staining, fixed with 75% ethanol, and subjected to flow analysis. As shown in FIG. 5, the blank group contained 62.57% of cells in G1/G0 phase, 34.22% of cells in S phase and 3.23% of cells in G2/M phase; the G2/M phase is 0 under the treatment of 0.010 percent and 0.025 percent of essential oil, the S phase cells are respectively reduced by 9.55 percent and 4.25 percent, and the G1/G0 phase is increased by 12.78 percent and 7.48 percent, which indicates that the two concentrations inhibit the DNA synthesis of B16-F10 and cause the G1/G0 phase to be blocked; the 0.050% and 0.075% treatments increased the G1/G0 phase of B16-F10 to 86.12% and 94.27%, respectively, and the S phase to 11.35% and 4.69%, respectively, further illustrating that complex essential oil treatment significantly inhibited B16-F10 proliferation by inhibiting DNA synthesis, causing cycle arrest of G1/G0.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. The rosemary compound essential oil for inhibiting skin melanoma cell proliferation is characterized by comprising the following raw materials in parts by weight: 25-30 parts of alpha-pinene type rosemary essential oil, 10-15 parts of verbenone type rosemary essential oil and 5-8 parts of 1, 8-cineole type rosemary essential oil, wherein the main components of the alpha-pinene type rosemary essential oil comprise 28.32% of alpha-pinene, 20.48% of 1, 8-cineole, 14.21% of verbenone and 5.22% of borneol; the verbenone type rosemary essential oil mainly comprises 24.46 percent of verbenone, 10.48 percent of 1, 8-cineole, 7.12 percent of alpha-terpineol and 5.88 percent of borneol; the 1, 8-cineole type rosemary essential oil mainly comprises 25.46% of 1, 8-cineole, 13% of verbenone, 11.96% of alpha-pinene and 11.38% of camphor.
2. The rosemary compound essential oil according to claim 1, wherein the main components are 18.56-20.59% of alpha-pinene, 16.70-19.67% of verbenone, 17.70-19.06% of 1, 8-cineole and 5.54-5.99% of borneol.
3. The rosemary compound essential oil according to claim 1 or 2, wherein the rosemary essential oil is prepared by extracting the aerial part of the rosemary by a steam distillation method, and the aerial part of the rosemary comprises branches, leaves and/or flowers.
4. The method for preparing rosemary compound essential oil according to claim 1 or 2, wherein the raw materials are mixed according to the mass part, and the compound essential oil is subjected to nano crushing, homogenizing, dispersing and sieving to form nano particles with the particle size of 10-30 nm.
5. Use of rosemary compound essential oil as claimed in claim 1 or 2 in the preparation of a medicament for inhibiting skin melanoma cell proliferation.
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