CN113181223B - Composition with nasopharyngeal carcinoma resisting effect - Google Patents
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Abstract
The invention relates to a composition with an anti-nasopharyngeal cancer effect. The composition with the effect of resisting nasopharyngeal carcinoma comprises two raw materials, namely podocarpus macrophyllus volatile oil and podocarpus macrophyllus general flavone, wherein the two raw materials comprise the following components in parts by weight: 10-30 parts of podocarpus macrophyllus volatile oil and 70-90 parts of podocarpus macrophyllus general flavone. Wherein, the podocarpus macrophyllus general flavone is obtained by reflux extraction with 75% ethanol and separation and enrichment through polyamide column chromatography, and the general flavone contains the following chemical components: the relative mass percentage of the podocarpine flavone A, the vitexin-2 ' -O-rhamnoside, the vitexin-4 ' -O-glucoside, the isoorientin and the orientin-2 ' -O-beta-L-galactoside is respectively 1.7%, 0.74%, 8.21%, 7.42%, 0.86% and 17.48%. The composition with the effect of resisting the nasopharyngeal carcinoma can obviously inhibit the growth of nasopharyngeal carcinoma CNE-2 cells, and is a pharmaceutical composition with further development value.
Description
Technical Field
The invention belongs to the fields of natural medicines, pharmacology and tumor treatment, and particularly relates to a composition with an anti-nasopharyngeal cancer effect.
Background
Nasopharyngeal carcinoma is one of high-incidence malignant tumors in China, the incidence rate in south is higher than that in north, the incidence rates in Hunan and Fujian are higher than that in other areas in China, and the life health of local people is seriously harmed. The onset of nasopharyngeal carcinoma is related to genetic factors, environmental factors, EB virus infection, etc. The treatment method mainly comprises radiotherapy, chemical therapy, combined radiotherapy and chemotherapy and operation treatment. Among them, radiation therapy is the first choice for nasopharyngeal carcinoma. However, because nasopharyngeal carcinoma is easy to recur and metastasize in early stage, the prognosis is often poor. The 5-year survival rate of radiation-insensitive squamous cell carcinoma is only 0-10%, and the 5-year survival rate of radiation-sensitive lymphatic epithelial carcinoma is also only about 30%. Chemical drug therapy such as cisplatin, carboplatin, fluorouracil and the like can only play an auxiliary treatment role, and has high drug toxicity and adverse reactions, so that patients are often difficult to tolerate. Therefore, the search for the medicine with good curative effect, low toxicity and small adverse reaction is still far in mind.
The Podocarpus macrophyllus fruit is fruit of Podocarpus macrophyllus and Podocarpus brachypodaus of Podocarpus, and is composed of receptacle and seed. The Arhat pine is widely distributed in southern areas of China, including Jiangsu, Fujian, Hubei, Guangxi and Guangdong. The northern sea city of Guangxi is the county of the Japanese pine, and has the largest Japanese pine planting base in China. The Podocarpus macrophyllus has the effects of tonifying kidney, benefiting lung and reinforcing primordial qi. In the field of podocarpus macrophyllus planting, people often pick podocarpus macrophyllus for eating. Few reports are reported at present about the research on the pharmacological action of the podocarpus macrophyllus and the extract thereof. Research reports that the podocarpus macrophyllus extract has the effects of resisting oxidation, protecting liver and resisting gastric cancer cells.
In the aspect of invention patents, the invention patents related to the kusamaki nut which are published in China have the following 10, and can be classified into two categories according to the patent protection scope and the different purposes:
food-related products
(1) Zhang Ming Kong (CN201110095608) discloses a preparation method of a Podocarpus macrophyllus wine;
(2) zhangming cave (CN201110095609) discloses a production method of a podocarpus macrophyllus extract and a product thereof, and application of an extracted concentrated solution in the field of food;
(3) lianglong (CN202011492129.0) discloses a fruit wine of Podocarpus macrophyllus and its preparation method;
(4) lianglong (CN202011492160.4) discloses a Podocarpus macrophyllus fruit vinegar and its preparation method;
II, related classes of drugs
(1) Plum blossom (CN201510167634.0) discloses a Chinese medicinal preparation containing Podocarpus macrophyllus fruits, which is a Chinese medicinal compound preparation mainly used for relieving qi stagnation type stomachache and gastric ulcer, and does not relate to the composition disclosed by the invention;
(2) huangzengqiong (CN201610460262) discloses a method for extracting and separating high-purity ligustrazine from Podocarpus macrophyllus seed with high efficiency;
(3) huang Zengqiong (CN201910050934.9) discloses a preparation method of podocarpus macrophyllus polysaccharide and an anti-liver cancer application thereof;
(4) huang Zengqiong (CN201910914249.6) discloses a quality control method of a podocarpus macrophyllus medicine material;
(5) huang Zengqiong (CN202011391408.8) discloses a composition of podocarpus macrophyllus polysaccharide and application thereof in the aspect of immunoregulation;
(6) shenjie (CN201910288948.4) discloses a response surface method for optimizing the extraction process of podocarpus macrophyllus flavone, the extraction of the podocarpus macrophyllus flavone in the patent is carried out by taking ultrapure water as a solvent and adopting an ultrasonic extraction method, and the extraction process is essentially different from the extraction method of the podocarpus macrophyllus total flavone in the composition disclosed by the invention.
The invention patents do not disclose the composition of the podocarpus macrophyllus seed volatile oil and the podocarpus macrophyllus seed total flavone and the application thereof in the aspect of resisting nasopharyngeal carcinoma. In the literature, as far as we know, the research report of anti-nasopharyngeal carcinoma of either podocarpus macrophyllus volatile oil or podocarpus macrophyllus total flavone or the combination of the two is not found so far.
Although many documents report the antitumor effect of plant volatile oil and total flavone, the volatile oil and total flavone from different sources do not necessarily have similar pharmacological effects due to different chemical components, and the combination of the two does not necessarily have similar or synergistic pharmacological effects. After the volatile oil and the total flavone are combined according to a specific proportion, whether the original pharmacological action of each component is still provided or not and how the action effect is, the effect cannot be predicted by the professional in the field.
Disclosure of Invention
The invention aims to provide a composition with remarkable nasopharyngeal carcinoma resisting effect.
The composition with the effect of resisting nasopharyngeal carcinoma comprises two raw materials, namely podocarpus macrophyllus volatile oil and podocarpus macrophyllus general flavone, wherein the two raw materials comprise the following components in parts by weight: 10-30 parts of podocarpus macrophyllus volatile oil and 70-90 parts of podocarpus macrophyllus general flavone.
In the composition with the effect of resisting nasopharyngeal carcinoma, the volatile oil and the total flavone respectively have the following characteristics:
1) the volatile oil is characterized in that: taking a podocarpus macrophyllus receptacle and seeds as raw materials, adding distilled water according to the mass-volume ratio of 1: 40, extracting for 9h by a steam distillation method, collecting distillate, separating the distillate by an oil-water separator, and taking upper layer light yellow oily liquid to obtain podocarpus macrophyllus volatile oil; the relative mass percentage of chemical components of the volatile oil, namely beta-caryophyllene, copaene, gamma-elemene and alpha-cubebene, are respectively 27.37 percent, 10.81 percent, 11.46 percent and 7.72 percent;
2) the total flavone is characterized in that:
(1) the preparation method comprises the following steps: reflux-extracting the podocarpus macrophyllus receptacle and seeds for 1 hour by using 75% ethanol, concentrating an extracting solution under reduced pressure to obtain clear paste with the relative density of 1.05-1.15 at 25 ℃, separating and enriching the obtained clear paste by using polyamide column chromatography, eluting by using 80% ethanol as an eluent, collecting eluent, recovering a solvent from the eluent under reduced pressure, and evaporating to dryness at 80 ℃ to obtain podocarpus macrophyllus fruit total flavonoids;
(2) the total flavonoids of the kusamaki fruit obtained by the preparation method in the step (1), wherein the mass percentage of the contained flavonoid components is more than or equal to 80 percent calculated by rutin;
(3) the podocarpus macrophyllus general flavone obtained by the preparation method in the step (1) contains the following chemical components: the relative mass percentage of the podocarpine flavone A, the vitexin-2 ' -O-rhamnoside, the vitexin-4 ' -O-glucoside, the isoorientin and the orientin-2 ' -O-beta-L-galactoside is respectively 1.7%, 0.74%, 8.21%, 7.42%, 0.86% and 17.48%.
The preferable scheme of the composition with the effect of resisting nasopharyngeal carcinoma provided by the invention comprises the following raw materials in parts by weight: 20 parts of podocarpus macrophyllus essential oil and 80 parts of podocarpus macrophyllus total flavonoids.
The composition with the effect of resisting nasopharyngeal carcinoma is prepared into tablets, capsules or pills.
The invention provides a preparation method of a tablet, a capsule or a pill of the composition with the effect of resisting nasopharyngeal carcinoma, which comprises the following steps: taking the podocarpus macrophyllus essential oil and the podocarpus macrophyllus essential flavone according to the parts by weight, crushing the podocarpus macrophyllus essential flavone, sieving by a 60-mesh sieve, adding auxiliary materials according to the requirements of formulation auxiliary materials, uniformly mixing, spraying 95% ethanol in which the podocarpus macrophyllus essential oil is dissolved, uniformly mixing, and preparing into tablets, capsules or pills according to the requirements of formulation processes.
The composition with the effect of resisting the nasopharyngeal carcinoma is proved to have the remarkable effect of resisting the nasopharyngeal carcinoma by in vitro cell experiments, and can remarkably inhibit the growth of nasopharyngeal carcinoma CNE-2 cells.
The invention will now be described in further detail with reference to specific examples, but the scope of the invention as claimed is not limited to the following examples.
Drawings
FIG. 1 is a graph showing the relationship between the different concentrations and different times of the drug and the growth inhibition rate of CNE-2 cells of nasopharyngeal carcinoma
FIG. 2 shows the growth inhibition rate of CNE-2 cells of nasopharyngeal carcinoma with different drug concentrations and different action times
Detailed Description
Example 1
A composition with an anti-nasopharyngeal cancer effect comprises the following components in parts by weight: 10 parts of podocarpus macrophyllus essential oil and 90 parts of podocarpus macrophyllus total flavonoids.
Example 2
A composition with an anti-nasopharyngeal cancer effect comprises the following components in parts by weight: 20 parts of podocarpus macrophyllus essential oil and 80 parts of podocarpus macrophyllus total flavonoids.
Example 3
A composition with an anti-nasopharyngeal cancer effect comprises the following components in parts by weight: 25 parts of podocarpus macrophyllus essential oil and 75 parts of podocarpus macrophyllus total flavone.
Example 4
A composition with nasopharyngeal carcinoma resisting effect is prepared into capsule by the following method.
Taking 800g of podocarpus macrophyllus and seeds, performing reflux extraction for 1 hour by 4800mL of 75% ethanol, filtering, taking filtrate, concentrating the filtrate at 60 ℃ under reduced pressure to obtain clear paste with the relative density of 1.15 at 25 ℃, adding the obtained clear paste into a polyamide column prepared in advance, eluting 5 column volumes by using 80% ethanol as an eluent, collecting eluent, recovering the solvent from the eluent at 60 ℃ under reduced pressure, evaporating to dryness at 80 ℃ to obtain podocarpus macrophyllus fruit total flavone, taking rutin as a reference substance, determining the mass percentage content of the total flavone by adopting a standard curve method, wherein the content of the flavone component is 87% in terms of rutin. According to the weight portion of the embodiment 2, 20g of the podocarpus macrophyllus volatile oil and 80g of the podocarpus macrophyllus general flavone are taken, firstly, the podocarpus macrophyllus general flavone is crushed, sieved by a 60-mesh sieve, added with 80g of starch, uniformly mixed, dissolved by 100mL of 95% ethanol, uniformly sprayed into the uniformly mixed materials, uniformly mixed, granulated, dried, granulated and filled into hard capsules.
Example 5
A composition with nasopharyngeal carcinoma resisting effect is prepared into tablet by the following method.
Taking 1000g of podocarpus macrophyllus and seeds, carrying out reflux extraction for 1 hour by using 8000mL of 75% ethanol, filtering, taking filtrate, carrying out reduced pressure concentration at 60 ℃ to obtain clear paste with the relative density of 1.05 at 25 ℃, adding the obtained clear paste into a polyamide column prepared in advance, eluting for 4 column volumes by using 80% ethanol as an eluent, collecting eluent, recovering a solvent from the eluent at 60 ℃ under reduced pressure, evaporating to dryness at 80 ℃ to obtain podocarpus macrophyllus fruit total flavone, using rutin as a reference substance, determining the mass percentage content of the total flavone by adopting a standard curve method, wherein the content of the flavone is 80% in terms of rutin. According to the weight portion of the embodiment 1, 10g of podocarpus macrophyllus essential oil and 90g of podocarpus macrophyllus total flavone are taken, the podocarpus macrophyllus total flavone is crushed, sieved by a 60-mesh sieve, added with 100g of starch, uniformly mixed, dissolved with 80mL of 95% ethanol, uniformly sprayed into the uniformly mixed materials, uniformly mixed, granulated, dried until the moisture content of the granules is more than 4% and less than 8%, granulated, tabletted and prepared into tablets.
Example 6
A composition with nasopharyngeal carcinoma resisting effect is prepared into dripping pill by the following method.
Taking 1000g of podocarpus macrophyllus and seeds, carrying out reflux extraction for 1 hour by using 6000mL of 75% ethanol, filtering, taking filtrate, carrying out reduced pressure concentration at 60 ℃ to obtain clear paste with the relative density of 1.10 at 25 ℃, adding the obtained clear paste into a polyamide column prepared in advance, eluting for 5 column volumes by using 80% ethanol as an eluent, collecting eluent, recovering a solvent from the eluent at 60 ℃ under reduced pressure, evaporating to dryness at 80 ℃ to obtain podocarpus macrophyllus fruit total flavone, taking rutin as a reference substance, determining the mass percentage content of the total flavone by adopting a standard curve method, wherein the content of the flavone is 85% in terms of rutin. According to the weight parts of the embodiment 1, 25g of podocarpus macrophyllus seed volatile oil and 75g of podocarpus macrophyllus seed total flavone are taken, the podocarpus macrophyllus seed total flavone is crushed, and the crushed podocarpus macrophyllus seed total flavone is sieved by a 60-mesh sieve for later use; respectively taking 100g of polyethylene glycol 4000 and polyethylene glycol 6000, heating and melting in water bath, adding 25g of the podocarpus macrophyllus volatile oil and 75g of the crushed and sieved podocarpus macrophyllus general flavone, uniformly stirring, preserving heat at 80 ℃, pouring into a heat-preserving hopper of a pill dropping machine, adjusting the dropping temperature to 80 ℃, the condensing temperature to 15 ℃, the dropping distance to 10cm and the dropping speed to 22-25 drops per minute, dropping into the dimethyl silicon oil condensate for cooling, collecting the cooled pill drops, removing the condensate on the surface of the pill drop, and naturally drying to prepare the pill drop.
Example 7
Experiment on influence of composition on growth and reproduction of nasopharyngeal carcinoma CNE-2 cells
First, experimental material
1. Preparation of complete culture solution
100mL of fetal bovine serum thawed overnight in a refrigerator at 4 ℃ was added to 900mL of an incomplete RPMI1640 medium to prepare a cell culture solution containing 10% fetal bovine serum. Subpackaging according to the experimental requirements, and storing at 4 ℃ for later use. During the experiment, the sample is placed in a constant-temperature water bath box at 37 ℃ for preheating. 5mL of the prepared culture solution is added into a sterile culture bottle, and the sterile culture bottle is placed in an incubator for 48 hours to carry out a sterile test.
2. Preparation of MTT solution
Dissolving 100mg MTT in 20mL of PBS buffer solution after autoclaving, filtering with 0.22 μm microporous membrane, subpackaging with 5mL of sterile centrifuge tube, wrapping the centrifuge tube with aluminum foil paper, and storing in a refrigerator at-20 deg.C for use.
3. Cell cryopreservation liquid
Mixing DMSO, fetal calf serum and RPMI1640 incomplete culture medium at a ratio of 1: 2: 7, and storing in a refrigerator at 4 deg.C for use, wherein the cell frozen stock solution is prepared on site.
4. Preparation of drugs
According to the weight portion of the embodiment 2, 20mg of the podocarpus macrophyllus essential oil and 80mg of the podocarpus macrophyllus general flavone are taken and evenly mixed to obtain the pharmaceutical composition, the pharmaceutical composition is prepared into mother solution with the mass concentration (calculated by the composition) of 1mg/mL by using dimethyl sulfoxide (DMSO), and then the mother solution is diluted into 6 concentrations by using complete culture solution, wherein the concentrations are as follows: 2.0. mu.g/mL, 5.0. mu.g/mL, 10.0. mu.g/mL, 20.0. mu.g/mL, 40.0. mu.g/mL, 50.0. mu.g/mL.
Second, Experimental methods
1. Inoculating cells: collecting CNE-2 cells in logarithmic growth phaseDigesting with trypsin containing 0.25% EDTA, adding appropriate amount of complete culture solution to stop digestion, centrifuging at 1000rpm for 5 min, discarding supernatant, adding complete culture solution, slightly blowing to obtain single cell suspension, counting cells, and regulating cell concentration to 5 × 104one/mL. And (3) taking a 96-well plate, adding 100 mu L of cell suspension into each well, seeding the 3 plates once, and culturing the 3 plates in an incubator at 37 ℃ and 5% carbon dioxide for 24 hours to allow the cells to adhere to the wall.
2. Experimental grouping and assay
After the cells are attached to the wall, the solution is changed, the drug concentration is set to be 2.0 mug/mL, 5.0 mug/mL, 10.0 mug/mL, 20.0 mug/mL, 40.0 mug/mL and 50.0 mug/mL, a cell control group (culture solution + cells) and a blank control group (only culture solution is added and no cells are added) are simultaneously set, 5 multiple wells are set for each group, the group is placed in an incubator with 37 ℃ and 5% carbon dioxide, and three time points of 24h, 48h and 72h are set. And taking out a 96-well plate at a set time point, adding 20 mu L of MTT solution into each well, continuously culturing for 4h, discarding the supernatant, adding 150 mu L of DMSO into each well, vibrating in a constant-temperature oscillator in a dark place for 10min, measuring the absorbance value (OD) of each group at the wavelength of 570nm on an enzyme-labeling instrument, repeating for 3 times, and taking the average value for statistics.
3. Calculation and statistical method
The cell growth inhibition rate IR ═ 100%
The intervention time is used as an abscissa, the growth inhibition rate is used as an ordinate, and the aging relationship is known.
The drug concentration is used as the abscissa and the growth inhibition rate is used as the ordinate to understand the dose-effect relationship, and the IC with different action time is obtained50。
All data were counted using statistical software SPSS20.0, the data were expressed as means. + -. standard deviation, and the comparisons between groups were performed using one-way analysis of variance. The test level α is 0.05, and differences of P < 0.05 are statistically significant.
Third, experimental results
The results are shown in Table 1. From the results, the growth of CNE-2 cells was significantly inhibited after the action of the pharmaceutical compositions at different concentrations, and the inhibition was more significant with increasing dose, with the difference having statistical significance (P < 0.05 or 0.01). After the same drug concentration acts for different time, the inhibition rate is obviously increased along with the prolonging of the time, and the difference has statistical significance (P is less than 0.05 or 0.01). Therefore, the pharmaceutical composition can obviously inhibit the growth of nasopharyngeal carcinoma CNE-2 cells within a certain concentration range, and is in a concentration and time dependence (see the attached figures 1 and 2 in the specification).
TABLE 1 growth inhibition of CNE-2 cells at different drug concentrations and at different times
Note: compared with the inhibition rate of the group of 0 mug/mL,*P＜0.05，**p is less than 0.01; the same concentration, compared with the inhibition rate of the group acting for 24h,#P＜0.05，##P＜0.01。
in conclusion, the composition with the effect of resisting the nasopharyngeal carcinoma can obviously inhibit the growth of the nasopharyngeal carcinoma CNE-2 cells, the inhibition rate can reach 76.63% at most, and the composition is a pharmaceutical composition with further development value.
Claims (4)
1. The composition with the effect of resisting nasopharyngeal carcinoma is characterized by comprising two raw materials, namely podocarpus macrophyllus volatile oil and podocarpus macrophyllus general flavone, wherein the two raw materials comprise the following components in parts by weight: 10-30 parts of podocarpus macrophyllus volatile oil and 70-90 parts of podocarpus macrophyllus general flavone; the volatile oil and the total flavone respectively have the following characteristics:
1) the volatile oil is characterized in that: taking a podocarpus macrophyllus receptacle and seeds as raw materials, adding distilled water according to the mass-volume ratio of 1: 40, extracting for 9h by a steam distillation method, collecting distillate, separating the distillate by an oil-water separator, and taking upper layer light yellow oily liquid to obtain podocarpus macrophyllus volatile oil; the relative mass percentage of chemical components of the volatile oil, namely beta-caryophyllene, copaene, gamma-elemene and alpha-cubebene, are respectively 27.37 percent, 10.81 percent, 11.46 percent and 7.72 percent;
2) the total flavone is characterized in that:
(1) the preparation method comprises the following steps: reflux-extracting the podocarpus macrophyllus receptacle and seeds for 1 hour by using 75% ethanol, concentrating an extracting solution under reduced pressure to obtain clear paste with the relative density of 1.05-1.15 at 25 ℃, separating and enriching the obtained clear paste by using polyamide column chromatography, eluting by using 80% ethanol as an eluent, collecting eluent, recovering a solvent from the eluent under reduced pressure, and evaporating to dryness at 80 ℃ to obtain podocarpus macrophyllus fruit total flavonoids;
(2) the total flavonoids of the kusamaki fruit obtained by the preparation method in the step (1), wherein the mass percentage of the contained flavonoid components is more than or equal to 80 percent calculated by rutin;
(3) the podocarpus macrophyllus general flavone obtained by the preparation method in the step (1) contains the following chemical components: the relative mass percentage of the podocarpine flavone A, the vitexin-2 ' -O-rhamnoside, the vitexin-4 ' -O-glucoside, the isoorientin and the orientin-2 ' -O-beta-L-galactoside is respectively 1.70%, 0.74%, 8.21%, 7.42%, 0.86% and 17.48%.
2. The composition of claim 1, wherein the volatile oil of Podocarpus macrophyllus is 20 parts by weight and the total flavonoids of Podocarpus macrophyllus is 80 parts by weight.
3. The composition for nasopharyngeal cancer according to claim 1 or 2, which is in the form of a tablet, capsule or pill.
4. The composition for treating nasopharyngeal carcinoma according to claim 3, wherein the preparation method of the tablet, capsule or pill comprises: taking the podocarpus macrophyllus essential oil and the podocarpus macrophyllus essential flavone according to the parts by weight, crushing the podocarpus macrophyllus essential flavone, sieving by a 60-mesh sieve, adding auxiliary materials according to the requirements of formulation auxiliary materials, uniformly mixing, spraying 95% ethanol in which the podocarpus macrophyllus essential oil is dissolved, uniformly mixing, and preparing into tablets, capsules or pills according to the requirements of formulation processes.
Priority Applications (1)
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