CN113713026B - Anti-tumor extract of daphne genkwa as well as preparation method and application thereof - Google Patents

Anti-tumor extract of daphne genkwa as well as preparation method and application thereof Download PDF

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CN113713026B
CN113713026B CN202110959914.0A CN202110959914A CN113713026B CN 113713026 B CN113713026 B CN 113713026B CN 202110959914 A CN202110959914 A CN 202110959914A CN 113713026 B CN113713026 B CN 113713026B
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melanoma
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daphne
ethyl acetate
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朱建勇
李媛
张春燕
聂依文
张瑞峰
夏伟
叶颖
俞雷
罗兰
翟晓翔
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Shanghai Seventh Peoples Hospital
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Abstract

The invention belongs to the technical field of anti-tumor medicines, and discloses a yellow lilac daphne flower bud extract, an anti-tumor medicine containing the extract, a preparation method and application. The preparation method of the yellow daphne flower bud extract comprises the following steps: extracting the daphne giraldii nitsche with micromolecule alcohol, removing alcohol liquid, extracting the obtained residual part with petroleum ether, removing petroleum ether extract, extracting the obtained residual part with ethyl acetate, concentrating the ethyl acetate extract, and drying to obtain the ethyl acetate extract of the daphne giraldii nitsche extract. The extract can inhibit tumor cell cycle (G) 0 /G 1 Phase or S phase), cell proliferation to play the role of inhibiting the activity of tumor cells, thereby promoting the apoptosis of the tumor cells, showing very good anti-proliferation and apoptosis-promoting activity, and having good industrialization prospect when being applied to the anti-tumor field.

Description

Anti-tumor extract of daphne genkwa as well as preparation method and application thereof
Technical Field
The invention belongs to the technical field of antitumor medicines, and relates to a flos daphnes genkwa antitumor extract, a preparation method thereof and application thereof in preparing antitumor medicines.
Background
About 2-3 million skin cancers are diagnosed each year worldwide, wherein melanoma is a common malignant tumor in skin cancers and accounts for 1% -3% of all malignant tumors. Melanoma (melanoma) is an epithelial malignancy originating from neural crest melanocytes, i.e., a maliciously changing pigmented nevus. In recent years, the onset age of melanoma is a trend of younger age, the incidence and mortality rate in the world are obviously increased, and the growth rate of melanoma is increased year by year at a growth rate of 6-7%, so that melanoma is one of the malignant tumors with the fastest increase in incidence rate. 23.2 million melanoma patients are newly added in the world every year, and the number of newly added deaths is 5.5 million, and the melanoma is the tumor with the highest disease death rate in skin cancer. More than 2 million people are newly added in China according to Chinese melanoma diagnosis and treatment guidelines (2015 edition), and the number of people is gradually increased in recent years. Melanoma has become one of the diseases seriously harming the health of people in China.
The current treatment of melanoma includes targeted therapy and immunotherapy in addition to traditional surgical therapy, radiotherapy and chemotherapy, the latter two of which have become the hot spots of clinical research. In the clinical application of targeted therapy, such as a BRAF inhibitor (such as vemurafenib) which is a medicament targeting a highly mutated gene in melanoma and a trametinib (GSK1120212) which is an inhibitor targeting MEK1/2, the effect is remarkable, but most patients can quickly develop drug resistance. While immunotherapy, such as the anti-CTLA-4 antibody ipilimumab and the anti-PD-1 antibody nivolumab, etc., produces satisfactory efficacy in only a fraction of patients. While new targeted therapies and immunotherapies provide higher patient response rates, long-term survival of patients remains low.
Therefore, the research on the aspects related to the treatment of melanoma needs to be strengthened urgently, and on the basis of understanding the potential related molecular mechanism in the development process of malignant melanoma, a new method for treating melanoma is explored, and a proper drug is developed and researched for treatment, so that the important significance in resisting the treatment of melanoma is achieved.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a lilac daphne flower bud anti-tumor extract and a preparation method and application thereof.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a lilac daphne flower bud anti-tumor extract which comprises the following components in parts by weight: 5-15 parts of isoquercitrin, 1-5 parts of eriodictyol, 40-65 parts of luteolin, 0.2-2 parts of apigenin, 1-3 parts of aureoflavone, 0.1-1 part of bimetacin A and 0.1-0.5 part of 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate.
In a preferred embodiment, the daphne genkwa antitumor extract comprises the following components in parts by weight: 11.1 parts of isoquercitrin, 2.8 parts of eriodictyol, 54.4 parts of luteolin, 0.9 part of apigenin, 1.7 parts of aureoflavone, 0.53 part of bimetacin A and 0.26 part of 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate.
The invention provides a preparation method of a lilac daphne flower bud anti-tumor extract, which comprises the following steps:
(1) percolating and extracting flos Wikstroemiae Chamaedaphnes with small molecular alcohol to obtain flos Wikstroemiae Chamaedaphnes small molecular alcohol extract;
(2) removal of small molecule alcohols
(3) Then adding petroleum ether for extraction;
(4) removing the petroleum ether extract;
(5) and adding an ethyl acetate solution into the residual solution from which the petroleum ether extraction liquid is removed for extraction to obtain an ethyl acetate extraction part of the daphne genkwa micromolecular alcohol extract, namely the daphne genkwa antitumor extract.
Specifically, the preparation method of the daphne genkwa antitumor extract comprises the following steps:
(1) preparation of small molecular alcohol extract of daphne genkwa
Taking Chinese medicinal material daphne genkwa, extracting by small molecule alcohol percolation, repeating for several times, and mixing repeatedly obtained small molecule alcohol percolation extract, namely daphne genkwa small molecule alcohol extract;
(2) removal of small molecule alcohols
Concentrating small molecular alcohol extract of flos Wikstroemiae Chamaedaphnes at low temperature, removing small molecular alcohol, and adding water into the obtained extract to obtain suspension;
(3) petroleum ether extraction of lilac daphne flower bud
Then adding petroleum ether for extraction;
(4) removing impurities
Removing petroleum ether extract (petroleum ether extraction part of small-molecule alcohol extract of flos Wikstroemiae Chamaedaphnes);
(5) preparation of ethyl acetate extract of small-molecule alcohol extract of daphne genkwa
Adding an ethyl acetate solution into the residual solution from which the petroleum ether extract is removed for extraction, and separating to obtain an ethyl acetate extract; repeating the operation, combining the ethyl acetate extract, and concentrating to obtain a yellow lilac daphne flower bud micromolecule alcohol extract ethyl acetate extract part extract, namely the yellow lilac daphne bud anti-tumor extract.
The invention also provides the lilac daphne flower bud anti-tumor extract obtained by the preparation method.
The invention also provides the application of the daphne genkwa antitumor extract in preparing antitumor drugs.
The invention also provides a pharmaceutical composition, which comprises the daphne genkwa antitumor extract, and a pharmaceutically acceptable carrier and a pharmaceutically acceptable medium.
The invention also provides application of the pharmaceutical composition in preparing antitumor drugs.
In the invention, the anti-tumor refers to the prevention and/or treatment of tumor; the tumor treatment refers to inhibiting proliferation, growth, migration and infiltration of tumors and promoting apoptosis of the tumors.
Compared with the prior art, the invention has the advantages that,
the raw materials of the lilac daphne flower bud anti-tumor extract are natural Chinese medicinal genuine medicinal materials, and the sources of the raw materials are wide.
The method combines the micromolecule alcohol extraction, the petroleum ether extraction and the ethyl acetate extraction to be applied to the yellow lilac daphne flower bud for extracting the anti-tumor active ingredients for the first time, and has the advantages of simple operation, less steps, low cost and more environmental protection.
In the method, the extraction liquid obtained by extracting the petroleum ether is not applied to the next ethyl acetate extraction, but the residual liquid is further extracted by ethyl acetate after the petroleum ether extraction liquid is removed, so that the extract with unexpected high anti-tumor activity is obtained.
The lilac daphne flower bud anti-tumor extract has higher activity of resisting tumor proliferation and growth and promoting tumor apoptosis, and can block tumor cells in S phase or G phase 0 /G 1 The invention can be used for preventing and treating tumors, particularly melanoma.
Drawings
FIG. 1 is a process diagram of preparation of anti-tumor extract of Daphne genkwa.
FIG. 2 liquid chromatogram of WCME and isolated compound; 1: isoquercitrin; 2: eriodictyol; 3: luteolin; 4: apigenin; 5: golden yellow alcohol; 6: a peptide A; 7: 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate; the horizontal coordinate values of fig. 2 are 0.00, 11.67, 23.34, 35.00, 46.67, 58.34 and 70.01 from left to right; the numerical values of the left ordinate are 0.00, 882.20, 1764.39, 2646.59 and 3528.79 from bottom to top in sequence; the ordinate on the right side is from S1 to S8 from bottom to top.
FIG. 3 measurement of the proliferation activity of cancer cells A375 and B16 in vitro by WCME at different concentrations.
FIG. 4 effect of different concentrations of WCME on A375 and B16 cell morphology; wherein (a) B16 is altered in cell morphology; (B) a375 cell morphology change.
FIG. 5 effect of different concentrations of WCME on the migratory capacity of A375 and B16 cells; wherein (A) B16 cell migration ability; (B) a375 cell migration ability.
FIG. 6 effect of different concentrations of WCME on A375 and B16 cell cycles;
wherein (A) the B16 cell cycle diagram; (control) G0/G1: 58.94%; 1.07 percent of G2/M; 39.99 percent of S; (WCME/100. mu.g/mL) G0/G1: 59.45%; 14.64 percent of G2/M; 25.91 percent of S; (WCME/200. mu.g/mL) G0/G1: 62.37%; 14.25 percent of G2/M; 23.38 percent of S; (WCME/300. mu.g/mL) G0/G1: 69.41%; 8.42 percent of G2/M; 22.17 percent of S; (WCME/400. mu.g/mL) G0/G1: 80.41%; 3.14 percent of G2/M; 16.45 percent of S; (DBZ/300. mu.M) G0/G1: 93.46%; 2.35 percent of G2/M; 4.19 percent of S. Wherein, the abscissa of each small graph in the upper half part of the graph A (B16) is 0, 50, 100, 150, 200 and 250 from left to right in sequence; the ordinate is 0, 200, 400, 600, 800 and 1000 from bottom to top in sequence.
(B) A375 cell cycle diagram. (control) G0/G1: 65.59%; 11.27 percent of G2/M; 23.14 percent of S; (WCME/100. mu.g/mL) G0/G1: 58.52%; 10.31 percent of G2/M; 31.17 percent of S; (WCME/200. mu.g/mL) G0/G1: 47.95%; 9.00 percent of G2/M; 43.05 percent of S; (WCME/300. mu.g/mL) G0/G1: 47.18%; 7.65 percent of G2/M; 45.17 percent of S; (WCME/400. mu.g/mL) G0/G1: 46.76%; 7.61 percent of G2/M; 45.63 percent of S; (DBZ/300. mu.M) G0/G1: 78.93%; 11.08 percent of G2/M; 9.99 percent of S. Wherein, the horizontal coordinates of each small graph in the upper half part of the B graph (A375) are 0, 50, 100, 150 and 200 from left to right in sequence; the ordinate is 0, 200, 400, 600, 800 and 1000 from bottom to top in sequence.
FIG. 7 effect of different concentrations of WCME on apoptosis of A375 and B16 cells; wherein, (A) B16 apoptosis pattern; (B) apoptosis pattern of a375 cells.
Detailed Description
The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
The invention obtains the daphne genkwa antitumor extract with higher antitumor activity by an improved preparation method. The method comprises the process steps of drying, crushing, solvent extraction, impurity removal, extraction, concentration and the like of the Chinese medicinal components of the yellow lilac daphne flower bud. The anti-tumor extract of daphne tinctoria has better curative effect on preventing and/or treating tumors, particularly melanoma. The method has the advantages of simple operation steps, low cost and no pollution, and provides a beneficial reference for further developing the value of the yellow daphne flower bud.
The anti-tumor extract of the daphne genkwa is an ethyl acetate extract of a small molecular alcohol extract of the daphne genkwa. Comprises the following components in parts by weight: 5-15 parts of isoquercitrin, 1-5 parts of eriodictyol, 40-65 parts of luteolin, 0.2-2 parts of apigenin, 1-3 parts of aureoflavone, 0.1-1 part of bimetacin A and 0.1-0.5 part of 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate. The Chinese and English comparison of each component is shown in Table 2.
In the daphne giraldii antineoplastic extract provided by the invention, the isoquercitrin is 5-15, or 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14 and 14-15.
In the daphne giraldii antitumor extract provided by the invention, 1-5 parts of eriodictyol, or 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5 and 4.5-5 parts of eriodictyol are included.
In the lilac daphne flower bud anti-tumor extract provided by the invention, 40-65 parts of luteolin can also be 40-42, 42-44, 44-46, 46-48, 48-50, 50-52, 52-54, 54-56, 56-58, 58-60, 60-62, 62-64 and 64-65.
In the lilac daphne flower bud anti-tumor extract provided by the invention, 0.2-2 parts of apigenin, or 0.2-0.5, 0.5-0.6, 0.6-0.9, 0.9-1.0, 1.0-1.3, 1.3-1.4, 1.4-1.7, 1.7-1.8 and 1.8-2 parts of apigenin.
In the daphne giraldii antineoplastic extract provided by the invention, 1-3 parts of the aureoflavone can be 1-1.2, 1.2-1.3, 1.3-1.5, 1.5-1.6, 1.6-1.8, 1.8-1.9, 1.9-2.1, 2.1-2.3, 2.3-2.5, 2.5-2.6, 2.6-2.8, 2.8-2.9 and 2.9-3.0.
In the lilac daphne flower bud anti-tumor extract provided by the invention, 0.1-1 part of the pyritinoid A can be 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9 and 0.9-1.0.
In the daphne giraldii antitumor extract provided by the invention, 0.1-0.5 part of 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate, or 0.1-0.2, 0.2-0.3, 0.3-0.4 and 0.4-0.5 parts of daphne giraldii antitumor extract.
In a preferred embodiment, the daphne genkwa antitumor extract comprises the following components in parts by weight: 10-13 parts of isoquercitrin, 2-3 parts of eriodictyol, 50-60 parts of luteolin, 0.8-1.2 parts of apigenin, 1.5-2 parts of aureoflavone, 0.40-0.65 part of bimetacin A and 0.2-0.4 part of 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate.
In a preferred embodiment, the daphne genkwa antitumor extract comprises the following components in parts by weight: 11.1 parts of isoquercitrin, 2.8 parts of eriodictyol, 54.4 parts of luteolin, 0.9 part of apigenin, 1.7 parts of aureoflavone, 0.53 part of bimetacin A and 0.26 part of 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate.
The structures of the main components are as follows:
Figure BDA0003221878290000051
the invention also provides a preparation method of the daphne genkwa antitumor extract, which comprises the following steps (as shown in figure 1):
(1) preparation of small molecular alcohol extract of daphne genkwa
Taking Chinese medicinal material daphne genkwa, carrying out small molecular alcohol percolation extraction repeatedly for several times, and combining repeatedly obtained small molecular alcohol percolation extract, namely the daphne genkwa small molecular alcohol extract.
(2) Removal of small molecule alcohols
Concentrating small molecular alcohol extract of flos Wikstroemiae Chamaedaphnes at low temperature, removing small molecular alcohol, and adding water into the obtained extract to obtain suspension;
(3) petroleum ether extraction of yellow lilac daphne
Then adding petroleum ether for extraction;
(4) removing impurities
Removing petroleum ether extract (petroleum ether extraction part of small molecular alcohol extract of flos Wikstroemiae Chamaedaphnes);
(5) preparation of ethyl acetate extract of small-molecule alcohol extract of daphne genkwa
Adding an ethyl acetate solution into the residual solution after the petroleum ether extraction liquid is removed for extraction, and separating to obtain an ethyl acetate extraction liquid; repeating the operation, mixing the ethyl acetate extractive solutions, and concentrating to obtain extract (active part extract, hereinafter abbreviated as WCME) of the extract of the small molecular alcohol extract of flos Wikstroemiae Chamaedaphnes.
In the step (1), the volume concentration of the small molecular alcohol is 75-99%; preferably, it is 95%.
In the step (1), the weight-volume ratio (g/mL) of the daphne genkwa flowers to the small molecular alcohol is 4-12 for each percolation; preferably, it is 8. The ratio of the amount of daphne giraldii to the amount of the small molecular alcohol used may be the same or different for each percolation, as long as it is within the range.
In the step (1), the temperature of each percolation is 15-40 ℃; preferably, it is 25 ℃. The temperature of each percolation may be the same or different, as long as it is within the range.
In the step (1), the time of each percolation is 5-8 days; preferably, it is 7 days. The time for each percolation may be the same or different, as long as it is within the stated range.
In the step (1), the times of percolating the micromolecule alcohol are 2-5; preferably 3 times.
In step (1), the percolation is carried out in a percolation vessel.
In the step (1), the small molecular alcohol is at least one selected from methanol, ethanol, propanol, ethylene glycol, 1, 2-propylene glycol, 1, 4-butanediol, 1, 6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, trimethylolpropane, glycerol and pentaerythritol; more preferably, the small molecule alcohol is selected from at least one of ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 4-butanediol, trimethylolpropane and glycerol; further preferably, the small molecule alcohol is selected from ethanol.
In the step (1), before the first percolation, the steps of cleaning and crushing the daphne genkwa are also included. The cleaning can be carried out by water until the impurities on the surface of the yellow lilac daphne flower bud are removed. The grinding is to grind the daphne genkwa into particles with the particle size of 1-3 mm.
In the step (2), the low-temperature concentration refers to the removal of small molecular alcohol by distillation, and the temperature of the low-temperature concentration refers to 50-70 ℃; preferably, the temperature of the cryoconcentration is 60 ℃.
In the step (2), the time of low-temperature concentration is 1-3 hours; preferably, the time for the cryoconcentration is 2 hours.
In the step (2), the low-temperature concentration is carried out under normal pressure or reduced pressure, wherein the reduced pressure is 0.09-0.1 MPa.
In the step (2), the low-temperature concentration is completed according to the following standard: concentrating to obtain an extract, and adding the daphne genkwa medicinal materials according to the mass ratio of 1:0.5-1: 2; preferably 1: 1.
in the step (2), the concentrated and distilled small molecular alcohol solution can be stored in a container for recycling.
In the step (2), the volume ratio of the extract to water is 1:0.5-1: 2; preferably 1: 1.
In the step (3), the temperature of petroleum ether extraction is 15-40 ℃; preferably, it is 25 ℃. The temperature of each petroleum ether extraction may be the same or different so long as it is within the range.
In the step (3), the extraction time of the petroleum ether is 1-4 hours; preferably, 2 hours. The time for each petroleum ether extraction may be the same or different, provided that it is within the stated range.
In the step (3), the number of times of petroleum ether extraction is 2-4; preferably 3 times.
In the step (3), the volume ratio of the suspension to the petroleum ether is 1:0.5-1: 2; preferably 1:1, the volume ratio of the suspension to the petroleum ether is 1: 1.
In step (4), the removed petroleum ether extract may be placed in a holding vessel for recovery.
In the step (4), the petroleum ether extract to be recovered can be further subjected to impurity removal, and the steps are as follows: concentrating and recovering petroleum ether, and directly discarding the residual impurities.
In the step (5), the volume ratio of the residual solution after the petroleum ether extraction liquid is removed to the ethyl acetate is 1:0.5-1: 2; preferably, it is 1: 1.
In the step (5), the temperature of the ethyl acetate extraction is 15-40 ℃; preferably, it is 25 ℃. The temperature for each extraction with ethyl acetate may be the same or different, provided that it is within the range.
In the step (5), the extraction time of the ethyl acetate is 5-10 hours; preferably, it is 8 hours. The time for each ethyl acetate extraction may be the same or different, provided that it is within the range.
In the step (5), the extraction frequency of the ethyl acetate is 2-4; preferably 3 times.
In the step (5), the concentration method is to concentrate under normal pressure or reduced pressure, wherein the reduced pressure is 0.09-0.1 Mpa.
In the step (5), the standard of the concentration completion is that the extract of the ethyl acetate extraction part of the small molecular alcohol extract of the lilac daphne has no ethyl acetate smell.
In one embodiment, the preparation method of the daphne genkwa antitumor extract specifically comprises the following steps: percolating Chinese medicinal material flos Wikstroemiae Chamaedaphnes with 95% small molecular alcohol for 3 times each time for one week, mixing the percolate for 3 times to obtain small molecular alcohol percolate (flos Wikstroemiae Chamaedaphnes small molecular alcohol extract).
Concentrating the small molecular alcohol percolate at low temperature, removing the small molecular alcohol, and adding water into the obtained extract to prepare suspension.
Then adding equal volume of petroleum for extraction, removing petroleum ether extract (petroleum ether extraction part of flos Wikstroemiae Chamaedaphnes micromolecule alcohol extract) and impurities therein, and repeating for 3 times.
Adding an equal volume of ethyl acetate solution into the residual solution from which the petroleum ether extract is removed for extraction, and separating the ethyl acetate extract; repeating for 3 times, mixing the 3 times of ethyl acetate extractive solutions, and concentrating to obtain extract (active part extract, WCME). The preparation process is shown in figure 1.
The invention also provides the lilac daphne flower bud anti-tumor extract obtained by the method, and the lilac daphne flower bud anti-tumor extract is a lilac daphne bud micromolecule alcohol extract ethyl acetate extract. In a preferred embodiment, the lilac daphne flower bud anti-tumor extract is an ethyl acetate extract of an ethanol extract of lilac daphne flower bud.
The invention also provides application of the daphne giraldii antitumor extract obtained by the method in preparation of antitumor drugs. The anti-tumor refers to the prevention and/or treatment of tumor. The tumor treatment refers to inhibiting proliferation, growth, migration and infiltration of tumors and promoting apoptosis of the tumors.
In the application of the present invention, the tumor includes lymphoma, hematological tumor, solid tumor, etc., and specifically, may include, but is not limited to, melanoma, adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid tumor, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, squamous cell carcinoma of head and neck, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myelogenous leukemia, low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymus cancer, endometrial carcinoma, uterine sarcoma, uveal melanoma, and the like, Multiple myeloma, acute lymphoid leukemia, chronic myeloid leukemia, T cell lymphoma, B cell lymphoma tumor cells in one or more combinations. Preferably, the tumor is melanoma.
In a preferred embodiment, the tumor is a human malignant melanoma or a mouse malignant melanoma. In another preferred embodiment, the tumor cells are human malignant melanoma cell A375 and mouse malignant melanoma cell B16
The invention also provides a pharmaceutical composition which comprises the daphne genkwa anti-tumor extract.
In one embodiment, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, vehicle, or the like. Such acceptable carriers, media such as sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders, and the like. Moreover, other low molecular weight polypeptides may also be present; proteins such as serum albumin, gelatin, and immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; saccharides or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol and sorbitol. When an aqueous solution for injection is prepared, for example, physiological saline, an isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, may be used in combination with an appropriate solubilizing agent such as alcohol (small molecular alcohol, etc.), polyhydric alcohol (propylene glycol, PEG, etc.), nonionic surfactant (Tween 80, HCO-50), etc.
The invention also provides application of the pharmaceutical composition in preparing anti-tumor products. The definition of the tumor is as indicated above. In the present invention, the anti-tumor means preventing and/or treating tumor. The tumor treatment refers to inhibiting the proliferation, growth, migration and infiltration of tumors and promoting the apoptosis of the tumors.
The present invention also provides a method for preventing and/or treating tumors, comprising administering to an individual (human or non-human animal) in need thereof a therapeutically effective amount of a daphne lilac anti-tumor extract as disclosed and/or claimed herein. Administration can be by one of a variety of means, including oral, intravenous, intramuscular, subcutaneous, rectal infusion, eye drops, nasal spray, oral spray (inhalant), topical (topical) or systemic (transdermal) administration to the skin, and the like.
Accordingly, the pharmaceutical composition may be formulated for convenient administration, for example, suitable for oral administration may include, but is not limited to, pills, tablets, granules, drops, chewables, capsules or syrups and the like, for further example, suitable for parenteral administration may include, but is not limited to, solutions, suspensions, reconstitutable dry preparations or sprays and the like, for further example, suitable for rectal administration may typically be suppositories and the like.
In the present invention, "subject" generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., which would benefit from treatment with the formulation, kit or combined formulation.
In the present invention, a "therapeutically effective amount" generally refers to an amount which, after an appropriate period of administration, is capable of achieving the effect of treating the diseases as listed above.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1 preparation of extract of active fraction of yellow lilac daphne
Percolating Chinese medicinal material flos Wikstroemiae Chamaedaphnes with 95% ethanol for 3 times each time for one week, and mixing 3 times of percolate to obtain ethanol percolate (flos Wikstroemiae Chamaedaphnes ethanol extract).
Concentrating the ethanol percolate at low temperature, removing ethanol, adding water into the obtained extract, and making into suspension.
Then adding equal volume of petroleum for extraction, removing petroleum ether extract (petroleum ether extract part of flos Wikstroemiae Chamaedaphnes ethanol extract), and repeating for 3 times.
Adding an equal volume of ethyl acetate solution into the residual solution after the petroleum ether extraction liquid is removed for extraction, and separating ethyl acetate extraction liquid; repeating for 3 times, mixing the 3 ethyl acetate extractive solutions, and concentrating to obtain extract (active part extract, WCME). Or, the steps are: adding an equal volume of n-butanol solution into the residual solution after the ethyl acetate extraction liquid is removed for extraction, and separating the n-butanol extraction liquid; repeating for 3 times, mixing the n-butanol extractive solutions for 3 times, and concentrating to obtain extract of n-butanol extractive part of flos Wikstroemiae Chamaedaphnes ethanol extract. The preparation process is shown in figure 1.
Example 2 high performance liquid chromatography analysis of the extract of ethyl acetate extract fraction of ethanol extract of daphne genkwa prepared in example 1 (active fraction extract)
Chromatographic conditions are as follows: the chromatographic column is ZORBAX Elipse XDB-C 18 (250 mm. times.4.6 mm,5 μm); mobile phase: methanol is taken as a mobile phase A, and 0.1% phosphoric acid water is taken as a mobile phase B; the gradient elution procedure is shown in table 1; flow rate: 1.0 ml/min; column temperature: 25 ℃; detection wavelength: 256 nm; sample injection amount: 10 μ l.
TABLE 1 gradient elution procedure
Figure BDA0003221878290000101
The high performance liquid analysis shows that the extract of the ethyl acetate extraction part of the daphne genkwa ethanol extract comprises the main components shown in the following structure (the component contents are shown in table 2):
Figure BDA0003221878290000111
TABLE 2. extract of flos Wikstroemiae Chamaedaphnes with ethanol extract containing main component and content of ethyl acetate
Figure BDA0003221878290000112
Example 3 determination of antitumor Activity of different fractions of extracts of Daphne genkwa
(3.1) cell culture
A375 (human malignant melanoma cells) and B16 (mouse malignant melanoma cells) were provided by the chinese academy of sciences cell bank (shanghai).
A375 cells in DMEM medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37 deg.C, 5% CO 2 And (5) culturing. Obtaining cells in exponential growth phase for next stepAnd (6) analyzing.
B16 cells in RPMI 1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37 deg.C, 5% CO 2 And (5) culturing. Cells in exponential growth phase were obtained for further analysis.
(3.2) determination of Activity of different extracts of daphne genkwa
Cell viability was measured using the MTT method. A375 and B16 cells were seeded in 96-well plates at a density of 5X 10 3 Individual cells/well, overnight culture.
The anti-tumor cell proliferation activity of the daphne genkwa ethanol extract, the daphne genkwa ethanol extract petroleum ether extract, the daphne genkwa ethanol extract ethyl acetate extract and the daphne genkwa ethanol extract n-butanol extract obtained in the embodiment 1 of the invention is determined: the different active site extracts were allowed to act on A375 and B16 cells for 24 hours, followed by incubation with MTT (5mg/mL in PBS) at 37 ℃ for 4 hours; then, after dissolving the crystals in dimethyl sulfoxide, absorbance at 570nm was measured using a spectrophotometer, and IC was calculated using GraphPad 8 50 。
The results of activity determination of the ethanol extract of daphne genkwa, the petroleum ether extract of the ethanol extract of daphne genkwa, the ethyl acetate extract of the ethanol extract of daphne genkwa (WCME), and the n-butanol extract of the ethanol extract of daphne genkwa on cells A375 and B16 are shown in Table 3, and the results show that WCme can obviously inhibit the proliferation of cells A375 and B16, while other extracts/extracts have no obvious antitumor activity (Table 3). In addition, as shown in FIG. 3, WCME has different inhibitory effects on A375 and B16 cell proliferation, IC 50 The values were 192.8. mu.g/mL (A375) and 156.2. mu.g/mL (B16), respectively. Therefore, the present inventors have conducted subsequent studies on the antitumor effects of WCME on A375 and B16 cells.
TABLE 3 screening of the activity of each part of Huanggenkwa
Figure BDA0003221878290000121
The results are shown in Table 3, wherein the extract of flos Genkwa is yellowFlos Genkwa ethanol extract petroleum ether extract part, flos Genkwa ethanol extract n-butanol extract part, and their activities in inhibiting two melanoma cells>300 mu g/mL, the three extracts are considered to have low activity, while the ethyl acetate extraction part of the Chinese lilac daphne flower bud ethanol extract has different inhibition effects on A375 and B16 cell proliferation, IC 50 The values were 192.8. mu.g/mL (A375) and 156.2. mu.g/mL (B16), respectively. The WCME activity of the invention is better than that of other part extracts.
Example 4 measurement of WCME-induced morphological changes in tumor cells by AO/PI staining
In this example, the concentrations of each drug added, unless otherwise specified, refer to the initial concentrations of the drug itself, with μ g/mL for WCME and μ M for DBZ.
Experimental groups A375 cells were treated with WCME at concentrations of 100, 200, 300 and 400. mu.g/mL for 24 hours, while the A375 cells treated with Dacarbazine (DBZ, Dacabazine) at a concentration of 300. mu.M served as positive controls and the group without any reagents served as blank controls (control); the A375 cells of each group were then stained with Acridine Orange (AO) and Propidium Iodide (PI), incubated for 5 minutes in the dark and unbound dye washed away with PBS. The stained cells were observed with a fluorescence microscope, and the morphological change of the cells after each group of treatments was observed.
Experimental groups treated B16 cells with WCME at concentrations of 100, 200, 300 and 400 μ g/mL for 24 hours, respectively, with B16 cells treated with Dacarbazine (DBZ, Dacarbazine) at a concentration of 300 μ M as a positive control group, and a group without any reagent as a blank control group (control); the groups of B16 cells were then stained with Acridine Orange (AO) and Propidium Iodide (PI), respectively, and incubated for 5 minutes in the absence of light, after which unbound dye was washed away with PBS. The stained cells were observed with a fluorescence microscope, and the morphological change of the cells after each group of treatments was observed.
The results are shown in FIG. 4, in which FIG. 4(A) shows the morphological change of B16 cells and FIG. 4(B) shows the morphological change of A375 cells. Compared with the positive control group, the WCME treatment group has obviously reduced cancer cell number and shows morphological changes of contraction, pyknosis and floating cell formation, which shows that the WCME of the invention can obtain better anti-tumor effect than the positive control group.
Example 5 scratch test
A375 cells were seeded in 6-well plates (1X 10) 6 Individual cells/well) to grow over 6-well plates. The cell migration was observed by scraping a cross-shaped wound line in the fused cells with 200. mu.L of the tip of a gun, the floating cells were washed with PBS, cell culture medium containing 2% FBS (blank control) or cell culture medium containing WCME at various concentrations of 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, and 200. mu.g/mL was added to the medium to act on A375 cells, and 48H (hours) was incubated, and the wound healing width was observed and photographed under a0, 24, and 48H microscope, respectively, and the wound healing rate was calculated.
B16 cells were seeded in 6-well plates (1X 10) 6 Individual cells/well) to grow over 6-well plates. Cell migration was observed by scraping a cross-shaped wound line in fused cells with 200. mu.L of the tip of a gun, floating cells were washed with PBS, cell culture medium containing 2% FBS (blank control) or WCME containing different concentrations of 30. mu.g/mL, 60. mu.g/mL, 90. mu.g/mL, and 120. mu.g/mL was added to the medium for B16 cells, 48H (hours) was incubated, wound healing width was observed, and photographs were taken under 0, 24, and 48H microscopes, respectively, and wound healing rate was calculated.
The results are shown in FIG. 5, in which FIG. 5(A) shows the migration ability of B16 cells; FIG. 5(B) shows the migration ability of A375 cells, and the experimental results show that WCME with a concentration of 90 μ g/mL can play a good role in inhibiting the migration of tumor cells 24 and 48 hours after WCME acts on B16 cells; after the WCME acts on A375 cells for 24 and 48 hours, the WCME with the concentration of 150 mu g/mL can play a good role in inhibiting the migration of tumor cells.
Example 6 cell cycle analysis
A375 and B16 cells were seeded in 6-well plates (1X 10) 6 Individual cells/well) and then added with 2% FBS in cell culture medium (blank control), WCME at different concentrations (100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL) and 300 μ M Dacarbazine (DBZ, Dacarbazine) as a positive control.
After 24 hours, a375 and B16 cells were harvested and washed twice with cold PBS. Cells were fixed with 75% frozen ethanol overnight at 4 ℃ and then with pre-cooled PBS suspension cells. Centrifuging again, adding dye, incubating at room temperature for 30 min, and detecting on a flow cytometer, wherein the result is shown in FIG. 6, in which FIG. 6(A) is a B16 cell cycle diagram, and FIG. 6(B) is an A375 cell cycle diagram; as a result, it was found that G in A375 cells was observed with the increase in the concentration of WCME 0 /G 1 Period and G 2 The cells in the M phase gradually decrease, and the cells in the S phase increase, which shows that the WCME can block the cells in the S phase to inhibit the cell proliferation; s phase and G phase in B16 cells with increasing concentration of WCME 2 Gradual decrease of cells in the/M phase, G 0 /G 1 Stage cytosis, suggesting that WCME can arrest cells at G 0 /G 1 To inhibit cell proliferation.
Example 7 flow cytometry analysis of apoptosis
The influence of WCME on apoptosis is detected by using Annexin V-FITC apoptosis detection method in combination with flow cytometry.
Cells A375 and B16 were treated with WCME or dacarbazine at various concentrations for 24h, washed with PBS, and then treated with 5. mu.Lannexin V-FITC and 5. mu.L PI at a concentration of 5X 10 4 cells/mL were mixed gently and allowed to wait 15 minutes at room temperature in the dark. Subsequently, cells were added to 400 μ L binding Buffer (1 × Buffer) and detected on a flow cytometer. The results are shown in FIG. 7, in which FIG. 7(A) is a B16 apoptosis graph; FIG. 7(B) is a graph of apoptosis of A375 cells; as a result, it was found that the proportion of apoptotic cells gradually increased with the increase of WCME concentration, which is consistent with the results of the fluorescence microscope in example 6 that most of the cells were in the late stage of apoptosis.
The above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; variations and advantages that may occur to those skilled in the art may be made without departing from the spirit and scope of the inventive concept, which is intended to be covered by the claims.

Claims (7)

1. The anti-melanoma extract of daphne tinctoria is characterized by comprising the following components in parts by weight: 5-15 parts of isoquercitrin, 1-5 parts of eriodictyol, 40-65 parts of luteolin, 0.2-2 parts of apigenin, 1-3 parts of aureoflavone, 0.1-1 part of bimetacin A, and 0.1-0.5 part of 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate;
the preparation method of the yellow lilac daphne flower bud anti-melanoma extract comprises the following steps:
(1) percolating flos Wikstroemiae Chamaedaphnes with small molecular alcohol to obtain flos Wikstroemiae Chamaedaphnes small molecular alcohol extract;
(2) removing the small molecular alcohol;
(3) then adding petroleum ether for extraction;
(4) removing the petroleum ether extract;
(5) and adding an ethyl acetate solution into the residual solution from which the petroleum ether extraction liquid is removed for extraction to obtain an ethyl acetate extraction part of the daphne genkwa micromolecular alcohol extract, namely the daphne genkwa anti-melanoma extract.
2. The daphne genkwa anti-melanoma extract as claimed in claim 1, which comprises the following components in parts by weight: 10-13 parts of isoquercitrin, 2-3 parts of eriodictyol, 50-60 parts of luteolin, 0.8-1.2 parts of apigenin, 1.5-2 parts of aureoflavone, 0.40-0.65 part of bimetacin A and 0.2-0.4 part of 6a,7 a-epoxy-5 b-hydroxy-12-deoxyphorbol-13-decanoate.
3. The preparation method of the daphne genkwa anti-melanoma extract as claimed in claim 1, which specifically comprises the following steps:
(1) taking Chinese medicinal material daphne genkwa, extracting by small molecule alcohol percolation, repeating for several times, and mixing repeatedly obtained small molecule alcohol percolation extract, namely daphne genkwa small molecule alcohol extract;
(2) concentrating small molecular alcohol extract of flos Wikstroemiae Chamaedaphnes at low temperature, removing small molecular alcohol, and adding water into the obtained extract to obtain suspension;
(3) then adding petroleum ether for extraction;
(4) removing the petroleum ether extract;
(5) adding an ethyl acetate solution into the residual solution from which the petroleum ether extract is removed for extraction, and separating to obtain an ethyl acetate extract; repeating the operation, combining the ethyl acetate extract, and concentrating to obtain extract of the ethyl acetate extraction part of the small molecular alcohol extract of the daphne genkwa, namely the anti-melanoma extract of the daphne genkwa.
4. The method for preparing an anti-melanoma extract from daphne genkwa as claimed in claim 3, wherein the small molecular alcohol is at least one selected from methanol, ethanol, propanol, ethylene glycol, 1, 2-propylene glycol, 1, 4-butylene glycol, 1, 6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, trimethylolpropane, glycerol and pentaerythritol.
5. Use of the daphne genkwa anti-melanoma extract as claimed in claim 1 or 2 in the preparation of anti-melanoma drugs.
6. The use according to claim 5, wherein the anti-melanoma is the prevention and/or treatment of melanoma;
the treatment of melanoma refers to inhibiting proliferation, growth, migration and infiltration of melanoma and promoting apoptosis of melanoma.
7. A pharmaceutical composition, which is characterized in that the pharmaceutical composition comprises the daphne genkwa anti-melanoma extract as claimed in claim 1 or 2, and a pharmaceutically acceptable carrier and a pharmaceutically acceptable medium.
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