CN115837051B - Composite cordycepin preparation, preparation method thereof and application thereof in preparation of small cell lung cancer resistant products - Google Patents
Composite cordycepin preparation, preparation method thereof and application thereof in preparation of small cell lung cancer resistant products Download PDFInfo
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- CN115837051B CN115837051B CN202210713732.XA CN202210713732A CN115837051B CN 115837051 B CN115837051 B CN 115837051B CN 202210713732 A CN202210713732 A CN 202210713732A CN 115837051 B CN115837051 B CN 115837051B
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Classifications
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- A61K36/066—Clavicipitaceae
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
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- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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- A—HUMAN NECESSITIES
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a compound cordycepin preparation, a preparation method thereof and application thereof in preparing anti-small cell lung cancer products; the compound cordycepin preparation comprises the following components in parts by weight: 65-80 parts of cordycepin, 10-25 parts of black nightshade extract and 1-20 parts of yew extract. The compound cordycepin preparation provided by the invention can not only enhance the efficacy of treating small cell lung cancer, but also reduce the side effects thereof.
Description
Technical Field
The invention belongs to the technical field of natural medicines, and particularly relates to a compound cordycepin preparation, a preparation method thereof and application thereof in preparation of anti-small cell lung cancer products.
Background
Small cell lung cancer accounts for about 15% -20% of bronchogenic lung cancer, and when small cell lung cancer is diagnosed, patients with tumor in local stage account for about 30%, and the rest in extensive stage (when tumor spreads beyond supraclavicular zone, it is extensive stage). Compared with other lung cancer types, the small cell lung cancer has better chemotherapy and radiotherapy curative effects. However, small cell lung cancer is often difficult to cure because the tumor is likely to have spread widely at the time of diagnosis of small cell lung cancer.
Cordycepin is also called cordycepin, and 3' -deoxyadenosine, and is the first nucleoside antibiotic separated from fungi. It has antibacterial, antiinflammatory, antiviral, antitumor, and immunoregulatory activities; cordycepin is a non-mutagenic and nontoxic compound, and has potential antiproliferative and anti-migration effects on small cell lung cancer cells.
The taxol is a natural secondary metabolite separated and purified from the bark of the gymnosperm yew, has good anti-tumor effect through clinical verification, and has special effects on lung cancer, ovarian cancer, uterine cancer, breast cancer and the like with higher incidence rate of cancer. Paclitaxel is the most popular anticancer drug in the international market in recent years, and is considered to be one of the most effective anticancer drugs in the future 20 years of humans. However, paclitaxel has strong side effects including anaphylaxis, neurotoxicity, cardiovascular reactions, intestinal reactions, hepatotoxicity, and local inflammatory reactions.
The steroid saponin and the steroid alkaloid can influence and reduce the expression of proteins on tumor cell membranes, such as reducing the content of sialic acid, inhibiting the ATPase activity and down-regulating the expression of N-acetyl transferase, thereby reducing the mobility of the tumor cell membranes and inhibiting the metabolic growth of the tumor cells, and can reduce the energy metabolism in cancer cells, prevent the growth of the cancer cells and inhibit the proliferation of the cancer cells. Meanwhile, the steroid saponin and the steroid alkaloid can activate the signal path of cancer cell apoptosis and promote the cancer cell apoptosis. However, it also has some side effects such as slight poisoning, or itching of the oral mucosa and throat, after several hours, it can be completely relieved by self metabolism, and the upper abdomen pain can occur for the person with heavy poisoning, and symptoms such as nausea, vomiting, diarrhea, etc. can occur. If the patients are further aggravated, dehydration, body temperature increase, pupil enlargement, photophobia, tinnitus, limb convulsion, dyspnea, blood pressure drop, shock and even death can be caused by vomiting and diarrhea, so that the serious patients should be sent to the hospital as early as possible.
Therefore, the invention provides a compound cordycepin preparation, a preparation method thereof and application thereof in preparing anti-small cell lung cancer products.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a compound cordycepin preparation aiming at the defects of the prior art.
The invention also solves the technical problem of providing a preparation method of the compound cordycepin preparation.
The invention further aims to provide the application of the compound cordycepin preparation.
In order to solve the first technical problem, the invention discloses a compound cordycepin preparation which comprises the following components in parts by weight:
65-80 parts of cordycepin
10-25 parts of black nightshade extract
1-20 parts of yew extract.
Preferably, the compound cordycepin preparation comprises the following components in parts by weight:
70-75 parts of cordycepin
15-22 parts of black nightshade extract
3-15 parts of yew extract.
Further preferably, the compound cordycepin preparation comprises any one of the following components in parts by weight:
still more preferably, the compound cordycepin formulation comprises any one of the following components in parts by weight:
wherein, the cordycepin is extracted from wild Cordyceps militaris and/or artificially cultured Cordyceps militaris.
Wherein the purity of the cordycepin is more than 50%.
Wherein the rest components of the cordycepin are polysaccharide compounds or other impurities; preferably, the rest of the cordycepin is mainly polysaccharide compound; further preferably, the rest of the cordycepin is polysaccharide compound.
Wherein the content of steroid in the herba Solani Nigri extract is above 50%.
Wherein the steroid compound is steroid saponin and/or steroid alkaloid.
Wherein the rest components of the black nightshade extract are polysaccharide compounds or other impurities; preferably, the rest components of the black nightshade extract are mainly polysaccharide compounds; further preferably, the rest components of the black nightshade extract are polysaccharide compounds.
Wherein the Taxus chinensis extract is extracted from natural wild and/or artificially cultivated Taxus chinensis.
Wherein the content of paclitaxel in the Taxus chinensis extract is above 50%.
Wherein the rest components of the yew extract are polysaccharide compounds or other impurities; preferably, the rest components of the yew extract mainly comprise polysaccharide compounds; further preferably, the rest components of the yew extract are polysaccharide compounds.
Wherein, the three components complement each other and act together; specifically, the cordycepin is an RNA synthesis inhibitor, can selectively inhibit RNA synthesis and then influence protein synthesis, has an anti-tumor effect, and has the effect of inducing apoptosis of tumor cells; the steroid compound in the black nightshade extract can reduce the membrane fluidity and the membrane protein level of tumor cells, and can also regulate the signal transduction pathway to promote apoptosis; the taxol in the taxus chinensis extract can promote the polymerization of the mesoscopin heterodimer into microtubules, and the microtubules are combined to promote the stability of the microtubules, so that the dynamic change of the microtubules is inhibited, the formation of a spinning body is interfered, the chromosome cannot be separated, and the mitosis is stopped in the middle stage.
Wherein, the dosage form of the compound cordycepin preparation is any one of powder, capsule, pill, granule, tablet and oral liquid.
In order to solve the second technical problem, the invention discloses a preparation method of the compound cordycepin preparation.
The preparation method comprises the steps of weighing the components according to a formula, mixing, dissolving in water, and preparing a liquid composition preparation.
The preparation method comprises the steps of weighing the components according to a formula, mixing, adding auxiliary materials required by preparation molding, and preparing a medicinal preparation by a conventional method.
In order to solve the third technical problem, the invention discloses application of the compound cordycepin preparation in preparation of anti-small cell lung cancer products.
Wherein the product is a pharmaceutical product.
In the invention, the purity and the content are both in mass percent.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) Compared with the traditional Chinese medicine composition for resisting small cell lung cancer, the compound cordycepin preparation provided by the invention is easier to be absorbed by human body, and is characterized in that the used extracts are anticancer active components of the medicine, the rest are saccharides with small molecular weight, the weight average molecular weight is about below 800, metabolism is not needed, and the compound cordycepin preparation can be directly absorbed by human body.
(2) The three extracts provided by the invention are strictly screened, and the three used medicinal components complement each other, so that the identification and synthesis of gene codes in the synthesis process of cancer cells can have an inhibition effect, thereby inhibiting proliferation of small cell cancers.
(3) The compound cordycepin preparation provided by the invention has obvious effects on the treatment of early cancers and the auxiliary treatment in the later chemotherapy process.
(4) The compound cordycepin preparation provided by the invention can not only enhance the efficacy of treating small cell lung cancer, but also reduce the side effects thereof.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 brain metastasis of H69 cells in zebra fish eggs under a fluorescence microscope in a zebra fish experiment stained with 0.01% acridine orange fluorescent dye.
FIG. 2 shows the tail transfer of H69 cells from zebra fish eggs under a fluorescence microscope after adding a filter in the zebra fish experiment.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
In the following examples, the purity of cordycepin was 95.5%, and the content of polysaccharide compound was 4.5%, which was mainly an oligosaccharide having a weight average molecular weight of 720 to 180.
In the following examples, reference to the extraction process of solanine in the solanum nigrum extract (extraction method of solanine in potato) is mainly solanine, the content of solanine is 83.8%, the content of sugar compound is 16.2%, which is mainly oligosaccharide with weight average molecular weight of 720-180.
In the following examples, the taxol content of the Taxus chinensis extract was 82.3% and the polysaccharide content was 17.7%, which were mainly oligosaccharides with weight average molecular weight of 720 to 180.
The experimental mice used in the invention are purchased from Nanjing university of agriculture animal center, lewis lung cancer cell strain is purchased from Nanjing university of agriculture animal center, and the mouse CD4 monoclonal antibody and CD8 monoclonal antibody are purchased from Simer Feishier technology (China) Co; TNF-alpha, IL-2, IL-7ELISA kit, abcam company, UK.
The invention adopts a Lewis lung cancer mouse model with deficiency of both qi and yin, and the preparation method refers to literature (the toxicity-reducing and efficacy-enhancing effects of the flavored Tortoise deer two-immortal glue soup on the chemotherapy of the Lewis tumor-bearing mice with deficiency of both qi and yin), and Chinese medicinal materials, 2014,37 (8): 1434-1437).
Animal feeding environment in the invention: SPF grade, room temperature (20.0-22.5 ℃) and relative humidity (54.4-68.4)% RH, and group cage feeding, 10/cage.
The invention detects through the following indexes:
1. observe the general condition of mice: the general condition of stool, hair color, etc. of each group of mice was observed daily after administration.
2. Weight loss rate= (day 1 weight-day 22 weight)/day 1 weight×100% weight.
3. Calculating the tumor inhibition rate: on day 22, mice were sacrificed by cervical dislocation and tumors were isolated.
Tumor inhibition rate = (mean tumor mass in model group-mean tumor mass in experimental group)/mean tumor mass in model group x 100%.
Example 1
(1) Compound cordycepin preparation
The mass of cordycepin, taxus chinensis extract and black nightshade extract is 750mg:30mg:220mg of each composition was weighed, mixed and dissolved in 1L of water as physiological salt.
(2) The lavage experiment was performed with 4 groups of mice. Each group of mice were 5 male and female mice. The high dose group, the medium dose group, the low dose group and the model group are respectively used. The mice in the high dose group were dosed at 80mg/kg, the mice in the medium dose group were dosed at 50mg/kg, and the mice in the low dose group were dosed at 20mg/kg. The model group is equal volume physiological saline. 2 times daily. For 22 days. The detection method refers to the method described above.
The experimental results are shown in tables 1-2:
TABLE 1
| Group of | Weight loss rate (%) | Hardness of feces | Hematochezia/occult blood | Dehairing device |
| Model group | 23 | Normal state | Normal state | Normal state |
| High dose group | 3 | Normal state | Normal state | Normal state |
| Medium dose group | 6 | Normal state | Normal state | Normal state |
| Low dose group | 10 | Normal state | Normal state | Normal state |
TABLE 2
| Group of | Tumor mass/g | Tumor inhibition rate/% |
| Model group | 3.56±0.45 | - |
| High dose group | 0.62±0.36 | 82.5％ |
| Medium dose group | 1.26±0.41 | 64.6％ |
| Low dose group | 2.04±0.45 | 42.7％ |
Example 2
Referring to example 1, only the proportions of the components in the compound cordycepin preparation were changed: the mass of cordycepin, taxus chinensis extract and black nightshade extract is 700mg:150mg:150mg, and the results of the gastric lavage experiments performed in the high dose group, the medium dose group, the low dose group, the model group and the blank group are shown in tables 3 to 4.
TABLE 3 Table 3
| Group of | Weight loss rate (%) | Hardness of feces | Hematochezia/occult blood | Dehairing device |
| Blank control group | 0 | Normal state | Normal state | Normal state |
| Model group | 22.9％ | Normal state | Normal state | Normal state |
| High dose group | 2.6％ | Normal state | Normal state | Normal state |
| Medium dose group | 5.4％ | Normal state | Normal state | Normal state |
| Low dose group | 9.3％ | Normal state | Normal state | Normal state |
TABLE 4 Table 4
| Group of | Tumor mass/g | Tumor inhibition rate/% |
| Blank control group | 0 | - |
| Model group | 3.61±0.12 | - |
| High dose group | 0.55±0.65 | 84.7％ |
| Medium dose group | 1.22±0.17 | 66.2％ |
| Low dose group | 2.01±0.44 | 44.3％ |
Example 3
Establishing a zebra fish brain transfer and tail transfer model of a small cell lung cancer cell strain H69
The test method comprises the following steps:
1. fish line hybridization and offspring cultivation
Putting female and male fishes 1:1 of a fish strain tg (flk-GFP) into the same mating box, inserting a partition board, separating the two fishes, changing water 9 hours a day after the water temperature is 28 ℃, illumination is 14L:10D, pH value is 6.5-7.5, pulling out the board, mating the two fishes, spawning, and recovering eggs after 1-2 hours. The received eggs are separated into plates, about 50 eggs are placed into a dish for culture at 28 ℃, and about 300 mu L of PTU is added into each dish at night on the day of egg collection to inhibit melanin formation. The water was changed once a day, and the PTU was added with care taken to change the water. Fluorescence was picked up at 2dpf (day post-transfer), embryos expressing flk-GFP were left, i.e., embryos expressing green fluorescence throughout the blood vessels, and embryos not expressing fluorescence were sacrificed (10 min on ice) and flk-GFP embryos were excised from the egg shells.
2. Cell passage and culture of tfRFP-B16-F10 cells
Cells in T25 cell flasks were washed once with 1mL PBS buffer, the buffer was discarded and repeated once. Adding 1mL of pancreatin preheated at 37 ℃ for digestion for 2min or until cells are found to be fully rounded under a microscope, adding an equal amount of complete culture solution for stopping digestion, gently blowing down digested cells by a pipette, transferring into a 15mL centrifuge tube, centrifuging at room temperature for 5min at 1000r/min, discarding supernatant, gently blowing up cells by 1mL of complete culture solution, mixing uniformly, sucking part of cell suspension into a new cell culture bottle, adding 4mL of fresh culture solution, gently shaking uniformly, placing at 37 ℃ and 5% CO 2 Culturing in an incubator.
3. Highly tumorigenic cell culture H69 cells
A fibrinogen, thrombin is prepared. Dissolving in ice for 30min, placing T7 buffer and E3 embryo culture medium, pancreatin, etc. in water bath at 37deg.C for preheating; the gun head and the culture plate are placed in an ultra-clean workbench for ultraviolet sterilization, and the ultraviolet irradiation is generally carried out for 30min. The tfRFP-B16-F10 cells were rinsed once with PBS, digested with 1mL pancreatin for 2min (37 ℃), then neutralized with 2mL E3 embryo culture medium, centrifuged for 2min at 1600r/min to obtain pellet, and the supernatant was discarded. The pellet was resuspended in 1mL of medium.
4. Cell microinjection
About 200H 69 cells were injected into the peri-egg space of 2dpf (two days after fertilization) using a microinjection apparatus, and brain and tail metastases of H69 cells in zebra fish eggs were observed using a fluorescence microscope until 3 dpf. The statistical result includes the sum of the number of brain metastases and tail metastases.
As shown in Table 5, the group Control (physiological saline), the CisPt group (0.9 mg CisPt) and the cordycepin preparation group (10 mg cordycepin preparation, 0.25 mg/mL) were set, and the brain transfer and tail transfer behaviors of H69 cells during 0-3dpt were observed.
Wherein, the cordycepin preparation group comprises cordycepin, yew extract and black nightshade extract according to the mass ratio of 70:15:15, weighing the components, mixing and dissolving in 1L of physiological salt water, wherein the concentration is 0.25mg of cordycepin, the taxus chinensis extract and the black nightshade extract/mL of water.
Experimental results:
TABLE 5
As shown in fig. 1 and 2 and table 5, both cisplatin and cordycepin preparation groups were effective in inhibiting brain and tail metastasis of H69 cells in zebra fish, but the effect of cordycepin preparation group was significantly better than cisplatin group. .
Comparative example 1
The tumor inhibition rates were measured in the same manner as in example 2, except that the proportions of the respective components in the compound cordycepin preparation were changed, and the results are shown in Table 6.
TABLE 6
| Quality of cordycepin, taxus chinensis extract and Solanum nigrum extract | Tumor mass/g | Tumor inhibition rate/% |
| Model group | 3.61±0.12 | - |
| 150mg：700mg：150mg | 0.99±0.46 | 72.6％ |
| 150mg：150mg：700mg | 0.85±0.38 | 76.5％ |
Comparative example 2
The tumor inhibition rates were measured at high doses by changing only the proportions of the respective components in the compound cordycepin preparation as in example 2, and the results are shown in tables 7 and 8.
TABLE 7
TABLE 8
From the above examples and comparative examples, it can be seen that, when cordycepin, taxus chinensis extract and Solanum nigrum extract (mass ratio 750mg:30mg:220mg, or 700mg:150 mg) are adopted at the same time, synergistic effect exists between the three, which not only can reduce side reaction of tumor drugs, but also can greatly improve tumor inhibition rate.
Example 4: toxicity test
(1) Acute toxicity test
The experimental method comprises the following steps: 6 experimental groups and 1 control group were set to correspond to 15g/kg, 12g/kg, 9g/kg, 6g/kg, 3g/kg and 0k/kg of the compound cordycepin preparation (cordycepin 750mg: taxus chinensis extract 30mg: solanum nigrum extract 220 mg)/mouse body weight, respectively. Dissolving in physiological saline to obtain gastric lavage liquid. Gastric lavage experiments were performed on 10 mice per group. The animals were fasted overnight and were free to drink water before being fed with the stomach twice for 24 h. Normal diet was given after gastric lavage, and 14 days was observed, and the poisoning characteristics and death were recorded.
Experimental phenomena: single feeding of 15g/kg of the compound cordycepin preparation can cause the death rate of mice to be 20%, and the other groups have no death condition. The group of mice corresponding to 15g/kg of the compound cordycepin preparation can have the conditions of watery stool and inappetence. The other mice were all stool normally and were more active and fed than the control group.
Experimental results: the Minimum Lethal Dose (MLD) of the compound cordycepin preparation for single oral dose of mice is 15g/kg (equivalent to 2.38g/kg of human equivalent dose), and the maximum drug tolerance (MID) is more than 12g/kg (equivalent to 2.14g/kg of human dose). The LD50 of the compound cordycepin preparation is more than 15g/kg and is far higher than 2000mg/kg specified by the OECD acute correlation principle. Therefore, the compound cordycepin preparation has the risk of severe acute poisoning.
(2) Chronic toxicity test
The experimental method comprises the following steps: the compound cordycepin preparation is continuously administered to mice for three months, and toxic reaction and severity thereof, main toxic target organs and damage condition of the mice are observed.
The compound cordycepin preparation was set in high dose group (80 mg/kg, 20 times of quasi-clinical dose), medium dose group (40 mg/kg, 10 times of quasi-clinical dose), low dose group (20 mg/kg, 5 times of quasi-clinical dose), and control group (physiological saline). The administration is performed by stomach irrigation once a day for 13 weeks.
Experimental results: during the dosing period, no significant abnormalities were seen in the appearance, behavioural activity, fecal character, and feeding. The long-term administration of cordycepin has no influence on the large tertiary weight. The blood index of the mice is not changed obviously, the AST in the large tertiary serum can be reduced obviously by the high and medium dose compound cordycepin preparation, and the AST of the serum is normal because the ASY value is lower than that of the serum which is just normal in liver function, and the AST of the serum does not have toxic action on liver and kidney functions of the mice.
The invention provides a compound cordycepin preparation, a preparation method thereof and an application thought and a method for preparing an anti-small cell lung cancer product, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by a person skilled in the art without departing from the principle of the invention, and the improvements and the modifications are also regarded as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (7)
1. The compound cordycepin preparation for resisting small cell lung cancer is characterized by being prepared from the following components in parts by weight:
65-80 parts of cordycepin
10-25 parts of black nightshade extract
1-20 parts of yew extract;
the content of steroid compounds in the black nightshade extract is more than 50%; wherein the steroid compound is steroid saponin and/or steroid alkaloid;
the content of paclitaxel in the Taxus chinensis extract is above 50%.
2. The compound cordycepin preparation of claim 1, which is characterized by comprising the following components in parts by weight:
70-75 parts of cordycepin
15-22 parts of black nightshade extract
3-15 parts of yew extract.
3. The compound cordycepin preparation of claim 1, which is characterized by comprising the following components in parts by weight:
cordycepin 75 parts
22 parts of black nightshade extract
3 parts of yew extract.
4. The compound cordycepin preparation of claim 1, which is characterized by comprising the following components in parts by weight:
cordycepin 70 parts
15 parts of black nightshade extract
15 parts of yew extract.
5. The compound cordycepin preparation of claim 1, wherein the compound cordycepin preparation is in the form of any one of powder, capsule, pill, granule, tablet, and oral liquid.
6. Use of a compound cordycepin formulation of any one of claims 1 to 5 in the manufacture of a product for the treatment of small cell lung cancer.
7. The use according to claim 6, wherein the product is a pharmaceutical product.
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