WO2024016142A1 - Use of bergapten and quercetin flavone composition in preparation of medicament for treating cancer - Google Patents
Use of bergapten and quercetin flavone composition in preparation of medicament for treating cancer Download PDFInfo
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- WO2024016142A1 WO2024016142A1 PCT/CN2022/106383 CN2022106383W WO2024016142A1 WO 2024016142 A1 WO2024016142 A1 WO 2024016142A1 CN 2022106383 W CN2022106383 W CN 2022106383W WO 2024016142 A1 WO2024016142 A1 WO 2024016142A1
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- WIPO (PCT)
- Prior art keywords
- cancer
- bergamot
- quercetin
- lactone
- preparation
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- 229930014626 natural product Natural products 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
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- 238000011127 radiochemotherapy Methods 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates to the technical field of anti-cancer drug preparation, and in particular to the application of a composition of bergamot lactone and quercetin flavonoids in the preparation of cancer treatment drugs.
- Radiochemotherapy has little effect in clinical use due to its major side effects.
- the probability of recurrence and metastasis is still relatively high. Therefore, patients need regular examinations and appropriate adjustments to their lifestyle and diet. .
- Standard postoperative adjuvant chemotherapy and radiotherapy can reduce the recurrence and metastasis of cancer or prolong the time of recurrence and metastasis, but there is still the possibility of recurrence and metastasis.
- cancer patients After surgery, cancer patients need to be reviewed every 3 to 4 months, given anti-cancer drugs intravenously, and hospitalized for about 10 days. This frequency should be maintained for at least two years; after two years, it can be changed to once every six months, and then continued Three years, so a total of five years of treatment is required.
- cancer surgery combined with radiotherapy and chemotherapy, the body is very weak. Generally speaking, it is difficult to prevent recurrence and restore health after cancer surgery.
- the object of the present invention is to provide the application of the composition of bergamot lactone and quercetin flavonoids in the preparation of cancer treatment drugs to solve the problems existing in the prior art.
- the present invention provides the following solutions:
- One of the objects of the present invention is to provide the application of the composition of bergamot lactone and quercetin flavonoid in the preparation of cancer treatment drugs.
- the bergamot lactone is a natural substance isolated from the hand of bergamot; the quercetin flavonoid It is a natural substance isolated from Sophora japonica;
- the mass ratio of bergamot lactone and quercetin flavonoids in the composition of bergamot lactone and quercetin flavonoids is 1:3 to 8;
- the cancer is lung cancer, liver cancer, cervical cancer or prostate cancer;
- the preparation method of bergamot lactone includes the following steps:
- the application also includes the preparation of drugs for auxiliary treatment of tumors and the preparation of anti-cancer reversal agents, that is, the application of drugs in enhancing the efficacy and reducing toxicity of tumor chemotherapy drugs, sensitizing tumor radiotherapy, and enhancing the immune function of the body.
- the therapeutically effective dose of the combination of bergamot lactone and quercetin flavonoids is 5 to 50 mg/kg.
- the therapeutically effective dose of the combination of bergamot lactone and quercetin flavonoids is 10 to 25 mg/kg.
- the second object of the present invention is to provide an anti-cancer drug, which uses a composition of bergamot lactone and quercetin flavonoids as active ingredients, and also contains a pharmaceutically acceptable carrier.
- the bergamot lactone is isolated from bergamot. Obtained natural substances;
- the quercetin flavonoid is a natural substance isolated from Sophora japonica
- the mass ratio of bergamot lactone and quercetin flavonoids in the composition of bergamot lactone and quercetin flavonoids is 1:3 to 8;
- the cancer is lung cancer, liver cancer, cervical cancer or prostate cancer.
- the therapeutically effective amount of the composition of bergamot lactone and quercetin flavonoids is 2.5-100 mg/kg, and the purity is greater than 80%.
- the bergamot lactone involved in the present invention is a high-purity natural compound bergamot lactone isolated from the fruit of the Rutaceae plant Citrus medica L. var. sarcodactylis Swingle. Its structures are:
- Quercetin flavonoid is a natural substance isolated from Sophora japonica. Its structures are:
- Quercetin (molecular formula: C 15 H 10 O 7 ).
- the pharmaceutically acceptable carrier includes one or more of fillers, binders, disintegrants, lubricants, flavoring agents and antioxidants.
- the filler includes mannitol, lactose or polyethylene glycol; the binder includes starch or microcrystalline cellulose; the disintegrant includes carboxymethyl cellulose or low-substituted hydroxypropyl cellulose. ; The lubricant includes talc or magnesium stearate.
- the mass ratio of the filler to the antioxidant is 2 to 5:1.
- anti-cancer drug dosage forms are oral preparations, injection oil preparations or external preparations.
- the oral preparation is a tablet, capsule, sustained-release tablet or dropping pill
- the external preparation is a cream, ointment, oil or glycerin preparation.
- the preparation method of the tablet is: weigh 10 to 70% of the composition of bergamot lactone and quercetin flavonoids in terms of mass percentage, add 30 to 90% of an acceptable carrier, and stir and mix. Granulate, dry at 60-70°C for 24 hours, and press to obtain the tablets, each tablet containing 2.5-20 mg of bergamot lactone and quercetin flavonoids, preferably 2.5 mg, 5 mg, 10 mg, 15 mg and 20 mg.
- the preparation method of the capsule is: weighing 10 to 70% of the composition of bergamot lactone and quercetin flavonoids in terms of mass percentage, adding 30 to 90% of an acceptable carrier, and stirring and mixing to prepare the capsule.
- the capsules are dried at 60 to 70° C. for 24 hours, and then filled into empty capsule shells to obtain the capsules.
- Each capsule contains 2 to 20 mg of bergamot lactone and quercetin flavonoids.
- the preparation method of the injection oil is: weighing 1 to 10% of the composition of bergamot lactone and quercetin flavonoids and 90 to 99% of the injection oil in terms of mass percentage, and then packaging.
- the injection oil is prepared by sterilization, and the injection oil is soybean oil or camellia oil for injection.
- the preparation method of the dripping pills is: weighing 10 to 60% of the composition of bergamot lactone and quercetin flavonoids in terms of mass percentage, adding 40 to 90% of an acceptable carrier, and heating and melting. , the melting temperature is 100-150°C, the raw materials and auxiliary materials are fully mixed and dropped into pills to obtain the dripping pills, each of which contains 2.5-20 mg of the composition of bergamot lactone and quercetin flavonoids.
- composition containing bergamot lactone and quercetin flavonoids of the present invention shows that it has obvious pharmacological activities such as anti-lung cancer, anti-liver cancer, anti-cervical cancer, etc.
- the composition is expected to be developed into a low-toxicity, high-efficiency and multi-functional New cancer prevention and treatment drugs, anti-cancer reversal agents and tumor auxiliary treatment drugs with pharmacological effects of natural traditional Chinese medicine ingredients.
- the material-liquid ratio of chayote powder and ethanol solution is 0.03g/mL.
- the sample solution is directly passed through the X-8 macroporous adsorption resin.
- the amount of raw solution is 110% of the static saturated adsorption capacity of bergamot lactone by the macroporous adsorption resin; the column diameter and column height ratio of the macroporous adsorption resin is 1:5.
- Quercetin flavonoid extraction method Add 200 ml of 0.4% boric acid aqueous solution to 20 g of sophora rice powder, and adjust the pH value to 8 to 9 with milk of lime. Stir and extract under slightly boiling conditions for 30 minutes. Adjust the filtrate to a pH value of 2 to 3 with concentrated hydrochloric acid. Let it stand for 12 hours and then centrifuge. The precipitate is washed once with concentrated hydrochloric acid of a pH value of 1 to 2, and washed with distilled water until neutral. The precipitate is washed with The crude rutin product was obtained by vacuum drying at 65°C. After purifying rutin, take 1 g, use 80 ml of 2% sulfuric acid solution, and heat and reflux for 90 minutes to convert it into quercetin flavonoids.
- Preparation of tablets In terms of mass percentage, weigh 50wt.% of the composition of bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5), Add 50wt.% mannitol, starch, carboxymethyl cellulose, and talc (the mass ratio of mannitol: starch: carboxymethyl cellulose: talc is 1:2:3:2), stir, mix and granulate. Dry at 70°C for 24 hours and press into tablets. Each tablet contains 10 mg of bergamot lactone and quercetin flavonoids.
- Preparation of capsules In terms of mass percentage, weigh 10 wt.% of the composition of bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5), and add 90wt.% lactose, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, magnesium stearate and flavoring (lactose, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, magnesium stearate and flavoring
- the mass ratio of the agent is 1: 2:1:2:1), stir, mix and granulate, dry at 70°C for 24 hours, and then fill into empty capsule shells.
- Each capsule contains bergamot lactone and quercetin flavonoids. 5mg.
- the flavoring agent is aspartame and lemon essence with a mass ratio of 40:1.
- Preparation of oil In terms of mass percentage, weigh 10 wt.% of the composition of bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5) and Mix 90wt.% injection oil, pack and sterilize to prepare the oil.
- Preparation of dripping pills In terms of mass percentage, weigh 60wt.% of the composition of bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5), Add 40wt.% polyethylene glycol and antioxidant (the mass ratio of polyethylene glycol and antioxidant is 4:1) for heating and melting. The melting temperature is 150°C. Drop it into pills. Each pill contains bergamot. Lactone and quercetin flavonoids 20mg.
- the antioxidants are vitamin C and sodium metabisulfite with a mass ratio of 4:1.
- composition of bergamot lactone and quercetin flavonoids has the following properties: it appears as light yellow needle-like crystals under a microscope and exhibits brown-yellow fluorescence under ultraviolet light.
- the TLC ratio shift value Rf is: 0.538 (petroleum ether:ethyl acetate 8:2).
- IR ⁇ KBr max (cm -1 ): 3370 (broad peak, OH), 1655 (keto group C O), 1600, 1560, 1510 (phenyl).
- the invention discloses that a composition containing bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5) can be used to prepare anti-tumor drugs and anti-cancer reversal agents.
- Applications, including anti-tumor activity include: applications in preparing drugs for the treatment of lung cancer, liver cancer, and cervical cancer.
- Inoculate cells Take logarithmic growth phase cells, digest them with 0.02% EDTA, add RPMI-1640 solution containing 10 to 15% fetal calf serum, dilute the cells into a single cell suspension, count and adjust the cell number, and Inoculate into a 96-well plate, and culture the inoculated cells in a 37°C carbon dioxide incubator for 24 hours.
- MTT MTT is dissolved in RPMI-1640, and 50ul is added to each well. Then place it at 37°C and continue culturing for 4 hours before taking it out.
- Hydroxycamptothecin produced by Hubei Huangshi Feiyun Pharmaceutical Co., Ltd.
- Lung adenocarcinoma spc-A-1 tumor line was provided by Shanghai Cancer Institute.
- DR-70 kit Lung cancer enzyme-linked quantitative kit, purchased from Beijing Medic Corporation.
- spc-A-1 cells were routinely cultured in cells containing 12% newborn calf serum, 0.25mmol/L Hepes, 100U/ml penicillin and 100U/ml chain In RP-MI1640 culture medium containing mycomycin, adjust the cell number to 5 ⁇ 10 5 /ml. After trypan blue staining, the cell viability was greater than 95%. Take 0.2ml of cell suspension and inoculate it subcutaneously on the back of nude mice.
- Tumor inhibition experiment 50 nude mice were subcutaneously inoculated with lung adenocarcinoma spc-A-1 cells and randomly divided into 5 groups, namely the treatment group (positive control group), the tumor control group (negative control group) and the experimental group (I - Group III), 10 animals in each group.
- the equivalent dose is 0.75mg/kg, 1.50mg/kg, 2.25mg/kg, and continue to administer for 10 days.
- the tumor control group was not given any medication and was only injected with the same volume of normal saline; the treatment group (positive control group) was given intraperitoneal injection of hydroxycamptothecin (OPT) 1.5 mg/kg for 10 consecutive days. After stopping the drug, the animals were sacrificed, the tumors were separated, the tumor weight and body weight were measured using an electronic balance, and the changes in body weight were recorded.
- OPT hydroxycamptothecin
- tumor growth inhibition rate (1-average tumor weight of the test group/average tumor weight of the control group) ⁇ 100%.
- DR-70 Tm for healthy people ⁇ 8ug/ml
- Inhibiting the growth of lung adenocarcinoma spc-A-1 The combination of bergamot lactone and quercetin flavonoids has a significant inhibitory effect on lung adenocarcinoma spc-A-1 at a dose of 0.75 to 2.25 mg/kg. It shows that the combination of bergamot lactone and quercetin flavonoids can significantly inhibit the growth of lung adenocarcinoma spc-A-1 in Kunming mice at a dose of 0.75-2.25 mg/kg, and the high-dose group has a tumor inhibition rate of 2.25 mg/kg.
- the positive control group significantly reduced the weight of mice, which was significantly different from that of the blank control group.
- the effects of the three dosage groups of bergamot lactone and quercetin flavonoid composition on the body weight of mice were significantly different from those of the blank control group. No significant difference. It shows that hydroxycamptothecin (OPT) causes greater damage to tumor-bearing mice; while the combination of bergamot lactone and quercetin flavonoids has lower toxic and side effects, which is the difference between it and chemotherapy drugs.
- the serum DR-70 levels in the three dosage groups of bergamot lactone and quercetin flavonoid combination were significantly reduced, indicating that the bergamot lactone and quercetin flavonoid combination has a certain inhibitory effect on the infiltration of cancer tissue into the body. .
- mice 60 Kunming mice, half male and half female, weighing 20 ⁇ 2g, purchased from the Experimental Animal Center of Nanjing University of Traditional Chinese Medicine.
- Liver cancer H22 tumor line was provided by Shanghai Cancer Institute.
- liver cancer H 22 tumor-bearing mice The laboratory was disinfected with ultraviolet light for 3 hours before dissection, and several passage ascites cancer mice that had grown for 6-7 days were selected. After dislocation and sacrifice, the abdomen was disinfected with alcohol and the better-growing ascites was taken out. The cancer was diluted at a ratio of 1:2, and the cancer cell count was 5.2 ⁇ 10 7 /ml. It was inoculated into the armpit of the right upper limb of the mice, and 0.2ml was inoculated into each mouse.
- mice inoculated with liver cancer cells H22 were randomly divided into 5 groups, namely the treatment group (positive control group), the tumor control group (negative control group) and the experimental group (groups I-III). Group of 10.
- the equivalent dose is 0.75mg/kg, 1.50mg/kg, 2.25mg/kg, and continue to administer for 10 days.
- the tumor control group was not given any medication and was only injected with the same volume of normal saline; the treatment group (positive control group) was given an intraperitoneal injection of cisplatin 4 mg/kg, and each animal was given 0.2 ml/d ip every 1 day, 3 times.
- tumor growth inhibition rate (1-average tumor weight of test group/average tumor weight of control group) ⁇ 100%.
- the composition of bergamot lactone and quercetin flavonoids has a significant inhibitory effect on liver cancer H 22 at a dose of 0.75 to 2.25 mg/kg. It shows that the combination of bergamot lactone and quercetin flavonoids can significantly inhibit the growth of Kunming mouse liver cancer H22 at a dose of 0.75-2.25 mg/kg, and the tumor inhibition rate of the medium-dose group at 1.50 mg/kg reached 62.13%.
- the bergamot lactone and quercetin flavonoid compositions at doses of 0.75 mg/kg, 1.50 mg/kg, and 2.25 mg/kg can significantly inhibit the growth of liver cancer H 22 .
- the positive control group significantly reduced the body weight, liver weight and immune indicators (spleen index and thymus index) of tumor-bearing mice, which was significantly different from the blank control group, while the combination of bergamot lactone and quercetin flavonoids There were no significant differences in these indicators between the object group and the blank control group. It shows that cisplatin causes great damage to the organs of tumor-bearing mice; while several dosage groups of bergamot lactone and quercetin flavonoid combinations have significant effects on the body weight, liver weight, spleen index and thymus index of tumor-bearing mice. There was no obvious change, indicating that the combination of bergamot lactone and quercetin flavonoids has low toxicity and high anti-tumor activity on tumors, which is a significant feature that is different from chemotherapy drugs.
- mice 60 Kunming mice, half male and half female, weighing 20 ⁇ 2g, purchased from the Experimental Animal Center of Nanjing University of Traditional Chinese Medicine.
- mice inoculated with cervical cancer U 14 were randomly divided into 5 groups, namely the treatment group (positive control group), the tumor control group (negative control group) and the experimental group (groups I-III). 10 per group.
- the equivalent dose is 0.75mg/kg, 1.50mg/kg, 2.25mg/kg, and continue to administer for 10 days.
- the tumor control group was not given any medication and was only injected with the same volume of normal saline; the treatment group (positive control group) was given intraperitoneal injection of cyclophosphamide (CPP) 20 mg/kg, and each animal was given 0.3 ml/d ip for 10 consecutive days. .
- CPP cyclophosphamide
- tumor growth inhibition rate (1-average tumor weight of the test group/average tumor weight of the control group) ⁇ 100%.
- Inhibiting the growth of cervical cancer U 14 The combination of bergamot lactone and quercetin flavonoids at doses of 0.75 mg/kg, 1.50 mg/kg, and 2.25 mg/kg can significantly inhibit the growth of cervical cancer U 14 .
- the low-dose group 0.75 mg/kg had the best tumor inhibitory effect, with a tumor inhibition rate of 60.62%, indicating that the combination of bergamot lactone and quercetin flavonoids has a certain dose selectivity in inhibiting different tumors; the positive control group cyclophosphate Amide (CPP) can significantly reduce the weight of tumor-bearing mice, while there is no significant difference between the bergamot lactone and quercetin flavonoid composition group and the blank control group.
- CPP cyclophosphate Amide
- cyclophosphamide has certain damage to tumor-bearing mice; while several dosage groups of bergamot lactone and quercetin flavonoid composition have no significant effect on the body weight of tumor-bearing mice, indicating that bergamot lactone and quercetin
- the cortin flavonoid composition has the characteristics of low toxicity and high efficiency.
Abstract
Disclosed is use of a bergapten and quercetin flavone composition in the preparation of a medicament for treating cancer. The present invention pertains to the technical field of anti-cancer drug preparation. The bergapten and quercetin flavone composition of the present invention has significant pharmacological activity in combating lung cancer, liver cancer, cervical cancer, and the like. The composition is expected to be developed into a new tumor prevention and treatment drug, an anti-cancer reversal agent, and a drug for adjuvant therapy of tumors which are low in toxicity and highly effective and have natural traditional Chinese medicine components with various pharmacological effects.
Description
本发明涉及抗癌药物制备技术领域，特别是涉及佛手柑内酯和槲皮素黄酮的组合物在制备治疗癌症药物中的应用。The present invention relates to the technical field of anti-cancer drug preparation, and in particular to the application of a composition of bergamot lactone and quercetin flavonoids in the preparation of cancer treatment drugs.
肿瘤的危害性仅次于心血管疾病，会对人类健康造成极大影响。现如今在人们的生活习性的改变和一些不良的生活习惯影响下，肿瘤的发病率呈上升和年轻化趋势，且该趋势愈发明显，这将会带来巨大的经济和医疗负担，因此癌症的预防和治疗需要予以高度重视并需要对其进一步攻克。The harm of tumors is second only to cardiovascular disease and will have a great impact on human health. Nowadays, under the influence of changes in people's living habits and some bad living habits, the incidence of tumors is increasing and getting younger, and this trend is becoming more and more obvious, which will bring huge economic and medical burdens, so cancer The prevention and treatment of it need to be paid great attention and need to be further conquered.
目前，肿瘤的治疗一般采用手术切除、放射治疗、化学药物治疗以及上述方法结合应用等治疗方法。放射化疗在临床使用上由于其较大的副作用而收效甚微，而手术切除两年内，其复发和转移的几率还是比较高的，所以，病人需要定期检查，生活、饮食也要做适当的调整。目前，还没有比较好的办法可以预防癌症术后的复发或转移，术后规范的辅助化疗和放疗可以减少癌症的复发转移或延长复发转移的时间，但仍有复发转移的可能，通常来说，术后的癌症病人需要每3～4个月就复查一次，并点滴治癌药物，住院10天左右，这样的频率起码要坚持两年；两年过后，可以改成半年一次，然后再坚持三年，这样一共需要治疗五年。另一方面，癌症术后，加之放疗、化疗，身体非常的虚弱。总的来看，癌症术后防止复发和恢复健康是一件比较艰难的事情。At present, tumors are generally treated with surgical resection, radiotherapy, chemical drug therapy, and a combination of the above methods. Radiochemotherapy has little effect in clinical use due to its major side effects. However, within two years of surgical resection, the probability of recurrence and metastasis is still relatively high. Therefore, patients need regular examinations and appropriate adjustments to their lifestyle and diet. . At present, there is no good way to prevent the recurrence or metastasis of cancer after surgery. Standard postoperative adjuvant chemotherapy and radiotherapy can reduce the recurrence and metastasis of cancer or prolong the time of recurrence and metastasis, but there is still the possibility of recurrence and metastasis. Generally speaking, After surgery, cancer patients need to be reviewed every 3 to 4 months, given anti-cancer drugs intravenously, and hospitalized for about 10 days. This frequency should be maintained for at least two years; after two years, it can be changed to once every six months, and then continued Three years, so a total of five years of treatment is required. On the other hand, after cancer surgery, combined with radiotherapy and chemotherapy, the body is very weak. Generally speaking, it is difficult to prevent recurrence and restore health after cancer surgery.
发明内容Contents of the invention
本发明的目的是提供佛手柑内酯和槲皮素黄酮的组合物在制备治疗癌 症药物中的应用，以解决现有技术存在的问题。The object of the present invention is to provide the application of the composition of bergamot lactone and quercetin flavonoids in the preparation of cancer treatment drugs to solve the problems existing in the prior art.
为实现上述目的，本发明提供了如下方案：In order to achieve the above objects, the present invention provides the following solutions:
本发明目的之一是提供佛手柑内酯和槲皮素黄酮的组合物在制备治疗癌症药物中的应用，所述佛手柑内酯是从佛手中分离得到的天然物质；所述槲皮素黄酮是从槐米中分离得到的天然物质；One of the objects of the present invention is to provide the application of the composition of bergamot lactone and quercetin flavonoid in the preparation of cancer treatment drugs. The bergamot lactone is a natural substance isolated from the hand of bergamot; the quercetin flavonoid It is a natural substance isolated from Sophora japonica;
所述佛手柑内酯和槲皮素黄酮的组合物中佛手柑内酯和槲皮素黄酮的质量比为1:3～8；The mass ratio of bergamot lactone and quercetin flavonoids in the composition of bergamot lactone and quercetin flavonoids is 1:3 to 8;
所述癌症为肺癌、肝癌、宫颈癌或前列腺癌；The cancer is lung cancer, liver cancer, cervical cancer or prostate cancer;
所述佛手柑内酯的制备方法，包括以下步骤：The preparation method of bergamot lactone includes the following steps:
(1)将40目的佛手粉与醋酸-醋酸钠缓冲液以质量比1:5混合，在40℃下酶解55min，得到酶解液；(1) Mix 40-mesh bergamot powder and acetic acid-sodium acetate buffer at a mass ratio of 1:5, and perform enzymatic hydrolysis at 40°C for 55 minutes to obtain an enzymatic hydrolyzate;
(2)在酶解液中加入体积分数为35％的乙醇溶液，在55℃下超声提取2次，每次40min，合并提取液，离心后，得到上清液；(2) Add an ethanol solution with a volume fraction of 35% to the enzymatic hydrolysis solution, conduct ultrasonic extraction twice at 55°C, 40 minutes each time, combine the extracts, and centrifuge to obtain the supernatant;
(3)将上清液经截留分子量为1000KD的聚砜膜或聚醚砜膜进行膜分离过滤后，得到样品原液；(3) After membrane separation and filtration of the supernatant through a polysulfone membrane or polyethersulfone membrane with a molecular weight cutoff of 1000KD, the sample stock solution is obtained;
(4)样品原液经过柱层析分离，得到洗脱液；(4) The sample stock solution is separated by column chromatography to obtain the eluent;
(5)将洗脱液干燥至恒重，得到所述佛手柑内酯。(5) Dry the eluate to constant weight to obtain the bergamot lactone.
进一步地，所述应用还包括肿瘤辅助治疗用药的制备、抗癌逆转剂的制备，即肿瘤化疗药物增效减毒、肿瘤放疗增敏、增强机体免疫功能等药物方面的应用。Furthermore, the application also includes the preparation of drugs for auxiliary treatment of tumors and the preparation of anti-cancer reversal agents, that is, the application of drugs in enhancing the efficacy and reducing toxicity of tumor chemotherapy drugs, sensitizing tumor radiotherapy, and enhancing the immune function of the body.
更进一步地，所述佛手柑内酯和槲皮素黄酮的组合物治疗的有效量为5～50mg/kg。Furthermore, the therapeutically effective dose of the combination of bergamot lactone and quercetin flavonoids is 5 to 50 mg/kg.
更进一步地，所述佛手柑内酯和槲皮素黄酮的组合物治疗的有效量为10～25mg/kg。Furthermore, the therapeutically effective dose of the combination of bergamot lactone and quercetin flavonoids is 10 to 25 mg/kg.
本发明目的之二是提供一种抗癌药物，以佛手柑内酯和槲皮素黄酮的组合物为活性成分，还含有药学上可接受的载体，所述佛手柑内酯是从佛手中分离得到的天然物质；The second object of the present invention is to provide an anti-cancer drug, which uses a composition of bergamot lactone and quercetin flavonoids as active ingredients, and also contains a pharmaceutically acceptable carrier. The bergamot lactone is isolated from bergamot. Obtained natural substances;
所述槲皮素黄酮是从槐米中分离得到的天然物质；The quercetin flavonoid is a natural substance isolated from Sophora japonica;
所述佛手柑内酯和槲皮素黄酮的组合物中佛手柑内酯和槲皮素黄酮的质量比为1:3～8；The mass ratio of bergamot lactone and quercetin flavonoids in the composition of bergamot lactone and quercetin flavonoids is 1:3 to 8;
所述癌症为肺癌、肝癌、宫颈癌或前列腺癌。The cancer is lung cancer, liver cancer, cervical cancer or prostate cancer.
所述佛手柑内酯和槲皮素黄酮的组合物的治疗有效量为2.5～100mg/kg，纯度大于80％。The therapeutically effective amount of the composition of bergamot lactone and quercetin flavonoids is 2.5-100 mg/kg, and the purity is greater than 80%.
本发明涉及的佛手柑内酯是从芸香科植物佛手(Citrus medica L.var.sarcodactylis Swingle)的果实中分离得到的高纯度天然化合物佛手柑内酯，其结构分别为：The bergamot lactone involved in the present invention is a high-purity natural compound bergamot lactone isolated from the fruit of the Rutaceae plant Citrus medica L. var. sarcodactylis Swingle. Its structures are:
槲皮素黄酮是从槐米中分离得到的天然物质，其结构分别为：Quercetin flavonoid is a natural substance isolated from Sophora japonica. Its structures are:
进一步地，所述药学上可接受的载体包括填充剂、粘合剂、崩解剂、 润滑剂、矫味剂和抗氧剂中的一种或多种。Further, the pharmaceutically acceptable carrier includes one or more of fillers, binders, disintegrants, lubricants, flavoring agents and antioxidants.
进一步地，所述填充剂包括甘露醇、乳糖或聚乙二醇；所述粘合剂包括淀粉或微晶纤维素；所述崩解剂包括羧甲基纤维素或低取代羟丙基纤维素；所述润滑剂包括滑石粉或硬脂酸镁。Further, the filler includes mannitol, lactose or polyethylene glycol; the binder includes starch or microcrystalline cellulose; the disintegrant includes carboxymethyl cellulose or low-substituted hydroxypropyl cellulose. ; The lubricant includes talc or magnesium stearate.
所述填充剂与抗氧剂的质量比为2～5：1。The mass ratio of the filler to the antioxidant is 2 to 5:1.
进一步地，所述抗癌药物剂型为口服制剂、注射油剂或外用制剂。Further, the anti-cancer drug dosage forms are oral preparations, injection oil preparations or external preparations.
进一步地，所述口服制剂为片剂、胶囊、缓释片或滴丸，所述外用制剂为霜剂、膏剂、油剂或甘油制剂。Further, the oral preparation is a tablet, capsule, sustained-release tablet or dropping pill, and the external preparation is a cream, ointment, oil or glycerin preparation.
进一步地，所述片剂的制备方法为：按质量百分比计，称取10～70％的佛手柑内酯和槲皮素黄酮的组合物，加入30～90％的可接受的载体，搅拌混合制粒，在60～70℃下干燥24小时，压制，得到所述片剂，每片含佛手柑内酯和槲皮素黄酮2.5～20mg，优选2.5mg、5mg、10mg、15mg和20mg。Further, the preparation method of the tablet is: weigh 10 to 70% of the composition of bergamot lactone and quercetin flavonoids in terms of mass percentage, add 30 to 90% of an acceptable carrier, and stir and mix. Granulate, dry at 60-70°C for 24 hours, and press to obtain the tablets, each tablet containing 2.5-20 mg of bergamot lactone and quercetin flavonoids, preferably 2.5 mg, 5 mg, 10 mg, 15 mg and 20 mg.
进一步地，所述胶囊的制备方法为：按质量百分比计，称取10～70％的佛手柑内酯和槲皮素黄酮的组合物，加入30～90％的可接受的载体，搅拌混合制粒，在60～70℃下干燥24小时，然后填充入空胶囊壳中，得到所述胶囊，每粒胶囊含佛手柑内酯和槲皮素黄酮2～20mg。Further, the preparation method of the capsule is: weighing 10 to 70% of the composition of bergamot lactone and quercetin flavonoids in terms of mass percentage, adding 30 to 90% of an acceptable carrier, and stirring and mixing to prepare the capsule. The capsules are dried at 60 to 70° C. for 24 hours, and then filled into empty capsule shells to obtain the capsules. Each capsule contains 2 to 20 mg of bergamot lactone and quercetin flavonoids.
进一步地，所述注射油剂的制备方法为：按质量百分比计，称取1～10％的佛手柑内酯和槲皮素黄酮的组合物与90～99％的注射用油混合，分装灭菌制得所述注射油剂，所述注射用油为注射用豆油或茶油。Further, the preparation method of the injection oil is: weighing 1 to 10% of the composition of bergamot lactone and quercetin flavonoids and 90 to 99% of the injection oil in terms of mass percentage, and then packaging. The injection oil is prepared by sterilization, and the injection oil is soybean oil or camellia oil for injection.
进一步地，所述滴丸的制备方法为：按质量百分比计，称取10～60％的佛手柑内酯和槲皮素黄酮的组合物，加入40～90％的可接受的载体进行加热熔融，熔融温度为100～150℃，使原料和辅料充分混熔，滴制成丸， 得到所述滴丸，每粒滴丸含佛手柑内酯和槲皮素黄酮的组合物2.5～20mg。Further, the preparation method of the dripping pills is: weighing 10 to 60% of the composition of bergamot lactone and quercetin flavonoids in terms of mass percentage, adding 40 to 90% of an acceptable carrier, and heating and melting. , the melting temperature is 100-150°C, the raw materials and auxiliary materials are fully mixed and dropped into pills to obtain the dripping pills, each of which contains 2.5-20 mg of the composition of bergamot lactone and quercetin flavonoids.
本发明的技术效果如下：The technical effects of the present invention are as follows:
本发明含有佛手柑内酯和槲皮素黄酮组合物药理活性的研究表明，其具有明显的抗肺癌、抗肝癌、抗宫颈癌等药理活性，该组合物有望研制成为低毒高效且具有多种药理功效的天然中药成分的肿瘤防治新药、抗癌逆转剂及肿瘤辅助治疗用药。Research on the pharmacological activity of the composition containing bergamot lactone and quercetin flavonoids of the present invention shows that it has obvious pharmacological activities such as anti-lung cancer, anti-liver cancer, anti-cervical cancer, etc. The composition is expected to be developed into a low-toxicity, high-efficiency and multi-functional New cancer prevention and treatment drugs, anti-cancer reversal agents and tumor auxiliary treatment drugs with pharmacological effects of natural traditional Chinese medicine ingredients.
本发明中，所有原料均为常规商购产品。In the present invention, all raw materials are conventional commercial products.
从佛手中制备佛手柑内酯的方法：Method for preparing bergamot lactone from bergamot:
(1)将干燥后的佛手经粉碎、过40目筛，得到佛手粉。(1) Crush the dried bergamot and pass it through a 40-mesh sieve to obtain bergamot powder.
(2)在佛手粉中加入其质量的5倍、含3％纤维素酶和果胶酶的pH值为4.0的醋酸-醋酸钠缓冲液，在温度为40℃的条件下酶解55min，得到酶解液。(2) Add 5 times its mass to the bergamot powder and an acetic acid-sodium acetate buffer with a pH value of 4.0 containing 3% cellulase and pectinase, and perform enzymatic hydrolysis at 40°C for 55 minutes to obtain Enzymatic solution.
(3)酶解液中加入体积分数为35％的乙醇溶液，在提取温度为55℃的条件下超声提取2次，每次40min，合并提取液，离心后，得到上清液。(3) Add an ethanol solution with a volume fraction of 35% to the enzymatic hydrolysis solution, conduct ultrasonic extraction twice at an extraction temperature of 55°C, 40 minutes each time, combine the extracts, and centrifuge to obtain the supernatant.
其中：佛手瓜粉与乙醇溶液的料液比为0.03g/mL。Among them: the material-liquid ratio of chayote powder and ethanol solution is 0.03g/mL.
(4)上清液经截留分子量为1000KD的聚砜膜或聚醚砜膜进行膜分离过滤后，得到样品原液。(4) After the supernatant is separated and filtered through a polysulfone membrane or a polyethersulfone membrane with a molecular weight cutoff of 1000KD, the sample stock solution is obtained.
(5)样品原液直接过X-8大孔吸附树脂，先用4倍柱床体积的水以2BV/h的流速洗至接近无色，弃去水洗液，再用体积浓度为20％的乙醇以1.5BV/h的流速洗脱，弃去洗脱液A，继以体积浓度为65％的乙醇以2.0BV/h的流速洗脱，收集洗脱液B。(5) The sample solution is directly passed through the X-8 macroporous adsorption resin. First, wash it with 4 times the column bed volume of water at a flow rate of 2BV/h until it is close to colorless. Discard the water wash, and then use ethanol with a volume concentration of 20%. Elute at a flow rate of 1.5 BV/h, discard eluent A, and then elute with ethanol with a volume concentration of 65% at a flow rate of 2.0 BV/h, and collect eluent B.
其中：原液量为大孔吸附树脂对佛手柑内酯静态饱和吸附量的110％；大孔吸附树脂的塔柱径和柱高比为1：5。Among them: the amount of raw solution is 110% of the static saturated adsorption capacity of bergamot lactone by the macroporous adsorption resin; the column diameter and column height ratio of the macroporous adsorption resin is 1:5.
(6)将洗脱液B回收乙醇，在真空度为0.06MPa、温度为40℃的条件下浓缩后，在温度为55℃下干燥至恒重，即得暗红色佛手柑内酯的提取物。(6) Recover ethanol from eluent B, concentrate it at a vacuum of 0.06MPa and a temperature of 40°C, and then dry it to a constant weight at a temperature of 55°C to obtain a dark red bergamot lactone extract. .
槲皮素黄酮提取方法：在20g的槐米粗粉加入0.4％的硼酸水溶液200ml，用石灰乳调节pH值8～9。在微沸条件下搅拌提取30min，将滤液用浓盐酸调节pH值2～3，静置12h后离心处理，沉淀用pH值1～2的浓盐酸洗涤一次，蒸馏水洗涤至中性，沉淀物经65℃真空干燥得到芦丁粗品。精制芦丁后取1g，用2％的硫酸溶液80ml，加热回流90min转化得到槲皮素黄酮。Quercetin flavonoid extraction method: Add 200 ml of 0.4% boric acid aqueous solution to 20 g of sophora rice powder, and adjust the pH value to 8 to 9 with milk of lime. Stir and extract under slightly boiling conditions for 30 minutes. Adjust the filtrate to a pH value of 2 to 3 with concentrated hydrochloric acid. Let it stand for 12 hours and then centrifuge. The precipitate is washed once with concentrated hydrochloric acid of a pH value of 1 to 2, and washed with distilled water until neutral. The precipitate is washed with The crude rutin product was obtained by vacuum drying at 65°C. After purifying rutin, take 1 g, use 80 ml of 2% sulfuric acid solution, and heat and reflux for 90 minutes to convert it into quercetin flavonoids.
实施例1Example 1
片剂的制备：按质量百分比计，称取50wt.％的佛手柑内酯和槲皮素黄酮的组合物(组合物中佛手柑内酯和槲皮素黄酮的质量比为1:5)，加入50wt.％的甘露醇、淀粉、羧甲基纤维素、滑石粉(甘露醇：淀粉：羧甲基纤维素：滑石粉的质量比为1：2：3：2)，搅拌混合制粒，在70℃下干燥24小时，压制成片剂，每片含佛手柑内酯和槲皮素黄酮10mg。Preparation of tablets: In terms of mass percentage, weigh 50wt.% of the composition of bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5), Add 50wt.% mannitol, starch, carboxymethyl cellulose, and talc (the mass ratio of mannitol: starch: carboxymethyl cellulose: talc is 1:2:3:2), stir, mix and granulate. Dry at 70°C for 24 hours and press into tablets. Each tablet contains 10 mg of bergamot lactone and quercetin flavonoids.
实施例2Example 2
胶囊的制备：按质量百分比计，称取10wt.％的佛手柑内酯和槲皮素黄酮的组合物(组合物中佛手柑内酯和槲皮素黄酮的质量比为1:5)，加入90wt.％的乳糖、微晶纤维素、低取代羟丙基纤维素、硬脂酸镁和矫味剂(乳糖、微晶纤维素、低取代羟丙基纤维素、硬脂酸镁和矫味剂的质量比为1： 2：1：2：1)，搅拌混合制粒，在70℃下干燥24小时，然后填充入空胶囊壳中，每粒胶囊含佛手柑内酯和槲皮素黄酮5mg。Preparation of capsules: In terms of mass percentage, weigh 10 wt.% of the composition of bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5), and add 90wt.% lactose, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, magnesium stearate and flavoring (lactose, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, magnesium stearate and flavoring The mass ratio of the agent is 1: 2:1:2:1), stir, mix and granulate, dry at 70°C for 24 hours, and then fill into empty capsule shells. Each capsule contains bergamot lactone and quercetin flavonoids. 5mg.
矫味剂为质量比为40：1的阿斯帕坦和柠檬香精。The flavoring agent is aspartame and lemon essence with a mass ratio of 40:1.
实施例3Example 3
油剂的制备：按质量百分比计，称取10wt.％的佛手柑内酯和槲皮素黄酮的组合物(组合物中佛手柑内酯和槲皮素黄酮的质量比为1:5)与90wt.％的注射用油混合，分装灭菌制得油剂。Preparation of oil: In terms of mass percentage, weigh 10 wt.% of the composition of bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5) and Mix 90wt.% injection oil, pack and sterilize to prepare the oil.
实施例4Example 4
滴丸的制备：按质量百分比计，称取60wt.％的佛手柑内酯和槲皮素黄酮的组合物(组合物中佛手柑内酯和槲皮素黄酮的质量比为1:5)，加入40wt.％的聚乙二醇和抗氧剂(聚乙二醇和抗氧剂的质量比为4：1)进行加热熔融，熔融温度为150℃，滴制成丸，每粒滴丸含佛手柑内酯和槲皮素黄酮20mg。Preparation of dripping pills: In terms of mass percentage, weigh 60wt.% of the composition of bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5), Add 40wt.% polyethylene glycol and antioxidant (the mass ratio of polyethylene glycol and antioxidant is 4:1) for heating and melting. The melting temperature is 150°C. Drop it into pills. Each pill contains bergamot. Lactone and quercetin flavonoids 20mg.
抗氧剂为质量比为4:1的维生素C和焦亚硫酸钠。The antioxidants are vitamin C and sodium metabisulfite with a mass ratio of 4:1.
效果验证一Effect verification one
(1)对佛手柑内酯和槲皮素黄酮的组合物进行加速实验考察：(1) Conduct accelerated experimental investigation on the composition of bergamot lactone and quercetin flavonoids:
在高温(60℃)下放置10d，无分解，纯度及理化指标不变；When placed at high temperature (60°C) for 10 days, there is no decomposition, and the purity and physical and chemical indicators remain unchanged;
在高湿(RH＞92％)下放置10d，无分解，纯度及理化指标不变；When placed under high humidity (RH>92%) for 10 days, there is no decomposition, and the purity and physical and chemical indicators remain unchanged;
在强光照射(4600Lx)下放置10d，无分解，纯度及理化指标不变；When placed under strong light irradiation (4600Lx) for 10 days, there is no decomposition, and the purity and physical and chemical indicators remain unchanged;
技术指标达到一类新药报批的要求。The technical indicators meet the requirements for approval of a Class I new drug.
(2)佛手柑内酯和槲皮素黄酮组合物经UV、IR、MS、
1H-NMR、
13C-NMR、熔点测定、TLC及性状分析与文献报道相一致。
(2) The composition of bergamot lactone and quercetin flavonoids was consistent with literature reports through UV, IR, MS, 1 H-NMR, 13 C-NMR, melting point measurement, TLC and property analysis.
佛手柑内酯和槲皮素黄酮组合物具有如下性质：在显微镜下其为淡黄色针状结晶，在紫外灯下显棕黄色荧光。The composition of bergamot lactone and quercetin flavonoids has the following properties: it appears as light yellow needle-like crystals under a microscope and exhibits brown-yellow fluorescence under ultraviolet light.
熔点Mp：181-193℃；Melting point Mp: 181-193℃;
TLC比移值Rf为：0.538(石油醚:乙酸乙酯8:2)。The TLC ratio shift value Rf is: 0.538 (petroleum ether:ethyl acetate 8:2).
佛手柑内酯光谱分析Spectral analysis of bergamot lactone
UVλ
EtOHmax(nm):243(肩峰),259,268,312
UVλ EtOH max(nm):243(shoulder peak),259,268,312
IRν
KBrmax(cm
-1):3100-3050(CH),1720(﹥C＝O)，1600，1480(苯环)。
IRν KBr max (cm -1 ): 3100-3050 (CH), 1720 (﹥C＝O), 1600, 1480 (benzene ring).
1H-NMR(CDCl
3):4.34(3H,s,C
5–OCH
3),6.20(1H,d,J＝10Hz,C
3-H),
1 H-NMR (CDCl 3 ): 4.34 (3H, s, C 5 –OCH 3 ), 6.20 (1H, d, J = 10Hz, C 3 -H),
7.16(1H,s,C
8-H),7.33(1H,d,J＝8.5Hz,C
4′-H),7.53(1H,d,J＝8.5Hz,C
5′-H),8.30
7.16(1H,s,C 8 -H),7.33(1H,d,J＝8.5Hz,C 4′ -H),7.53(1H,d,J＝8.5Hz,C 5′ -H),8.30
(1H,d,J＝10Hz,C
4-H)。
(1H,d,J=10Hz,C 4 -H).
13C-NMR:65(1-OCH
3),106(呋喃环碳)，115(苯环碳)，137(共轭碳)，
13 C-NMR: 65 (1-OCH 3 ), 106 (furan ring carbon), 115 (phenyl ring carbon), 137 (conjugated carbon),
151(羰基碳)。151 (carbonyl carbon).
MS m/z：216(M
+)，201，188，173，145。
MS m/z: 216(M + ), 201, 188, 173, 145.
槲皮素光谱分析Spectral analysis of quercetin
UVλ
MeOHmax(nm):256,370；UVλ
NaOMemax(nm):276,330,414
UVλ MeOH max(nm):256,370; UVλ NaOMe max(nm):276,330,414
IRν
KBrmax(cm
-1):3370(宽峰，OH),1655(酮基C＝O)，1600，1560,1510(苯基)。
IRν KBr max (cm -1 ): 3370 (broad peak, OH), 1655 (keto group C=O), 1600, 1560, 1510 (phenyl).
MS m/z：301(M
+)，285，218，193，160。
MS m/z: 301(M + ), 285, 218, 193, 160.
以上光谱数据与文献比较，并与标准品一起测混合熔点不下降，故确定该化合物为佛手柑内酯和槲皮素黄酮。The above spectral data were compared with the literature, and the melting point of the mixture when measured with the standard product did not decrease, so the compound was determined to be bergamot lactone and quercetin flavonoids.
效果验证二Effect Verification 2
本发明公开了含有佛手柑内酯和槲皮素黄酮的组合物(组合物中佛手 柑内酯和槲皮素黄酮的质量比为1:5)在用于制备抗肿瘤药物和抗癌逆转剂方面的应用，其中抗肿瘤活性包括：制备治疗肺癌、肝癌、宫颈癌药物方面的应用。The invention discloses that a composition containing bergamot lactone and quercetin flavonoids (the mass ratio of bergamot lactone and quercetin flavonoids in the composition is 1:5) can be used to prepare anti-tumor drugs and anti-cancer reversal agents. Applications, including anti-tumor activity, include: applications in preparing drugs for the treatment of lung cancer, liver cancer, and cervical cancer.
一、急性毒性试验1. Acute toxicity test
选取昆明种健康小鼠[体重(20±2)g]50只，由南京中医药大学实验动物中心提供。进行分组，雌雄各半，按体重、性别随机分组，每组10只。Fifty healthy Kunming mice [body weight (20±2)g] were selected and provided by the Experimental Animal Center of Nanjing University of Traditional Chinese Medicine. Divide the animals into groups, half male and half female, and randomly group them according to weight and sex, with 10 animals in each group.
精密称取抗肿瘤成分C(佛手柑内酯和槲皮素黄酮的组合物)10mg，以DMSO为溶剂配制成5mg/ml的高浓度药物溶液，再用生理盐水配成不同浓度的水溶液，并用其对小鼠进行半数致死量(LD
50)急性毒性实验，对照品为0.9％氯化钠溶液。
Precisely weigh 10 mg of the anti-tumor ingredient C (the combination of bergamot lactone and quercetin flavonoids), use DMSO as the solvent to prepare a high-concentration drug solution of 5 mg/ml, and then use physiological saline to prepare aqueous solutions of different concentrations, and use It conducted an acute toxicity test on mice with a median lethal dose (LD 50 ), and the control substance was 0.9% sodium chloride solution.
结果：抗肿瘤成分C的LD
50(11.37±3.23)mg/g。结论：抗肿瘤成分C几乎无毒性。
Results: The LD 50 (11.37±3.23) mg/g of anti-tumor component C. Conclusion: Anti-tumor component C has almost no toxicity.
二、体外抑瘤实验2. In vitro tumor inhibition experiments
(1)接种细胞：取对数生长期细胞，经0.02％EDTA消化后加入含有10～15％胎牛血清的RPMI-1640溶液，将细胞稀释成单细胞悬液，计数并调整细胞数，并接种于96孔板，将接种后的细胞置37℃二氧化碳培养箱内培养24小时。(1) Inoculate cells: Take logarithmic growth phase cells, digest them with 0.02% EDTA, add RPMI-1640 solution containing 10 to 15% fetal calf serum, dilute the cells into a single cell suspension, count and adjust the cell number, and Inoculate into a 96-well plate, and culture the inoculated cells in a 37°C carbon dioxide incubator for 24 hours.
(2)加药：将佛手柑内酯和槲皮素黄酮组合物设置成1.56、3.125、6.25、12.5、25、50、100、200、400ug/ml等不同浓度，每浓度设4个复孔，对照设8个以上复孔。被检样品用RPMI-1640稀释，用DMSO助溶。加药后仍将细胞置37℃二氧化碳培养箱内继续培养72小时。(2) Dosing: Set the bergamot lactone and quercetin flavonoid composition to different concentrations such as 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400ug/ml, etc., and set 4 duplicate holes for each concentration. , control with more than 8 duplicate holes. The sample to be tested was diluted with RPMI-1640 and DMSO was used to help dissolve it. After adding the drug, the cells were placed in a 37°C carbon dioxide incubator and continued to be cultured for 72 hours.
(3)加MTT：MTT以RPMI-1640溶解，每孔加50ul。再置于37℃ 下继续培养4小时后取出。(3) Add MTT: MTT is dissolved in RPMI-1640, and 50ul is added to each well. Then place it at 37°C and continue culturing for 4 hours before taking it out.
(4)测OD值：弃除96孔板内的上清液，每孔加入DMSO150ul，在酶标仪630nm处测OD值。(4) Measure the OD value: Discard the supernatant in the 96-well plate, add 150ul of DMSO to each well, and measure the OD value at 630nm on a microplate reader.
评价指标：细胞生长抑制率：抑制率＝(1-实验组平均OD值/对照组平均OD值)×100％，以直线回归方法计算IC
50。
Evaluation index: Cell growth inhibition rate: Inhibition rate = (1-average OD value of the experimental group/average OD value of the control group) × 100%, and the IC 50 was calculated using the linear regression method.
结果：对人肺腺癌细胞系A549的细胞毒性作用，IC
50为58.76ug/ml，对肝癌细胞系Bel-7402的细胞毒性作用，IC
50为96.67ug/ml。
Results: The cytotoxic effect on human lung adenocarcinoma cell line A549, IC 50 was 58.76ug/ml, and the cytotoxic effect on liver cancer cell line Bel-7402, IC 50 was 96.67ug/ml.
三、体内抑瘤实验3. In vivo tumor inhibition experiments
(一)对小鼠肺腺癌spc-A-1的抑制作用：(1) Inhibitory effect on mouse lung adenocarcinoma spc-A-1:
1.材料1.Materials
1.1实验动物：6-7周龄雄性BALB/C裸鼠，购自南京中医药大学实验动物中心。1.1 Experimental animals: 6-7 week old male BALB/C nude mice were purchased from the Experimental Animal Center of Nanjing University of Traditional Chinese Medicine.
羟基喜树碱：湖北黄石飞云制药有限公司生产。Hydroxycamptothecin: produced by Hubei Huangshi Feiyun Pharmaceutical Co., Ltd.
1.2肿瘤细胞：肺腺癌spc-A-1瘤株由上海肿瘤研究所提供。1.2 Tumor cells: Lung adenocarcinoma spc-A-1 tumor line was provided by Shanghai Cancer Institute.
1.3 DR-70试剂盒：即肺癌酶联定量试剂盒，购自北京美迪克公司。1.3 DR-70 kit: Lung cancer enzyme-linked quantitative kit, purchased from Beijing Medic Corporation.
2.方法2.Method
2.1肺腺癌(spc-A-1)荷瘤小鼠的建立：spc-A-1细胞常规培养于含12％新生小牛血清、0.25mmol/L Hepes、100U/ml青霉素和100U/ml链霉素的RP-MI1640培养基中，调整细胞数为5×10
5/ml。经台盼兰染色，细胞存活力大于95％。取细胞悬液0.2ml/只接种于裸鼠背部皮下。
2.1 Establishment of lung adenocarcinoma (spc-A-1) tumor-bearing mice: spc-A-1 cells were routinely cultured in cells containing 12% newborn calf serum, 0.25mmol/L Hepes, 100U/ml penicillin and 100U/ml chain In RP-MI1640 culture medium containing mycomycin, adjust the cell number to 5×10 5 /ml. After trypan blue staining, the cell viability was greater than 95%. Take 0.2ml of cell suspension and inoculate it subcutaneously on the back of nude mice.
2.2药物配制：取含量为98.7％的佛手柑内酯和槲皮素黄酮组合物，研细并配制成浓度为0.05mg/ml、0.10mg/ml、0.15mg/ml的混悬液，溶剂为 蒸馏水，给药时摇匀。2.2 Drug preparation: Take the bergamot lactone and quercetin flavonoid composition with a content of 98.7%, grind it finely and prepare it into a suspension with a concentration of 0.05mg/ml, 0.10mg/ml, and 0.15mg/ml. The solvent is Distilled water, shake well before administration.
2.3抑瘤实验：皮下接种肺腺癌spc-A-1细胞的50只裸鼠，随机分为5组，即治疗组(阳性对照组)、肿瘤对照组(阴性对照组)和试验组(Ⅰ-Ⅲ组)，每组10只。接种后第2天，开始口服灌胃给药0.3ml/只，折合剂量为0.75mg/kg，1.50mg/kg，2.25mg/kg，连续给药10天。肿瘤对照组不给药，只注射同体积的生理盐水；治疗组(阳性对照组)给予腹腔注射羟基喜树碱(OPT)1.5mg/kg连续10天。停药后分别处死动物，分离瘤体，用电子天平称瘤重和体重，记录体重变化情况。2.3 Tumor inhibition experiment: 50 nude mice were subcutaneously inoculated with lung adenocarcinoma spc-A-1 cells and randomly divided into 5 groups, namely the treatment group (positive control group), the tumor control group (negative control group) and the experimental group (Ⅰ - Group III), 10 animals in each group. On the second day after vaccination, start oral administration of 0.3ml/animal, the equivalent dose is 0.75mg/kg, 1.50mg/kg, 2.25mg/kg, and continue to administer for 10 days. The tumor control group was not given any medication and was only injected with the same volume of normal saline; the treatment group (positive control group) was given intraperitoneal injection of hydroxycamptothecin (OPT) 1.5 mg/kg for 10 consecutive days. After stopping the drug, the animals were sacrificed, the tumors were separated, the tumor weight and body weight were measured using an electronic balance, and the changes in body weight were recorded.
3.疗效评价3.Effectiveness evaluation
3.1肿瘤生长抑制率3.1 Tumor growth inhibition rate
计算公式：肿瘤生长抑制率＝(1-试验组平均瘤重/对照组平均瘤重)×100％。Calculation formula: tumor growth inhibition rate = (1-average tumor weight of the test group/average tumor weight of the control group) × 100%.
3.2测定DR-70：用小鼠血清10ul按说明书在波长λ＝450nm处测OD值。在试验中，采10只健康裸鼠血清测定DR-70的OD值，以其平均值作为裸鼠的正常值。(健康人的DR-70
Tm<8ug/ml)。
3.2 Determination of DR-70: Use 10ul of mouse serum to measure the OD value at wavelength λ = 450nm according to the instructions. In the experiment, the serum of 10 healthy nude mice was collected to measure the OD value of DR-70, and the average value was used as the normal value of nude mice. (DR-70 Tm for healthy people <8ug/ml).
4.结果4.Results
抑制肺腺癌spc-A-1的生长：佛手柑内酯和槲皮素黄酮组合物在剂量为0.75～2.25mg/kg之间对肺腺癌spc-A-1有明显的抑制作用。表明佛手柑内酯和槲皮素黄酮组合物在0.75～2.25mg/kg剂量能较明显地抑制昆明小鼠肺腺癌spc-A-1的生长，其中高剂量组2.25mg/kg抑瘤率达到71.56％，并随着剂量的增加对小鼠肺腺癌spc-A-1的抑制作用逐渐增强，呈现一定的剂量关系。阳性对照组(OPT)使小鼠的体重明显下降，与空白对照组 比较具有显著差异，而佛手柑内酯和槲皮素黄酮组合物的3个剂量组对小鼠体重的影响与空白对照组无显著差异。表明羟基喜树碱(OPT)对荷瘤小鼠的损伤较大；而佛手柑内酯和槲皮素黄酮组合物毒副作用较低，这是其区别与化疗药物的不同之处。并且佛手柑内酯和槲皮素黄酮组合物3个剂量组与对照组比较，血清DR-70水平明显降低，表明佛手柑内酯和槲皮素黄酮组合物对癌组织浸润机体有一定抑制作用。Inhibiting the growth of lung adenocarcinoma spc-A-1: The combination of bergamot lactone and quercetin flavonoids has a significant inhibitory effect on lung adenocarcinoma spc-A-1 at a dose of 0.75 to 2.25 mg/kg. It shows that the combination of bergamot lactone and quercetin flavonoids can significantly inhibit the growth of lung adenocarcinoma spc-A-1 in Kunming mice at a dose of 0.75-2.25 mg/kg, and the high-dose group has a tumor inhibition rate of 2.25 mg/kg. reached 71.56%, and as the dose increased, the inhibitory effect on mouse lung adenocarcinoma spc-A-1 gradually increased, showing a certain dose relationship. The positive control group (OPT) significantly reduced the weight of mice, which was significantly different from that of the blank control group. The effects of the three dosage groups of bergamot lactone and quercetin flavonoid composition on the body weight of mice were significantly different from those of the blank control group. No significant difference. It shows that hydroxycamptothecin (OPT) causes greater damage to tumor-bearing mice; while the combination of bergamot lactone and quercetin flavonoids has lower toxic and side effects, which is the difference between it and chemotherapy drugs. Moreover, compared with the control group, the serum DR-70 levels in the three dosage groups of bergamot lactone and quercetin flavonoid combination were significantly reduced, indicating that the bergamot lactone and quercetin flavonoid combination has a certain inhibitory effect on the infiltration of cancer tissue into the body. .
(二)对小鼠肝癌H
22的抑制作用：
(2) Inhibitory effect on mouse liver cancer H22 :
1.材料1.Materials
1.1实验动物：所用实验动物为昆明种小鼠60只，雌雄各半，体重为20±2g，购自南京中医药大学实验动物中心。1.1 Experimental animals: The experimental animals used were 60 Kunming mice, half male and half female, weighing 20±2g, purchased from the Experimental Animal Center of Nanjing University of Traditional Chinese Medicine.
1.2肿瘤细胞：肝癌H
22瘤株由上海肿瘤研究所提供。
1.2 Tumor cells: Liver cancer H22 tumor line was provided by Shanghai Cancer Institute.
2.方法2.Method
2.1肝癌H
22荷瘤小鼠的建立：解剖前实验室用紫外灯消毒3小时，选择生长6-7天的传代腹水癌小鼠数只，脱臼处死后酒精消毒腹部，取出生长较好的腹水癌按1：2的比例稀释，癌细胞计数为5.2×10
7个/ml接种在小鼠的右上肢腋下，每只接种0.2ml。
2.1 Establishment of liver cancer H 22 tumor-bearing mice: The laboratory was disinfected with ultraviolet light for 3 hours before dissection, and several passage ascites cancer mice that had grown for 6-7 days were selected. After dislocation and sacrifice, the abdomen was disinfected with alcohol and the better-growing ascites was taken out. The cancer was diluted at a ratio of 1:2, and the cancer cell count was 5.2×10 7 /ml. It was inoculated into the armpit of the right upper limb of the mice, and 0.2ml was inoculated into each mouse.
2.2药物配制：取含量为98.7％的佛手柑内酯和槲皮素黄酮组合物，研细并配制成浓度为0.05mg/ml、0.10mg/ml、0.15mg/ml的混悬液，溶剂为蒸馏水，给药时摇匀。2.2 Drug preparation: Take the bergamot lactone and quercetin flavonoid composition with a content of 98.7%, grind it finely and prepare it into a suspension with a concentration of 0.05mg/ml, 0.10mg/ml, and 0.15mg/ml. The solvent is Distilled water, shake well before administration.
2.3抑瘤实验：接种肝癌细胞H
22的50只小鼠，随机分为5组，即治疗组(阳性对照组)、肿瘤对照组(阴性对照组)和试验组(Ⅰ-Ⅲ组)，每组10只。接种后第2天，开始口服灌胃给药0.3ml/只，折合剂量为 0.75mg/kg，1.50mg/kg，2.25mg/kg，连续给药10天。肿瘤对照组不给药，只注射同体积的生理盐水；治疗组(阳性对照组)给予腹腔注射顺铂4mg/kg，每间隔1天每只ip给药0.2ml/d，给药3次。
2.3 Tumor inhibition experiment: 50 mice inoculated with liver cancer cells H22 were randomly divided into 5 groups, namely the treatment group (positive control group), the tumor control group (negative control group) and the experimental group (groups I-III). Group of 10. On the second day after vaccination, start oral administration of 0.3ml/animal, the equivalent dose is 0.75mg/kg, 1.50mg/kg, 2.25mg/kg, and continue to administer for 10 days. The tumor control group was not given any medication and was only injected with the same volume of normal saline; the treatment group (positive control group) was given an intraperitoneal injection of cisplatin 4 mg/kg, and each animal was given 0.2 ml/d ip every 1 day, 3 times.
3.观察指标：停药后分别处死动物，分离瘤体、肝、脾及胸腺，分别用电子天平称重和体重，记录体重变化情况，计算抑瘤率、脾指数和胸腺指数，统计体重增长和肝重。3. Observation indicators: After stopping the drug, the animals were sacrificed respectively, and the tumors, liver, spleen and thymus were separated, weighed and body weighted respectively with an electronic balance, the body weight changes were recorded, the tumor inhibition rate, spleen index and thymus index were calculated, and the body weight growth was counted. and liver weight.
肿瘤生长抑制率计算公式：肿瘤生长抑制率＝(1-试验组平均瘤重/对照组平均瘤重)×100％。The calculation formula of tumor growth inhibition rate: tumor growth inhibition rate = (1-average tumor weight of test group/average tumor weight of control group) × 100%.
4.结果4.Results
4.1抑制肝癌H
22的生长：佛手柑内酯和槲皮素黄酮组合物在剂量为0.75～2.25mg/kg之间对肝癌H
22有明显的抑制作用。表明佛手柑内酯和槲皮素黄酮组合物在0.75～2.25mg/kg剂量能较明显地抑制昆明小鼠肝癌H
22的生长，其中中剂量组1.50mg/kg抑瘤率达到62.13％。剂量为0.75mg/kg，1.50mg/kg，2.25mg/kg的佛手柑内酯和槲皮素黄酮组合物能较明显地抑制肝癌H
22的生长。
4.1 Inhibit the growth of liver cancer H 22 : The composition of bergamot lactone and quercetin flavonoids has a significant inhibitory effect on liver cancer H 22 at a dose of 0.75 to 2.25 mg/kg. It shows that the combination of bergamot lactone and quercetin flavonoids can significantly inhibit the growth of Kunming mouse liver cancer H22 at a dose of 0.75-2.25 mg/kg, and the tumor inhibition rate of the medium-dose group at 1.50 mg/kg reached 62.13%. The bergamot lactone and quercetin flavonoid compositions at doses of 0.75 mg/kg, 1.50 mg/kg, and 2.25 mg/kg can significantly inhibit the growth of liver cancer H 22 .
4.2佛手柑内酯和槲皮素黄酮组合物对肝癌H
22小鼠各脏器指标的影响
4.2 Effects of bergamot lactone and quercetin flavonoid composition on various organ indicators in mice with liver cancer H 22
阳性对照组(顺铂)使荷瘤小鼠的体重、肝重及免疫指标(脾指数和胸腺指数)明显下降，与空白对照组比较具有显著差异，而佛手柑内酯和槲皮素黄酮组合物组的这些指标与空白对照组无显著差异。表明顺铂对荷瘤小鼠的脏器有较大损伤；而佛手柑内酯和槲皮素黄酮组合物的几个剂量组对荷瘤小鼠的体重、肝重、脾指数和胸腺指数均无明显变化，表明佛手柑内酯和槲皮素黄酮组合物对肿瘤具有低毒、高效的抑瘤活性，这是其不 同与化疗药物的显著特点。The positive control group (cisplatin) significantly reduced the body weight, liver weight and immune indicators (spleen index and thymus index) of tumor-bearing mice, which was significantly different from the blank control group, while the combination of bergamot lactone and quercetin flavonoids There were no significant differences in these indicators between the object group and the blank control group. It shows that cisplatin causes great damage to the organs of tumor-bearing mice; while several dosage groups of bergamot lactone and quercetin flavonoid combinations have significant effects on the body weight, liver weight, spleen index and thymus index of tumor-bearing mice. There was no obvious change, indicating that the combination of bergamot lactone and quercetin flavonoids has low toxicity and high anti-tumor activity on tumors, which is a significant feature that is different from chemotherapy drugs.
(三)对小鼠宫颈癌U
14的抑制作用：
(3) Inhibitory effect on mouse cervical cancer U 14 :
1.材料1.Materials
1.1实验动物：所用实验动物为昆明种小鼠60只，雌雄各半，体重为20±2g，购自南京中医药大学实验动物中心。1.1 Experimental animals: The experimental animals used were 60 Kunming mice, half male and half female, weighing 20±2g, purchased from the Experimental Animal Center of Nanjing University of Traditional Chinese Medicine.
1.2肿瘤细胞：宫颈癌U
14瘤株由上海肿瘤研究所提供。
1.2 Tumor cells: Cervical cancer U14 tumor line was provided by Shanghai Cancer Institute.
2.方法2.Method
2.1宫颈癌U
14荷瘤小鼠的建立：解剖前实验室用紫外灯消毒3小时，选择生长6-7天的传代腹水癌小鼠数只，脱臼处死后酒精消毒腹部，取出生长较好的腹水癌按1：2的比例稀释，癌细胞计数为3.5×10
7个/ml接种在小鼠的左侧腋窝处皮下，每只接种0.2ml。
2.1 Establishment of cervical cancer U 14 tumor-bearing mice: The laboratory was disinfected with ultraviolet light for 3 hours before dissection, and several passage ascites cancer mice that had grown for 6-7 days were selected. After dislocation and sacrifice, the abdomen was disinfected with alcohol, and the better-growing mice were taken out. Ascites cancer was diluted at a ratio of 1:2 and the cancer cell count was 3.5×10 7 /ml and inoculated subcutaneously in the left axilla of mice, with 0.2ml inoculated into each mouse.
2.2药物配制：取含量为98.7％的佛手柑内酯和槲皮素黄酮组合物，研细并配制成浓度为0.05mg/ml、0.10mg/ml、0.15mg/ml的混悬液，溶剂为蒸馏水，给药时摇匀。2.2 Drug preparation: Take the bergamot lactone and quercetin flavonoid composition with a content of 98.7%, grind it finely and prepare it into a suspension with a concentration of 0.05mg/ml, 0.10mg/ml, and 0.15mg/ml. The solvent is Distilled water, shake well before administration.
2.3抑瘤实验：接种宫颈癌U
14的50只小鼠，随机分为5组，即治疗组(阳性对照组)、肿瘤对照组(阴性对照组)和试验组(Ⅰ-Ⅲ组)。每组10只。接种后第2天，开始口服灌胃给药0.3ml/只，折合剂量为0.75mg/kg，1.50mg/kg，2.25mg/kg，连续给药10天。肿瘤对照组不给药，只注射同体积的生理盐水；治疗组(阳性对照组)给予腹腔注射环磷酰胺(CPP)20mg/kg，每只ip给药0.3ml/d，连续给药10天。
2.3 Tumor inhibition experiment: 50 mice inoculated with cervical cancer U 14 were randomly divided into 5 groups, namely the treatment group (positive control group), the tumor control group (negative control group) and the experimental group (groups I-III). 10 per group. On the second day after vaccination, start oral administration of 0.3ml/animal, the equivalent dose is 0.75mg/kg, 1.50mg/kg, 2.25mg/kg, and continue to administer for 10 days. The tumor control group was not given any medication and was only injected with the same volume of normal saline; the treatment group (positive control group) was given intraperitoneal injection of cyclophosphamide (CPP) 20 mg/kg, and each animal was given 0.3 ml/d ip for 10 consecutive days. .
3.观察指标：停药后分别处死动物，分离瘤体、肝、脾及胸腺，分别用电子天平称重和体重，记录体重变化情况，计算肿瘤生长抑制率。3. Observation indicators: After stopping the drug, the animals were sacrificed respectively, and the tumors, liver, spleen and thymus were separated, weighed and body weighted respectively with an electronic balance, the changes in body weight were recorded, and the tumor growth inhibition rate was calculated.
3.1肿瘤生长抑制率3.1 Tumor growth inhibition rate
计算公式：肿瘤生长抑制率＝(1-试验组平均瘤重/对照组平均瘤重)×100％。Calculation formula: tumor growth inhibition rate = (1-average tumor weight of the test group/average tumor weight of the control group) × 100%.
4.结果4.Results
抑制宫颈癌U
14的生长：剂量为0.75mg/kg，1.50mg/kg，2.25mg/kg的佛手柑内酯和槲皮素黄酮组合物能较明显地抑制宫颈癌U
14的生长。小剂量组0.75mg/kg抑瘤效果最佳，抑瘤率为60.62％，表明佛手柑内酯和槲皮素黄酮组合物对不同肿瘤的抑制作用具有一定的剂量选择性；阳性对照组环磷酰胺(CPP)能够使荷瘤小鼠的体重明显下降，而佛手柑内酯和槲皮素黄酮组合物组与空白对照组无显著差异。表明环磷酰胺对荷瘤小鼠有一定的损伤；而佛手柑内酯和槲皮素黄酮组合物的几个剂量组对荷瘤小鼠的体重均无明显影响，表明佛手柑内酯和槲皮素黄酮组合物具有低毒、高效的特点。
Inhibiting the growth of cervical cancer U 14 : The combination of bergamot lactone and quercetin flavonoids at doses of 0.75 mg/kg, 1.50 mg/kg, and 2.25 mg/kg can significantly inhibit the growth of cervical cancer U 14 . The low-dose group 0.75 mg/kg had the best tumor inhibitory effect, with a tumor inhibition rate of 60.62%, indicating that the combination of bergamot lactone and quercetin flavonoids has a certain dose selectivity in inhibiting different tumors; the positive control group cyclophosphate Amide (CPP) can significantly reduce the weight of tumor-bearing mice, while there is no significant difference between the bergamot lactone and quercetin flavonoid composition group and the blank control group. It shows that cyclophosphamide has certain damage to tumor-bearing mice; while several dosage groups of bergamot lactone and quercetin flavonoid composition have no significant effect on the body weight of tumor-bearing mice, indicating that bergamot lactone and quercetin The cortin flavonoid composition has the characteristics of low toxicity and high efficiency.
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。The above-described embodiments only describe preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.
Claims (9)
- 佛手柑内酯和槲皮素黄酮的组合物在制备治疗癌症药物中的应用，其特征在于，所述佛手柑内酯是从佛手中分离得到的天然物质；所述槲皮素黄酮是从槐米中分离得到的天然物质；The application of the composition of bergamot lactone and quercetin flavonoids in the preparation of drugs for treating cancer, which is characterized in that the bergamot lactone is a natural substance isolated from the hands of bergamot; the quercetin flavonoids are obtained from Sophora japonica Natural substances isolated from rice;所述佛手柑内酯和槲皮素黄酮的组合物中佛手柑内酯和槲皮素黄酮的质量比为1:3～8；The mass ratio of bergamot lactone and quercetin flavonoids in the composition of bergamot lactone and quercetin flavonoids is 1:3 to 8;所述癌症为肺癌、肝癌、宫颈癌或前列腺癌；The cancer is lung cancer, liver cancer, cervical cancer or prostate cancer;所述佛手柑内酯的制备方法，包括以下步骤：The preparation method of bergamot lactone includes the following steps:(1)将40目的佛手粉与醋酸-醋酸钠缓冲液以质量比1:5混合，在40℃下酶解55min，得到酶解液；(1) Mix 40-mesh bergamot powder and acetic acid-sodium acetate buffer at a mass ratio of 1:5, and perform enzymatic hydrolysis at 40°C for 55 minutes to obtain an enzymatic hydrolyzate;(2)在酶解液中加入体积分数为35％的乙醇溶液，在55℃下超声提取2次，每次40min，合并提取液，离心后，得到上清液；(2) Add an ethanol solution with a volume fraction of 35% to the enzymatic hydrolysis solution, conduct ultrasonic extraction twice at 55°C, 40 minutes each time, combine the extracts, and centrifuge to obtain the supernatant;(3)将上清液经截留分子量为1000KD的聚砜膜或聚醚砜膜进行膜分离过滤后，得到样品原液；(3) After membrane separation and filtration of the supernatant through a polysulfone membrane or polyethersulfone membrane with a molecular weight cutoff of 1000KD, the sample stock solution is obtained;(4)样品原液经过柱层析分离，得到洗脱液；(4) The sample stock solution is separated by column chromatography to obtain the eluate;(5)将洗脱液干燥至恒重，得到所述佛手柑内酯。(5) Dry the eluate to constant weight to obtain the bergamot lactone.
- 一种抗癌药物，其特征在于，以佛手柑内酯和槲皮素黄酮的组合物为活性成分，还含有药学上可接受的载体；An anti-cancer drug, characterized in that it uses a combination of bergamot lactone and quercetin flavonoids as active ingredients, and also contains a pharmaceutically acceptable carrier;所述佛手柑内酯是从佛手中分离得到的天然物质；所述槲皮素黄酮是从槐米中分离得到的天然物质；The bergamot lactone is a natural substance isolated from bergamot; the quercetin flavonoid is a natural substance isolated from Sophora japonica;所述佛手柑内酯和槲皮素黄酮的组合物中佛手柑内酯和槲皮素黄酮的质量比为1:3～8；The mass ratio of bergamot lactone and quercetin flavonoids in the composition of bergamot lactone and quercetin flavonoids is 1:3 to 8;所述癌症为肺癌、肝癌、宫颈癌或前列腺癌；The cancer is lung cancer, liver cancer, cervical cancer or prostate cancer;所述佛手柑内酯的制备方法，包括以下步骤：The preparation method of bergamot lactone includes the following steps:(1)将40目的佛手粉与醋酸-醋酸钠缓冲液以质量比1:5混合，在40℃下酶解55min，得到酶解液；(1) Mix 40-mesh bergamot powder and acetic acid-sodium acetate buffer at a mass ratio of 1:5, and perform enzymatic hydrolysis at 40°C for 55 minutes to obtain an enzymatic hydrolyzate;(2)在酶解液中加入体积分数为35％的乙醇溶液，在55℃下超声提取2次，每次40min，合并提取液，离心后，得到上清液；(2) Add an ethanol solution with a volume fraction of 35% to the enzymatic hydrolysis solution, conduct ultrasonic extraction twice at 55°C, 40 minutes each time, combine the extracts, and centrifuge to obtain the supernatant;(3)将上清液经截留分子量为1000KD的聚砜膜或聚醚砜膜进行膜分离过滤后，得到样品原液；(3) After membrane separation and filtration of the supernatant through a polysulfone membrane or polyethersulfone membrane with a molecular weight cutoff of 1000KD, the sample stock solution is obtained;(4)样品原液经过柱层析分离，得到洗脱液；(4) The sample stock solution is separated by column chromatography to obtain the eluent;(5)将洗脱液干燥至恒重，得到所述佛手柑内酯。(5) Dry the eluate to constant weight to obtain the bergamot lactone.
- 根据权利要求2所述的抗癌药物，其特征在于，所述药学上可接受的载体包括填充剂、粘合剂、崩解剂、润滑剂、矫味剂和抗氧剂中的一种或多种。The anticancer drug according to claim 2, wherein the pharmaceutically acceptable carrier includes one of fillers, binders, disintegrants, lubricants, flavoring agents and antioxidants, or Various.
- 根据权利要求3所述的抗癌药物，其特征在于，所述填充剂包括甘露醇、乳糖或聚乙二醇。The anticancer drug according to claim 3, wherein the filler includes mannitol, lactose or polyethylene glycol.
- 根据权利要求3所述的抗癌药物，其特征在于，所述粘合剂包括淀粉或微晶纤维素。The anticancer drug according to claim 3, wherein the binder includes starch or microcrystalline cellulose.
- 根据权利要求3所述的抗癌药物，其特征在于，所述崩解剂包括羧甲基纤维素或低取代羟丙基纤维素。The anticancer drug according to claim 3, wherein the disintegrant includes carboxymethylcellulose or low-substituted hydroxypropylcellulose.
- 根据权利要求3所述的抗癌药物，其特征在于，所述润滑剂包括滑石粉或硬脂酸镁。The anticancer drug according to claim 3, wherein the lubricant includes talc or magnesium stearate.
- 根据权利要求2所述的抗癌药物，其特征在于，所述抗癌药物剂型为口服制剂、注射油剂或外用制剂。The anti-cancer drug according to claim 2, wherein the anti-cancer drug dosage form is an oral preparation, an injection oil or an external preparation.
- 根据权利要求8所述的抗癌药物，其特征在于，所述口服制剂为片剂、胶囊、缓释片或滴丸，所述外用制剂为霜剂、膏剂、油剂或甘油制剂。The anti-cancer drug according to claim 8, wherein the oral preparation is a tablet, capsule, sustained-release tablet or dropping pill, and the external preparation is a cream, ointment, oil or glycerin preparation.
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