WO2024114248A1 - 一种柑橘属果实提取物及其制备方法与应用 - Google Patents

一种柑橘属果实提取物及其制备方法与应用 Download PDF

Info

Publication number
WO2024114248A1
WO2024114248A1 PCT/CN2023/128344 CN2023128344W WO2024114248A1 WO 2024114248 A1 WO2024114248 A1 WO 2024114248A1 CN 2023128344 W CN2023128344 W CN 2023128344W WO 2024114248 A1 WO2024114248 A1 WO 2024114248A1
Authority
WO
WIPO (PCT)
Prior art keywords
citrus fruit
estrogen receptor
breast cancer
extract
fruit extract
Prior art date
Application number
PCT/CN2023/128344
Other languages
English (en)
French (fr)
Inventor
邓张双
秦烨
王羿语
孙梦
杜姝
Original Assignee
三峡大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 三峡大学 filed Critical 三峡大学
Publication of WO2024114248A1 publication Critical patent/WO2024114248A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/40—Separation, e.g. from natural material; Purification

Definitions

  • the present invention relates to the field of medical technology, and in particular to a citrus fruit extract and a preparation method and application thereof.
  • Estrogen receptor-positive breast cancer is a common type of breast cancer.
  • the condition is affected by estrogen, and endocrine therapy is usually an effective treatment. Since it is impossible to completely inhibit ER signal transduction, it is difficult to effectively inhibit the proliferation of cancer cells. In addition, the problem of drug resistance limits clinical drug use. Therefore, ER-positive breast cancer urgently needs new small molecule drugs for treatment.
  • the current representative therapeutic drugs are tamoxifen and fulvestrant.
  • the problem of tamoxifen resistance has gradually emerged clinically.
  • fulvestrant has higher efficacy and lower side effects than tamoxifen, and can effectively prolong the survival of patients, its wide application is limited by its high price, as well as problems such as intramuscular injection, poor solubility and lack of oral availability.
  • estrogen receptor degraders for breast cancer has become more and more concentrated on small molecule synthetic compounds or pharmaceutical compositions (CN 110343101A, CN 111646972A, CN 112041307A, CN 112424205A, CN 112638916A, CN 112912078A, CN 113614076A, CN 114302879A, CN 114502539A, CN 114656452A and CN 114667147A).
  • small molecule synthetic compounds or pharmaceutical compositions CN 110343101A, CN 111646972A, CN 112041307A, CN 112424205A, CN 112638916A, CN 112912078A, CN 113614076A, CN 114302879A, CN 114502539A, CN 114656452A and CN 114667147A.
  • Citrus is the world's largest type of fruit that can be used as both medicine and food. It is an important branch of the Rutaceae family. It has rich varieties and is widely distributed. It is mainly concentrated in the provinces south of the Yangtze River, such as Hubei, Hunan, Guangdong, and Fujian. It is the fruit with the largest planting area.
  • the chemical components of citrus fruits are diverse, mainly including flavonoids, terpenes, coumarins, alkaloids, etc., with flavonoids as the main component. It has a wide range of biological activities. At present, the compounds of citrus plants have been studied in many aspects, such as anti-tumor, anti-inflammatory, antioxidant, antiviral and prevention of cardiovascular diseases.
  • the purpose of the present invention is to overcome the lack of estrogen receptor degraders with natural sources, low toxicity and side effects and for endocrine therapy, and provide a method for treating breast cancer using a citrus fruit extract as an estrogen receptor degrader.
  • a citrus fruit extract and a preparation method thereof are provided, as well as the use of the extract in treating estrogen receptor-positive breast cancer, that is, degrading estrogen receptors through the ubiquitin-proteasome pathway, thereby significantly inhibiting the growth of estrogen receptor-positive breast cancer.
  • the present invention provides a citrus fruit extract, wherein the main component of the extract is polymethoxyflavonoids;
  • the polymethoxyflavones include one or more of 5,7,8,4',5'-pentamethoxyflavones, 5,6,7,4',5'-pentamethoxyflavones, 5,6,7,8,3',4'-hexamethoxyflavones, 3,5,6,7,8,3',4'-heptamethoxyflavones and 5,6,7,8,4'-pentamethoxyflavones.
  • the present invention also provides a method for preparing the citrus fruit extract, comprising the following steps:
  • step (2) Wash the material 2 obtained in step (1) with water, centrifuge three times to obtain a precipitate, add anhydrous ethanol to extract 2 to 4 times, combine the extracts, concentrate under reduced pressure, and dry to obtain a citrus fruit extract.
  • the mixing ratio of the citrus fruit and water is 8-12 g:1 mL; the rotation speeds of the first centrifugation and the second centrifugation are independently 8000-12000 rpm, and the time is independently 8-12 min.
  • the mass volume ratio of the material 2 to water is 1 g: 8-12 mL, and the washing is performed 3 times; the rotation speed of the three centrifugations is 8000-12000 rpm, and the time is 8-12 min; for each extraction, the mass volume ratio of the precipitate to anhydrous ethanol is 1 g: 1.5-2.5 mL, the extraction temperature is 22-28° C., and the extraction time is 12-18 min.
  • the citrus fruit includes tangerine, orange or mandarin.
  • the citrus fruit comprises the whole fruit, peel or pulp.
  • the present invention further provides the citrus fruit extract, and the use of the citrus fruit extract prepared by the preparation method of the citrus fruit extract in preparing drugs for treating breast cancer.
  • the drug is an estrogen receptor degrader.
  • the breast cancer is estrogen receptor positive breast cancer.
  • the present invention has the following beneficial effects:
  • the model constructed in the present invention uses more than two estrogen receptor-positive breast cancer cell lines for verification, and the animal experiments used at the same time further clarify the efficacy of the anti-tumor drug mixed extract on estrogen receptor-positive breast cancer and the degradation effect on estrogen receptors.
  • the present invention provides a citrus fruit extract as an estrogen receptor degrader, which is a natural ingredient derived from plants of the genus Citrus in the family Rutaceae, and can be used as an additive to prepare foods, health products and medicines.
  • the present invention also provides the use of such extracts as estrogen receptor degraders for treating breast cancer, thereby making up for the lack of estrogen receptor degraders from natural sources.
  • FIG1 is a crystal violet live cell staining result after the extract of Example 1 was co-incubated with human breast cancer MCF7 cells;
  • FIG2 is a growth inhibition curve of the extract of Example 1 on human breast cancer MCF7 cells
  • FIG3 shows the effect of the extract of Example 1 on the estrogen receptor protein level of human estrogen receptor positive breast cancer cells MCF7 over time and concentration gradient
  • Figure 4 shows the effect of the estrogen receptor degrader of Example 1 on the estrogen receptor protein levels of human and mouse estrogen receptor-positive breast cancer cells MCF7, T47D and SSM2 at different time gradients after MG132 (proteasome pathway inhibitor) and BAF-A1 (lysosome pathway inhibitor) were used to inhibit protein degradation-related pathways in Example 4, respectively.
  • FIG5 is a real-life tumor image of the SSM2-129 mouse homograft tumor model in Example 5;
  • FIG6 is the statistical result of tumor volume of the SSM2-129 mouse homograft tumor model in Example 5.
  • FIG7 is the statistical result of tumor weight of SSM2-129 mouse homograft tumor model in Example 5.
  • FIG. 8 shows the effect of tumor estrogen receptor protein levels in the SSM2-129 mouse homograft tumor model in Example 5.
  • FIG9 is a high performance liquid chromatogram and main chemical components of extract 3-1 of Example 1.
  • the HPLC analysis conditions were as follows: the mobile phase was methanol-water, with gradient elution from 10% methanol to 60% methanol for 5 min, from 60% methanol to 80% methanol for 20 min, from 80% methanol to 100% methanol for 2 min, and 100% methanol for 3 min; the detection wavelength was 254 nm; the flow rate was 1 mL/min; the injection volume was 10 uL; the chromatographic column was InertSustainAQ-C18, 5 ⁇ m, 4.6 ⁇ 250 mm (UP); and the chromatograph was Shimadzu LC-2030C 3D Plus.
  • the present invention provides a citrus fruit extract, wherein the main component of the extract is polymethoxyflavonoids;
  • the polymethoxyflavones include one or more of 5,7,8,4',5'-pentamethoxyflavones, 5,6,7,4',5'-pentamethoxyflavones, 5,6,7,8,3',4'-hexamethoxyflavones, 3,5,6,7,8,3',4'-heptamethoxyflavones and 5,6,7,8,4'-pentamethoxyflavones; preferably 5,7,8,4',5'-pentamethoxyflavones, 5,6,7,4',5'-pentamethoxyflavones, 5,6,7,8,3',4'-heptamethoxyflavones, 3,5,6,7,8,3',4'-heptamethoxyflavones and 5,6,7,8,4'-pentamethoxyflavones.
  • the present invention also provides a method for preparing the citrus fruit extract, comprising the following steps:
  • step (2) washing the material 2 obtained in step (1) with water, centrifuging three times to obtain a precipitate, adding anhydrous ethanol to extract 2 to 4 times, combining the extracts, concentrating under reduced pressure, and drying to obtain a citrus fruit extract; preferably 3 times.
  • the mixing ratio of the citrus fruit and water in step (1) is 8-12 g:1 mL; preferably 9-11 g:1 mL; more preferably 10 g:1 mL.
  • the speed of the first centrifugation and the second centrifugation in step (1) is independently 8000-12000 rpm, and the time is independently 8-12 min; preferably, the speed is independently 9000-11000 rpm, and the time is independently 9-11 min; more preferably, the speed is independently 10000 rpm, and the time is independently 10 min.
  • the volume ratio of the material 1 to the hydrochloric acid in step (1) is 1:1.5-2.5, preferably 1:2.
  • the standing time in step (1) is 12 to 16 hours, preferably 13 to 15 hours, and more preferably 14 hours.
  • the mass volume ratio of the material 2 to water in step (2) is 1 g: 8-12 mL, washed 3 times; preferably 1 g: 9-11 mL; more preferably 1 g: 10 mL.
  • the rotation speed of the three centrifugations is 8000-12000 rpm, and the time is 8-12 min; preferably, the rotation speed is 9000-11000 rpm, and the time is 9-11 min; more preferably, the rotation speed is 10000 rpm, and the time is 10 min.
  • the mass volume ratio of the precipitate to anhydrous ethanol is 1 g: 1.5-2.5 mL; preferably 1 g: 2 mL.
  • the extraction temperature in step (2) is 22-28°C; preferably 23-27°C; more preferably 24-26°C; and more preferably 25°C.
  • the extraction time in step (2) is 12 to 18 minutes, preferably 13 to 17 minutes, more preferably 14 to 16 minutes, and more preferably 15 minutes.
  • the citrus fruit includes tangerine, orange or mandarin orange; preferably tangerine.
  • the citrus fruit includes the whole fruit, peel or pulp; preferably the whole fruit.
  • the present invention further provides the citrus fruit extract, and the use of the citrus fruit extract prepared by the preparation method of the citrus fruit extract in preparing drugs for treating breast cancer.
  • the drug is an estrogen receptor degrader.
  • the breast cancer is estrogen receptor positive breast cancer.
  • the breast cancer models are MCF7 and SSM2 cell models and SSM2-129 mouse tumor-bearing models.
  • the detection substance selected in the model is estrogen receptor, which is specifically described as degrading estrogen receptor through ubiquitin-proteasome pathway.
  • MCF7, SSM2, and T47D related estrogen receptor positive breast cancer cell lines were used as model cells.
  • 129 strain mice were used as model animals, and SSM2 cell lines were transplanted into the fourth mammary pad on the right side.
  • This embodiment provides a method for preparing a citrus fruit extract, comprising the following steps:
  • the extract mainly includes the following components: 5,7,8,4',5'-pentamethoxyflavone, 5,6,7,4',5'-pentamethoxyflavone, 5,6,7,8,3',4'-hexamethoxyflavone, 3,5,6,7,8,3',4'-heptamethoxyflavone and 5,6,7,8,4'-pentamethoxyflavone.
  • the extract in Example 1 can significantly inhibit the growth of tumor cells when co-incubated with human estrogen receptor-positive breast cancer cells MCF7 and SSM2.
  • the inhibitory activity of typical human breast cancer MCF7 cells is now described as follows.
  • the extract in Example 1 was co-incubated with human estrogen receptor-positive breast cancer cells MCF7 cells for 72 hours.
  • the specific method is as follows: the estrogen receptor-positive breast cancer cells MCF7 cells were inoculated in DMEM culture medium, and the DMEM culture medium was added with 10% fetal bovine serum, 100 units/mL penicillin and 100 ⁇ g/mL streptomycin, and cultured in a 37°C, 5% CO2 incubator. The cells were seeded into 96-well plates at a density of 5000 cells per well overnight and waited for the cells to fully adhere to the wall. Then, estrogen receptor degraders were added and incubated with the fully adhered cells for 72 hours.
  • the blank control group was used without adding drugs, and the positive control group was used with the endocrine therapy drug tamoxifen.
  • the cells after co-incubation in each group were stained with crystal violet solution for living cells.
  • the staining results are shown in Figure 1.
  • the estrogen receptor degraders were co-incubated with human estrogen receptor-positive breast cancer cells MCF7, which can significantly inhibit the growth of tumor cells.
  • the measurement results are shown in Figure 2.
  • the half-inhibitory concentration IC 50 value of the estrogen receptor degrader and human estrogen receptor-positive breast cancer cells MCF7 cells is 0.06 mg/mL.
  • the extracts in Example 1 have an effect on the protein level of the estrogen receptor associated with the estrogen receptor positive breast cancer cell line MCF7.
  • the estrogen receptor degrader is co-incubated with human estrogen receptor positive breast cancer cells MCF7 and SSM2, and the protein level of the estrogen receptor can be significantly down-regulated.
  • the protein level of the estrogen receptor in typical human breast cancer MCF7 cells is down-regulated as an example as follows.
  • Example 1 The extract in Example 1 was incubated with human estrogen receptor-positive breast cancer cells MCF7 for 24, 48, and 72 hours, and then the protein was extracted. The protein level of estrogen receptor was detected by Western blot.
  • the specific method is as follows: estrogen receptor-positive breast cancer cells MCF7 in the logarithmic growth phase were taken, digested with 0.125% trypsin, and then inoculated in a 6-well culture plate containing DMEM medium.
  • the DMEM medium was added with 10% fetal bovine serum, 100 units/mL penicillin and 100 ⁇ g/mL streptomycin, and placed in a 37°C, 5% CO 2 incubator for culture.
  • the cells were inoculated into a 96-well plate culture plate at a density of 2 ⁇ 10 5 cells per well overnight to wait for the cells to attach completely, and then the estrogen receptor degrader was added to the fully attached cells and incubated for 24, 48, and 72 hours as a time gradient.
  • the blank control group was used without adding drugs, and the concentration of the extracts corresponding to the half-maximal inhibition concentration of the drugs obtained in the implementation case 2 was used as a concentration gradient.
  • the cells were washed with 4°C pre-cooled PBS, an appropriate amount of RIPA cell lysis buffer was added, and the cells were scraped with a cell scraper and the lysis buffer containing cell fragments was aspirated. After an ice bath for 30 minutes, the cells were centrifuged at 4°C, 12000 rpm, for 30 minutes, and the supernatant was taken as the whole-cell protein extraction sample.
  • the specific method of Western blot is as follows: prepare 10% separation gel and 5% concentrated gel, and configure SDS-PAGE gel. Extract protein from cell samples, use BCA kit (BCA Protein Assay Reagent Kit) for protein quantification, prepare sample and denature at 100°C for 10min. Then load 20 ⁇ g protein in total and perform SDS-PAGE electrophoresis (constant voltage 120V) for about 2h. After electrophoresis, peel off the gel and cut off the concentrated gel. Put the NC membrane, filter paper and gel in the transfer solution for assembly. On the electrotransfer plate, stack from the black side (negative electrode) to the red side (positive electrode): sponge pad, 3 filter papers, gel, NC membrane, 3 filter papers, and finally sponge pad.
  • the electrotransfer tank After installing in order, insert the electrotransfer tank, pour in the newly prepared transfer solution, plug in the electrode, and transfer the membrane for 90min under the condition of steady current 200mA, thereby transferring the protein in the gel to the NC membrane.
  • the NC membrane was removed and briefly rinsed in TBST solution.
  • the NC membrane was gently shaken with 5% skim milk (prepared with TBST) on a shaker at room temperature for 1 hour, and then washed 3 times with TBST, each time for 5 minutes.
  • the primary antibody (rabbit source) was incubated overnight on a 4°C chromatography cabinet shaker. The membrane was washed with TBST, 5 minutes/time, and washed 3 times in total.
  • the diluted, HRP-labeled goat anti-rabbit IgG was co-incubated with the NC membrane and incubated on a shaker at room temperature for 1 hour.
  • the membrane was washed with TBST, 5 minutes/time, and washed 3 times in total.
  • the developer A and B were mixed in a 1:1 ratio, the NC membrane was removed, and the mixture was evenly added to the membrane.
  • the gel imaging system was used to take pictures and save the results.
  • the Western blot development results are shown in Figure 3.
  • the estrogen receptor degrader downregulated the protein level of the estrogen receptor in the human estrogen receptor-positive breast cancer cell MCF7 in a concentration- and time-dependent manner.
  • the extract in Example 1 down-regulates the protein level of estrogen receptor in estrogen receptor-positive breast cancer cell lines MCF7, T47D, and SSM2 to further explore the relevant mechanism.
  • MCF7 menoplasmic cancer
  • T47D breast cancer cell lines
  • SSM2 serotonin-associated breast cancer cell lines
  • MG132 proteasome pathway inhibitor
  • BAF-A1 lysosomal pathway inhibitor
  • the effect of the extract on the estrogen receptor protein level is shown in Figure 4.
  • BAF-A1 lysosomal pathway inhibitor
  • the extract can still down-regulate the estrogen receptor protein level at different time gradients.
  • the extract After using MG132 (proteasome pathway inhibitor) to inhibit the proteasome pathway of protein degradation, the extract has a significant rebound in its down-regulating effect on the estrogen receptor protein level. As shown in Figure 4, the down-regulation level of the extract on the estrogen receptor in the estrogen receptor-positive breast cancer cell line is promoted by the proteasome pathway, not by the lysosomal pathway.
  • the specific method is the same as that of implementation case 3.
  • the pharmacodynamics of the extract (3-1) in Example 1 on the tumor inhibition rate of estrogen receptor-positive breast cancer and its effect on the expression of estrogen receptors in tumor tissues were evaluated by the SSM2-129 breast cancer tumor-bearing mouse model.
  • the tumor inhibition rate of the extract in Example 1 on estrogen receptor-positive breast cancer was about 50% after 10 days of administration, indicating that it has a significant inhibitory effect on estrogen receptor-positive breast cancer, and has no obvious damage to important organs, and can also significantly downregulate the protein level of its estrogen receptor.
  • SSM2 cells were inoculated at a density of 1 ⁇ 10 6 per mouse in the fourth mammary pad on the right side of the same species 129 mice (18-20g).
  • the specific method is as follows: SSM2 cells in the logarithmic growth phase were digested with 0.125% trypsin and centrifuged after cell counting. The cells were resuspended with PBS and diluted to the required density. The SSM2 cell suspension was inoculated at a density of 1 ⁇ 10 6 per mouse in the fourth mammary pad on the right side of the 129 mice. Subcutaneous tumor nodules with a diameter of about 0.5 cm were observed in 2 weeks, indicating that the tumor mouse model was successfully established.
  • mice were divided into 4 groups, 5 in each group, and the drug administration test was started 2 weeks after tumor inoculation.
  • the solvent without drug administration was used as the solvent control group, and the extract concentration in the drug administration example 1 was selected as 25mg/kg, 50mg/kg, and 100mg/kg as the drug treatment experimental group.
  • the weight of mice was recorded on the 0th, 1st, 3rd, 5th, 7th, 9th, and 11th days, and the tumor volume was measured and recorded using a vernier caliper and the tumor inhibition rate was calculated. The results are shown in Figure 6.
  • the tumor weight was weighed after killing, and the results are shown in Figures 5 and 7.
  • the tumors and important organs (heart, liver, spleen, lung, and kidney) of each group of mice were fixed with tissue fixative and then paraffin sections were sliced, and H&E staining was performed to observe the effects of the drug on the important organs of mice.
  • the protein level of estrogen receptor in tumor tissue was detected by Western blot.
  • the results are shown in Figure 8.
  • the expression level of estrogen receptor in SSM2 tumor tissue of mice in the drug-treated group decreased, and the decrease was obvious at the 50mg/kg dosage.
  • medium and high concentrations of extracts can significantly inhibit the tumor growth of the SSM2-129 mouse homologous transplant tumor model without obvious damage to important organs, and significantly reduce the expression level of estrogen receptors in the tumor site.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

本发明涉及医药技术领域，尤其涉及一种柑橘属果实提取物及其制备方法与应用。本发明提供了一种柑橘属果实提取物，所述提取物主要成分为多甲氧基黄酮。本发明的目的在于克服天然来源、低毒副作用和用于内分泌治疗的雌激素受体降解剂的缺乏问题，提供了一种柑橘属果实提取物作为雌激素受体降解剂治疗乳腺癌的方法。具体是提供了一种柑橘属果实提取物及其制备方法，以及提取物在治疗雌激素受体阳性乳腺癌上的用途，即通过泛素-蛋白酶体途径降解雌激素受体，进而显著抑制雌激素受体阳性乳腺癌的生长。

Description

一种柑橘属果实提取物及其制备方法与应用 技术领域
本发明涉及医药技术领域，尤其涉及一种柑橘属果实提取物及其制备方法与应用。
背景技术
癌症是威胁人类健康的重大杀手之一，其中乳腺癌患者发病率高居第一位。雌激素受体阳性乳腺癌是乳腺癌常见类型，病情受到雌激素影响，通常内分泌治疗是有效的治疗手段。由于无法完全抑制ER信号传导，导致难以有效抑制癌细胞增殖，加上耐药问题，临床用药受限，因此ER阳性乳腺癌亟需新的治疗小分子药物。
现有代表治疗药物为他莫昔芬和氟维司群。临床上他莫昔芬耐药问题逐渐显现出来。氟维司群虽具有比他莫昔芬更高的的疗效及更低的副作用，可有效延长患者的生存期，但由于价格昂贵，加上肌肉注射、溶解性差和口服利用度缺乏等问题，限制了它的广泛应用。近年来，用于乳腺癌的雌激素受体降解剂的开发越来越多，更多集中于小分子合成化合物或药物组合物(CN 110343101A，CN 111646972A，CN 112041307A，CN 112424205A，CN 112638916A，CN 112912078A，CN 113614076A，CN 114302879A，CN 114502539A，CN 114656452A和CN 114667147A)。而天然来源的雌激素受体降解剂报道较少，为充分开发丰富植物资源，以方便、快捷、经济为目的，寻找天然来源的雌激素受体降解剂具有重要意义。
柑橘是世界最大类药食两用水果，是芸香科植物的重要分支，品种资源丰富，分布广泛，主要集中在湖北、湖南、广东、福建等长江流域以南各省，是种植面积最大的水果。柑橘属果实化学成分类型多样，主要包括黄酮类、萜类、香豆素类、生物碱类等，以黄酮成分为主；生物活性广泛，目前已对柑橘属植物的化合物进行了抗肿瘤、抗炎、抗氧化、抗病毒和预防心血管疾病等多方面的研究。文献[Journal of Agricultural and Food Chemistry,2011,59(6):2314-2323.]研究发现，与其他柠檬苦素类化合物以及合成柠檬苦素衍生物相比，去乙酰诺米林对雌激素受体阳性乳腺癌细胞抑制率要高出400倍，是最有效的雌激素受体阳性乳腺癌细胞抑制剂。这一研究给发明者提供了一种思路，从柑橘属果实中寻找更安全、更高效的雌激素受体降解剂治疗乳腺癌成为一种可能。
因此，针对上述科学问题，进一步挖掘柑橘属果实活性物质成分，并拓展其作为新的乳腺癌治疗方案，推进柑橘精深加工往大健康产业发展，具有重要的基础和应用基础研究价值。
发明内容
本发明的目的在于克服天然来源、低毒副作用和用于内分泌治疗的雌激素受体降解剂的缺乏问题，提供了一种柑橘属果实提取物作为雌激素受体降解剂治疗乳腺癌的方法。具体是提供了一种柑橘属果实提取物及其制备方法，以及提取物在治疗雌激素受体阳性乳腺癌上的用途，即通过泛素-蛋白酶体途径降解雌激素受体，进而显著抑制雌激素受体阳性乳腺癌的生长。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种柑橘属果实提取物，所述提取物主要成分为多甲氧基黄酮；
所述多甲氧基黄酮包括5,7,8,4',5'-五甲氧基黄酮、5,6,7,4',5'-五甲氧基黄酮、5,6,7,8,3',4'-六甲氧基黄酮、3,5,6,7,8,3',4'-七甲氧基黄酮和5,6,7,8,4'-五甲氧基黄酮中的一种或多种。
本发明还提供了所述的柑橘属果实提取物的制备方法，包括如下步骤：
(1)将柑橘属果实与水混合后粉碎，一次离心去渣，得物料1，将物料1与盐酸混合，静置冷却至室温，二次离心，收集沉淀，为物料2；
(2)将步骤(1)所得物料2用水洗涤，三次离心得沉淀，加入无水乙醇浸提2～4次，合并浸提液，减压浓缩，干燥，得柑橘属果实提取物。
优选的，步骤(1)所述柑橘属果实与水混合比例为8～12g：1mL；所述一次离心和二次离心的转速独立为8000～12000rpm，时间独立为8～12min。
优选的，步骤(1)所述物料1与盐酸的体积比为1：1.5～2.5；所述静置的时间为12～16h。
优选的，步骤(2)所述物料2与水的质量体积比为1g：8～12mL，洗涤3次；所述三次离心的转速为8000～12000rpm，时间为8～12min；每次浸提，所述沉淀与无水乙醇的质量体积比为1g：1.5～2.5mL，所述浸提的温度为22～28℃，浸提的时间为12～18min。
优选的，所述柑橘属果实包括橘、橙或柑。
优选的，所述柑橘属果实包括全果、果皮或果肉。
本发明还进一步提供了所述的柑橘属果实提取物，所述的柑橘属果实提取物的制备方法制备得到的柑橘属果实提取物在制备治疗乳腺癌药物中的应用。
优选的，所述药物为雌激素受体降解剂。
优选的，所述乳腺癌为雌激素受体阳性乳腺癌。
与现有技术相比，本发明具有如下的有益效果：
(1)本发明构造模型所使用的是两种以上细胞雌激素受体阳性乳腺癌细胞株进行验证，同时使用的动物实验进一步明确所述抗肿瘤药物混合提取物对雌激素受体阳性乳腺癌的疗效及对雌激素受体的降解作用。
(2)本发明提供所述一种柑橘属果实提取物作为雌激素受体降解剂是来源于芸香科柑橘属植物的天然成分，可作为添加成分制备食品、保健品和药物，还提供了该类提取物作为雌激素受体降解剂治疗乳腺癌的用途，弥补了天然来源的雌激素受体降解剂的缺失。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为实施例1的提取物与人乳腺癌MCF7细胞共孵育后结晶紫活细胞染色结果；
图2为实施例1的提取物对人乳腺癌MCF7细胞生长抑制曲线；
图3为实施例1的提取物随着时间梯度及浓度梯度对人雌激素受体阳性乳腺癌细胞MCF7的雌激素受体蛋白水平的影响；
图4为实施例4使用MG132(蛋白酶体途径抑制剂)，BAF-A1(溶酶体途径抑制剂)分别抑制蛋白降解相关途径后，实施例1的雌激素受体降解剂在不同时间梯度对人及鼠的雌激素受体阳性乳腺癌细胞MCF7、T47D和SSM2的雌激素受体蛋白水平的影响。
图5为实施例5中SSM2-129小鼠同种移植瘤模型的肿瘤实拍结果；
图6为实施例5中SSM2-129小鼠同种移植瘤模型的肿瘤体积统计结果；
图7为实施例5中SSM2-129小鼠同种移植瘤模型的肿瘤重量统计结果；
图8为实施例5中SSM2-129小鼠同种移植瘤模型的肿瘤雌激素受体蛋白水平影响。
图9为实施例1的提取物3-1的高效液相色谱图和主要化学成分。
高效液相色谱分析条件为：高效液相色谱分析条件为：流动相为甲醇-水，从10％甲醇5min梯度洗脱到60％甲醇，从60％甲醇20min梯度洗脱到80％甲醇，从80％甲醇2min梯度洗脱到100％甲醇，100％甲醇洗脱3min；检测波长254nm；流速1mL/min；进样量10uL；色谱柱为InertSustainAQ‐C18，5μm，4.6×250mm(UP)；色谱仪为岛津LC‐2030C 3D Plus。
具体实施方式
本发明提供了一种柑橘属果实提取物，所述提取物主要成分为多甲氧基黄酮；
所述多甲氧基黄酮包括5,7,8,4',5'-五甲氧基黄酮、5,6,7,4',5'-五甲氧基黄酮、5,6,7,8,3',4'-六甲氧基黄酮、3,5,6,7,8,3',4'-七甲氧基黄酮和5,6,7,8,4'-五甲氧基黄酮中的一种或多种；优选为5,7,8,4',5'-五甲氧基黄酮、5,6,7,4',5'-五甲氧基黄酮、5,6,7,8,3',4'-六甲氧基黄酮、3,5,6,7,8,3',4'-七甲氧基黄酮和5,6,7,8,4'-五甲氧基黄酮。
本发明还提供了所述的柑橘属果实提取物的制备方法，包括如下步骤：
(1)将柑橘属果实与水混合后粉碎，一次离心去渣，得物料1，将物料1与盐酸混合，静置冷却至室温，二次离心，收集沉淀，为物料2；
(2)将步骤(1)所得物料2用水洗涤，三次离心得沉淀，加入无水乙醇浸提2～4次，合并浸提液，减压浓缩，干燥，得柑橘属果实提取物；优选为3次。
在本发明中，步骤(1)所述柑橘属果实与水混合比例为8～12g：1mL；优选为9～11g：1mL；进一步优选为10g：1mL。
在本发明中，步骤(1)所述一次离心和二次离心的转速独立为8000～12000rpm，时间独立为8～12min；优选转速独立为9000～11000rpm，时间独立为9～11min；进一步优选为转速独立为10000rpm，时间独立为10min。
在本发明中，步骤(1)所述物料1与盐酸的体积比为1：1.5～2.5；优选为1：2。
在本发明中，步骤(1)所述静置的时间为12～16h；优选为13～15h；进一步优选为14h。
在本发明中，步骤(2)所述物料2与水的质量体积比为1g：8～12mL，洗涤3次；优选为1g：9～11mL；进一步优选为1g：10mL。
所述三次离心的转速为8000～12000rpm，时间为8～12min；优选转速为9000～11000rpm，时间为9～11min；进一步优选为转速为10000rpm，时间为10min。
在本发明中，步骤(2)每次浸提，所述沉淀与无水乙醇的质量体积比为1g：1.5～2.5mL；优选为1g：2mL。
在本发明中，步骤(2)所述浸提的温度为22～28℃；优选为23～27℃；进一步优选为24～26℃；更优选为25℃。
在本发明中，步骤(2)所述浸提的时间为12～18min；优选为13～17min；进一步优选为14～16min；更优选为15min。
在本发明中，所述柑橘属果实包括橘、橙或柑；优选为橘。
在本发明中，所述柑橘属果实包括全果、果皮或果肉；优选为全果。
本发明还进一步提供了所述的柑橘属果实提取物，所述的柑橘属果实提取物的制备方法制备得到的柑橘属果实提取物在制备治疗乳腺癌药物中的应用。
在本发明中，所述药物为雌激素受体降解剂。
在本发明中，所述乳腺癌为雌激素受体阳性乳腺癌。
在本发明中，所述乳腺癌模型为MCF7和SSM2细胞模型及SSM2-129小鼠荷瘤模型。
在本发明中，所述模型中选择的检测物质为雌激素受体，具体表述为通过泛素-蛋白酶体途径降解雌激素受体。
在本发明中，使用MCF7、SSM2、T47D相关雌激素受体阳性乳腺癌细胞株作为模型细胞。
在本发明中，使用129品系小鼠，右侧第四乳垫同种移植SSM2细胞株作为模型动物。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
本实施例提供了一种柑橘属果实提取物制备方法，包括以下步骤：
取新鲜成熟夏橙，洗净晾干称重178.8g，加入18mL自来水粉碎，10000rpm离心10min去渣，得100mL液体，加入200mL浓度为1mol/L的盐酸溶液，80℃，酸解14h，静置冷却至室温，在10000rpm条件下离心10min，收集沉淀，将所述沉淀用水洗涤3次，离心，按1g：2mL配比往沉淀中加入无水乙醇5mL，浸提3次，每次浸提15min，合并浸提液，减压浓缩，干燥，称重得到189.6mg柑橘果实提取物，命名为3-1。该提取物主要包括以下成分：5,7,8,4',5'-五甲氧基黄酮、5,6,7,4',5'-五甲氧基黄酮、5,6,7,8,3',4'-六甲氧基黄酮、3,5,6,7,8,3',4'-七甲氧基黄酮和5,6,7,8,4'-五甲氧基黄酮。
实施例2
本实施案例1中的提取物与与人雌激素受体阳性乳腺癌细胞共孵育MCF7、SSM2，均能显著抑制肿瘤细胞生长。现以典型人乳腺癌MCF7细胞抑制活性为例说明如下。
将实施例1中的提取物与人雌激素受体阳性乳腺癌细胞MCF7细胞共孵育72小时，具体方法如下：将雌激素受体阳性乳腺癌细胞MCF7细胞接种在于DMEM培养基中，DMEM培养基在加入10％胎牛血清的同时，加入100units/mL青霉素和100μg/mL链霉素，置于37℃、5％CO2培养箱中培养。 以每孔5000个细胞的密度接种到96孔板过夜等待细胞贴壁完全，然后分别加入雌激素受体降解剂与完全贴壁细胞共孵育72h。以不加药物分别作为空白对照组，以加入内分泌治疗药物他莫昔芬分别作为阳性对照组，将各个组共孵育后的细胞使用结晶紫溶液染色活细胞，染色结果如图1所示。由图1可知，雌激素受体降解剂与人雌激素受体阳性乳腺癌细胞MCF7共孵育，均能显著抑制肿瘤细胞生长。
将实施例1中的提取物与人雌激素受体阳性乳腺癌细胞MCF7细胞共孵育后的结晶紫活细胞染色结果进行量化，采用柠檬酸三钠溶液置于室温摇床进行洗脱溶解一个小时，在570nm的吸收波长测定吸光度。测定结果如图2所示。由图2可知，雌激素受体降解剂与人雌激素受体阳性乳腺癌细胞MCF7细胞半数抑制浓度IC50值为0.06mg/mL。
实施例3
本实施案例中多种以实施例1中的提取物对雌激素受体阳性乳腺癌细胞株MCF7相关的雌激素受体的蛋白水平影响，雌激素受体降解剂与人雌激素受体阳性乳腺癌细胞共孵育MCF7、SSM2，均能显著下调其雌激素受体的蛋白水平。现以典型人乳腺癌MCF7细胞下调其雌激素受体的蛋白水平为例说明如下。
将实施例1中的提取物分别与人雌激素受体阳性乳腺癌细胞MCF7细胞共孵育24、48、72小时后提取蛋白，通过Western blot检测雌激素受体的蛋白水平，具体方法如下：取处于对数生长期的雌激素受体阳性乳腺癌细胞MCF7细胞，使用0.125％胰蛋白酶消化后，将其接种在于含有DMEM培养基的6孔培养板中，DMEM培养基在加入10％胎牛血清的同时，加入100units/mL青霉素和100μg/mL链霉素，置于37℃、5％CO2培养箱中培养。以每孔2×105个细胞的密度接种到96孔板培养板过夜等待细胞贴壁完全，然后分别加入雌激素受体降解剂与完全贴壁细胞共孵育24、48、72小时作为时间梯度。以不加药物分别作为空白对照组，依据实施案例2中所得药物对应的半数抑制浓度的二分之一倍、一倍、两倍的提取物浓度作为浓度梯度。完全贴壁细胞共孵育24、48、72小时后，弃去上清，使用4℃预冷的PBS清洗，加入适量的RIPA细胞裂解液，再使用细胞刮刀刮取细胞并吸取含有细胞碎片的裂解液，至于冰浴30分钟后，4℃离心，12000rpm，30分钟，取上清为全细胞蛋白提取样品。
Western blot具体方法如下：配10％分离胶和5％的浓缩胶，配置SDS-PAGE胶。将细胞样本提取蛋白，用BCA试剂盒(BCA Protein Assay Reagent Kit)蛋白定量，制备上样样品并经100℃高温变性10min。然后按照20μg蛋白总量上样，进行SDS-PAGE电泳(恒压120V)，时间约为2h。电泳完毕后剥下凝胶，切去浓缩胶部分。将NC膜、滤纸和凝胶置于转膜液中进行组装。在电转板上由黑面(负极)至红面(正极)依次叠放：海绵垫、3张滤纸、凝胶、NC膜、3张滤纸、最后是海绵垫。按顺序安装好后插入电转槽，倒入新配制的转膜液，插上电极，在稳流200mA条件下转膜90min，由此将凝胶中的蛋白质转移至NC膜上。电转结束后，将NC膜取出，置于TBST溶液中进行短暂漂洗。用5％脱脂牛奶(TBST配制)于室温摇床上轻摇封闭NC膜1h，再用TBST洗3次，每次5min。在4℃层析柜摇床上一抗(兔源)孵育过夜。用TBST洗膜，5min/次，共洗3次。将稀释的、HRP标记的羊抗兔IgG与NC膜共孵育，室温摇床孵育1h。用TBST洗膜，5min/次，共洗3次。将显影液A和B以1:1混合，取出NC膜，将混合液均匀滴加到膜上，凝胶成像系统拍照并保存结果。Western blot显影结果如图3所示。由图3可知，雌激素受体降解剂随浓度和时间依赖的下调人雌激素受体阳性乳腺癌细胞MCF7中雌激素受体的蛋白水平。
实施例4
本实施案例中，进行实施例1中提取物下调雌激素受体阳性乳腺癌细胞株MCF7、T47D、SSM2雌激素受体的蛋白水平的相关机制进行进一步的探索。现以典型人乳腺癌MCF7细胞为例说明如下。利用MG132(蛋白酶体途径抑制剂)，BAF-A1(溶酶体途径抑制剂)分别抑制蛋白降解相关途径后，提取物对雌激素受体蛋白水平的影响，结果如图4所示。利用BAF-A1(溶酶体途径抑制剂)抑制蛋白降解的溶酶体途径后，提取物仍然能在不同时间梯度下调雌激素受体蛋白水平。而在利用MG132(蛋白酶体途径抑制剂)抑制蛋白降解的蛋白酶体途径后，提取物对其雌激素受体蛋白水平的下调作用有了明显回升。结果如图4所示，提取物对雌激素受体阳性乳腺癌细胞株雌激素受体的下调水平是通过蛋白酶体途径促进其降解，并非通过溶酶体途径。具体方法，同实施案例3。
实施例5
本实施案例中通过SSM2-129乳腺癌荷瘤小鼠模型的药效学评价实施例1中的提取物(3-1)对雌激素受体阳性乳腺癌的肿瘤抑制率，其对肿瘤组织的雌激素受体表达的影响。实施例1中的提取物在给药处理10天后对雌激素受体阳性乳腺癌的肿瘤抑制率约为50％，表明对雌激素受体阳性乳腺癌具有显著的抑制效果，且对重要脏器无明显损伤，同时也可明显下调其雌激素受体的蛋白水平。
肿瘤小鼠模型建立：将人雌激素受体阳性乳腺癌细胞SSM2细胞以1×106个每只小鼠的密度接种于同种129小鼠(18～20g)右侧第四乳垫处。具体方法如下：将处于对数生长期的SSM2细胞，使用0.125％胰蛋白酶进行消化并进行细胞计数后离心，使用PBS将细胞进行重悬并稀释至所需密度。将SSM2细胞悬液以1×106个每只小鼠的密度接种于129小鼠右侧第四乳垫处。2周可观察到直径约0.5cm的皮下肿瘤结节，表明肿瘤小鼠模型建立成功。
分组与给药处理：根据处理方式不同将小鼠分为4组，每组5只，并于肿瘤接种后2周开始给药试验。以给药不加药物的溶剂作为溶剂对照组，以给药实施例1中的提取物浓度选择为25mg/kg、50mg/kg、100mg/kg作为用药治疗实验组。在开始给药后，第0，1，3，5，7，9，11天时记录小鼠体重，及使用游标卡尺测量记录肿瘤体积并计算抑瘤率，结果如图6所示。处死后称量肿瘤重量，结果如图5、7所示。取各组小鼠肿瘤、重要脏器(心、肝、脾、肺、肾)用组织固定液进行固定后石蜡切片，进行H&E染色以观察药物对小鼠各重要脏器的影响。此外，用Western blot检测肿瘤组织中雌激素受体的蛋白水平，结果如图8所示，经药物处理组的小鼠SSM2肿瘤组织的雌激素受体表达水平下降，在50mg/kg给药剂量时下降明显。图5-8所示，中、高浓度的提取物可显著抑制SSM2-129小鼠同种移植瘤模型的肿瘤生长且对重要脏器无明显损伤，显著降低其肿瘤部位的雌激素受体表达水平。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种柑橘属果实提取物，其特征在于，所述提取物主要成分为多甲氧基黄酮；
    所述多甲氧基黄酮包括5,7,8,4',5'-五甲氧基黄酮、5,6,7,4',5'-五甲氧基黄酮、5,6,7,8,3',4'-六甲氧基黄酮、3,5,6,7,8,3',4'-七甲氧基黄酮和5,6,7,8,4'-五甲氧基黄酮中的一种或多种。
  2. 权利要求1所述的柑橘属果实提取物的制备方法，包括如下步骤：
    (1)将柑橘属果实与水混合后粉碎，一次离心去渣，得物料1，将物料1与盐酸混合，静置冷却至室温，二次离心，收集沉淀，为物料2；
    (2)将步骤(1)所得物料2用水洗涤，三次离心得沉淀，加入无水乙醇浸提2～4次，合并浸提液，减压浓缩，干燥，得柑橘属果实提取物。
  3. 根据权利要求2所述的方法，其特征在于，步骤(1)所述柑橘属果实与水混合比例为8～12g：1mL；所述一次离心和二次离心的转速独立为8000～12000rpm，时间独立为8～12min。
  4. 根据权利要求2所述的方法，其特征在于，步骤(1)所述物料1与盐酸的体积比为1：1.5～2.5；所述静置的时间为12～16h。
  5. 根据权利要求2所述的方法，其特征在于，步骤(2)所述物料2与水的质量体积比为1g：8～12mL，洗涤3次；所述三次离心的转速为8000～12000rpm，时间为8～12min；每次浸提，所述沉淀与无水乙醇的质量体积比为1g：1.5～2.5mL，所述浸提的温度为22～28℃，浸提的时间为12～18min。
  6. 根据权利要求2所述的方法，其特征在于，所述柑橘属果实包括橘、橙或柑。
  7. 根据权利要求2所述的方法，其特征在于，所述柑橘属果实包括全果、果皮或果肉。
  8. 权利要求1所述的柑橘属果实提取物，权利要求2～7任一项所述的柑橘属果实提取物的制备方法制备得到的柑橘属果实提取物在制备治疗乳腺癌药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述药物为雌激素受体降解剂。
  10. 根据权利要求8或9所述的应用，其特征在于，所述乳腺癌为雌激素受体阳性乳腺癌。
PCT/CN2023/128344 2022-11-29 2023-10-31 一种柑橘属果实提取物及其制备方法与应用 WO2024114248A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211509125.8 2022-11-29
CN202211509125.8A CN115772145A (zh) 2022-11-29 2022-11-29 一种柑橘属果实提取物及其制备方法与应用

Publications (1)

Publication Number Publication Date
WO2024114248A1 true WO2024114248A1 (zh) 2024-06-06

Family

ID=85390554

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/128344 WO2024114248A1 (zh) 2022-11-29 2023-10-31 一种柑橘属果实提取物及其制备方法与应用

Country Status (2)

Country Link
CN (1) CN115772145A (zh)
WO (1) WO2024114248A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115772145A (zh) * 2022-11-29 2023-03-10 三峡大学 一种柑橘属果实提取物及其制备方法与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021137A1 (en) * 1999-09-21 2001-03-29 Rutgers, The State University Extracts of orange peel for prevention and treatment of cancer
CN114645069A (zh) * 2022-03-28 2022-06-21 三峡大学 一种多甲氧基黄酮及其全水相制备方法与应用
CN115772145A (zh) * 2022-11-29 2023-03-10 三峡大学 一种柑橘属果实提取物及其制备方法与应用

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101947215A (zh) * 2010-06-21 2011-01-19 澳门大学 陈皮中黄酮类化合物在制备抑制血管新生药物中的应用
CN102344429B (zh) * 2011-07-22 2013-11-20 浙江省农业科学院 柑桔多甲氧基黄酮的提取及纯化方法
CN102875509B (zh) * 2012-11-02 2014-09-17 陕西慧科植物开发有限公司 一种从柑橘类果皮中提取高纯度川陈皮素的方法
KR101605122B1 (ko) * 2015-12-16 2016-03-23 명지대학교 산학협력단 감귤류에서 노빌레틴 및 탄제레틴 함량이 높은 추출물을 추출하는 방법과 추출물의 용도
KR20190125041A (ko) * 2018-04-27 2019-11-06 건국대학교 글로컬산학협력단 노빌레틴을 유효성분으로 포함하는 유방암 예방 및 억제 약학 조성물
TW202017583A (zh) * 2018-11-05 2020-05-16 大江生醫股份有限公司 柑橘幼果的萃取物及其用途
CN113801084B (zh) * 2021-10-14 2023-11-28 三峡大学 一种橘醋发酵底泥提取的多甲氧基黄酮及提取方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021137A1 (en) * 1999-09-21 2001-03-29 Rutgers, The State University Extracts of orange peel for prevention and treatment of cancer
CN114645069A (zh) * 2022-03-28 2022-06-21 三峡大学 一种多甲氧基黄酮及其全水相制备方法与应用
CN115772145A (zh) * 2022-11-29 2023-03-10 三峡大学 一种柑橘属果实提取物及其制备方法与应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MANTHEY JOHN A, GUTHRIE NAJLA: "Antiproliferative Activities of Citrus Flavonoids against Six Human Cancer Cell Lines", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 50, no. 21, 1 October 2002 (2002-10-01), US , pages 5837 - 5843, XP093176500, ISSN: 0021-8561, DOI: 10.1021/jf020121d *
QIZHEN DU ET AL: "The methoxyflavones in Citrus reticulata Blanco cv. Ponkan and their antiproliferative activity against cancer cells", FOOD CHEMISTRY, vol. 119, 24 June 2009 (2009-06-24), pages 567 - 572, XP026709140, DOI: 10.1016/j.foodchem.2009.06.059 *
SERGEEV IGOR N, HO CHI‐TANG; LI SHIMING; COLBY JULIE; DUSHENKOV SLAVIK: "Apoptosis‐inducing activity of hydroxylated polymethoxyflavones and polymethoxyflavones from orange peel in human breast cancer cells", MOLECULAR NUTRITION & FOOD RESEARCH, WILEY - VCH VERLAG, WEINHEIM, DE, vol. 51, no. 12, 1 December 2007 (2007-12-01), DE , pages 1478 - 1484, XP093176503, ISSN: 1613-4125, DOI: 10.1002/mnfr.200700136 *

Also Published As

Publication number Publication date
CN115772145A (zh) 2023-03-10

Similar Documents

Publication Publication Date Title
Randriamboavonjy et al. Cardiac protective effects of Moringa oleifera seeds in spontaneous hypertensive rats
WO2024114248A1 (zh) 一种柑橘属果实提取物及其制备方法与应用
CN103222988B (zh) 一种美洲大蠊提取物及其制备方法和应用
CN103687606B (zh) 特定草药制剂的治疗性组合物及其用途
JP2014087364A (ja) 食用アピオス花、食品素材、血糖値上昇抑制効果具有物、血糖値上昇抑制物質およびアピオス花の使用方法
Katsuki et al. Aged garlic extract has chemopreventative effects on 1, 2-dimethylhydrazine-induced colon tumors in rats
CN102281888A (zh) Gip升高抑制剂
JP5622222B2 (ja) 血脂降下組成物及びその使用
Jin et al. Study the mechanism of peimisine derivatives on NF‐κB inflammation pathway on mice with acute lung injury induced by lipopolysaccharide
Yeh et al. Identification of scoparone from Chinese olive fruit as a modulator of macrophage polarization
CN110327399A (zh) 裸花紫珠提取物在制备降脂药物中的应用
CN101653447A (zh) 三七中原人参二醇型皂苷在制备抗动脉粥样硬化药物中的应用
Cui et al. 3-O-Acetyloleanolic acid exhibits anti-angiogenic effects and induces apoptosis in human umbilical vein endothelial cells
Lassila et al. Alternative options to manage menopausal symptoms with a focus on melatonin and osteoporosis
KR20070068837A (ko) 영실로부터 얻은 지방세포분화 억제용 활성 분획 조성물
EP2246061B1 (en) Brachystemma calycinum extract for osteoarthritis
CN115813991A (zh) 一种柑橘属果实提取物在制备治疗乳腺癌内分泌药物中的应用
Jin et al. Exploration of phytochemicals and biological functions of Kadsura coccinea pericarpium based on LC-MS and network pharmacology analysis and experimental validation
KR20030075947A (ko) 신 혈관형성을 억제하는 물질
KR101781186B1 (ko) 유산균(Lactobacillus rhamnosus HK-9) 발효 길경추출물을 유효성분으로 포함하는 비만 개선 및 치료용 조성물
Ren et al. Identification and mechanism elucidation of medicative diet for food therapy XQCSY in NAFLD prevention: an integrative in silico study
Zhang et al. Ginseng Soluble Dietary Fiber Enhances Spermatogenic Potential in Obese Mice via the MAPK Signaling Pathway
CN115969838A (zh) 一种天然组合物作为雌激素受体降解剂的应用
Osman et al. Impact of Methanolic Extract of Pomegranate (Punica granatum L.) Seeds on Serum Biomarkers in Wistar Rats Fed High Cholesterol and Fructose Diet
CN118542864A (zh) 一种缓解动脉粥样硬化的肌肽组合物及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23896402

Country of ref document: EP

Kind code of ref document: A1