阅读说明：本技术 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 (Polymethoxyflavone extracted from orange vinegar fermentation substrate sludge, and extraction method and application thereof ) 是由 邓张双 胡飞飞 秦烨 杜姝 王应喜 于 2021-10-14 设计创作，主要内容包括：本发明属于制药技术领域,公开了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。本发明将橘醋发酵底泥利用有机溶剂浸提,离心,收集有机相,将固相重复浸提2～6次,合并有机相,减压浓缩得到多甲氧基黄酮。该方法成本低廉、工艺简单、经济环保,富集程度高,便于后续针对单体成分纯化,避免了橘醋陈酿过程中发酵底泥作为废弃物丢弃对环境造成的污染,也避免了以果实直接提取带来的工序繁杂、高成本、高有机溶剂使用等弊端。本发明的方法能从橘醋发酵底泥中有效剥离促肿瘤细胞生长的成分,而保留显著抑制肿瘤细胞生长的成分,即多甲氧基黄酮。(The invention belongs to the technical field of pharmacy, and discloses polymethoxylated flavone extracted from orange vinegar fermentation substrate sludge, and an extraction method and application thereof. According to the invention, the orange vinegar fermentation substrate sludge is extracted by using an organic solvent, centrifuged, an organic phase is collected, a solid phase is repeatedly extracted for 2-6 times, the organic phases are combined, and the polymethoxyflavone is obtained by decompression and concentration. The method has the advantages of low cost, simple process, economy, environmental protection and high enrichment degree, is convenient for subsequent monomer component purification, avoids the environmental pollution caused by discarding the fermentation substrate sludge as waste in the aging process of the orange vinegar, and also avoids the defects of complicated working procedures, high cost, high organic solvent use and the like caused by direct extraction of fruits. The method can effectively strip the components promoting the growth of the tumor cells from the orange vinegar fermentation substrate sludge, and retain the components obviously inhibiting the growth of the tumor cells, namely the polymethoxyflavone.)

1. A method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge is characterized by comprising the following steps:

extracting and centrifuging the orange vinegar fermentation substrate sludge by using an organic solvent, collecting an organic phase, repeatedly extracting a solid phase for 2-6 times, combining the organic phases, and concentrating under reduced pressure to obtain polymethoxyflavone.

2. The method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge according to claim 1, wherein the mass volume ratio of the orange vinegar fermentation substrate sludge to the organic solvent is 1 g: 1-5 mL.

3. The method for extracting polymethoxylated flavones from the fermented sediment of orange vinegar according to claim 1 or 2, wherein said organic solvent is alcohol solvent, ethyl acetate or acetone.

4. The method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge according to claim 3, wherein the step of immersing the orange vinegar fermentation substrate sludge in an organic solvent further comprises washing the orange vinegar fermentation substrate sludge with water and centrifuging.

5. The method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge according to claim 1, 2 or 4, wherein the leaching temperature is 20-70 ℃ and the leaching time is 3-48 h.

6. The method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge according to claim 5, wherein the reduced pressure concentration time is 3-4 h, and the temperature is 50-60 ℃.

7. The polymethoxyflavone prepared by the method for extracting polymethoxyflavone from orange vinegar fermentation bottom mud according to any one of claims 1 to 6, wherein the polymethoxyflavone comprises one or more of 5,7,4' -trihydroxyflavone, 5,7,3' -trihydroxy-4 ' -methoxyflavone, 3,5,7,4' -tetrahydroxy-8, 3' -dimethoxyflavone, 5,7,8,4' -tetramethoxyflavone, 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone.

8. The use of polymethoxylated flavone as claimed in claim 7, wherein one or more of polymethoxylated flavone as claimed in claim 7 is used for preparing food, health product or medicine for treating tumor diseases.

9. The use of claim 8, wherein the neoplastic disease comprises breast cancer, lung cancer, colon cancer, prostate cancer, melanoma or liver cancer.

Technical Field

The invention relates to the technical field of pharmacy, in particular to polymethoxylated flavone extracted from orange vinegar fermentation substrate sludge, an extraction method and application.

Background

The fruit and peel of Citrus plant of Rutaceae are rich in flavonoids, wherein polymethoxyflavone is specific to the plant, and has flavone mother-nucleus structure, and hydroxyl on benzene ring is methylated, and has remarkable physiological activities of resisting inflammation, resisting virus, protecting acute liver injury, protecting nerve, inhibiting platelet function, reducing thrombosis, treating diabetic complication, reducing blood lipid and blood sugar, and resisting atherosclerosis, such as Nobiletin (Nobiletin), Tangeretin (tageretin), etc. The substances have low content and small polarity in plants, are difficult to dissolve in water, have complex enrichment and purification procedures and high treatment cost, and are limited in development and application at present. For example, CN108727324A discloses a method for extracting polymethoxylated flavones from citrus peel, which uses an organic solvent to extract, and then the crude extract is subjected to enrichment, impurity removal and desorption by a chromatographic column to obtain a mixture of polymethoxylated flavones, which has complicated process and increased treatment cost. For another example, in the prior art CN102875509A, citrus peel is extracted at 70-85 ℃ after being crushed, and then steps such as pH adjustment and degreasing are required, so that the purification process of polymethoxylated flavone is complicated, and is not beneficial to industrial application.

The orange vinegar is prepared by taking fruit juice or pericarp water extract of Citrus plant of Rutaceae as matrix, performing secondary fermentation, and aging and refining in the way of 'sun drying in summer and ice removing in winter'. In the aging process, sediment substances in the orange vinegar gradually settle to cause the accumulation of an aging tank to generate a large amount of tank bottom mud, and the tank bottom mud is generally discarded after the orange vinegar is recovered, so that resource waste and environmental pollution are caused. Moreover, in the prior art, the research on resource utilization of the orange vinegar fermentation substrate sludge is less.

Therefore, aiming at the two problems, how to utilize the orange vinegar fermentation substrate sludge as resources is developed, and the method for extracting the polymethoxylated flavone from the orange vinegar fermentation substrate sludge, which has the advantages of low cost, simple process, economy and environmental protection, is suitable for industrial production, and has important research value.

Disclosure of Invention

The invention aims to provide polymethoxylated flavone extracted from orange vinegar fermentation substrate sludge, an extraction method and application, and solves the defects that the prior art has low resource utilization degree of the fermentation substrate sludge in the orange vinegar aging process, the enrichment, purification and development processes of the polymethoxylated flavone of a rue citrus plant are complicated, the cost is high, the development and application prospects are limited, and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge, which comprises the following steps:

extracting the orange vinegar fermentation substrate sludge by using an organic solvent, centrifuging, collecting an organic phase, repeatedly extracting a solid phase for 2-6 times, combining the organic phases, and concentrating under reduced pressure to obtain polymethoxyflavone.

Preferably, in the method for extracting polymethoxylated flavones from the orange vinegar fermentation substrate sludge, the mass volume ratio of the orange vinegar fermentation substrate sludge to the organic solvent is 1 g: 1-5 mL.

Preferably, in the method for extracting polymethoxylated flavones from the orange vinegar fermentation substrate sludge, the organic solvent is an alcohol solvent, ethyl acetate or acetone.

Preferably, in the method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge, the leaching temperature is 20-70 ℃ and the leaching time is 3-48 h.

Preferably, in the method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge, the reduced pressure concentration time is 3-4 h, and the temperature is 50-60 ℃.

Preferably, in the method for extracting polymethoxylated flavones from the orange vinegar fermentation substrate sludge, the step of immersing the orange vinegar fermentation substrate sludge in an organic solvent further comprises the steps of washing the orange vinegar fermentation substrate sludge with water and centrifuging.

The invention also provides polymethoxyflavone prepared by the method for extracting polymethoxyflavone from the orange vinegar fermentation substrate sludge, wherein the polymethoxyflavone comprises one or more of 5,7,4' -trihydroxyflavone, 5,7,3' -trihydroxy-4 ' -methoxyflavone, 3,5,7,4' -tetrahydroxy-8, 3' -dimethoxyflavone, 5,7,8,4' -tetramethoxyflavone, 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone.

The invention also provides application of one or more of the polymethoxylated flavones in preparation of foods, health-care products or medicines for treating tumor diseases.

Preferably, in the above application, the tumor disease includes breast cancer, lung cancer, colon cancer, prostate cancer, melanoma or liver cancer. In the invention, the polymethoxyflavone or the compound combination thereof can induce tumor cells to generate G2/M phase block by inhibiting the polymerization of tumor cell tubulin, thereby causing cell cycle disorder and playing a role in inhibiting the growth of the tumor cells.

Through the technical scheme, compared with the prior art, the invention has the following beneficial effects:

(1) the invention provides a method for extracting polymethoxylated flavone from orange vinegar fermentation substrate sludge, which has the advantages of low cost, simple process, economy, environmental protection and high enrichment degree, is convenient for subsequent monomer component purification, avoids the pollution to the environment caused by discarding the fermentation substrate sludge as waste in the orange vinegar aging process, and also avoids the defects of complicated working procedures, high cost, high organic solvent use and the like caused by the direct extraction of the traditional polymethoxylated flavone from fruits.

(2) By the method, components for promoting the growth of the tumor cells can be effectively stripped from the orange vinegar fermentation substrate sludge, and components for obviously inhibiting the growth of the tumor cells, namely polymethoxyflavone, are reserved.

(3) The polymethoxyflavone prepared by the method belongs to medicine-food homologous components, can be used as an additive component for preparing food, health care products and medicines, is used for preventing and treating tumor-related diseases, and induces tumor cells to generate G2/M phase block by inhibiting polymerization of tumor cell tubulin through polymethoxyflavone or compound combination thereof, so as to cause cell cycle disorder and play a role in inhibiting growth of the tumor cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a full ingredient analysis spectrum of polymethoxylated flavone HPLC of example 1;

FIG. 2 is a high performance liquid chromatography analysis chart of polymethoxylated flavones in examples 1-3;

FIG. 3 shows the result of cell staining after co-incubation of polymethoxyflavone of example 1 with MCF-7 cells of human breast cancer;

FIG. 4 shows the result of cytometric examination of polymethoxylated flavones of example 1 incubated with MCF-7 cells of human breast cancer;

wherein a is a cell microscopic examination result after JCCD-2 treatment; b is the result of the cytological microscopy after JCCD-EXT treatment;

FIG. 5 is the MCF-7 cell growth inhibition curve for human breast cancer of polymethoxyflavone of example 1;

wherein a is a growth inhibition curve of JCCD-1 to MCF-7 cells; b is a growth inhibition curve of JCCD-2 to MCF-7 cells; c is a growth inhibition curve of JCCD-EXT to MCF-7 cells;

FIG. 6 is a cycle analysis of polymethoxyflavone-treated human breast cancer MCF-7 cells of example 1 after propidium iodide (PI staining);

wherein a is MCF-7 cell cycle analysis under JCCD-EXT treatment; b is the ratio analysis of G1 phase, S phase and G2/M phase cells under JCCD-EXT treatment;

FIG. 7 is a graph showing the effect of polymethoxyflavone of example 1 on tubulin and nucleus of human breast cancer MCF-7 cells.

Detailed Description

The invention provides a method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge, which comprises the following steps:

extracting the orange vinegar fermentation substrate sludge by using an organic solvent, centrifuging, collecting an organic phase, repeatedly extracting a solid phase for 2-6 times, combining the organic phases, and concentrating under reduced pressure to obtain polymethoxyflavone.

In the invention, the preparation method of the orange vinegar fermentation substrate sludge comprises the following steps: processing whole citrus, fermenting, making vinegar, filtering, and recovering orange vinegar to obtain orange vinegar fermentation bottom mud.

In the invention, the mass volume ratio of the orange vinegar fermentation substrate sludge to the organic solvent is preferably 1 g: 1-5 mL; more preferably 1 g: 1.5-4 mL; more preferably 1 g: 2.5 mL.

In the present invention, the organic solvent is preferably an alcohol solvent, ethyl acetate or acetone; further preferred is an alcohol solvent; more preferably ethanol or methanol.

In the invention, the leaching time is preferably 3-48 h, and the temperature is preferably 20-70 ℃; further preferably, the leaching time is 6-36 h, and the temperature is 23-50 ℃; more preferably, the leaching time is 12h and the temperature is 25 ℃.

In the invention, the centrifugal speed is preferably 1000-10000 rpm, and the time is preferably 10-120 min; further preferably, the rotating speed is 3500-7500 rpm, and the time is 20-60 min; more preferably, the rotation speed is 5000rpm and the time is 30 min.

In the invention, the time of reduced pressure concentration is preferably 3-4 h, and the temperature is preferably 50-60 ℃; further preferably, the time is 3.2-3.8 h, and the temperature is 52-57 ℃; more preferably, the time is 3.5h and the temperature is 55 ℃.

In the invention, before leaching, the orange vinegar fermentation substrate sludge is washed and centrifuged; preferably, the centrifugal speed is 1000-10000 rpm, and the time is 10-120 min; further preferably, the rotating speed is 3500-7500 rpm, and the time is 20-60 min; more preferably, the rotation speed is 5000rpm and the time is 30 min. The purpose of the water washing of the present invention is to remove part of the organic acids.

The invention also provides polymethoxyflavone prepared by the method for extracting the polymethoxyflavone from the orange vinegar fermentation substrate sludge.

In the present invention, the polymethoxyflavone is preferably one or more of 5,7,4' -trihydroxyflavone, 5,7,3' -trihydroxy-4 ' -methoxyflavone, 3,5,7,4' -tetrahydroxy-8, 3' -dimethoxyflavone, 5,7,8,4' -tetramethoxyflavone, 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone; further preferably one or more of 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone; more preferably a mixture of 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone.

The invention also provides the application of the polymethoxylated flavone in preparing foods, health-care products or medicines for treating tumor diseases.

In the invention, the application of the mixture of 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone in preparing food, health products or medicines for treating tumor diseases is preferred.

In the present invention, the tumor disease is preferably breast cancer, lung cancer, colon cancer, prostate cancer, melanoma or liver cancer. The polymethoxyflavone or its compound combination can inhibit the polymerization of tumor cell tubulin to induce tumor cell to block in G2/M phase, so as to cause cell cycle disorder and inhibit the growth of tumor cell.

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The embodiment provides a method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge, which comprises the following steps:

washing 55g of orange vinegar fermentation substrate sludge with 150mL of water, centrifuging at 5000rpm for 30min, leaching with 110mL of analytically pure methanol at 25 ℃ for 3h, centrifuging at 6000rpm for 35min, collecting an organic phase, repeatedly leaching the solid phase for 3 times, combining the organic phases, and concentrating under reduced pressure to obtain 10g of polymethoxylated flavone.

Example 2

The embodiment provides a method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge, which comprises the following steps:

washing 55g of orange vinegar fermentation substrate sludge with 200mL of water, centrifuging at 7000rpm for 60min, leaching with 55mL of analytically pure ethanol at 50 ℃ for 4h, centrifuging at 8000rpm for 20min, collecting an organic phase, leaching the solid phase for 6 times, combining the organic phases, and concentrating under reduced pressure to obtain 9.7g of polymethoxyflavone.

Example 3

The embodiment provides a method for extracting polymethoxylated flavones from orange vinegar fermentation substrate sludge, which comprises the following steps:

washing 55g of orange vinegar fermentation substrate sludge with 200mL of water, centrifuging at 3000rpm for 80min, leaching with 165mL of analytically pure ethyl acetate at 70 ℃ for 6h, centrifuging at 7000rpm for 100min, collecting an organic phase, repeatedly leaching the solid phase for 4 times, combining the organic phases, and concentrating under reduced pressure to obtain 10.2g of polymethoxyflavone.

Example 4

Separating and identifying components of polymethoxylated flavone:

the polymethoxyflavone of example 1 was subjected to preliminary separation and purification by reverse phase silicA gel column chromatography using Japanese YMC ODS-A reverse phase C-18 packing as A stationary phase and water-methanol as A mobile phase at A flow rate: 6mL/min, and gradient elution is sequentially carried out by using 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% methanol solution in 5 times of column volume. The eluted phases were analyzed by HPLC, the same fractions were combined and concentrated under reduced pressure to give 4 fractions (Fr.1, Fr.8, Fr.10 and Fr.12). The fractions Fr.8, Fr.10 and Fr.12 were separately purified on an Ultimate XB-C18(10 μm, 4.6X 250mm) semi-preparative chromatography column in 100 μ L and the mobile phases were 60%, 72% and 80% aqueous methanol, respectively, at a flow rate of 2 mL/min; detection wavelength: 220. 254 nm. Fractions were collected according to the peak time and purified to give compounds 1(10.4mg), 2(3.5mg), 5(9.7mg), 6(8.9mg) and 7(4.6 mg). Wherein Fr.8-3 and Fr.10-2 were prepared twice to give compounds 3(2.8mg) and 4(2.0mg), respectively. The full component analysis spectrum of the high performance liquid chromatograph is shown in figure 1.

The product after separation and purification is subjected to structure identification by methods such as high resolution mass spectrum, nuclear magnetic resonance spectrum and the like, and the result is 7 compounds. Compound nuclear magnetic data are as follows:

compound 1:

a light yellow powder; molecular formula C₁₅H₁₂O₅.¹H NMR(400MHz,DMSO-d₆,δppm):12.18(1H,dr,s,5-OH),10.31(1H,dr,s,7-OH),9.10(1H,dr,s,4'-OH),7.31(2H,d,J＝8.4Hz,2'-Hand6'-H),6.78(2H,d,J＝8.4Hz,3'-Hand5'-H),5.80(2H,s,6-Hand8-H),5.40(1H,dd,J＝3.2,12.8Hz,2-H),3.21(1H,dd,J＝12.8,17.2Hz,2-H),2.64(1H,dd,J＝3.2,17.2Hz,3-H).¹³C-NMR(100MHz,DMSO-d₆δ ppm 195.5(C-4),168.3(C-7),163.4(C-5),162.7(C-9),157.6(C-4'),128.9(C-1'),128.2(C-2' and C-6'),115.1(C-3' and C-6'),101.1(C-10),96.1(C-6),95.3(C-8),78.2(C-2),41.9(C-3). according to the above data, Compound 1 was identified as 5,7,4' -trihydroxyflavone, naringenin.

Compound 2:

a light yellow powder; molecular formula C₁₆H₁₄O₆。¹H NMR(400MHz,DMSO-d₆,δppm):12.19(1H,s,5-OH),10.32(1H,dr,s,7-OH),9.43(1H,dr,s,3'-OH),6.87(3H,m,H-2',5',6'),5.75(2H,d,J＝6.8Hz,H-6and8),5.37(1H,dd,J＝2.3,12.4Hz,2-H),3.76(3H,s,4'-OCH₃),3.12(1H,dd,J＝12.4,16.8Hz,3-H),2.64(1H,dd,J＝3.2,17.2Hz,3-H).¹³C-NMR(100MHz,DMSO-d₆,δppm):192.7(C-4),163.4(C-7),162.5(C-5),161.8(C-9),147.7(C-4'),146.4(C-3'),131.3(C-1'),117.4(C-6'),113.9(C-2'),111.9(C-5'),100.8(C-10),96.2(C-6),95.5(C-8),77.8(C-2),55.6(7-OCH₃) Compound 2 was identified as 5,7,3 '-trihydroxy-4' -methoxyflavone, hesperetin, according to the above data.

Compound 3:

a light yellow powder; molecular formula C₁₇H₁₄O₈.¹H NMR(400MHz,DMSO-d₆,δppm):12.14(1H,s,5-OH),10.68(1H,s,7-OH),9.82(1H,s,4'-OH),9.54(1H,s,3-OH),7.75(2H,m,2'-H and 6'-H),6.97(1H,d,J＝5'-H),6.27(1H,s,6-H),3.85(6H,3',8-OCH₃).¹³C-NMR(100MHz,DMSO-d₆,δppm):176.5(C-4),156.9(C-7),155.8(C-5),146.9(C-9),149.3(C-3'),148.8(C-4'),147.8(C-2),136.3(C-3),127.8(C-8),122.6(C-1'),122.1(C-6'),116.1(C-5'),111.6(C-2'),103.4(C-10),98.8(C-6),61.3(8-OCH₃),56.0(3'-OCH₃) Compound 3 was identified as 3,5,7,4 '-tetrahydroxy-3', 8-dimethoxyflavone, i.e. limonin, based on nmr data.

Compound 4:

a light yellow powder; molecular formula C₁₉H₁₈O₆。¹H NMR(400MHz,DMSO-d₆,δppm):7.98(2H,d,J＝9.2Hz,2'-Hand6'-H),7.13(2H,d,J＝8.8Hz,3'-Hand5'-H),6.68(2H,d,J＝1.6Hz,3-Hand6-H),3.98(3H,s,7-OCH₃),3.88(3H,s,5-OCH₃),3.85(3H,s,4'-OCH₃),3.84(3H,s,8-OCH₃).¹³C-NMR(100MHz,DMSO-d₆,δppm):175.7(C-4),161.8(C-4'),159.5(C-2),156.2(C-7),155.6(C-5),150.0(C-9),129.9(C-8),27.5(C-2'andC-6'),123.1(C-1'),114.6(C-3'andC-5'),108.0(C-10),106.2(C-3),93.7(C-6),60.9(8-OCH₃),56.3(5-OCH₃),56.2(7-OCH₃),55.4(4'-OCH₃) Compound 4 was identified as 5,7,8,4' -tetramethoxyflavone according to the above data.

Compound 5:

a light yellow powder; molecular formula C₂₁H₂₂O₈。¹H NMR(400MHz,DMSO-d₆,δppm):7.65(1H,dd,J＝2,8.4Hz,H-6'),7.54(1Hd,J＝2,H-2'),7.16(1H,d,J＝8.4,H-5'),6.86(1H,s,H-3),4.03(3H,s,7-OCH₃),3.97(3H,s,5-OCH₃),3.88(3H,s,3'-OCH₃),3.85(3H,s,4'-OCH₃),3.84(3H,s,6-OCH₃),3.78(3H,s,8-OCH₃).¹³C-NMR(100MHz,DMSO-d₆,δppm):56.2(6-OCH₃),56.23(8-OCH₃),61.8(4'-OCH₃),61.9(3'-OCH₃),62.2(7'-OCH₃),62.3(5-OCH₃) 106.8(C-3),109.5(C-2'),112.4(C-5'),114.7(C-10),119.8(C-6'),123.6(C-1'),138.1(C-6),144.0(C-5),147.6(C-9),148.0(C-8),149.5(C-3'),151.4(C-7),152.3(C-4'),160.7(C-2),175.2(C-4). Compound 5 was identified as 5,6,7,8,3',4' -hexamethoxyflavone, i.e., nobiletin, in combination with NMR spectroscopic data.

Compound 6:

a light yellow powder; molecular formula C₂₂H₂₄O₉。¹H NMR(400MHz,DMSO-d₆,δppm):7.71(1H,dd,J＝2,8.4Hz,H-6'),7.65(1H,d,J＝2.4Hz,H-2'),7.19(1H,d,J＝8.8,H-5'),4.02(3H,s,7-OCH₃),3.95(3H,s,5-OCH₃),3.86(3H,s,3'-OCH₃),3.85(3H,s,4'-OCH₃),3.84(3H,s,6-OCH₃),3.81(3H,s,3'-OCH₃),3.79(3H,s,8-OCH₃).¹³C-NMR(100MHz,DMSO-d₆,δppm):55.4(4'-OCH₃),55.6(3'-OCH₃),59.2(6-OCH₃),61.3(8-OCH₃),61.4(3-OCH₃),61.6(7-OCH₃),61.8(5-OCH₃) 110.7(C-2'),111.7(C-5'),114.3(C-10),121.4(C-6'),122.5(C-1'),137.3(C-6),139.9(C-8),143.2(C-3),146.1(C-5),147.2(C-9),148.4(C-3'),150.7(C-2),150.8(C-7),152.3(C-4'),172.2(C-4). Compound 6 was identified as 3,5,6,7,8,3',4' -heptamethoxyflavone according to the above data.

Compound 7:

a light yellow powder; molecular formula C₂₀H₂₀O₇。¹H NMR(400MHz,DMSO-d₆,δppm):8.00(2H,d,J＝8.8Hz,H-2'andH-6'),7.14(2H,d,J＝8.8Hz,H-3'andH-5'),6.77(1H,s,H-3),4.02(3H,s,7-OCH₃),3.99(3H,s,5-OCH₃),3.96(3H,s,4'-OCH₃),3.85(3H,s,6-OCH₃),3.84(3H,s,8-OCH₃).¹³C-NMR(100MHz,DMSO-d₆,δppm):56.0(6-OCH₃),61.8(8-OCH₃),61.9(4'-OCH₃),62.3(7-OCH₃),62.4(5-OCH₃) 106.57(C-3),114.8(C-10),115.1(C-3',5'),123.5(C-1'),128.2(C-2',6'),138.2(C-8),144.0(C-6),147.6(C-9),148.0(C-5),151.4(C-4'),160.8(C-2),162.5(C-7),176.2 (C-4.) Compound 7 was identified as 5,6,7,8,4' -pentamethoxyflavone, i.e. hesperetin, according to the above data.

The polymethoxylated flavones extracted by different organic solvents in examples 1-3 were subjected to high performance liquid chromatography, and the analysis conditions were as follows: the mobile phase is methanol/water, and the gradient elution is carried out from 10% methanol for 40min to 100% methanol, and finally 100% methanol is carried out for 5 min; the detection wavelength is 220 nm; the flow rate is 1 mL/min; the sample volume is 10 mu L; the chromatographic column is Welch Ultimate XB-C18, 5 mu m, 4.6X 250 mm; the chromatograph is a Dyan Ultimate 3000. The HPLC analysis spectrum is shown in FIG. 2. As can be seen from FIG. 2, the extracts of different solvents have different components, and the variety of the extracted products is more abundant when methanol is used for extraction.

Example 5

Evaluation of inhibition of tumor cell growth by polymethoxyflavone:

the cancer cell strain used by the invention comprises human breast cancer MCF-7 cells, human lung cancer A549 cells, human colon cancer HCT116 cells, human prostate cancer DU145 cells, human melanoma SK-MEL-1 cells and human liver cancer HepG2 cells, and the polymethoxyflavone in the embodiment 1 has half inhibition concentration IC on the tumor cells₅₀The value is 0.03 to 0.48. mu.g/. mu.L. The inhibitory activity of MCF-7 cells of a typical human breast cancer is described below by way of example.

The polymethoxyflavone (JCCD-EXT) of example 1 and human breast cancer MCF-7 cells were co-incubated by the following specific method: inoculating MCF-7 cells of human breast cancer into DMEM medium containing 10% fetal calf serum, adding 100units/mL penicillin and 100mg/mL streptomycin, and standing at 37 deg.C and 5% CO₂Culturing in an incubator. The 96-well plates were seeded with 5000 cells per well overnight and then incubated with JCCD-EXT for 72 h. The insoluble fraction (JCCD-2) of the fermented substrate sludge of orange vinegar (JCCD-1) and the fermented substrate sludge of orange vinegar (JCCD-2) of example 1 after methanol extraction were used as control groups, and the cells obtained after 3 groups of co-incubation were stained, and the staining results are shown in FIG. 3. As can be seen from FIG. 3, JCCD-EXT can significantly inhibit the growth of tumor cells by co-incubation with human breast cancer cells MCF-7; JCCD-1 and JCCD-2 did not inhibit tumor cell growth.

The results of the cytological microscopy after the co-incubation of the JCCD-EXT and the JCCD-2 and the human breast cancer MCF-7 cells are shown in figure 4. As can be seen in FIG. 4, a large number of viable tumor cells were observed after JCCD-2 treatment, and no significant viable tumor cells were observed after JCCD-EXT treatment.

And (3) co-incubating JCCD-EXT, JCCD-1 and JCCD-2 with human breast cancer MCF-7 cells, and detecting the proliferation curve of the human breast cancer MCF-7 cells by adopting a tetramethyl azoazolate trace enzyme reaction colorimetric method. The specific method comprises the following steps: cells in logarithmic growth phase are taken, digested by 0.25% trypsin, prepared into cell suspension by RPMI 1640 medium containing 10% fetal bovine serum, inoculated in a 96-well plate and cultured for 24 h. After the sample to be tested is dissolved by DMSO, the sample is diluted by a culture medium to form different concentration gradients. The negative control group is culture medium with equal volume, after culturing for 72h, 10 μ L MTT solution is added, 5% CO at 37 ℃₂And culturing for 4 h. The 96-well plate was removed and the medium supernatant was aspirated off. DMSO was added and shaken for 10min to completely dissolve the product of the MTT reaction. The OD value at 490nm was read with a microplate reader, the results were repeated three times, and the average value was taken. Inhibition rate of cell proliferation ═ a_{Blank space}-A_{Experiment of})/A_{Blank space}X 100%, regression analysis was performed using the concentration of each compound and the cell growth inhibition rate of the corresponding series as variables, and the results are shown in fig. 5. As shown in FIG. 5, JCCD-EXT and human milkThe cancer cell MCF-7 co-incubation can obviously inhibit the growth of the cancer cell, and the half inhibition concentration IC thereof₅₀The value was 0.03. mu.g/. mu.L; JCCD-1 can not inhibit the growth of cancer cells; JCCD-2 can promote the growth of cancer cells.

Cycle analysis and proportion analysis of cells at G1, S and G2/M phases were performed on JCCD-EXT treated human breast cancer cells MCF-7 after propidium iodide (PI staining), and the results are shown in FIG. 6. As can be seen from FIG. 6, the proportion of G2/M cells increased significantly with the increase of the sample concentration, and the proportion of the remaining cycles decreased, and the proportion of G2/M cells was significantly higher than that of the control group without JCCD-EXT treatment. Analysis of the cell ratios at the G1, S and G2/M phases under JCCD-EXT treatment showed that the G2/M cell ratio of the control group was 11.14% for 24h, while the G2/M cell ratio increased to 46.04% (p <0.01) under JCCD-EXT at a concentration of 160G/mL.

After human breast cancer cells MCF-724h were treated with JCCD-EXT, the effect of JCCD-EXT on tubulin and nucleus of MCF-7 cells was determined by tubulin Tracker Green reagent and Hoechst 33258, and the results are shown in FIG. 7. As shown in FIG. 7, the cells of the control group without JCCD-EXT treatment had intact nuclei and uniform chromatin, and exhibited uniform weak blue fluorescence under UV light, whereas some cells exhibited nuclear pyknosis after JCCD-EXT (160. mu.g/L) treatment, small nuclear volume, enhanced blue fluorescence intensity, and loose chromatin structure, which exhibited typical apoptotic morphology.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.