CN110305094B

CN110305094B - Two flavonoid compounds in purslane and extraction and separation method and application thereof

Info

Publication number: CN110305094B
Application number: CN201910640045.8A
Authority: CN
Inventors: 英锡相; 段阳; 张文洁
Original assignee: Liaoning University of Traditional Chinese Medicine
Current assignee: Liaoning University of Traditional Chinese Medicine
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2022-06-17
Anticipated expiration: 2039-07-16
Also published as: CN110305094A

Abstract

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to two new flavonoid compounds extracted, separated and identified from purslane and an extraction and separation method thereof. The molecular formulas of the two new flavone compounds are sequentially C₁₈H₁₆O₅、C₁₈H₁₈O₅The names are 3- (2-hydroxybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroxybenzyl) -6,8-dimethoxychroman-4-one, respectively. Also provides an extraction and separation method of the new compound, which sequentially adopts water decoction extraction, silica gel column chromatography, polyamide column chromatography, ODS medium-pressure column purification, Sephadex LH-20 and recrystallization for separation, purification and preparation. The structure adopts HR-ESI-TOF-MS,¹H‑NMR、¹³Two new flavonoids compounds are identified by analysis methods of C-NMR and two-dimensional nuclear magnetic spectrum. The neoflavone compound has potential activities of anti-inflammation, anti-tumor, antioxidation and the like, and the neoflavone compound and the salt or the derivative thereof can be used as a lead compound for synthesizing other compounds and a raw material for developing new medicines and researching pharmacological activity and can be used for preparing medicines for anti-inflammation, anti-tumor and antioxidation.

Description

Two flavonoid compounds in purslane and extraction and separation method and application thereof

Technical Field

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to a flavonoid compound extracted, separated and identified from a purslane medicinal material and an extraction and separation method thereof.

Background

Herba Portulacae (Portulaca oleracea L.), also called herba Portulacae and herba Portulacae, is a plant of Portulacaceae. Purslane has the advantages of drought and waterlogging resistance, light and yin resistance, wide distribution and rich resources, and is concerned as a wild plant for both medicine and food. 2015, pharmacopoeia of the people's republic of China, which contains dry aerial parts of herba Portulacae, has effects of clearing away heat and toxic materials, cooling blood, stopping bleeding, and stopping dysentery, and can be used for treating toxic heat, bloody dysentery, carbuncle, furuncle, eczema, erysipelas, snake and insect bite, hematochezia, hemorrhoidal bleeding, metrorrhagia, metrostaxis, etc.

Modern pharmacological research of purslane shows that the purslane has the effects of resisting inflammation, relieving pain, resisting bacteria and viruses, reducing blood pressure, reducing blood fat, resisting oxidation and cancers, relaxing skeletal muscles and smooth muscles, regulating immune function and the like. Research shows that numerous chemical components of purslane provide material basis for various pharmacological actions of purslane, and the main chemical components of purslane comprise flavonoids, coumarins, terpenes, steroids, organic acids, volatile oil, alkaloids, amino acids, various pigments, minerals and the like. Wherein alkaloids are a main chemical component in purslane, and the alkaloid components reported at present comprise norepinephrine, dopamine, a small amount of dopa, adenosine, uracil, adenine, N-dicyclohexylurea, allantoin and N-trans-feruloyltyramine; cyclic dipeptide alkaloids and amide alkaloids are also present: oleracein A-I, K, L, N-S.

Most of the chemical components separated from purslane are known and have low structural novelty, so the development and separation of new compounds in purslane are urgently needed.

Disclosure of Invention

In order to solve the problems, the invention provides two novel flavonoid compounds extracted from purslane, and researches show that the two novel compounds have anti-inflammatory, anti-tumor and anti-oxidation effects, and simultaneously provides a simple, convenient, rapid, environment-friendly and high-purity extraction and separation method for the novel compounds.

In order to achieve the above objects, the present invention provides two flavonoid compounds separated from purslane, wherein the molecular formulas are respectively C₁₈H₁₆O₅、C₁₈H₁₈O₅They are respectively named as 3- (2-hydroxybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroxybenzyl) -6, 8-dimethoxychrome-4-one, and the chemical structural formulas are respectively as follows:

in order to achieve the above object, the present invention further provides a method for extracting and separating flavonoid compounds from purslane, comprising the following steps:

step 1: taking a purslane dry medicinal material, extracting twice with water (the water dosage is 8-16 times of the medicinal material), filtering the water extract, combining the filtrates, directly heating and concentrating, and cooling to room temperature to obtain a liquid medicine for later use.

Step 2: evaporating the liquid medicine obtained in the step 1 to dryness, then loading the liquid medicine to a silica gel column, eluting the liquid medicine by using ethyl acetate, and recovering the ethyl acetate to obtain an extract under reduced pressure to obtain an ethyl acetate extract.

And step 3: and (3) separating the ethyl acetate extract obtained in the step (2) by using a polyamide column, performing gradient elution by using ethanol-water, evaporating 50% ethanol part to dryness, then applying to a silica gel column, sequentially performing elution by using ethyl acetate, and recovering the ethyl acetate to obtain an extract to obtain the ethyl acetate extract.

And 4, step 4: and (3) performing chromatographic separation on the product obtained in the step (3) by using a pretreated ODS (octadecylsilane chemically bonded silica) column, performing gradient elution by using methanol-water to obtain a plurality of elution parts, performing detection and color development by using a thin-layer chromatography, and respectively concentrating the elution parts subjected to color development under reduced pressure until the elution parts are dried to obtain a concentrate for later use.

And 5: separating the product obtained in step 4 by pretreated Sephadex LH-20 column chromatography, eluting with methanol to obtain multiple elution parts, detecting by thin layer chromatography, developing, mixing the developed elution parts, and concentrating under reduced pressure to dry.

Step 6: standing the obtained product in the step 5 to precipitate crystals, washing the crystals with methanol for multiple times, volatilizing the methanol, detecting by using thin-layer chromatography, and developing to obtain the two novel flavonoid compounds.

The pretreatment process of the ODS and the sephadex comprises the steps of soaking in methanol for 24 hours, loading on a column, washing with methanol until no turbidity exists in dropping water, and then balancing with an initial mobile phase.

Compared with the prior art, the invention has the beneficial effects that: the separation and pharmacological activity research of the purslane neoflavonoid compounds in the invention is not reported in the current journal of papers. The invention provides two flavonoid compounds derived from purslane and an extraction and separation method aiming at the novel compound, which are used for separation, purification and preparation by sequentially adopting water decoction and extraction, silica gel column chromatography, polyamide column chromatography, ODS medium-pressure column purification, Sephadex LH-20 and recrystallization to successfully extract and separate the two novel flavonoid compounds. The method has the advantages of only six operating steps, simple and quick operation method, environment-friendly process method and high purity of the compound separated by the method, wherein the extraction and separation process mainly adopts water decoction and extraction and ethyl acetate elution, and the purity of the compound is higher than 90%. The research shows that the compounds have the functions of anti-inflammation, anti-tumor and anti-oxidation, so that the novel compounds, the salts and the derivatives thereof can be used as the synthetic leads of other compounds, the raw materials for the development of new drugs and the research of pharmacological activity, and can also be used for preparing anti-inflammation, anti-tumor and anti-oxidation drugs.

Drawings

FIG. 1 is a high-resolution mass spectrum of the novel flavone compound 3- (2-hydroxybenzyl) -6,8-dimethoxy-4H-chromen-4-one of the present invention.

FIG. 2 is a high-resolution mass spectrum of the novel flavone compound 3- (2-hydroxybenzyl) -6, 8-dimethylchromanan-4-one of the present invention.

FIG. 3 shows the synthesis of 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroybenzyl) -6, 8-dimethoxychrome-4-one, which are novel flavonoid compounds of the present invention¹H-NMR spectrum chart.

FIG. 4 shows the synthesis of 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroybenzyl) -6, 8-dimethoxychrome-4-one, which are novel flavonoid compounds of the present invention¹³C-NMR spectrum chart.

FIG. 5 is a nuclear magnetic resonance carbon spectrum (DEPT) spectrum of the novel flavone compounds 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroybenzyl) -6, 8-dimethoxychrome-4-one of the present invention.

FIG. 6 shows NMR spectra of the novel flavone compounds 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroybenzyl) -6, 8-dimethoxychrome-4-one of the present invention¹H-¹HCOSY spectrum.

FIG. 7 is a nuclear magnetic resonance HMBC spectrogram of the novel flavone compounds 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroybenzyl) -6,8-dimethoxychroman-4-one of the present invention.

FIG. 8 is a nuclear magnetic resonance HSQC spectrum of the novel flavone compounds 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroybenzyl) -6,8-dimethoxychroman-4-one of the present invention.

FIG. 9 is a NMR ROESY spectrum of the novel flavone compounds 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroybenzyl) -6,8-dimethoxychroman-4-one of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific embodiments.

The invention provides two new compounds, the molecular formulas of which are respectively C₁₈H₁₆O₅、C₁₈H₁₈O₅They are respectively named as 3- (2-hydroxybenzyl) -6,8-dimethoxy-4H-chromen-4-one and 3- (2-hydroxybenzyl) -6, 8-dimethoxychrome-4-one, and the chemical structural formulas are respectively as follows:

the two new compounds are respectively named as 3- (2-hydroxybenzyl) -6,8-dimethoxy-4H-chromen-4-one, 3- (2-hydroxybenzyl) -6,8-dimethoxychroman-4-one according to the structures, and the tables 1 and 2 respectively represent the nuclear magnetic data of the two flavone compounds:¹H-NMR of¹³C-NMR in CCl₃And D, preparing the intermediate product.

TABLE 1 NMR data on the novel compound 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one of the present invention

Serial number	δ_C	Type (B)	δ_H(J in Hz)
				1		O
2	151.11	CH	7.87，s
				3	125.51	C
4	178.73	C
				5	92.58	CH	6.42，d，(2.2)
6	164.76	C
				6-OMe	55.81	CH₃	3.83，s
7	96.60	CH	6.35，d，(2.2)
				8	161.31	C
8-OMe	56.61	CH₃	3.9，s
				9	160.65	C
10	108.89	C
				11	27.48	CH₂	3.66，s
1′	126.21	C
				2′	155.43	C
2′-OH			8.23，brs
				3′	118.69	CH	6.94，q，(8.1，14.1)
4′	128.56	CH	7.12，m
				5′	120.31	CH	6.82，t，(7.1)
6	130.24	CH	7.04，dd，(0.9，7.1)

3- (2-hydroxybenzyl) -6, 8-dimethoxy-4H-chromen-4-one: white powder, poorly soluble in methanol, and readily soluble in chloroform. After spotting on a silica gel thin layer plate, spraying the test solution of vanillyl phenol green to make the spot light yellow. HRESI (+) TOFMS gives m/z: 313.1071[ M + H]⁺Has an excimer ion peak of 312.0998 molecular weight. Bonding of¹H-NMR，¹³C-NMR and DEPT data, presuming that the possible molecular formula of the compound is C₁₈H₁₆O₅The unsaturation degree was 11.¹³The C-NMR spectrum and DEPT spectrum showed 18 carbon signals, respectively 2 CH₃(delta: 55.81, 56.61), 1 CH₂(delta: 27.48), 7 CH (delta: 92.58, 96.60, 118.69, 120.31, 128.56, 130.24, 151.11), 8 quaternary carbons (one carbonyl carbon, delta: 178.73; four double-bonded carbons to O, delta: 155.43, 160.65, 161.31, 164.76; three double-bonded carbons, delta: 108.89, 125.51, 126.21).

¹The H-NMR spectrum showed 1 active H signal, delta 8.23(1H, brs), indicating the possible presence of a hydroxyl group; 2 methyl signals, δ 3.83(3H, s) and δ 03.93(3H, s); 1 methylene signal, δ 3.66(2H, s); the 7 methine signals are δ 6.35(1H, d, J ═ 2.2), δ 6.42(1H, d, J ═ 2.2), δ 6.82(1H, t, J ═ 7.1), δ 6.94(1H, q, J ═ 8.1, 14.1), δ 7.04(1H, dd, J ═ 0.9, 7.1), δ 7.12(1H, m), δ 7.87(1H, s), respectively. According to¹H-¹H COSY spectrum can show that methylene delta 3.66 is coupled with methine delta 7.87; two methines δ 6.35 and δ 6.42 are coupled; methine groups δ 6.82, δ 7.04, δ 7.12, δ 6.94 are coupled to each other, indicating the presence of a benzene ring. According to HMBC spectrum correlation peaks, H-5 and H-7 are coupled with C-9 and C-10 respectively, and H-5 and H-7 are coupled with each other, which indicates that C-5 is associated with C-7 and C-5 and C-7 are associated with C-9 and C-10 respectively; delta in two methoxy groups_H3.83，δ_H3.93 to C-6, C-8 respectively, and C-6 (delta 164.76), C-8 (delta 161.31) in the low field region, suggesting that O is connected, indicating that two methoxy groups are connected to C-6, C-8 on the benzene ring respectively; two methoxy groups are shown according to related peaks of a ROESY spectrumDelta in the radical_H3.83，δ_H3.93 to H-5, H-7, respectively, indicating that C-6 is associated with C-5 and C-8 is associated with C-7; h-5 is coupled to C-9 and C-9 (delta 160.65) is located in the low field region, indicating that C-5 is associated with C-9 and C-9 is connected to O; meanwhile, H-5 and H-7 are related to C-10, which indicates that C-5 and C-7 are related to C-10; h-2 is coupled to H-11 and H-2, H-11 is coupled to C-3, indicating that C-2, C-11 is associated with C-3, while C-2 (delta 151.11) is located in the low field region, indicating attachment to O, while H-2 is coupled to C-9, indicating attachment to O in the middle of C-2, C-9; h-2, H-5, H-11 is coupled with C-4 carbonyl C, indicating that C-2, C-5, C-11 is associated with C-4. H-3 ', H-4 ', H-5 ' and H-6 ' are coupled to each other, wherein H-3 ', H-5 ', H-6 ' are coupled to C-1 ', H-3 ', H-4 ', H-5 ', H-6 ' are coupled to C-2 ', indicating the presence of a benzene ring and the ortho-substitution of C-1 ' and C-2 '; wherein C-2 '(delta 155.43) is in the low field region, indicating that it is linked to O, indicating that C-2' is linked to a hydroxyl group; h-2, H-11 are coupled to C-1 ', indicating that C-2, C-11 are associated with C-1 ', H-11 is coupled to C-2 ', C-6 ', indicating that C-11 is associated with C-2 ', C-6 ', H-6 ' is coupled to C-1 ', indicating that C-6 ' is associated with C-1 ', summarizing that C-11 is associated with C-1 '. From the above information, the novel compounds can be identified as having the above structure.

TABLE 2 Nuclear magnetic data of 3- (2-hydroxybenzyl) -6,8-dimethoxychroman-4-one, a novel compound of the present invention

3- (2-hydroxybenzyl) -6, 8-dimethylychroman-4-one: white powder, poorly soluble in methanol, and readily soluble in chloroform. After spotting on a silica gel thin layer plate, spraying the test solution of vanillyl phenol green to make the spot light yellow. HRESI (+) TOFMS gives m/z: 315.1224[ M + H]⁺Has an excimer ion peak of 314.1154 molecular weight. Bonding of¹H-NMR，¹³C-NMR and DEPT data, and the conversion was estimatedPossible compounds have the formula C₁₈H₁₈O₅The unsaturation degree was 10.¹³The C-NMR spectrum and DEPT spectrum showed 18 carbon signals, respectively 2 CH₃(delta: 55.97, 56.33), 2 CH₂(delta: 26.85, 70.52), 7 CH (delta: 47.80, 93.14, 93.40, 117.76, 120.43, 128.62, 131.04), 7 quaternary carbons (one carbonyl carbon, delta: 194.09; four double-bonded carbons to O, delta: 155.12, 162.79, 165.56, 166.70; two double-bonded carbons, delta: 105.45, 125.17).

¹The H-NMR spectrum showed 1 active H signal delta 9.65(1H, brs), indicating the possible presence of a hydroxyl group; 2 methyl signals, δ 3.88(6H, s); 2 methylene signals, δ 02.79(1H, q, J ═ 6.6, 16.7), δ 13.06(1H, m); δ 4.17(1H, t, J ═ 11.3), δ 4.53(1H, dd, J ═ 4.9, 11.2); the 7 methine signals are δ 3.04(1H, m), δ 6.05(2H, q, J ═ 2.3, 3.7), δ 6.82(1H, t, J ═ 7.1), δ 6.94(1H, q, J ═ 8.1, 14.1), δ 7.11(1H, m), δ 7.12(1H, m), respectively. According to¹H-¹H COSY spectrum shows that H delta 4.17, delta 4.53, delta 02.79 and delta 13.06 in methylene are respectively coupled with methine delta 3.04; two methines δ 6.05 and δ 6.05 are coupled; methine groups δ 6.82, δ 6.94, δ 7.11, δ 7.12 are coupled to each other, indicating the presence of a benzene ring. The correlation peaks according to the HMBC spectrum show that H-5 and H-7 are respectively coupled with C-9 and C-10, and H-5 and H-7 are mutually coupled, which shows that C-5 is associated with C-7, and C-5 and C-7 are respectively associated with C-9 and C-10; delta in two methoxy groups_H3.88(6H, s) is coupled to C-6, C-8, and C-6 (delta 166.70) and C-8 (delta 162.79) are located in the low field region, indicating that attachment to O indicates that the two methoxy groups are attached to C-6, C-8, respectively, on the phenyl ring; according to the ROESY spectrum, delta in two methoxy groups_H3.88(6H, s) is coupled to H-5, H-7, indicating that C-6, C-8 is associated with C-5, C-7; h-5 is coupled to C-9 and C-9 (delta 160.65) is located in the low field region, indicating that C-5 is associated with C-9 and C-9 is connected to O; h-2, H-3 and H-11 are mutually coupled, and C-2 (delta 70.52) is positioned in a low field region to indicate that the H-2 is connected with O, and meanwhile, the H-2 is coupled with C-9 to indicate that the middle of the C-2 and the C-9 is connected with O; h-2, H-3, H-11 is coupled to C-4 carbonyl C, indicating that C-2, C-3, C-11 is associated with C-4. H-3 ', H-4 ', H-5 ' and H-6 ' are coupled to each other, wherein H-3 ', H-5 ', H-6 ' are coupled to C-1 ', H-3 ', H-4 ', H-5 ', H-6 ' are coupled to C-2 ', indicating the presence of a phenyl ring and ortho-substitution of C-1 ' and C-2 '; wherein C-2 '(delta 155.12) is in the low field region, indicating that it is linked to O, indicating that C-2' is linked to a hydroxyl group; h-2, H-3, H-11 is coupled to C-1 ', indicating that C-2, C-3, C-11 is associated with C-1', H-11 is coupled to C-2 ', C-6', indicating that C-11 is associated with C-2 ', C-6', H-6 'is coupled to C-1', indicating that C-11 is associated with C-1 ', in summary, indicating that C-11 is coupled to C-1'. From the above information, the novel compounds can be identified as having the above structure.

The invention also provides an extraction and separation method of the two flavone compounds, which comprises the following steps:

step 1: weighing 150kg of purslane dry medicinal material, adopting water for reflux extraction, wherein the water dosage (v/v) is 10 times of that of the medicinal material, carrying out reflux extraction twice for 2h each time, heating and concentrating, and cooling to room temperature to obtain liquid medicine for later use.

Step 2: evaporating the liquid medicine obtained in the step 1 to dryness, performing chromatographic separation by using a silica gel column, isocratically eluting by using ethyl acetate (115L), wherein the silica gel is 100-200 meshes, and recovering the ethyl acetate to obtain an extract under reduced pressure below 40 ℃ to obtain an ethyl acetate extract.

And step 3: separating the ethyl acetate extract obtained in the step 2 by using a polyamide column, performing gradient elution by using ethanol-water (0/100, 30/70, 50/50, 70/30, 100/0, v/v), evaporating 50% (volume percentage) ethanol to dryness, and performing chromatographic separation by using a silica gel column, wherein the silica gel is 200-300 meshes, performing gradient elution by using ethyl acetate and ethyl acetate-methanol (5/1, 2/1, 1/2, v/v) in sequence to obtain 19 parts (namely obtaining 19 bottles in total and 300mL in each bottle), detecting by using a thin-layer chromatography, developing, combining the parts obtained by ethyl acetate elution, and concentrating under reduced pressure below 40 ℃ at room temperature until the parts are dry for later use.

And 4, step 4: and (3) performing pretreated ODS (octadecylsilane chemically bonded silica) medium-pressure column chromatography separation on the product obtained in the step (3), wherein the granularity of the filler is 20-40 mu m, performing gradient elution (pressurizing to enable the flow rate to be 1mL/min and the temperature to be room temperature) by using methanol-water (60/40, 70/30, 80/20, 90/10, 100/0, v/v) to obtain 16 parts (namely performing gradient elution to obtain 16 bottles, wherein each bottle is 100mL), detecting by using thin-layer chromatography, developing, retaining 3-5 parts for developing, and concentrating under reduced pressure below 50 ℃ until the parts are dry for later use.

And 5: and (3) separating the product obtained in the step (4) by pretreated Sephadex LH-20 column chromatography, eluting by methanol to obtain 26 elution parts (namely, 26 bottles are obtained in total, and each bottle is 50mL), detecting by thin-layer chromatography, developing, retaining the developed 13 and 14 parts, and concentrating under reduced pressure below 50 ℃ until the parts are dry for later use to obtain a new compound.

Step 6: and (4) standing the substance obtained in the step (5), detecting and developing a light yellow crystal through thin-layer chromatography, washing the crystal with methanol for multiple times, and evaporating the methanol to dryness after the supernatant of the methanol is colorless, thereby finally obtaining the two new flavone compounds.

The pretreatment process of ODS and sephadex is soaking in methanol for 24h, and loading on column to balance with initial mobile phase.

The anti-inflammatory effect experiment of the novel flavone compound is carried out.

1 main material

1.1 drugs and reagents: the two new flavone compounds used in the experiment are prepared by the method, the purity of the two new flavone compounds is 90-99%, the two new flavone compounds are precisely weighed and diluted by DMSO to be the solution required by each dosage group. DMEM high-glucose medium, fetal bovine serum (Hyclone, usa); penicillin, streptomycin (Hangzhou Sijiqing Co.); LPS (Sigma, usa); IL-6, TNF-alpha, PGE₂ELISA kit of (Cayman, USA); cell lysate, Griess reagent (bi yun tian biotechnology limited).

1.2 cell lines: RAW264.7 macrophages (us ATCC cell bank).

1.3 grouping: the test group was divided into a control group, an LPS group and an experimental group.

2 method of experiment

2.1 cell culture: DMEM high-glucose medium, added with 0% fetal calf serum, antibiotics 100U/mL penicillin and 100. mu.g/mL streptomycin, and placed in a medium containing 37.5% CO₂Culturing in an incubator.

2.2MTT colorimetry for determining cell viability: the three groups are respectively inoculated with RAW264.7 macrophage in logarithmic growth phase in 96-well culture plate with cell density of 1 × 10⁴one/mL, 100. mu.L per well, temperature 37 ℃, 5% CO₂After overnight culture under the conditions, the experimental group was added with different concentrations of the two new flavonoid compounds of the present invention, 3- (2-hydroxybenzyl) -6,8-dimethoxy-4H-chromen-4-one (1-100. mu.M) or 3- (2-hydroxybenzyl) -6, 8-dimethoychroman-4-one (1-100. mu.M), after 1H incubation, LPS was added to the LPS group and the experimental group to a final concentration of 1. mu.g/mL, respectively, and a null-adjusting group (culture solution containing DMSO solvent) was provided, each group was provided with 3 duplicate wells, and the effect on cells after addition of the drug was examined. After culturing the above groups of cells for 24 hours, 5mg/mL MTT 20. mu.L was added to each well of cells at 37 ℃ with 5% CO₂After incubation for 4h, terminating the culture, absorbing the liquid in the wells, adding 100 μ L of dimethyl sulfoxide (DMSO) into each well, oscillating for 10min to dissolve the intracellular crystal, and measuring the light absorption value of each well at 570nm wavelength of an enzyme-labeling instrument.

2.3 measurement of NO content by Griess method, the inhibitory action of the novel flavone compounds of the present invention on the NO production of LPS-induced mouse macrophage RAW264.7 was examined. Mouse macrophage RAW264.7 passage, culturing in high glucose cell culture medium DMEM containing 10% fetal bovine serum, adding different concentrations of two new flavone compounds of the present invention, 3- (2-hydroxyphenyl) -6,8-dimethoxy-4H-chromen-4-one (1-50 μ M) or 3- (2-hydroxyphenyl) -6, 8-dimethoxy-4-one (1-50 μ M), at 37 deg.C and 5% CO₂After incubation for 1h under conditions, inflammatory responses were induced with LPS (final concentration 1. mu.g/mL), and after 24h supernatants were collected and 3 wells were repeated for each group. The Griess method measures the content of NO in cell supernatant, and the influence of two neoflavone compounds of the invention on the NO release of LPS-induced RAW264.7 cells is used for reflecting the NO level according to different concentrations.

2.4 measurement of inflammatory factors IL-6, TNF-alpha and inflammatory mediators PGE by ELISA₂: RAW264.7 macrophages in logarithmic growth phase were seeded in 24-well culture plates at a cell density of 1X 10⁵1 mL/mL per well, 37 ℃ temperature, 5% CO₂Culturing overnight under the condition, adding two new xanthones of the invention into experimental groupsThe compound 3- (2-hydroxybenzoyl) -6, 8-dimethy-4H-chromen-4-one (1-50. mu.M) or 3- (2-hydroxybenzoyl) -6, 8-dimethy hroman-4-one (1-50. mu.M) was added to each well after 1 hour of incubation, LPS (final concentration: 1. mu.g/mL) was incubated for 24 hours, and 3 wells were repeated for each treatment. ELISA method for determining IL-6, TNF-alpha and PGE secreted by RAW264.7 macrophage after treatment of two purslane-derived neoflavone compounds₂The content of (a).

3 results of the experiment

The experimental result shows that the two new flavone compounds have no influence on the proliferation of macrophage RAW264.7 induced by LPS, and are safe and nontoxic; and can effectively inhibit excessive inflammatory cytokines IL-6, TNF-alpha and inflammatory mediators NO and PGE generated by macrophage RAW264.7 induced by LPS₂And is concentration dependent.

The results of the cell relative survival experiments are shown in table 3.

TABLE 3 Effect of the invention on the relative survival of RAW264.7 macrophages

Note:^*P<0.05 comparison with control group (significant difference in high concentration group)

The results of the experiments for determining the NO content by the Griess method are shown in Table 4.

Table 4 effect of the invention on LPS-induced NO release from RAW264.7 cells (mean ± sd, n ═ 3)

Note:^*P<0.05 compared with the control group,^#P<0.05 compared to the LPS group.

ELISA method for measuring inflammatory factors IL-6, TNF-alpha and inflammatory mediator PGE₂The results are shown in Table 5.

TABLE 5 IL-6, TNF-alpha and PGE secreted by RAW264.7 cells induced by LPS according to the invention₂Influence of the content (mean. + -. standard deviation, n. about.3)

The new flavone compound has the anti-tumor effect.

1 main material

1.1 drugs and reagents: the two new flavone compounds used in the experiment are prepared by the method, the purity of the two new flavone compounds is 90-99%, the two new flavone compounds are precisely weighed and diluted by DMSO to be the solution required by each dosage group. DMEM high-glucose medium, fetal bovine serum (Hyclone, usa); penicillin and streptomycin (Hangzhou Sijiqing Co., Ltd.).

1.2 cell lines: human colon cancer cells Caco-2, human breast cancer cells MCF-7, human gastric cancer cells BGC-823, human lung adenocarcinoma cells SPC-A1, human liver cancer cells BEL-7402, human cervical cancer cells Hela-229, ovarian cancer cells Ho-8910, and human oral epidermoid carcinoma cells KB (Shanghai cell Bank of Chinese academy of sciences).

1.3 grouping: divided into a control group, an experimental group and a zero-adjustment group (culture solution containing DMSO solvent).

2 method of experiment

2.1 cell culture: DMEM high-sugar medium, added with 0% fetal calf serum, 100% antibiotics (100U/mL penicillin and 100. mu.g/mL streptomycin), and placed at 37 ℃ in 5% CO₂Culturing in an incubator.

2.2MTI method for detecting cell proliferation: inoculating cells in logarithmic growth phase into 96-well culture plate with cell density of 1 × 10⁴one/mL, 100. mu.L per well, temperature 37 ℃, 5% CO₂After overnight incubation under conditions, the experimental groups were added with the differenceThe two new flavone compounds 3- (2-hydroybenzyl) -6,8-dimethoxy-4H-chromen-4-one or 3- (2-hydroybenzyl) -6, 8-dimethoxychrome-4-one with concentration are arranged in each group with 3 multiple holes, the mixture is placed at 37 ℃ and 5 percent CO after being added with medicine₂Culturing in an incubator for 48 h. Absorbing the culture solution containing the medicine, and adding the mixture into the culture solution in a volume ratio of 4: 1 and MTT (5 mg/L) for further incubation for 4h, carefully removing supernatant, adding 150 mu L of DMSO into each well, placing the well on a shaker to shake so as to completely dissolve crystals (5min), and detecting the absorbance (A) value of each well by a microplate reader at a wavelength of 570 nm. Then, the inhibition rate of each concentration compound on cell growth is calculated, SPSS software is used for processing data, the inhibition rate is plotted against the drug concentration, and IC is calculated₅₀The value is obtained.

The formula of the inhibition rate is as follows: inhibition of cell growth rate ═ 1-A_{Medicine feeding hole}/A_{Control well})×100％

3 results of the experiment

Experimental results show that the two novel flavone compounds have an inhibition effect on proliferation of human colon cancer cells Caco-2, human breast cancer cells MCF-7, human gastric cancer cells BGC-823, human lung adenocarcinoma cells SPC-A1, human liver cancer cells BEL-7402, human cervical cancer cells Hela-229, ovarian cancer cells Ho-8910 and human oral epidermoid cancer cells KB, and the inhibition rate is obviously increased along with the increase of the drug concentration, namely the inhibition rate is concentration dependent. The two new flavone compounds of the invention are used for treating the eight tumor cells IC₅₀The values are shown in Table 6.

TABLE 6 inhibition of tumor cells by two neoflavone compounds of the invention

The antioxidant effect experiment of the new flavone compound is carried out.

1 main material

1.1 drugs and reagents: the two new flavone compounds used in the experiment are prepared by the method, the purity of the two new flavone compounds is 90-99%, the two new flavone compounds are precisely weighed and diluted by methanol to be the solution required by each dosage group. DPPH (1, 1-diphenyl-2-picrylhydrazyl radical) (Sigma-Fluka corporation); BHA (tert-butyl hydroxyanisole) (auspicious shanghai science and technology ltd); methanol, pure chromatography (Changtaixing, Inc.).

1.2 grouping: the test group is divided into a control group, an experimental group and a blank group.

2 method of experiment

Colorimetric determination of the ability to eliminate DPPH free radicals: 1mL of DPPH solution (126.80. mu.M) is added to a 4mL cuvette, and 1mL of a sample solution of 3- (2-hydroxybenzenyl) -6,8-dimethoxy-4H-chromen-4-one (8.32, 16.61, 33.31, 50.02, 66.61. mu.M) or 3- (2-hydroxybenzenyl) -6,8-dimethoxychroman-4-one (8.32, 16.61, 33.31, 50.02, 66.61. mu.M) at different concentrations is added; adding 1mL of methanol solution into a 4mL cuvette in the control group, and then adding 1mL of sample solutions with different concentrations; a blank 1mL of DPPH solution was added to a 4mL cuvette, followed by 1mL of methanol solution. Mixing the three groups, standing at room temperature in dark for 10min, measuring light absorption value at 517nm, standing for 30min, and performing the same method. Three average determinations were made for each sample, and the positive controls were BHA solutions of different concentrations. The DPPH free radical clearance rate of the sample is calculated according to the following formula, and the free radical clearance rate IC is further calculated₅₀The value is obtained.

DPPH clearance (%) < 1- (A)₁－A₂)/A₀×100％

Wherein A is₀Absorbance values for the blank set; a. the₁Is the absorbance value of the sample set; a. the₂Absorbance values for the control group.

3 results of the experiment

The experimental result shows that the two new flavone compounds have the effect of removing DPPH free radicals, and the removal rate is obviously increased along with the increase of the concentration of the medicament. The invention relates to two new flavone compounds on DPPH free radical IC₅₀The values are shown in Table 7.

TABLE 7 DPPH radical scavenging action of two neoflavone compounds of the invention

In conclusion, the invention provides two new flavone compounds and an extraction and separation method thereof, wherein the two new flavone compounds are successfully separated to obtain the two new compounds by sequentially adopting water decoction extraction, silica gel column chromatography, polyamide column chromatography, silica gel column chromatography, ODS medium-pressure column chromatography and recrystallization for separation, purification and preparation. The method is simple, convenient, rapid and environment-friendly, and the compound separated by the method has higher purity. The obtained compound has unique chemical structure, is extracted from common traditional Chinese medicine purslane, and has the functions of anti-inflammation, anti-tumor and anti-oxidation, so the two new flavone compounds, the salts and the derivatives thereof can be used as natural products to develop new traditional Chinese medicines, and have wide prospects.

Claims

1. The method for extracting and separating two flavonoid compounds separated from the purslane medicinal material is characterized by comprising the following specific steps:

step 1: taking dry purslane medicinal materials, decocting and extracting the medicinal materials by adopting water, and cooling the medicinal materials to room temperature to obtain liquid medicine for later use;

and 2, step: evaporating the liquid medicine obtained in the step (1) to dryness, then putting the liquid medicine into a silica gel column, eluting the liquid medicine by using ethyl acetate, and recovering the ethyl acetate under reduced pressure to obtain an extract so as to obtain an ethyl acetate extract;

and step 3: separating the ethyl acetate extract obtained in the step 2 by using a polyamide column, performing gradient elution by using ethanol-water, evaporating 50% ethanol part to dryness, then applying to a silica gel column, performing gradient elution by using ethyl acetate and ethyl acetate-methanol in sequence, and recovering ethyl acetate to obtain an extract to obtain an ethyl acetate extract;

and 4, step 4: separating the product obtained in the step 3 by pretreated ODS column chromatography, performing gradient elution with methanol-water to obtain a plurality of elution parts, detecting by thin-layer chromatography, developing, and concentrating the developed elution parts under reduced pressure to dryness to obtain a concentrate for later use;

and 5: separating the product obtained in step 4 by pretreated sephadex column chromatography, eluting with methanol to obtain multiple elution parts, detecting by thin layer chromatography, developing, mixing the developed elution parts, and concentrating the mixed elution parts under reduced pressure to dry;

and 6: standing in the step 5 to precipitate crystals, washing the crystals with methanol for multiple times, volatilizing the methanol, detecting by thin-layer chromatography, and developing to obtain two flavonoid compounds with the following chemical structural formulas:

。

2. the extraction and separation method according to claim 1, wherein the water is decocted and extracted twice in the step 1, each time for 2 hours, and the water amount is 8-16 times of that of the medicinal materials.

3. The extraction separation method according to claim 1, wherein the mobile phase elution procedure used in step 2 is isocratic elution.

4. The extraction separation method of claim 1, wherein step 3 is performed by gradient elution with water and ethanol at a volume ratio of 100/0, 70/30, 50/50, 30/70 and 0/100; the ethyl acetate elution procedure used in the step 3 is isocratic elution, and the volume ratio of ethyl acetate to methanol is 5/1, 2/1 and 1/2; and step 4, carrying out gradient elution by using methanol and water with the volume ratio of 50/50, 60/40, 70/30 and 80/20.

5. The extraction separation method according to claim 1, wherein the pretreatment of ODS and sephadex used in steps 4 and 5 is carried out by soaking in methanol for 24 hours, loading on a column, washing with methanol until the dropping water is free from turbidity, and equilibrating with an initial mobile phase.

6. The extraction separation method according to claim 1, wherein the methanol elution procedure in step 5 is isocratic elution.