CN111217773A

CN111217773A - Furan ring compound in purslane and extraction and separation method and application thereof

Info

Publication number: CN111217773A
Application number: CN202010170157.4A
Authority: CN
Inventors: 英锡相; 刘锡龙; 张文洁
Original assignee: Liaoning University of Traditional Chinese Medicine
Current assignee: Liaoning University of Traditional Chinese Medicine
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2020-06-02
Anticipated expiration: 2040-03-12
Also published as: CN111217773B

Abstract

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to a novel compound extracted, separated and identified from a purslane medicinal material, an extraction and separation method and application thereof, and specifically relates to an extraction and separation method and application of a furan cyclic compound in purslane. The new compound is prepared by sequentially adopting ethanol extraction, silica gel column chromatography, polyamide column chromatography, macroporous resin column chromatography, ODS medium-pressure column and Sephadex LH-20 purification and liquid phase separation, and the structure adopts UHPLC-ESI-TOF-MS,¹H‑NMR、¹³The furan nucleus compound is determined to be a new furan nucleus compound by a C-NMR and two-dimensional nuclear magnetic spectrum analysis method. The compound has potential anti-inflammatory and neuroprotective activities, and the new compound and its salt or derivative can be used as a lead for other compound synthesis toAnd raw materials for new drug development and pharmacological activity research, and provide a guide and theoretical basis for developing new drugs and new components.

Description

Furan ring compound 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 novel compound extracted, separated and identified from a purslane medicinal material and an extraction and separation method thereof, and specifically relates to an extraction and separation method and application of a furan ring compound in purslane.

Background

Herba Portulacae (Portulaca oleracea L.), also called herba Portulacae and herba Portulacae, is a plant of Portulacaceae. Purslane has drought and waterlogging resistance, light and yin resistance, wide distribution and rich resources, is taken as a medicinal and edible wild plant, takes dry overground parts of purslane as a medicament in 2015 edition pharmacopoeia of the people's republic of China, has the effects of clearing heat and detoxicating, cooling blood and stopping bleeding, stopping dysentery and the like, and is used for treating heat toxin and bloody dysentery, carbuncle swelling and furuncle, eczema, erysipelas, snake and insect bite, hematochezia, hemorrhoidal bleeding, metrorrhagia and metrostaxis and the like.

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, 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-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 a method for extracting and separating furan nucleus compounds, in particular to a novel compound extracted from purslane.

In order to achieve the purpose, the invention provides the following technical scheme:

horse gearA furan cyclic compound in Amaranthus mangostanus is named olerafuran, and its molecular formula is C₁₁H₁₀O₂The chemical structural formula is as follows:

the extraction and separation method of furan ring-containing compound olerafuran in purslane comprises the following steps:

step 1, extracting dry purslane medicinal materials by using ethanol, filtering ethanol extract, combining filtrates, directly heating and concentrating, and cooling to room temperature to obtain liquid medicine for later use;

step 2, evaporating the concentrated solution obtained in the step 1 to dryness, putting the evaporated concentrated solution on a silica gel column, eluting the silica gel column by using ethyl acetate, and recovering the ethyl acetate under reduced pressure to obtain an extract to obtain an ethyl acetate extract;

step 3, separating the ethyl acetate extract obtained in the step 2 by using a polyamide column, performing ethanol-water gradient elution, evaporating a cold water part to dryness, putting the cold water part on a macroporous resin column, sequentially performing ethanol-water gradient elution, and evaporating 70% ethanol and 100% ethanol part to dryness for later use;

step 4, subjecting the product obtained in the step 3 to chromatographic separation by a pretreated ODS (Octadecylsilyl silica gel filler), performing gradient elution by 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, carrying out isocratic elution on the pretreated Sephadex LH-20 (hydroxypropyl Sephadex) of the concentrate obtained in the step 4 by using methanol, detecting by using a thin-layer chromatography, developing, and respectively concentrating the developed elution parts under reduced pressure until the developed elution parts are dried to obtain the concentrate for later use.

And 6, carrying out HPLC (high performance liquid phase) separation preparation on the concentrate obtained in the step 5, and preparing by taking acetonitrile and 0.1% formic acid with the volume ratio of 25:75 as a mobile phase to finally obtain the novel compound.

Further, 50% ethanol is refluxed for each extraction in the step 1, and the reflux is performed for 2 hours each time, wherein the ethanol amount is 8-16 times of that of the medicinal materials.

Further, the pretreatment process of ODS and Sephadex LH-20 gel is that methanol is soaked for 24 hours, loaded on a column, washed by methanol until no turbidity is caused in the dropping water, and then equilibrated by an initial mobile phase.

Further, the mobile phase elution procedure used in step 2 is isocratic elution.

Further, the step 3 uses ethanol-water gradient elution, which is cold water, hot water, 70% ethanol, 100% ethanol gradient elution.

Further, the methanol-water gradient elution used in step 4 was carried out with methanol and water in a volume ratio of 60:40, 80:20 and 100: 0.

The application of furan ring compound olerafuran in the purslane extracted by the invention in preparing anti-inflammatory products.

Further, the anti-inflammatory product includes, but is not limited to, an anti-inflammatory drug, an anti-inflammatory health product, or an anti-inflammatory cosmetic.

The application of furan ring compound olerafuran in the purslane extracted by the invention in preparing neuroprotective products.

Further, the neuroprotective product includes, but is not limited to, neuroprotective drugs, neuroprotective nutraceuticals.

Compared with the prior art, the invention has the beneficial effects.

The separation and pharmacological activity research of the novel purslane compound is not reported in the journal of the prior paper; the invention provides a new compound from purslane and an extraction and separation method aiming at the new compound, which adopts ethanol extraction, silica gel column chromatography, polyamide column chromatography, macroporous resin column chromatography, ODS medium-pressure column, Sephadex LH-20 and HPLC to separate, purify and prepare, so as to successfully extract and separate the new compound, the method has seven operation steps, the operation method is simple and rapid, the extraction and separation process mainly adopts ethanol extraction and ethyl acetate extraction, the process method is environment-friendly, and the purity of the compound separated by the method is higher and is more than 90 percent, in addition, the research shows that the compound has the anti-inflammatory and neuroprotective effects, so the new compound and the salt and the derivative thereof can be used as the synthesis guide of other compounds, and the raw materials for new drug development and pharmacological activity research, and can also be used for preparing the anti-inflammatory and neuroprotective effects, Neuroprotective drugs.

Drawings

FIG. 1 is a UV spectrum of the novel compound olerafuran of the present invention.

FIG. 2 is a high resolution mass spectrum of the novel compound olerafuran of the present invention.

FIG. 3 shows the novel compound olerafuran of the present invention¹H-NMR spectrum chart.

FIG. 4 shows the novel compound olerafuran of the present invention¹³C-NMR spectrum chart.

FIG. 5 is a nuclear magnetic resonance carbon spectrum (DEPT) spectrum of the novel compound olerafuran of the present invention.

FIG. 6 shows the NMR of the novel compound olerafuran of the present invention¹H-¹H COSY spectrogram.

FIG. 7 is a nuclear magnetic resonance HMBC spectrum of the novel compound olerafuran.

FIG. 8 is a nuclear magnetic resonance HSQC spectrum of the novel compound olerafuran of the present invention.

FIG. 9 is a ROESY spectrum of the novel compound olerafuran of the present invention.

Detailed Description

The present invention provides novel compounds of formula C₁₁H₁₀O₂The chemical structural formula is as follows:

the new compound is named 4-methyl-5-phenylfuran-2-ol according to the structure, the common name is olerafuran, and the nuclear magnetic data of the new compound are shown in the table 1:¹H-NMR of¹³C-NMR in DMSO.

TABLE 1 Nuclear magnetic data for novel compounds of the invention

Position	δ_C	Type	δ_H mult.(J in Hz)
				2	172.2	C
3	97.4	CH	6.53d(1.08)
				4	124.1	C
Me-C₄	9.9		2.00d(0.84)
				5	154.9	C
1′	131.1	C
				2′/6′	128.5	CH	7.62m
3′/5′	128.7	CH	7.52m
				4′	129.7	CH	7.47m

olerafuran: yellow oil, readily soluble in methanol and slightly soluble in chloroform. UV (MeOH) λ_max: 207.7, 273.5 nm. UHPLC-ESI (+) -TOF-MS gave M/z 175.0754, [ M + H]⁺Has an excimer ion peak of 174.1990 molecular weight. Bonding of¹H-NMR，¹³C-NMR and DEPT data, presuming that the possible molecular formula of the compound is C₁₁H₁₀O₂The unsaturation degree was 7.¹³The C-NMR spectrum and DEPT spectrum showed 11 carbon signals, respectively, of 1 CH₃(delta: 9.91), 6 CH (both olefinic carbons, delta: 97.40; 128.47; 128.69; 129.71; where 128.47 and 128.69 are overlapping peaks), 4 quaternary carbons (both olefinic carbons, delta: 124.13; 131.10; 154.94; 172.15).

¹The H-NMR spectrum shows 10 hydrogen signals, 1 methyl signal δ 2.00(3H, d, J ═ 0.84Hz), 6 methine signals, δ 6.53(1H, d, J ═ 1.08Hz), δ 7.47(1H, m), δ 7.52(2H, m), δ 7.62(2H, m), with 7.52 and 7.62 overlapping peaks.

According to pairs¹³C NMR and HMBCCareful analysis of the spectra, the presence of 6 olefin carbons, including 5 methine and 1 quaternary carbon, suggests the presence of a mono-substituted benzene ring structure [ delta ]_H7.47(1H，m，H-4′)，δ_H7.62(2H，m，H-2′/6′)，δ_H7.52(2H，m，H-3′/5′)；δ_C129.71(C-4′)，δ_C128.47(C-2 'overlaps with C-6'), delta_C128.69(C-3 'overlaps C-5'), delta_C131.1(C-1′)]. According to the related peak on the HMBC spectrum, H-2 '/6 ' is coupled with C-5, and C-1 ' is connected with C-5; according to the related peak on the HMBC spectrum, H-3 is coupled with C-2, C-4 and C-5, and C-2 and C-5 are in a low field, which indicates that the connection with O is carried out, and a furan ring structure exists; according to the related peak on HMBC spectrum, methyl hydrogen is coupled with C-3, C-4 and C-5, which shows that methyl is connected with C-4, and the new compound is determined to be the structure.

The invention also provides an extraction and separation method of the novel compound, which comprises the following specific steps.

Step 1: weighing 150kg of purslane dry medicinal materials, performing reflux extraction by adopting 50% ethanol, wherein the dosage of the 50% ethanol is 8-16 times of that of the medicinal materials, performing reflux extraction twice, each time for 2 hours, recovering ethanol under reduced pressure, and cooling to room temperature to obtain liquid medicine for later use.

Step 2: evaporating part of 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, wherein the silica gel is 100-200 meshes, and recovering the ethyl acetate to obtain an extract under reduced pressure at the temperature of below 40 ℃ to obtain an ethyl acetate extract.

And step 3: separating the ethyl acetate extract obtained in step 2 by using a polyamide column, performing gradient elution by using cold water, hot water, 70% ethanol and 100% ethanol, evaporating the cold water part to dryness at 90-100 ℃, performing column chromatography separation by using macroporous resin, sequentially performing gradient elution by using cold water, hot water, 70% ethanol and 100% ethanol, and evaporating the 70% ethanol and 100% ethanol part to dryness at 90-100 ℃ for later use.

And 4, step 4: and (3) separating the product obtained in the step (3) by pretreated ODS medium-pressure column chromatography, wherein the filler particle size is 40-70 mu m, performing gradient elution (pressurizing to ensure that the flow rate is 1mL/min and the temperature is room temperature) by using methanol-water (60/40, 80/20, 100/0, v/v) to obtain 11 parts (namely performing gradient elution to obtain 11 bottles, wherein each bottle has 500mL), detecting by using thin-layer chromatography, developing, reserving No. 5 part for developing, and concentrating under reduced pressure below 50 ℃ until the part is dry for later use. The pretreatment process of the ODS 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 balancing with an initial mobile phase.

And 5: and (3) carrying out column chromatography on the developed part obtained in the step (4) by pretreated Sephadex LH-20 column, carrying out isocratic elution by methanol to obtain 10 parts (namely 10 bottles are obtained by gradient elution, and each bottle is 100mL), carrying out detection by thin-layer chromatography, developing, combining the developed No. 6 parts, and concentrating under reduced pressure below 50 ℃ until the parts are dry for later use. The pretreatment process of the Sephadex LH-20 gel comprises the steps of soaking for 24 hours in methanol, loading on a column, washing with the methanol until no turbidity exists in dripping water, and balancing with an initial mobile phase.

Step 6: separating and preparing the chromogenic site obtained in the step 5 by HPLC, taking acetonitrile and 0.1% formic acid with the volume ratio of 25:75 as a mobile phase, detecting the wavelength of 210 and 280nm, and separating and preparing to obtain the novel compound, wherein the purity is 90-99% by a normalization method.

The novel compounds of the present invention have anti-inflammatory properties.

1. The main material.

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

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 experimental methods.

2.1 cell culture, DMEM high-sugar medium, with addition of l 0% fetal bovine serum, l% antibiotics (100U/mL penicillin and 100. mu.g/mL streptomycin), 37.5% CO₂Culturing in an incubator.

2.2MTT colorimetric method for determining cell viability, and respectively inoculating RAW264.7 macrophage in logarithmic growth phase into 96-well culture plateIn the medium, the cell density is 1X 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 novel compound olerafuran of the present invention (50nM, 100nM, 1. mu.M, 20. mu.M, 50. 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 additionally provided, each group was provided with 3 duplicate wells, and the effect on cells after drug addition was examined. After culturing the above groups of cells for 24 hours, 20. mu.L of MTT 5mg/mL 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.

3, experimental results.

The experimental result shows that the special compound has the inhibition effect on the proliferation of macrophage RAW264.7 induced by LPS and has the anti-inflammatory effect at 50 mu M.

The results of the cell relative survival experiments are shown in table 2.

Table 2 effect of the invention on relative survival of RAW264.7 macrophages.

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

neuroprotective effects of the novel compounds of the present invention.

1 main material.

1.1 drugs and reagents: the new compound used in the experiment is prepared by the method, the purity of the new compound is 90-99%, the new compound is precisely weighed and diluted by DMSO to be a solution required by each dosage group. DMEM high-glucose medium, fetal bovine serum (Hyclone, usa); penicillin, streptomycin (Hangzhou Sijiqing Co.), Phosphate Buffered Saline (PBS), (Wuhan Boshi De Co., Ltd.), ROS detection kit (Haimebi Yuntian reagent Co., Ltd.)

1.2 cell lines: human neuroblastoma cell line (SH-SY5Y, IMR-32) (Shanghai cell of Chinese academy of sciences)

1.3 grouping: divided into control group, H₂O₂Injury model group and experimental group.

2 experimental methods.

2.1 cell culture, DMEM high sugar medium, 10% fetal bovine serum, l% antibiotics (100U/mL penicillin and 100. mu.g/mL streptomycin), at 37 deg.C and 5% CO₂Culturing in an incubator.

2.2MTT colorimetric method for determining cell viability, the three groups are respectively inoculated in a 96-hole culture plate by SH-SY5Y cells and IMR-32 cells in logarithmic growth phase, and the cell density is 1 multiplied by 10⁴one/mL, 100. mu.L per well, temperature 37 ℃, 5% CO₂After overnight incubation under these conditions, the experimental groups were incubated for 1H with different concentrations of the novel compound olerafuran of the present invention (50nM, 100nM, 1. mu.M, 20. mu.M, 50. mu.M)₂O₂Group and experimental group were each added with H at a final concentration of 800. mu.M/L₂O₂And a zero-adjustment group (culture solution containing DMSO solvent) is additionally arranged, each group is provided with 3 multiple holes, and the influence on cells after the drugs are added is inspected. After culturing the above groups of cells for 24 hours, 20. mu.L of MTT 5mg/mL was added to each well of cells at 37 ℃ with 5% CO₂After further incubation for 4h under the conditions, the culture was terminated, the liquid in the wells was aspirated, 100. mu.L of dimethyl sulfoxide (DMSO) was added to each well, shaking was carried out for 10min to dissolve the intracellular crystals sufficiently, the absorbance (A) value of each well was measured at a wavelength of 450nm with a microplate reader, the cell survival rate was calculated, and the cell survival rate was (AH ═ cell survival rate)₂O₂Lesion-a blank)/(a control-a blank).

2.3 detecting ROS in SH-SY5Y cells and IMR-32 cells by DCFH-DA method, incubating each group of cells for 24h after corresponding substances are given, incubating for 30min before incubation is finished, adding DCFH-DA into each hole to enable the final concentration to be 10 mu mol/L, continuing incubating for 30min at 37 ℃, collecting cells, washing for 2 times by PBS, counting the cells, and preparing each group of cells into cell suspension with the same concentration. And (3) taking 100 mu L of cell suspension to detect the fluorescence intensity, wherein the excitation wavelength is 485nm, and the emission wavelength is 538 nm. The change in intracellular ROS was calculated by comparing the fluorescence intensity of the control group to 100% and the fluorescence intensity of the remaining groups to that of the control group.

2.4 measurement of LDH Release amount by INT color reaction method, except for the above control group、H₂O₂And (3) additionally setting a blank control group (the blank control group is not inoculated with cells) outside the damage model group and the experimental group, adding corresponding substances into the cells of each group for culturing for 24h, taking 120 mu L of supernatant of each well to a new 96-well plate, adding 60 mu L of prepared LDH detection working solution, incubating for 30min at the dark room temperature, measuring the A value by using a multifunctional microplate reader at 490nm, and calculating the percentage of LDH release amount relative to a control tube. LDH release rate ═ (a dose-a blank)/(a control-a blank)

3, experimental results.

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

TABLE 3 Effect of the present invention on the relative survival rates of human neuroblastoma cell lines SH-SY5Y and IMR-32

Note:^*P<0.05 and H₂O₂And comparing the damage model groups.

The results of ROS measurement in SH-SY5Y cells and IMR-32 cells are shown in Table 4.

TABLE 4 Effect of the present invention on the intracellular ROS levels of human neuroblastoma cell lines SH-SY5Y and IMR-32

Note:^*P<0.05 compared with the control group,^#P<0.05 and H₂O₂The results of the injury model groups comparing the effects of LDH release in SH-SY5Y cells and IMR-32 cells are shown in Table 5.

TABLE 5 Effect of the present invention on the intracellular LDH Release from human neuroblastoma cell lines SH-SY5Y and IMR-32

Note:^*P<0.05 compared with the control group,^#P<0.05 and H₂O₂And comparing the damage model groups.

In conclusion, the invention provides a new compound and an extraction and separation method thereof, ethanol extraction, silica gel column chromatography, polyamide column chromatography, macroporous resin column chromatography, ODS medium-pressure column chromatography, Sephadex LH-20 column chromatography and HPLC separation are sequentially adopted for preparation, a new compound is successfully separated and obtained, the method is simple, convenient, rapid and environment-friendly, the purity of the compound obtained by separation is high, and the obtained compound has a unique chemical structure, is extracted from the common traditional Chinese medicine purslane and has anti-inflammatory and neuroprotective effects, so the new compound, the salt and the derivative thereof can be used as a natural product for developing new traditional Chinese medicines, and have wide prospects.

Claims

1. A furan cyclic compound in herba Portulacae is named olerafuran, and its molecular formula is C₁₁H₁₀O₂The chemical structural formula is as follows:

。

2. the method for extracting and separating the furan nucleus compound in the purslane according to claim 1, which comprises the following specific steps:

step 5, carrying out isocratic elution on the pretreated Sephadex LH-20 (hydroxypropyl Sephadex) of the concentrate obtained in the step 4 by using methanol, detecting by using a thin-layer chromatography, developing, and respectively concentrating the developed elution parts under reduced pressure until the developed elution parts are dried to obtain a concentrate for later use;

3. The extraction and separation method of claim 2, wherein 50% ethanol is refluxed for each extraction in step 1, and the amount of ethanol is 8-16 times of that of the medicinal materials, and the reflux time is 2 hours.

4. The extraction separation method as claimed in claim 2, wherein the pretreatment process of ODS and Sephadex LH-20 gel is soaking in methanol for 24 hours, loading on column, washing with methanol until no turbidity is observed in the dropping water, and equilibrating with initial mobile phase.

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

6. The extraction separation method according to claim 2, wherein the ethanol-water gradient elution used in step 3 is cold water, hot water, 70% ethanol, 100% ethanol gradient elution.

7. The extraction separation method according to claim 2, wherein the methanol-water gradient elution used in the step 4 has methanol to water volume ratios of 60:40, 80:20 and 100: 0.

8. The use of a furan ring compound in purslane of claim 1 in the preparation of an anti-inflammatory product.

9. The use of claim 8, wherein the anti-inflammatory product comprises an anti-inflammatory drug, an anti-inflammatory nutraceutical, and an anti-inflammatory cosmetic.

10. Use of a furan ring compound in purslane according to claim 1 in the preparation of neuroprotective products; the nerve protection product comprises a nerve protection medicament and a nerve protection health product.