CN113912657A

CN113912657A - Three indole alkaloids in purslane, extraction and separation method and application thereof

Info

Publication number: CN113912657A
Application number: CN202111394363.4A
Authority: CN
Inventors: 兰秀娟; 英锡相; 张文洁; 郭胜男; 宋铭扬
Original assignee: Liaoning University of Traditional Chinese Medicine
Current assignee: Liaoning University of Traditional Chinese Medicine
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2022-01-11
Anticipated expiration: 2041-11-23
Also published as: CN113912657B

Abstract

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to three new indole alkaloid compounds extracted, separated and identified from purslane and an extraction and separation method thereof. The new alkaloid compound has the molecular formula of C₂₄H₂₅NO₁₀，C₂₃H₂₃NO₉，C₂₉H₃₃NO₁₄Named, oleraindone E, oleraindone F and oleraindone G. Also provides an extraction and separation method of the novel alkaloid compound, which sequentially adopts 50 percent ethanol reflux extraction, silica gel column chromatography, polyamide column chromatography, ODS medium-pressure column and Sephadex LH-20 purification and liquid phase separation for preparation. The structure of the indole alkaloid compound is determined to be three new indole alkaloid compounds by adopting methods of mass spectrum, hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectrum analysis. Novel alkaloidsThe new alkaloid compound and the salt or the derivative thereof can be used as a raw material for synthesizing a guide substance of other compounds, developing a new medicament and researching pharmacological activity, and can be used for preparing anti-inflammatory medicaments or health-care products.

Description

Three indole alkaloids in purslane, 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 alkaloid compound extracted, separated and identified from a purslane medicinal material and an extraction and separation method thereof.

Background

Purslane (purslane)Portulaca oleraceaL.), herba Portulacae and herba Portulacae (Portulacaceae). Purslane is fertile and fertile in soil, drought-resistant and waterlogging-resistant, strong in vitality, wide in distribution and rich in resources. The purslane can be used as a medicine and can be eaten, and is one of wild plants which are determined by the Ministry of health and have homology of medicine and food. The 2020 edition of pharmacopoeia of the people's republic of China accepts dry aerial parts of purslane as medicine, has sour and cold taste, enters liver and large intestine channels, has the effects of clearing away heat and toxic materials, cooling blood and stopping bleeding, and can be used for treating heat toxin and bloody dysentery, carbuncle, furuncle, eczema, erysipelas, snake and insect bite, hematochezia, hemorrhoidal bleeding, metrorrhagia and metrostaxis and bleeding.

Modern pharmacological studies show that purslane has the effects of resisting inflammation, tumors, oxidation and atherosclerosis, reducing blood fat, reducing blood sugar, relaxing or exciting smooth muscles, enhancing immunity and the like. Research shows that various chemical components contained in purslane are closely related to various pharmacological actions of purslane, and the main chemical components of the purslane comprise: alkaloids, flavonoids, coumarins, organic acids, terpenes, volatile oil, polysaccharides, amino acids, various pigments and minerals. Wherein the alkaloid is a major active component of 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

Aiming at the problems, the invention provides three indole alkaloid compounds extracted from purslane, researches show that the indole alkaloid of the invention has cholinesterase resisting activity, and simultaneously provides a simple, convenient, rapid, environment-friendly and high-purity extraction and separation method aiming at the three new alkaloid compounds.

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

The invention provides three indole alkaloid compounds extracted from purslane, which are characterized in that the molecular formulas of the three indole alkaloids are as follows: c₂₄H₂₅NO₁₀，C₂₃H₂₃NO₉，C₂₉H₃₃NO₁₄Named as oleerandole E, oleerandole F and oleerandole G, the chemical structural formulas are respectively as follows:

。

the invention also provides three methods for extracting and separating indole alkaloid compounds in purslane, which specifically comprise the following steps:

step 1: extracting herba Portulacae dry medicinal material with 50% ethanol under reflux, concentrating the extractive solution, and cooling to room temperature to obtain medicinal liquid;

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

and step 3: separating the ethyl acetate extract in the step 2 by using a polyamide column, performing gradient elution by using ethanol-water, merging the color development parts eluted by 100% ethanol, evaporating to dryness, putting the mixture on a silica gel column, performing gradient elution by using ethyl acetate and ethyl acetate-methanol, detecting by using a thin-layer chromatography, developing color, merging the color development parts of the ethyl acetate part, and concentrating under reduced pressure to be dry for later use;

and 4, step 4: 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, detecting by using a thin-layer chromatography, developing, and concentrating the developed elution parts under reduced pressure until the developed elution parts are dry to obtain a concentrate for later use;

and 5: carrying out chromatographic separation on the concentrate obtained in the step 4 by pretreated Sephadex LH-20 (hydroxypropyl Sephadex), eluting by methanol, detecting by thin-layer chromatography, developing, and respectively concentrating the developed elution parts under reduced pressure to dryness to obtain a concentrate for later use;

step 6: separating and preparing the concentrate obtained in the step 5 by HPLC (high performance liquid chromatography), and carrying out isocratic elution by using methanol-0.1% formic acid (volume percentage) as a mobile phase to finally obtain the three novel indole alkaloids.

Further, in the

step

1, 50% ethanol is extracted twice under reflux, each time lasts for 2 hours, and the dosage is 8-16 times of that of the medicinal materials.

Further, the ethyl acetate mobile phase elution procedure used in the step 2 is isocratic elution; the mesh number of the silica gel is 100 meshes and 200 meshes.

Further, the volume ratio of ethanol to water used in the step 3 is 0: 100. 30: 70. 50: 50. 70: 30 and 100: 0; the volume ratio of ethyl acetate to methanol is 5: 1. 2:1 and 1: 2; the mesh number of the silica gel is 200 meshes and 300 meshes.

Further, the volume ratio of the methanol to the water used in the step 4 is 60: 40. 70: 30. 80: 20. 90: 10 and 100: 0; the ODS particle size is 40-70 μm.

Further, the pretreatment process of ODS and Sephadex LH-20 gel in

said steps

4 and 5 is methanol soaking for 24 hours, washing with methanol after column loading until no turbidity is formed in the dropping water, and then balancing with the initial mobile phase.

Further, the methanol elution procedure used in step 5 is isocratic elution.

Further, the volume ratio of methanol to 0.1% formic acid used in the step 6 is 48: the retention times of the three compounds were 16.36min, 11.67min and 10.045 min, respectively.

The invention also provides application of the three indole alkaloid compounds separated from the purslane medicinal material in preparation of anti-inflammatory drugs or health products.

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

The separation and pharmacological activity research of the purslane new indole alkaloid compound is not reported in the journal of the prior paper; the invention provides three indole alkaloid compounds derived from purslane and an extraction and separation method for the new compound, which successively adopts 50 percent ethanol reflux extraction, silica gel column chromatography, polyamide column, ODS medium-pressure column, Sephadex LH-20 and high performance liquid chromatograph for separation, purification and preparation, so as to successfully extract and separate the three new indole alkaloid compounds, the method has six steps of operation, simple and quick operation method, the extraction and separation process mainly adopts 50 percent ethanol extraction and ethyl acetate elution, 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 compounds have anti-inflammatory activity, so that the three new alkaloid compounds, the salts and the derivatives thereof can be used as raw materials for other compound synthesis pilots, new drug development and pharmacological activity research, and can also be used for preparing anti-inflammatory drugs.

Drawings

FIG. 1 shows the synthesis of the novel alkaloid compound, olerainole E¹H-NMR spectrum chart.

FIG. 2 shows the synthesis of the novel alkaloid compound, olerainole E¹³C-NMR spectrum chart.

FIG. 3 is a DEPT spectrum of the novel alkaloid compound, oleeraindole E.

FIG. 4 is a spectrum of HSQC of the novel alkaloid compound, oleeraindole E, of the present invention.

FIG. 5 is a HMBC spectrum of the novel alkaloid compound, oleeraindole E, of the present invention.

FIG. 6 shows the synthesis of the novel alkaloid compound, olerainole E¹H-¹H COSY spectrogram.

FIG. 7 is a ROESY spectrum of the novel alkaloid compound, oleeraindole E, of the present invention.

FIG. 8 is a high resolution mass spectrum of the novel alkaloid compound, oleeraindole E, of the present invention.

FIG. 9 shows the synthesis of the novel alkaloid compound, olerainole F, of the present invention¹H-NMR spectrum chart.

FIG. 10 shows the synthesis of the novel alkaloid compound, oleeraindole F¹³C-NMR spectrum chart.

FIG. 11 is a DEPT spectrum of the novel alkaloid compound, oleeraindole F.

FIG. 12 is an HSQC spectrum of the novel alkaloid compound, oleeraindole F, of the present invention.

FIG. 13 is an HMBC spectrum of the novel alkaloid compound, oleeraindole F, of the present invention.

FIG. 14 shows the synthesis of the novel alkaloid compound, oleeraindole F¹H-¹H COSY spectrogram.

FIG. 15 is a ROESY spectrum of the novel alkaloid compound, oleeraindole F.

FIG. 16 is a high resolution mass spectrum of the novel alkaloid compound, oleeraindole F.

FIG. 17 shows the preparation of the novel alkaloid compound, oleadindole G¹H-NMR spectrum chart.

FIG. 18 shows the synthesis of the novel alkaloid compound, olerainole G¹³C-NMR spectrum chart.

FIG. 19 is a DEPT spectrum of the novel alkaloid compound, oleeraindole G.

FIG. 20 is an HSQC spectrum of the novel alkaloid compound, oleeraindole G, of the present invention.

FIG. 21 is an HMBC spectrum of the novel alkaloid compound, oleeraindole G, of the present invention.

FIG. 22 shows the synthesis of the novel alkaloid compound, olerainole G¹H-¹H COSY spectrogram.

FIG. 23 is a ROESY spectrum of the novel alkaloid compound, oleeraindole G.

FIG. 24 is a high resolution mass spectrum of the novel alkaloid compound, oleeraindole G.

Detailed Description

The following examples will help to understand the present invention, but they are only for illustrative purposes and the present invention is not limited to these contents. The methods of operation in the examples are conventional in the art.

The invention provides three newIndole alkaloid compounds with molecular formula of C₂₄H₂₅NO₁₀，C₂₃H₂₃NO₉，C₂₉H₃₃NO₁₄Named as oleerandole E, oleerandole F and oleerandole G, the chemical structural formulas are respectively as follows:

。

the three alkaloid compounds are named as oleaindole E, oleaindole F and oleaindole G according to the structures, the tables 1, 2 and 3 respectively represent nuclear magnetic data of the three alkaloid compounds, and the solvent used for NMR is CD₃OD。

Table 1: nuclear magnetic data for the novel alkaloid Compound, olerainpole E of the invention

。

Oleraindole E: yellow powder, which is easily soluble in methanol and slightly soluble in water, is sprayed with diluted bismuth potassium iodide after being spotted on a silica gel thin-layer plate, and the spot appears orange, which indicates that the compound is an alkaloid component. Bonding of¹H-NMR，¹³C-NMR and HR-ESI-TOF-MS signals, and the possible molecular formula of the compound is presumed to be C₂₄H₂₅NO₁₀The unsaturation degree was 13. HR-ESI-TOF-MS gives M/z 486.1406 [ M-H [ ] -]^-Has an excimer ion peak of 486.1405 molecular weight. According to¹³C-NMR Signal delta_C 77.93 (C-3''')，δ_C 75.15 (C-4''')，δ_C 71.18 (C-5''')，δ_C 78.47 (C-6''')，δ_C62.39 (C-7' ' ') and δ_C105.27 (C-2' ' '), suggesting the Presenceβ-structure of D-glucose. In that¹In H-NMR,. delta._H8.43 (1H, s, H-7) and δ_H7.02 (1H, s, H-4) is two singlet peaks and HMBC shows H-2 (. delta.) (_H 7.96, d, J=3.6 Hz) and C-3a (δ)_C128.31)、C-7a (δ_C131.02), H-3 (delta)_H 6.59, d, J=3.6 Hz) with C-7a, H-4 with C-3 (delta)_C 109.73)、C-6 (δ_C145.57), C-7a, H-7 and C-5 (. delta.) (delta.))_C146.32) and C-3a, indicating the presence of a 5, 6-disubstituted indole ring structure in the structure. And C-5 is located in the low field region, so that the C-5 position is substituted by a hydroxyl group. HMBC showed H-2' ″ correlation with C-6, describedβ-D-glucose is linked to C-6. According to¹H-NMRδ_H 7.39 (1H, d, J=15.6 Hz, H-2') and delta_H 7.89 (1H, d, JH-3') indicating that the structure contains a trans double bond structure, and in HMBC, H-2' and C-1' (δ) are present_C166.68), H-3' with C-1' and C-2' (delta)_C115.06) indicating that C-2 'is linked to C-1', and, in addition, ROESY indicates that H-2 is linked to H-2 'indicating that C-1' is linked to N-1.¹H-NMR Signal delta_H 7.41 (1H, s, H-2'')，δ_H 6.86 (1H, d, J=7.8 Hz, H-5'')，δ_H 7.24 (1H, d, JH-6 =7.8 Hz) indicates the presence of a 1, 3, 4-trisubstituted benzene ring structure, delta_C56.78/δ_H3.95 (3H, s) signals the presence of a methoxy group. In HMBC, H-2'' and C-4'' (delta)_C 151.23)、C-6'' (δ_C125.15) related, H-5'' and C-1'' (delta)_C128.37)、C-3'' (δ_C149.69), H-6'' and C-2'' (delta)_C112.33), C-4'' related, hydrogen in methoxy and delta_C149.69, it is known that the C-3 'position is substituted by methoxy group and the C-4' position is substituted by hydroxy group. HMBC shows H-2' associated with C-1' and H-3' associated with C-2', C-6', indicating that C-1' is linked to C-3' of the trans double bond. From the above information, the novel compounds can be identified as having the above structure.

Table 2: nuclear magnetic data of novel alkaloid compound oleandindole F of the invention

。

Oleraindole F: white powder, which is easily soluble in methanol and slightly soluble in water, is sprayed with diluted bismuth potassium iodide after being spotted on a silica gel thin-layer plate, and the spot appears orange, which indicates that the compound is an alkaloid component. Bonding of¹H-NMR，¹³C-NMR and HR-ESI-TOF-MS signals, presumably from this compoundMolecular formula C₂₃H₂₃NO₉The unsaturation degree was 13. HR-ESI-TOF-MS gives M/z 456.1295 [ M-H [ ] -]^-Has an excimer ion peak of 456.1295 molecular weight. According to¹³C-NMR Signal delta_C 105.08 (C-2''')，δ_C 74.07 (C-3''')，δ_C 75.13 (C-4''')，δ_C 71.21 (C-5''')，δ_C 78.46 (C-6''')，δ_C62.38 (C-7' ' '), suggesting the Presenceβ-structure of D-galactose. In that¹In H-NMR, H-7 (. delta.)_H8.34, 1H, s) and H-4 (Δ 6.99, 1H, s) are two singlet peaks, HMBC shows H-3 (Δ 6.45, d,J=3.6 Hz) and C-7a (delta)_C130.25), H-4 and C-3 (. delta.))_C 109.95)、C-6 (δ_C 145.62)、C-7a (δ_C130.25), H-7 and C-3a (delta)_C128.47)、C-5 (δ_C146.43) indicating the presence of a 5, 6-disubstituted indole ring structure in the structure. And C-5 is located in the low field region, so that the C-5 position is substituted by a hydroxyl group. HMBC showed H-2' ″ correlation with C-6, describedβ-D-galactose is linked to C-6. According to¹H-NMR δ_H 6.36 (1H, d, J=12.6 Hz, H-2') and delta_H 6.97 (1H, d, J=12.6 Hz, H-3') indicating that the structure contains a trans double bond structure, from¹³C-2' (delta) can be seen in C-NMR_C119.11), C-3' (delta)_C140.85) indicating a C-2 'and carbonyl group (C-1', delta)_C168.64), and in addition, ROESY showed that H-2 is associated with H-2', indicating that C-1' is associated with N-1.¹H-NMR Signal delta_H 7.31 (2H, d, J=9.0 Hz, H-2'', H-6'') and delta_H 6.66 (2H, d, J=8.4 Hz, H-3'', H-5'') indicates the presence of an AA 'BB' spin system in the structure, and C-4'' is located in the low field region, so that the C-4'' position is substituted by a hydroxyl group. HMBC show H-2' and C-1 ″ (delta)_C127.72) and H-3' is related to C-2' and C-6', indicating that C-1' is linked to C-3' of the trans double bond. From the above information, the novel compounds can be identified as having the above structure.

Table 3: nuclear magnetic data of novel alkaloid compound oleandindole G of the invention

。

Oleraindole G: yellow powder, which is easily soluble in methanol and slightly soluble in water, is sprayed with diluted bismuth potassium iodide after being spotted on a silica gel thin-layer plate, and the spot appears orange, which indicates that the compound is an alkaloid component. Bonding of¹H-NMR，¹³C-NMR and UHPLC-ESI-QTOF/MS signals, and the possible molecular formula of the compound is presumed to be C₂₉H₃₃NO₁₄The unsaturation degree was 14. UHPLC-ESI-QTOF/MS gives M/z 456.1293 [ M-C [ ]₆H₁₁O₅]^-Has an excimer ion peak of 486.1405 molecular weight. According to¹³C-NMR Signal delta_C105.22 (C-2''')，δ_C 77.93 (C-3''')，δ_C 74.78 (C-4''')，δ_C 71.62 (C-5''')，δ_C 78.96 (C-6''')，δ_C 69.58 (C-7''')，δ_C102.92 (C-2'''')，δ_C76.13 (C-3'''')，δ_C 75.79 (C-4'''')，δ_C72.00 (C-5'''')，δ_C 77.96 (C-6'''')，δ_C62.84 (C-7'' '') indicates that there are twoβ-D-glucose and is linked at C-7' ' '. In that¹In H-NMR, H-7 (. delta.)_H8.45, 1H, s) and H-4 (. delta.) (delta.)_H7.04, 1H, s) are two singlet peaks, HMBC shows H-3 (δ 6.57, d,J=3.7 Hz) and C-7a (delta)_C131.13), H-4 and C-3 (. delta.))_C 109.71)、C-6 (δ_C 144.50)、C-7a (δ_C131.13), H-7 and C-3a (delta)_C129.27)、C-5 (δ_C146.72) indicating the presence of a 5, 6-disubstituted indole ring structure in the structure. And C-5 is located in the low field region, so that the C-5 position is substituted by a hydroxyl group. HMBC showed that H-2' ' ' correlates with C-6, indicating twoβ-D-glucose is linked to C-6. According to¹H-NMR δ_H7.33 (1H, d, J=15.4 Hz, H-2') and δ_H7.91 (1H, d, J=15.3 Hz, H-3') indicating that the structure contains a trans double bond structure, from¹³C-2' (delta) can be seen in C-NMR_C114.95), C-3' (δ)_C148.24) indicating a C-2 'and carbonyl group (C-1', delta)_C166.77), and in addition, ROESY showed that H-2 is associated with H-2', indicating that C-1' is associated with N-1.¹H-NMR Signal delta_H 7.62 (2H, d, J=8.6 Hz, H-2'', H-6'') and delta_H 6.83 (2H, d, J=8.6 Hz, H-3'', H-5'') indicates the presence of an AA 'BB' spin system in the structure, and C-4'' is located in the low field region, so that the C-4'' position is substituted by a hydroxyl group. HMBC show H-2' and C-1 ″ (delta)_C128.00) and H-3 'with C-2' 'and C-6' ', indicating that C-1' 'is attached to C-3' of the trans double bond. 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 three alkaloid compounds, which comprises the following specific steps:

step 1: weighing 250kg of dry purslane medicinal material, performing reflux extraction by adopting 50% ethanol, performing extraction twice with the dosage of 10 times of the medicinal material, performing extraction for 2 hours each time, combining the extracting solutions, 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, performing chromatographic separation by using a silica gel column, and isocratically eluting by using ethyl acetate (115L), wherein the silica gel is 100-200 meshes, the temperature is higher than room temperature, and the ethyl acetate is recovered to an extract under reduced pressure below 40 ℃ to obtain an ethyl acetate extract;

and step 3: separating the ethyl acetate extract 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), merging chromogenic parts of 100% (volume percentage) ethanol parts, evaporating to dryness, performing chromatography 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, detecting by using a thin-layer chromatography, developing, merging the chromogenic parts of the ethyl acetate, and concentrating to be dry at the temperature of more than room temperature and under the reduced pressure of 40 ℃ for later use;

and 4, step 4: separating the product obtained in the step 3 by pretreated ODS medium-pressure column chromatography, wherein the filler particle size is 40-70 μm, performing gradient elution (pressurizing to make the flow rate be 1 mL/min and the temperature be room temperature) by using methanol-water (60/40, 70/30, 80/20, 90/10, 100/0, v/v), detecting by thin-layer chromatography, developing color to obtain 7 parts, and concentrating the part 1 at 50 ℃ under reduced pressure until the part is dry for later use. The pretreatment process of the ODS comprises the steps of soaking in methanol for 24 hours, washing with methanol after column loading until no turbidity exists in dripped water, and balancing with an initial mobile phase;

and 5: subjecting the fraction obtained in step 4 to Sephadex LH-20 column chromatography, isocratic eluting with methanol, detecting by thin layer chromatography, developing to obtain 4 fractions, and concentrating the fraction 4 at 50 deg.C under reduced pressure to dry. The pretreatment process of the Sephadex LH-20 gel comprises the steps of soaking for 24 hours in methanol, washing with the methanol after column loading until no turbidity exists in dropwise added water, and balancing with an initial mobile phase;

step 6: separating and preparing the part obtained in the step 5 by HPLC, taking methanol and 0.1% formic acid with the volume ratio of 48:52 as a mobile phase, and separating and preparing three new alkaloid compounds with the detection wavelength of 210 nm and 280 nm, wherein the purity is 90-99% by a normalization method.

Example 2 the anti-inflammatory action of the novel alkaloid 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 is 90-99%, the new compound is precisely weighed and diluted by DMSO to 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-1β、TNF-αELISA kit of (A) (Cayman, USA); cell lysate.

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

1.3 grouping: divided into a control group, an LPS group and an 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), 5% CO at 37 ℃₂Culturing in an incubator.

2.2 measurement by CCK8 methodCell viability, the three groups are respectively inoculated in 96-well culture plates with RAW264.7 macrophage in logarithmic growth phase, and the cell density is 1 × 10⁴one/mL, 100. mu.L per well, temperature 37 ℃, 5% CO₂After overnight culture under the condition, the experimental group is added with three alkaloid compounds, namely, oleandindole E, oleandindole F and oleandindole G (10 mu M-100 mu M) with different concentrations, after 1h of incubation, LPS with the concentration of 1 mu G/mL is respectively added into an LPS group and the experimental group, a zero-adjusting 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 24h, 10. mu.L of CCK8 was added to each well of cells at 37 ℃ with 5% CO₂After incubation for 4h under the condition, the absorbance of each hole is measured at the wavelength of 450nm by an enzyme-labeling instrument.

2.3 measurement of inflammatory factor IL-1 by ELISAβAnd TNF-α: RAW264.7 macrophages in logarithmic growth phase were seeded in 24-well culture plates at a cell density of 1X 10⁵one/mL, 1mL per well, temperature 37 ℃, 5% CO₂Culturing overnight under the condition, adding the alkaloid compounds of the invention, namely, oleaindole E, oleaindole F and oleaindole G into experimental groups, culturing for 1h, adding LPS (with the final concentration of 1 mu G/mL) into each hole, incubating for 24h, and repeating 3 holes in each group. ELISA method for determining IL-1 secreted by RAW264.7 macrophage after treatment of purslane-derived new compoundβAnd TNF-αThe content of (a).

3, experimental results.

The experimental result shows that the new alkaloid compound has no influence on the proliferation of macrophage RAW264.7 induced by LPS, and is safe and nontoxic; and can effectively inhibit excessive inflammatory cytokine IL-1 produced by macrophage RAW264.7 induced by LPSβAnd TNF-alpha inflammatory mediators, and is concentration dependent.

The results of the cell relative survival experiments are shown in table 4.

Table 4: effect of Compounds of the invention on the relative survival of RAW264.7 macrophages

。

ELISA methodDetermination of the inflammatory factor IL-1β、TNF-αThe results are shown in Table 5.

Table 5: IL-1 secreted by LPS-induced RAW264.7 cells by the compounds of the inventionβ、TNF-αInfluence of the content (mean. + -. standard deviation, n = 3)

。

In conclusion, the invention provides three new indole alkaloid compounds and an extraction and separation method thereof, which are prepared by sequentially adopting 50% ethanol extraction, silica gel column chromatography, polyamide column chromatography, ODS medium-pressure column, Sephadex LH-20 purification and liquid phase separation to successfully obtain the three new alkaloid compounds.

Claims

1. Three indole alkaloids separated from purslane medicinal materials are characterized in that the molecular formulas of the three indole alkaloids are as follows: c₂₄H₂₅NO₁₀，C₂₃H₂₃NO₉，C₂₉H₃₃NO₁₄Designated oleraceole E, oleraceole F and oleraceole G, having the following chemical structures:

。

2. the method for extracting and separating alkaloid according to claim 1, which comprises the following steps:

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: carrying out chromatographic separation on the concentrate obtained in the step 4 by pretreated Sephadex LH-20, eluting by methanol, detecting by thin-layer chromatography, developing, and respectively concentrating the developed elution parts under reduced pressure to dryness to obtain a concentrate for later use;

step 6: and (3) carrying out HPLC separation preparation on the concentrate obtained in the step (5), and carrying out isocratic elution by using methanol-0.1% formic acid as a mobile phase to prepare three indole alkaloid compounds.

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

4. The extraction separation method according to claim 2, wherein the ethyl acetate mobile phase elution procedure used in the step 2 is isocratic elution; the mesh number of the silica gel is 100 meshes and 200 meshes.

5. The extraction separation method according to claim 2, wherein the volume ratio of ethanol to water used in the step 3 is 0: 100. 30: 70. 50: 50. 70: 30 and 100: 0; the volume ratio of ethyl acetate to methanol is 5: 1. 2:1 and 1: 2; the mesh number of the silica gel is 200 meshes and 300 meshes.

6. The extraction separation method according to claim 2, wherein the volume ratio of methanol to water used in the step 4 is 60: 40. 70: 30. 80: 20. 90: 10 and 100: 0; the ODS particle size is 40-70 μm.

7. The extraction separation method as claimed in claim 2, wherein the pretreatment process of ODS and Sephadex LH-20 gel in step 4 and step 5 is methanol soaking for 24 hours, loading on column, washing with methanol until no turbidity is observed in the dropping water, and balancing with initial mobile phase.

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

9. The extraction separation method according to claim 2, wherein the methanol-0.1% formic acid volume ratio used in the step 6 is 48: the retention times of the three compounds were 16.36min, 11.67min and 10.045 min, respectively.

10. The application of the three indole alkaloid compounds separated from the purslane medicinal material as claimed in claim 1 in preparing anti-inflammatory drugs or health products has potential.