CN109824568B

CN109824568B - Two indole novel alkaloid compounds in purslane and extraction and separation method and application thereof

Info

Publication number: CN109824568B
Application number: CN201910269477.2A
Authority: CN
Inventors: 英锡相; 赵程程; 张文洁; 马懿飞
Original assignee: Liaoning University of Traditional Chinese Medicine
Current assignee: Liaoning University of Traditional Chinese Medicine
Priority date: 2019-04-03
Filing date: 2019-04-03
Publication date: 2021-07-27
Anticipated expiration: 2039-04-03
Also published as: CN109824568A

Abstract

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to two indole novel alkaloid compounds extracted, separated and identified from purslane, and an extraction and separation method and application thereof. The two new indole alkaloid compounds separated from herba Portulacae have molecular formulas of C₁₇H₁₃NO₄And C₁₈H₁₅NO₅And are named as olerainpole a and olerainpole B, respectively. Also provides the extraction and separation method of the two new alkaloid compounds, which is prepared by sequentially adopting water decoction extraction, silica gel column chromatography, polyamide column chromatography, ODS medium-pressure column and Sephadex LH-20 purification and liquid phase separation; the structure is determined by adopting a method of ultraviolet, infrared, mass spectrum, hydrogen spectrum, carbon spectrum and two-dimensional nuclear magnetic spectrum analysis. The two indole novel alkaloid compounds extracted from the purslane have the effects of anti-inflammation, anti-tumor and neuroprotection, and the salts or derivatives thereof can be used as a lead compound for synthesizing other compounds and a raw material for developing new drugs and researching pharmacological activity and can be used for preparing anti-inflammation, anti-tumor and neuroprotection drugs.

Description

Two indole novel alkaloid 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 novel alkaloid 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 is fertile and fertile in soil, drought 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. 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 studies show that the purslane has the effects of resisting inflammation, oxidation and tumors, resisting 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 effects of purslane, and the main chemical components of the purslane comprise: alkaloids, flavonoids, terpenoids, coumarins, organic acids, volatile oil, polysaccharides, amino acids, various pigments and minerals. Wherein the alkaloid is a major active 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-feruloyl tyramine; 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 indole alkaloid compounds extracted from purslane, researches show that the indole alkaloid has the effects of anti-inflammation, anti-tumor and neuroprotection, and simultaneously provides a simple, convenient, rapid, environment-friendly and high-purity extraction and separation method aiming at the two new alkaloid compounds.

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

An indole-type novel alkaloid compound oleandindole A in purslane is characterized in that the molecular formula is as follows: c₁₇H₁₃NO₄The chemical structural formula is as follows:

an indole-type novel alkaloid compound oleandindole B in purslane is characterized in that the molecular formula is as follows: c₁₈H₁₅NO₅Chemical structure ofThe formula is as follows:

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

step 1, taking dry purslane medicinal materials, decocting and extracting the medicinal materials by adopting water, filtering water extract, combining the filtrates, directly heating and concentrating the combined filtrates, and cooling the combined filtrates to room temperature to obtain liquid medicine for later use.

And 2, applying the concentrated solution obtained in the step 1 to a silica gel column, eluting with ethyl acetate, and recovering ethyl acetate under reduced pressure to obtain an extract so as to obtain an ethyl acetate extract.

And 3, separating the ethyl acetate extract obtained in the step 2 by using a polyamide column, performing gradient elution by using ethanol-water, merging the color development parts eluted by 70% 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 pure ethyl acetate parts, and concentrating under reduced pressure until the color development parts are dry for later use.

And 4, carrying out chromatographic separation on the product obtained in the step 3 by using a pretreated ODS (Octadecylsilyl silica gel filler), carrying out 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 elution parts are dried 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 until the developed elution parts are dried to obtain the concentrate for later use.

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

The pretreatment process of the ODS and Sephadex LH-20 gel comprises the steps of soaking in methanol for 24 hours, loading on a column, washing with methanol until no turbidity exists in dripping water, and balancing with an initial mobile phase.

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

The separation and pharmacological activity research of the purslane indole novel alkaloid compound is not reported by the prior art; the invention provides two indole alkaloid compounds from purslane and an extraction and separation method aiming at the new compound, which successively adopts water decoction 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 two new indole alkaloid compounds, the method has six steps of operation, simple and quick operation method, the extraction and separation process mainly adopts water extraction and ethyl acetate elution, the process method is environment-friendly, the purity of the compound obtained by the separation method is higher than 90%, and in addition, the research shows that the compounds have the effects of anti-inflammation, anti-tumor and neuroprotection, therefore, the two new alkaloid compounds, the salts and the derivatives thereof can be used as the synthesis leads of other compounds, and the raw materials for new drug development and pharmacological activity research, can also be used for preparing anti-inflammatory, anti-tumor and neuroprotective drugs.

Drawings

FIG. 1 is a UV spectrum of the novel alkaloid compound, oleeraindole A.

FIG. 2 is an infrared spectrum of the novel alkaloid compound, oleeraindole A.

FIG. 3 is a high resolution mass spectrum of the novel alkaloid compound, oleeraindole A, of the present invention.

FIG. 4 shows the synthesis of the novel alkaloid compound, olerainole A¹H-NMR spectrum chart.

FIG. 5 shows the synthesis of the novel alkaloid compound, olerainole A¹³C-NMR spectrum chart.

FIG. 6 is a nuclear magnetic resonance carbon spectrum (DEPT) spectrum of the novel alkaloid compound, oleeraindole A.

FIG. 7 shows the NMR of the novel alkaloid compound, oleeraindole A¹H-¹HCOSY spectrum.

FIG. 8 is a nuclear magnetic resonance HMBC spectrum of the novel alkaloid compound, oleeraindole A.

FIG. 9 shows the spectrum of HSQC of new alkaloid compound oleeraindole A of the present invention.

FIG. 10 is a NOESY spectrum of the new alkaloid compound, oleerandole A, by nuclear magnetic resonance.

FIG. 11 is a UV spectrum of the novel alkaloid compound, oleeraindole B.

FIG. 12 is an infrared spectrum of the novel alkaloid compound, oleeraindole B.

FIG. 13 is a high resolution mass spectrum of the novel alkaloid compound, oleeraindole B, of the present invention.

FIG. 14 shows the synthesis of the novel alkaloid compound, oleeraindole B¹H-NMR spectrum chart.

FIG. 15 shows the synthesis of the novel alkaloid compound, olerainole B¹³C-NMR spectrum chart.

FIG. 16 is a nuclear magnetic resonance carbon spectrum (DEPT) spectrum of the novel alkaloid compound, oleeraindole B.

FIG. 17 shows NMR of the novel alkaloid compound, oleeraindole B¹H-¹HCOSY spectrum.

FIG. 18 is a nuclear magnetic resonance HMBC spectrum of the novel alkaloid compound, oleeraindole B.

FIG. 19 is a spectrum of nuclear magnetic resonance HSQC of the novel alkaloid compound oleeraindole B of the present invention.

FIG. 20 is a NOESY spectrum of the new alkaloid compound, oleerandole B, in nuclear magnetic resonance.

Detailed Description

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

Example 1.

The invention provides two new indole alkaloid compounds with molecular formulas of C₁₇H₁₃NO₄，C₁₈H₁₅NO₅Named as oleraindone A and oleraindone B, and has the chemical structural formula:

the two alkaloid compounds are named according to structures, olearandole a and olearandole B, and table 1 is the nuclear magnetic data of the two alkaloid compounds:¹H-NMR of¹³C-NMR in DMSO.

Table 1: nuclear magnetic data for the novel alkaloid compounds, oleandindole A and oleandindole B, of the present invention

The invention relates to the structure identification and derivation of a novel alkaloid compound, namely oleandindole A.

Oleraindole A: yellow powder, easily soluble in methanol, insoluble, slightly soluble in water. After the sample is applied to a silica gel thin layer plate, a spot sprayed with diluted bismuth potassium iodide solution shows orange color, which indicates that the compound is an alkaloid component. UV (MeOH) lambda_max:210nm，324nm，IR(KBr)ν_max 1659，1606，1515,1468，1387,1265，1168，821cm^-1. HRESI (+) TOFMS gave M/z 296.0922[ M + H]⁺Has an excimer ion peak of 295.0845 molecular weight. Bonding of¹H-NMR，¹³C-NMR and DEPT data, presuming that the possible molecular formula of the compound is C₁₇H₁₃NO₄The unsaturation degree was 12.¹³The C-NMR spectrum and the DEPT spectrum showed 17 carbon signals, 10 CH (delta: 103.58,105.72,108.16,114.01,115.74,124.06,130.89,145.61, where 115.74,130.89 is the overlapping peak), 7 quaternary carbons (one carbonyl, delta: 163.97; six olefinic carbons, delta: 122.76,125.59,129.09,143.24,144.04,160.08), respectively.

¹H-NMR spectrum signals δ 7.47(d,1H, J ═ 15.24), δ 7.78(d,1H, J ═ 15.18), corresponding to¹³C-NMR spectral signal delta_C114.01 and delta_C145.61, showing the presence of a trans olefinic double bond;¹³C-NMR spectral signal delta_C163.97 combine the infrared spectra to determine the presence of a carbonyl group.¹H-NMR spectrum signals δ 6.84(d,2H, J ═ 8.64), δ 7.75(d,2H, J ═ 8.58) and¹³C-NMR spectral signal delta_C115.74(C-3 ', C-5', overlap), delta_C130.89(C-2 ', C-6', overlap), indicating the presence of an AA 'BB' system. According to¹The H-NMR spectrum signals show the correlation of two aromatic protons delta 6.91(s,1H), delta 7.97(s,1H) and HMBC spectra, H-2 is related to C-3, C-3a and C-7a, H-4 is related to C-3 and C-7a, and H-7 is related to C-3a and C-7a, and the existence of a 5, 6-disubstituted indole ring structure is established. The signals associated with H-3 ' and C-1 ' on the HMBC spectrum indicate that the carbonyl carbon is attached to one end of the trans-olefinic double bond, while the signals associated with H-3 ' and C-2 ", C-6", H-2 "/6" and C-3 ', H-2 ' and C-1 ", and the other end of the trans-olefinic double bond is attached to the AA ' BB ' system. From the HMBC, H-2 associated with C-1 ', and the NOESY, H-2 associated with H-2', it is known that the 5, 6-disubstituted indole ring structure is attached to the carbonyl carbon. Finally, the formula of the binding compound and¹H-NMR spectrum signals delta 9.02(s,2H) and delta 10.06(s,1H), and the compound is known to have three hydroxyl groups which are respectively positioned at C-5, C-6 and C-4'.

The invention relates to the structure identification and derivation of a new alkaloid compound, namely oleandindole B.

Oleraindole B: yellow powder, easily soluble in methanol, insoluble, slightly soluble in water. After the sample is applied to a silica gel thin layer plate, a spot sprayed with diluted bismuth potassium iodide solution shows orange color, which indicates that the compound is an alkaloid component. UV (MeOH) lambda_max:206nm，345nm，IR(KBr)ν_max 1668，1596，1515,1465，1387,1275，1028，843cm^-1. HRESI (+) TOFMS gave M/z 326.1018[ M + H]⁺Has an excimer ion peak of 325.0950 molecular weight. Bonding of¹H-NMR，¹³C-NMR and DEPT data, presuming that the possible molecular formula of the compound is C₁₈H₁₅NO₅The unsaturation degree was 12. By comparing the spectrum data of olerainole B and olerainole a, it was found that both have the identical structural part. Of Oleraidole B¹³The C-NMR spectrum and the DEPT spectrum showed 18 carbon signals, respectively 1-OCH₃(delta: 55.84), 9 CH (delta: 103.5)8,105.72,108.08,111.57,114.08,115.48,124.04,124.16,146.01), 8 quaternary carbons (one carbonyl, δ: 163.95; 7 olefin carbons, δ: 122.76,126.02,129.09,143.24,144.04,147.97,149.65).

¹H-NMR spectrum signals δ 7.49(d,1H, J ═ 15.18), δ 7.77(d,1H, J ═ 15.18), corresponding to¹³C-NMR spectral signal delta_C114.08 and delta_C146.01, showing the presence of a trans olefinic double bond;¹³C-NMR spectral signal delta_C163.95 combining the infrared spectrum signals determines the presence of a carbonyl group. In addition to this, the present invention is,¹³C-NMR spectral signal delta_C55.84 and¹H-NMR spectrum signal delta 3.88(s,3H) to determine a-OCH₃Presence of groups and spectrum of-OCH by HMBC₃With respect to the hydrogen of C-3', determining-OCH₃Should be connected to C-3 ".¹H-NMR spectrum signals δ 6.84(d,1H, J ═ 8.16), δ 7.28(dd,1H, J ═ 1.74,8.16), δ 7.53(d,1H, J ═ 1.68), corresponding to those of the corresponding NMR spectra¹³C-NMR spectral signal delta_C 115.48,δ_C 124.16,δ_C111.57 showing the existence of an ABX system, which is characterised by the spectral correlation of HMBC, H-2 "being related to C-3", C-4 ", C-6"; h-5 "is related to C-1", C-3 ", C-4"; and H-6 is related to C-2 and C-4, and the ABX system is determined to be a1, 3, 4-trisubstituted benzene ring structure. According to¹The correlation of two aromatic protons delta 6.91(s,1H), delta 7.98(s,1H) and HMBC spectra shown by H-NMR spectrum signals, H-2 is related to C-3, C-3a, C-7a, H-3 is related to C-2, C-3a, C-7a, H-4 is related to C-3, C-7a, H-7 is related to C-3a, C-7a, and the existence of a 5, 6-disubstituted indole ring structure is established. The signals associated with H-2 '/3' and C-1 'on the HMBC spectrum indicate that the carbonyl carbon is attached to one end of the trans-olefinic double bond, while the signals associated with H-3' and C-1 ", C-2", H-2 "and C-3 ', H-2' and C-1", and the other end of the trans-olefinic double bond is attached to the ABX system. From the NOESY spectrum signals, H-2 correlates with H-2' to indicate that the 5, 6-disubstituted indole ring structure is attached to the carbonyl carbon. The molecular formula of the binding compound and¹the signals delta 8.85(s,1H), delta 9.12(s,1H) and delta 9.70(s,1H) of the H-NMR spectrum show that the compound has three hydroxyl groups which are respectively positioned at C-5, C-6 and C-4.

Based on the above information, two new alkaloids were identified as the structures described above.

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

step 1: weighing 150kg of dry herba Portulacae, decocting and extracting with water at 90-100 deg.C 10 times of the medicinal materials for 2 times, each time for 2 hr, filtering the water extract, mixing filtrates, heating and concentrating, and cooling to room temperature to obtain medicinal liquid.

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), combining the chromogenic parts of 70% (volume percentage) ethanol parts, evaporating to dryness at 90-100 ℃, performing chromatography separation by using a silica gel column, wherein the silica gel is 200-mesh and 300-mesh, performing gradient elution by using ethyl acetate and ethyl acetate-methanol (5:1, 2:1, 1:2, v: v), performing detection by using a thin layer chromatography, performing color development, combining the chromogenic 3-12 elution parts, and concentrating to dryness at room temperature and below 40 ℃ under reduced pressure for later use, wherein the 15 parts obtained by eluting the ethyl acetate (namely, 300mL in each bottle) are obtained.

And 4, step 4: and (3) performing chromatographic separation on the product obtained in the step (3) by using a pretreated ODS medium-pressure column, wherein the granularity of a filler 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 (70: 30, 80: 20, 85: 15, 90: 10 and 100: 0, v/v) to obtain 12 parts (namely performing gradient elution to obtain 12 bottles with 100mL per bottle), detecting by using a thin-layer chromatography, developing, combining 3-10 parts for developing, and concentrating under reduced pressure below 50 ℃ until the parts are 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 25 parts (namely, carrying out gradient elution to obtain 25 bottles, wherein each bottle is 50mL), carrying out detection by thin-layer chromatography, developing, combining the developed 14-20 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 30:70 as a mobile phase, detecting the wavelength of 210 and 280nm, separating and preparing to obtain two new alkaloid compounds, wherein the purity of the two new alkaloid compounds is 90-99% by a normalization method.

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

1. The main material.

1.1, drugs and reagents: the new alkaloid compound used in the experiment is prepared by the method, the purity of the compound is 90-99%, the 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); IL-6, TNF-alpha, PGE₂ELISA kit of (A) (Cayman, USA); cell lysate, Griess reagent (Biyuntian Biotechnology Co., Ltd.)

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 colorimetry for determining cell viability, inoculating RAW264.7 macrophage in logarithmic growth phase into 96-well culture plate respectively, wherein the cell density is 1 × 104/mL, each well is 100 μ L, the temperature is 37 ℃, and 5% CO is adopted₂After overnight culture under the conditions, the experimental groups added two new alkaloid compounds of the invention with different concentrationsle A (10-100. mu.M) or oleandindole B (10-100. mu.M), after 1h of incubation, LPS was added to the LPS group and the experimental group to a final concentration of 1. mu.g/mL, and a zero-adjustment group (culture solution containing DMSO solvent) was provided, each group having 3 multiple wells, and the effect on the cells after addition of the drug 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.

2.3 determination of NO content by Griess method, two novel alkaloid compounds of the present invention were examined for their inhibitory effect on the NO production of LPS-induced mouse macrophage RAW 264.7. Mouse macrophage RAW264.7 is cultured in high glucose cell culture medium DMEM containing 10% fetal calf serum after passage, two new alkaloid compounds of the invention, namely, oleandindole A (10-50 mu M) or oleandindole B (10-50 mu M) with different concentrations are added into experimental groups, and the temperature is 37 ℃ and the temperature is 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 is used for measuring the content of NO in cell supernatant, and the influence of two new alkaloid compounds on NO release of LPS-induced RAW264.7 cells is reflected according to different concentrations so as to reflect the NO level.

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 10⁵one/mL, 1mL per well, temperature 37 ℃, 5% CO₂Culturing overnight under the condition, adding two new alkaloid compounds, namely, oleandindole A (10-50 mu M) or oleandindole B (10-50 mu M) into experimental groups, adding LPS (with the final concentration of 1 mu g/mL) into each hole after culturing for 1h, incubating for 24h, and repeating 3 holes in each group of treatment. ELISA method for determining IL-6, TNF-alpha and PGE secreted by RAW264.7 macrophage after treatment of two purslane-derived new alkaloids₂The content of (a).

3, experimental results.

The experimental result shows that the two new alkaloid compounds of the invention induce LPS hugeThe proliferation of phagocytic cells RAW264.7 has no influence, and is 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 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),

the results of the experiments for determining the NO content by the Griess method are shown in Table 3.

Table 3: influence of the invention on NO release from LPS-induced RAW264.7 cells (mean. + -. standard deviation, 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 4.

Table 4: the invention relates to IL-6, TNF-alpha and PGE secreted by RAW264.7 cells induced by LPS₂Influence of the content (mean ± sd, n ═ 3).

The new alkaloid compounds of the present invention have anti-tumor effects.

1 main material.

1.1 drugs and reagents: the new alkaloid compound used in the experiment is prepared by the method, the purity of the compound is 90-99%, the 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.);

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

2.1 cell culture, DMEM high sugar medium, added with l 0% fetal bovine serum, l% antibiotics (100U/mL penicillin and 100. mu.g/mL streptomycin), placed at 37 ℃ with 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% CO₂Culturing overnight under the condition, adding two new alkaloid compounds with different concentrations into experimental groups, each group having 3 multiple holes, adding medicine, placing at 37 deg.C and 5% CO₂Culturing 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/mL) for 4 hours, carefully absorbing the supernatant, adding 150 mu L of DMSO into each hole, placing the hole on a shaker to shake so as to completely dissolve crystals (5min), and detecting the absorbance (A) value of each hole by a microplate reader at the wavelength of 570 nm. Then, the inhibition rate of each concentration of compound on cell growth is calculated, and the inhibition rate formula is as follows: inhibition of cell growth rate ═ 1-A_{Medicine feeding hole}/A_{Control well}) X 100%, processing data with SPSS software, plotting inhibition rate against drug concentration, and calculating IC₅₀The value is obtained.

3, experimental results.

The experimental result shows that the two new alkaloid compounds of the invention are used for treating human colon cancer cells Caco-2 and humanThe proliferation of the breast cancer cell MCF-7, the human gastric cancer cell BGC-823, the human lung adenocarcinoma cell SPC-A1, the human liver cancer cell BEL-7402, the human cervical cancer cell Hela-229, the ovarian cancer cell Ho-8910 and the human oral epidermoid cancer cell KB has an inhibiting effect, and the inhibiting rate is obviously increased along with the increase of the drug concentration, namely the concentration is dependent. The two new alkaloid compounds of the invention have effects on the eight tumor cells IC₅₀The values are shown in Table 5.

Table 5 inhibitory effect of two novel alkaloid compounds of the present invention on tumor cells.

The novel alkaloid compounds of the present invention have neuroprotective effects.

1 main material.

1.1 drugs and reagents: the new alkaloid compound used in the experiment is prepared by the method, the purity of the compound is 90-99%, the 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, added with 0% fetal bovine serum, l% antibiotics (100U/mL penicillin and 100. mu.g/mL streptomycin), placed in a 37 ℃, 5% CO2 incubator for culture.

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 10⁴Each well at 100. mu.L/mL, temperature 37 ℃ and 5% CO2 overnight, the experimental groups added different concentrations of two novel compounds of the inventionAlkaloid compound oleaindole A (5-50 μ M) or oleaindole B (5-50 μ M), incubating for 1 hr, and adding₂O₂Group 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 control group and 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 results.

The results of the cell relative survival experiments are shown in table 6.

Table 6: the invention has the influence on the relative survival rate of human neuroblastoma cell strains SH-SY5Y and IMR-32 cells.

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 7.

Table 7: the invention has the influence on the intracellular ROS amount of human neuroblastoma cell strains SH-SY5Y and IMR-32.

Note:^*P<0.05 compared with the control group,^#P<0.05 and H₂O₂Damage model group comparison

The results of the effects of LDH release in SH-SY5Y cells and IMR-32 cells are shown in Table 8.

Table 8: the invention has the influence on the release of LDH in human neuroblastoma cell strains SH-SY5Y and IMR-32 cells.

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

Claims

1. An indole alkaloid compound olearandole A in purslane is characterized in that the molecular formula is as follows: c₁₇H₁₃NO₄The chemical structural formula is as follows:

。

2. an indole alkaloid compound olearandole B in purslane is characterized in that the molecular formula is as follows: c₁₈H₁₅NO₅The chemical structural formula is as follows:

。

3. the method for extracting and separating indole alkaloid compounds according to any of claims 1 and 2, comprising the steps of:

step 1, taking dry purslane medicinal materials, decocting and extracting the medicinal materials by adopting water, filtering water extract, combining filtrates, directly heating and concentrating the combined filtrates, and cooling the combined filtrates to room temperature to obtain liquid medicine for later use;

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 3, separating the ethyl acetate extract obtained in the step 2 by using a polyamide column, wherein the volume ratio is 0: 100, 30:70, 50: 50, 70: 30 and 100: performing gradient elution with 0 ethanol-water, mixing the color parts eluted by 70% ethanol, evaporating to dryness, loading on a silica gel column, performing gradient elution with ethyl acetate and ethyl acetate-methanol, wherein the volume ratio of ethyl acetate to methanol is 5:1,2: 1 and 1: detecting by thin-layer chromatography, developing, mixing the developed parts of ethyl acetate part, and concentrating under reduced pressure to dry;

and 4, separating the product obtained in the step 3 by pretreated ODS column chromatography, wherein the volume ratio is 70: 30, 80: 20, 85: 15, 90: 10, 100: performing gradient elution with methanol-water to obtain multiple elution parts, detecting by thin layer chromatography, developing, and concentrating the developed elution parts under reduced pressure to dry to obtain concentrate;

step 5, eluting the pretreated Sephadex LH-20 of the concentrate obtained in the step 4 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;

and 6, carrying out HPLC separation preparation on the concentrate obtained in the step 5, wherein the volume ratio is 30:70 acetonitrile-0.1% formic acid is used as a mobile phase for isocratic elution to prepare two indole alkaloid compounds.

4. The method for extracting and separating indole alkaloid compounds according to claim 3, wherein the water is decocted and extracted for 2 times in the step 1, each time for 2 hours, and the amount of water is 8-16 times of that of the medicinal materials.

5. The method for extracting and separating indole alkaloid compounds according to claim 3, wherein the ethyl acetate mobile phase elution procedure used in step 2 is isocratic elution; the mesh number of the silica gel is 100 meshes and 200 meshes.

6. The method for extracting and separating indole alkaloid compounds as claimed in claim 3, wherein the silica gel used in step 3 has a mesh size of 200-300.

7. The method for extracting and separating indole alkaloid compounds according to claim 3, wherein said pretreatment of ODS and Sephadex LH-20 gel in steps 4 and 5 comprises soaking in methanol for 24 hours, loading onto a column, washing with methanol until the dropping water is free from turbidity, and equilibrating with an initial mobile phase.

8. The method for extracting and separating indole alkaloid compounds according to claim 3, wherein the filler used in step 4 has a particle size of 40 to 70 μm.

9. The method for extracting and separating indole alkaloid compounds according to claim 3, wherein the methanol elution procedure used in step 5 is isocratic elution;

the retention time of the two compounds used in the step 6 is 7.53min and 8.86min respectively.

10. The indole alkaloid compounds according to any of claims 1 and 2 for use in the preparation of anti-inflammatory, anti-tumor and neuroprotective medicaments.