CN109824568A - Two kinds of indoles new alkaloids compounds and its extraction separation method and application in purslane - Google Patents
Two kinds of indoles new alkaloids compounds and its extraction separation method and application in purslane Download PDFInfo
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Abstract
The present invention relates to traditional Chinese medicine extractions, separation field, more particularly to extracting and developing and the two kinds of indoles new alkaloids compounds and its extraction separation method that identify and application from purslane.The new indoles alkaloid compound that described two kinds are isolated from purslane medicinal material, molecular formula is respectively C17H13NO4And C18H15NO5, and it is respectively designated as oleraindole A and oleraindole B.The extraction separation method of above two new alkaloids compound is also provided, successively using compression leg in water boiling and extraction, silica gel column chromatography, polyamide column chromatography, ODS and Sephadex LH-20 purifying, liquid phase separation preparation；Its structure is determined using ultraviolet, infrared, mass spectrum, hydrogen spectrum, carbon spectrum and the method for two-dimensional nuclear magnetic spectrum parsing.Two kinds of indoles new alkaloids compounds that the present invention is extracted from purslane have anti-inflammatory, antitumor and neuroprotection; its salt or derivative can be used as other compound synthesis primers; and the raw material of new drug development and pharmacology activity research, it is used to prepare anti-inflammatory, antitumor and neuroprotection drug.
Description
Technical field
The present invention relates to traditional Chinese medicine extraction, separation field, more particularly to extracting and developing and identifies from purslane medicinal material
New alkaloids compound and its extraction separation method.
Background technique
Purslane (Portulaca oleracea L.) also known as long life dish, horse three-coloured amaranth are portulacaceous plant.Purslane
Property happiness rich soil, drought-enduring also waterlogging, vitality is strong, widely distributed, resourceful.Purslane can not only be used as medicine, but also edible, be
One of the wild plant of integration of drinking and medicinal herbs that the Ministry of Public Health, China delimit.Purslane is recorded in 2015 editions Pharmacopoeias of the People's Republic of China
Dry aerial parts be used as medicine, there is clearing heat and detoxicating, cooling blood and hemostasis, stop dysentery and other effects, for toxic-heat and blood stasis, carbuncle swells furunculosis, wet
Rash, erysipelas, snakebite and bugbite, hematochezia, hemorrhoid blood, metrostaxis etc..
Modern pharmacology research shows that purslane has anti-inflammatory, anti-oxidant, antitumor, antiatherosclerosis, drop blood
Rouge, hypoglycemic, loose or excited smooth muscle and strengthen immunity and other effects.Research shows that contained a variety of chemical components in purslane
With its multiplicity pharmacological action it is closely bound up, main chemical compositions include: alkaloids, flavonoids, terpene, Coumarins,
Organic acid, volatile oil, polysaccharide, amino acid, various pigments and minerals class etc..Wherein alkaloid is one big in purslane
Active component, composition of alkaloids reported at present have norepinephrine, dopamine, a small amount of DOPA, adenosine, urine phonetic
Pyridine, adenine, N, N- dicyclohexylurea (DCU), allantoin, N- be trans--asafoetide acyl group tyrasamine；There are also Cyclic dipeptides alkaloid and amides
Alkaloid: oleracein A-I, K, L, N-S.
Most of chemical component isolated from purslane at present is known, and structure novel is lower, therefore, right
The exploitation and separation of noval chemical compound urgently need in purslane.
Summary of the invention
In view of the above-mentioned problems, the present invention provides the two kinds of indoles alkaloid compounds extracted from purslane, through studying
It was found that indoles alkaloid of the invention has the function of anti-inflammatory, antitumor and neuroprotection, while providing a kind of for this hair
The extraction separation method easy, quick, environmentally friendly, with high purity of bright two kinds of new alkaloids compounds.
Above-mentioned purpose to realize the present invention, it is as follows that the present invention provides technical solution.
A kind of indoles new alkaloids compound oleraindole A in purslane, which is characterized in that molecular formula are as follows:
C17H13NO4, chemical structural formula is as follows:
A kind of indoles new alkaloids compound oleraindole B in purslane, which is characterized in that molecular formula are as follows:
C18H15NO5, chemical structural formula is as follows:
Above-mentioned purpose to realize the present invention, the present invention also provides indoles new alkaloids compounds in a kind of purslane
Extraction separation method, comprising the following steps:
Step 1 takes the dry medicinal material of purslane, and using water boiling and extraction, Aqueous extracts filtration, merging filtrate directly heats dense
Contracting, cools to room temperature, it is spare to obtain medical fluid.
Step 2, by silicagel column on concentrate in step 1, eluted with ethyl acetate, ethyl acetate be recovered under reduced pressure to medicinal extract,
Obtain ethyl acetate extract.
Step 3 separates ethyl acetate extract in step 2 through polyamide column, will using alcohol-water gradient elution
Upper silicagel column after the coloured moiety merging of 70% ethanol elution is evaporated, with ethyl acetate, acetate-methanol gradient elution, warp
The coloured moiety at pure ethyl acetate position is merged, is concentrated to dryness by thin-layer chromatography detection, colour developing, spare.
Step 4, by gains in step 3 pretreated ODS column (Octadecylsilyl, octadecylsilane key again
Close silica filler) chromatography obtains several elution positions, detected through thin-layer chromatography with methanol-water gradient elution, it shows
The elution position of colour developing is concentrated to dryness, it is spare to obtain concentrate by color.
Step 5, by step 4 gained concentrate pretreated Sephadex LH-20 (hydroxypropyl sephadex) layer
Analysis separation, is eluted with methanol, is detected through thin-layer chromatography, develops the color, the elution position of colour developing is concentrated to dryness respectively, is obtained
Concentrate is spare.
Step 6 prepares gained concentrate in step 5 by HPLC (efficient liquid phase) separation, with -0.1% formic acid of acetonitrile
(percentage by volume) is that mobile phase carries out isocratic elution, finally obtains two kinds of new indoles alkaloids of the present invention.
The preprocessing process of the ODS and Sephadex LH-20 gel is that methanol impregnated 24 hours, and upper prop uses methanol
It is washed till to instill in water and balance each other without muddiness, then with initial flow.
Beneficial effects of the present invention compared with prior art.
The separation of heretofore described purslane indoles new alkaloids compound and pharmacology activity research be not by existing skill
Art is reported；The present invention provides two kinds of indoles alkaloid compounds and one kind from purslane for the new chemical combination of the present invention
The extraction separation method of object, successively using compression leg, Sephadex in water boiling and extraction, silica gel column chromatography, polyamide column, ODS
LH-20 and high performance liquid chromatograph are isolated and purified and are prepared, and are successfully extracted and are isolated two kinds of new indoles alkaloids
Object is closed, this method operating procedure is only six steps, and operating method is easy and quickly, extracts separation process and mainly adopts and is extracted with water and second
Acetoacetic ester elution, process environmental protection, and it is all larger than 90% through the isolated compound purity of this method is higher, furthermore through grinding
Study carefully and shows that above each compound has anti-inflammatory, antitumor and neuroprotection, therefore two kinds of new alkaloids compounds of the invention
And its salt and derivative can be used as the raw material of other compound synthesis primers and new drug development and pharmacology activity research,
Also it can be used for preparing anti-inflammatory, antitumor and neuroprotection drug.
Detailed description of the invention
Fig. 1 is the ultraviolet spectrogram of new alkaloids compound oleraindole A of the present invention.
Fig. 2 is the infrared spectrogram of new alkaloids compound oleraindole A of the present invention.
Fig. 3 is the high resolution mass spectrum figure of new alkaloids compound oleraindole A of the present invention.
Fig. 4 is new alkaloids compound oleraindole A's of the present invention1H-NMR spectrogram.
Fig. 5 is new alkaloids compound oleraindole A's of the present invention13C-NMR spectrogram.
Fig. 6 is carbon-13 nmr spectra (DEPT) spectrogram of new alkaloids compound oleraindole A of the present invention.
Fig. 7 is the nuclear magnetic resonance of new alkaloids compound oleraindole A of the present invention1H-1HCOSY spectrogram.
Fig. 8 is the nuclear magnetic resonance HMBC spectrogram of new alkaloids compound oleraindole A of the present invention.
Fig. 9 is the nuclear magnetic resonance HSQC spectrogram of new alkaloids compound oleraindole A of the present invention.
Figure 10 is the nuclear magnetic resonance NOESY spectrogram of new alkaloids compound oleraindole A of the present invention.
Figure 11 is the ultraviolet spectrogram of new alkaloids compound oleraindole B of the present invention.
Figure 12 is the infrared spectrogram of new alkaloids compound oleraindole B of the present invention.
Figure 13 is the high resolution mass spectrum figure of new alkaloids compound oleraindole B of the present invention.
Figure 14 is new alkaloids compound oleraindole B's of the present invention1H-NMR spectrogram.
Figure 15 is new alkaloids compound oleraindole B's of the present invention13C-NMR spectrogram.
Figure 16 is carbon-13 nmr spectra (DEPT) spectrogram of new alkaloids compound oleraindole B of the present invention.
Figure 17 is the nuclear magnetic resonance of new alkaloids compound oleraindole B of the present invention1H-1HCOSY spectrogram.
Figure 18 is the nuclear magnetic resonance HMBC spectrogram of new alkaloids compound oleraindole B of the present invention.
Figure 19 is the nuclear magnetic resonance HSQC spectrogram of new alkaloids compound oleraindole B of the present invention.
Figure 20 is the nuclear magnetic resonance NOESY spectrogram of new alkaloids compound oleraindole B of the present invention.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.
Embodiment 1.
The present invention mentions two kinds of indoles new alkaloids compounds, and molecular formula is respectively C17H13NO4, C18H15NO5, it is named as
Oleraindole A and oleraindole B, chemical structural formula are as follows:
Described two alkaloid compounds are named as oleraindole A and oleraindole B according to structure, and table 1 is
The nuclear magnetic data of two kinds of alkaloid compounds:1H-NMR with13C-NMR is in DMSO.
Table 1: the nuclear magnetic data of new alkaloids compound oleraindole A and oleraindole B of the present invention
The Structural Identification of new alkaloids compound oleraindole A of the present invention a kind of and derivation.
Oleraindole A: yellow powder object is soluble in methanol, insoluble, be slightly soluble in water.Point sample is in silica gel thin-layer plate
Afterwards, it sprays dilute bismuth potassium iodide test solution spot and shows crocus, prompting the compound is alkaloid component.UV(MeOH)λmax: 210nm,
324nm, IR (KBr) νmax1659,1606,1515,1468,1387,1265,1168,821cm-1.HRESI (+) TOFMS is provided
m/z:296.0922[M+H]+Quasi-molecular ion peak, molecular weight 295.0845.In conjunction with1H-NMR,13C-NMR and DEPT number
According to, thus it is speculated that the possible molecular formula of the compound is C17H13NO4, degree of unsaturation 12.13C-NMR spectrum and DEPT spectrum 17 carbon of display
Signal, respectively 10 CH (δ: 103.58,105.72,108.16,114.01,115.74,124.06,130.89,145.61,
Wherein 115.74,130.89 be overlap peak), 7 quaternary carbons (carbonyl, δ: 163.97；Six alkene carbon, δ: 122.76,
125.59,129.09,143.24,144.04,160.08)。
1H-NMR spectrum signal δ 7.47 (d, 1H, J=15.24), δ 7.78 (d, 1H, J=15.18), it is corresponding13C-NMR spectrum letter
Number δC114.01 and δC145.61, show the presence of a trans olefins double bond；13C-NMR spectrum signal δC163.97 combinations are red
External spectrum signal determines the presence of a carbonyl.1H-NMR spectrum signal δ 6.84 (d, 2H, J=8.64), δ 7.75 (d, 2H, J=
8.58) and13C-NMR spectrum signal δC115.74 (C-3 ", C-5 ", overlappings), δC130.89 (C-2 ", C-6 ", overlappings), display
The presence of one AA ' BB ' system.Foundation1Two aromatic δ 6.91 (s, 1H) that H-NMR spectrum signal is shown, δ 7.97 (s,
1H) and the correlation of HMBC spectrum, H-2 and C-3, C-3a, C-7a are related, and H-4 and C-3, C-7a are related, H-7 and C-3a, C-7a
Correlation establishes the presence of 5, a 6- disubstituted indole ring structure.The coherent signal of H-3 ' and C-1 ', illustrates carbonyl in HMBC spectrum
Carbon is connected with one end of trans olefins double bond, while the coherent signal of H-3 ' and C-2 ", C-6 ", H-2 "/6 " are relevant to C-3 '
Signal, H-2 ' signal relevant to C-1 " add the other end of bright trans olefins double bond and are connected with AA ' BB ' system.Foundation
HMBC spectrum signal, H-2 is related to C-1 ' and NOESY spectrum signal, and H-2 is related to H-2 ', learns the disubstituted indole ring of 5,6-
Structure is connected with carbonyl carbon.Finally, binding compounds molecular formula and1H-NMR spectrum signal δ 9.02 (s, 2H), δ 10.06 (s,
1H), it is known that there are three hydroxyl groups for compound, they are respectively at C-5, the position C-6, C-4 ".
The Structural Identification of new alkaloids compound oleraindole B of the present invention a kind of and derivation.
Oleraindole B: yellow powder object is soluble in methanol, insoluble, be slightly soluble in water.Point sample is in silica gel thin-layer plate
Afterwards, it sprays dilute bismuth potassium iodide test solution spot and shows crocus, prompting the compound is alkaloid component.UV(MeOH)λmax: 206nm,
345nm, IR (KBr) νmax1668,1596,1515,1465,1387,1275,1028,843cm-1.HRESI (+) TOFMS is provided
m/z:326.1018[M+H]+Quasi-molecular ion peak, molecular weight 325.0950.In conjunction with1H-NMR,13C-NMR and DEPT number
According to, thus it is speculated that the possible molecular formula of the compound is C18H15NO5, degree of unsaturation 12.By comparing oleraindole B and
The spectroscopic data of oleraindoleA, both discoveries have identical structure division.Oleraindole B's13C-NMR spectrum
18 carbon signals of display, respectively 1-OCH are composed with DEPT3(δ: 55.84), 9 CH (δ: 103.58,105.72,108.08,
111.57,114.08,115.48,124.04,124.16,146.01), 8 quaternary carbons (carbonyl, δ: 163.95；7 alkene
Carbon, δ: 122.76,126.02,129.09,143.24,144.04,147.97,149.65).
1H-NMR spectrum signal δ 7.49 (d, 1H, J=15.18), δ 7.77 (d, 1H, J=15.18), it is corresponding13C-NMR spectrum letter
Number δC114.08 and δC146.01, show the presence of a trans olefins double bond；13C-NMR spectrum signal δC163.95 combinations are red
External spectrum signal determines the presence of a carbonyl.In addition,13C-NMR spectrum signal δC55.84 with1H-NMR spectrum signal δ 3.88 (s,
3H) determine-an OCH3The presence of group, and by-OCH in HMBC spectrum3Hydrogen and C-3 " coherent signal, determine-OCH3It answers
It is connected with C-3 ".1H-NMR spectrum signal 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), it is corresponding13C-NMR spectrum signal δC 115.48,δC 124.16,δC111.57, show depositing for an ABX system
, by HMBC spectrum correlation, H-2 " and C-3 ", C-4 ", C-6 " correlation；H-5 " and C-1 ", C-3 ", C-4 " are related；And H-6 "
With C-2 ", C-4 " is related, determines that this ABX system is one 1,3,4- trisubstituted benzene ring structures.Foundation1H-NMR spectrum signal is aobvious
The two aromatic δ 6.91 (s, 1H) shown, the correlation of δ 7.98 (s, 1H) and HMBC spectrum, H-2 and C-3, C-3a, C-7a
Correlation, H-3 and C-2, C-3a, C-7a are related, and H-4 and C-3, C-7a are related, and H-7 and C-3a, C-7a are related, establish one 5,6-
The presence of disubstituted indole ring structure.The coherent signal of H-2 '/3 ' and C-1 ' in HMBC spectrum illustrates that carbonyl carbon and trans olefins are double
One end of key is connected, while the coherent signal of H-3 ' and C-1 ", C-2 ", H-2 " signal relevant to C-3 ', H-2 ' and C-1 " phase
The signal of pass, the other end for adding bright trans olefins double bond are connected with ABX system.According to NOESY spectrum signal, H-2 and H-2 ' phase
It closes, learns that the disubstituted indole ring structures of 5,6- are connected with carbonyl carbon.Binding compounds molecular formula and1H-NMR spectrum signal δ
8.85 (s, 1H), δ 9.12 (s, 1H), δ 9.70 (s, 1H), it is known that for compound there are three hydroxyl groups, they are respectively at C-
The position 5, C-6, C-4 ".
According to information above, determine that two kinds of new alkaloids are above structure.
The present invention also provides the extraction separation method of above two alkaloid compound, specific steps are as follows:
Step 1: weighing the dry medicinal material 150kg of purslane, using 90-100 DEG C of water boiling and extraction, water consumption is the 10 of medicinal material
Times, it decocts and extracts 2 times, decoct 2h, Aqueous extracts filtration every time, merging filtrate heats concentration, cools to room temperature, it is spare to obtain medical fluid.
Step 2: separating after gained medical fluid in step 1 is evaporated through silica gel column chromatography, washed with ethyl acetate (115L) is isocratic
De-, wherein silica gel is 100-200 mesh, and more than room temperature, 40 DEG C or less are recovered under reduced pressure ethyl acetate to medicinal extract, obtain ethyl acetate and mention
Take object.
Step 3: ethyl acetate extract in step 2 is separated through polyamide column, using alcohol-water (0:100,30:70,
50:50,70:30,100:0, v:v) gradient elution, the coloured moiety at 70% (percentage by volume) ethyl alcohol position is merged 90~
100 DEG C be evaporated after separated through silica gel column chromatography, wherein silica gel be 200-300 mesh, successively use ethyl acetate, acetate-methanol
(5:1,2:1,1:2, v:v) gradient elution, by 15 positions that ethyl acetate affords (obtain 15 bottles, every bottle
300mL), it is detected, is developed the color through thin-layer chromatography, merge 3~12 elution positions of colour developing, and more than room temperature, 40 DEG C or less
It is concentrated to dryness, it is spare.
Step 4: by gains in step 3, pretreated ODS medium pressure column chromatography is separated again, wherein filler particle size be 40~
70 μm, with methanol-water (70:30,80:20,85:15,90:10 and 100:0, v/v) gradient elution, (pressurization, makes flow velocity 1mL/
Min, temperature are room temperature), 12 positions (i.e. gradient elution obtains 12 bottles, every bottle of 100mL) is obtained, is examined through thin-layer chromatography
It surveys, colour developing merges 3~10 positions of colour developing, and 50 DEG C or less are concentrated to dryness, spare.The preprocessing process of the ODS is
Methanol impregnated for 24 hours, upper prop, is washed till to instill in water with methanol and be balanced each other without muddiness, then with initial flow.
Step 5: the pretreated Sephadex LH-20 column in gained colour developing position in step 4 is chromatographed, it is isocratic with methanol
Elution, obtains 25 positions (i.e. gradient elution obtains 25 bottles, every bottle of 50mL), is detected through thin-layer chromatography, develops the color, will develop the color
14~20 positions merge, 50 DEG C or less are concentrated to dryness, spare.The preprocessing process of the Sephadex LH-20 gel
It was impregnated for 24 hours for methanol, upper prop, and was washed till to instill in water with methanol and be balanced each other without muddiness, then with initial flow.
Step 6: gained colour developing position in step 5 being separated through HPLC and is prepared, is with acetonitrile and 0.1% formic acid volume ratio
Two kinds of new alkaloids compounds of the invention, normalizing is prepared as mobile phase, Detection wavelength 210,280nm, separation in 30:70
It is 90~99% that method, which measures purity,.
The anti-inflammatory effect of new alkaloids compound of the present invention.
1, main material.
1.1, it drug and reagent: tests new alkaloids compound used and is prepared by the above method, purity is 90~99%, essence
It is close to weigh, solution needed for being diluted to following each dosage groups with DMSO.(U.S. Hyclone is public for DMEM high glucose medium, fetal calf serum
Department)；Penicillin, streptomysin (Hangzhou Chinese holly company)；LPS (Sigma Co., USA)；IL-6,TNF-α,PGE2ELISA examination
Agent box (Cayman company, the U.S.)；Cell pyrolysis liquid, Griess reagent (green skies Bioisystech Co., Ltd)
1.2 cell strains: RAW264.7 macrophage (U.S.'s ATCC cell bank).
1.3 groupings: being divided into control group, LPS group and experimental group, and each one group.
2 experimental methods.
The fetal calf serum of l0%, l% antibiotic (100U/mL penicillin is added in 2.1 cell culture, DMEM high glucose medium
With 100 μ g/mL streptomysins), it is placed in 37.5%, CO2It is cultivated in incubator.
2.2MTT colorimetric method for determining cell viability, above-mentioned three groups of difference logarithmic growth phase RAW264.7 macrophage inoculation
In 96 well culture plates, cell density be 1 × 104/mL, every 100 μ L of hole, 37 DEG C of temperature, 5%CO2Under the conditions of overnight incubation
Afterwards, experimental group be added various concentration two kinds of new alkaloids compound oleraindole A (10-100 μM) of the present invention or
The LPS of final concentration of 1 μ g/mL is separately added into after oleraindole B (10-100 μM), incubation 1h to LPS group and experimental group, separately
If zeroing group (culture solution of the solvent containing DMSO), every group sets 3 multiple holes, investigates the influence being added after drug to cell.It is above-mentioned each
After group cell culture for 24 hours, the addition 20 μ L of 5mg/mL MTT in each hole cell, 37 DEG C of temperature, 5%CO2Under the conditions of continue to be incubated for
After 4h, culture is terminated, inhales and abandons liquid in hole, 100 μ L dimethyl sulfoxides (DMSO) are added in every hole, vibrate 10min, make to tie into the cell
It is brilliant sufficiently to dissolve, each hole light absorption value of measurement at microplate reader 570nm wavelength.
2.3 measure the content of NO using Ge Lisi (Griess) method, investigate two kinds of new alkaloids compounds pair in the present invention
The inhibiting effect of the NO yield of the mouse macrophage RAW264.7 of LPS induction.After mouse macrophage RAW264.7 passage
It is cultivated in the sugared cell culture medium DMEM of height containing 10% fetal calf serum, two kinds of new lives of the present invention of various concentration are added in experimental group
Alkaloids compound oleraindole A (10-50 μM) or oleraindole B (10-50 μM), at 37 DEG C, 5%CO2Under the conditions of
It is incubated for after 1h and induces inflammatory reaction with LPS (final concentration of 1 μ g/mL), collect supernatant afterwards for 24 hours, every group of processing repeats 3 holes.
Griess method measures the content of NO in cell supernatant, is lured according to various concentration two kinds of new alkaloids compounds of the invention LPS
The influence of the RAW264.7 cell release NO led, to reflect NO level.
2.4ELISA method measures inflammatory factor IL-6, TNF-α and inflammatory mediator PGE2: logarithmic growth phase RAW264.7 is huge
Phagocyte is inoculated in 24 well culture plates, and cell density is 1 × 105A/mL, every hole 1mL, 37 DEG C of temperature, 5%CO2Under the conditions of train
It supports overnight, two kinds of new alkaloids compound oleraindole A (10-50 μM) of the invention or oleraindole is added in experimental group
B (10-50 μM) after cultivating 1h, is added LPS (final concentration of 1 μ g/mL) in every hole, is incubated for altogether for 24 hours, every group of processing repeats 3 holes.
ELISA method measure the IL-6 of two breeds of horses bitterroot source new alkaloids treated RAW264.7 macrophages secrete, TNF-α and
PGE2Content.
3 experimental results.
The experimental results showed that proliferation of the two kinds of new alkaloids compounds of the invention to the LPS macrophage RAW264.7 induced
It is safe and non-toxic without influence；And excess inflammatory cytokine produced by the macrophage RAW264.7 of LPS induction can be effectively suppressed
IL-6, TNF-α and inflammatory mediator NO, PGE2, and be in concentration dependant.
Comparative survival rate of cells experimental result is as shown in table 2.
Table 2: influence of the present invention to RAW264.7 macrophage relative survival rate.
Note:*P < 0.05 compared with the control group (high concentration group has significant difference),
3 are shown in Table using the content experimental result of Ge Lisi (Griess) method measurement NO.
Table 3: influence (mean ± standard deviation, n=3) of the present invention to the LPS RAW264.7 cell release NO induced
Note:*P < 0.05 compared with the control group,#P < 0.05 is compared with LPS group.
ELISA method measures inflammatory factor IL-6, TNF-α and inflammatory mediator PGE2The results are shown in Table 4.
Table 4: IL-6, TNF-α and PGE of the present invention to the LPS RAW264.7 cell secretion induced2The influence of content is (
Number ± standard deviation, n=3).
Note:*P < 0.05 compared with the control group,#P < 0.05 is compared with LPS group.
The antitumor action of new alkaloids compound of the present invention.
1 main material.
1.1 drugs and reagent: it tests new alkaloids compound used and is prepared by the above method, purity is 90~99%, essence
It is close to weigh, solution needed for being diluted to following each dosage groups with DMSO.(U.S. Hyclone is public for DMEM high glucose medium, fetal calf serum
Department)；Penicillin, streptomysin (Hangzhou Chinese holly company)；
1.2 cell strains: Human colon adenocarcinoma cell line Caco-2, human breast cancer cell line Bcap-37, human gastric cancer cells BGC-823, people's lung
Adenocarcinoma cell SPC-A1, human liver cancer cell BEL-7402, human cervical carcinoma cell Hela-229, ovarian cancer cell Ho-8910, the mankind
Oral cavity epidermoid carcinoma cell KB (Chinese Academy of Sciences's Shanghai cell bank).
1.3 groupings: it is divided into control group, experimental group and zeroing group (culture solution of the solvent containing DMSO).
2 experimental methods.
The fetal calf serum of l0%, l% antibiotic (100U/mL penicillin is added in 2.1 cell culture, DMEM high glucose medium
With 100 μ g/mL streptomysins), it is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
2.2MTI method detect cell Proliferation, logarithmic growth phase cell inoculation in 96 well culture plates, cell density be 1 ×
104A/mL, every 100 μ L of hole, 37 DEG C of temperature, 5%CO2Under the conditions of after overnight incubation, the present invention of various concentration is added in experimental group
Two kinds of new alkaloids compounds, every group sets 3 multiple holes, and dosing is placed on 37 DEG C, 5%CO248h is cultivated in incubator.By drug containing
Culture solution sucks, and serum-free medium and MTT (whole mass concentration is 5mg/mL) total 100mL that volume ratio is 4:1 is added, and continues
It is incubated for 4h, after carefully sucking supernatant, 150 μ L of DMSO is added in every hole, is put on oscillator and is shaken so that crystallization is completely dissolved
(5min), microplate reader detect absorbance (A) value in each hole under 570nm wavelength.Then, it is raw to calculate each concentration compound on intracellular
Long inhibiting rate, inhibiting rate formula: inhibitory rate of cell growth=(1-AMedicine feeding hole/AControl wells) × 100% reapplies the processing of SPSS software
Inhibiting rate is made curve to drug concentration, calculates IC by data50Value.
3 experimental results.
The experimental results showed that two kinds of new alkaloids compounds of the invention are to Human colon adenocarcinoma cell line Caco-2, human breast cancer cell
MCF-7, human gastric cancer cells BGC-823, human lung adenocarcinoma cell SPC-A1, human liver cancer cell BEL-7402, human cervical carcinoma cell
Hela-229, ovarian cancer cell Ho-8910, Human Oral Cavity epidermoid carcinoma cell KB, proliferation are inhibited, and with drug
Concentration increases, and inhibiting rate is also significantly raised, that is, is in concentration dependant.Two kinds of new alkaloids compounds of the invention are to above-mentioned eight kinds of tumours
Cell IC50Value is shown in Table 5.
The inhibiting effect of the two kinds of new alkaloids compound on tumor cell of the invention of table 5.
The neuroprotection of new alkaloids compound of the present invention.
1 main material.
1.1 drugs and reagent: it tests new alkaloids compound used and is prepared by the above method, purity is 90~99%, essence
It is close to weigh, solution needed for being diluted to following each dosage groups with DMSO.(U.S. Hyclone is public for DMEM high glucose medium, fetal calf serum
Department)；Penicillin, streptomysin (Hangzhou Chinese holly company), phosphate buffer (PBS), (Wuhan doctor's moral Co., Ltd), ROS
Detection kit (the green skies Reagent Company in Haimen)
1.2 cell strains: human neuroblastoma cells' strain (SH-SY5Y, IMR-32) (Chinese Academy of Sciences's Shanghai cell
1.3 groupings: it is divided into control group, H2O2Damage model group and experimental group.
2 experimental methods.
The fetal calf serum of l0%, l% antibiotic (100U/mL penicillin is added in 2.1 cell culture, DMEM high glucose medium
With 100 μ g/mL streptomysins), it is placed in 37 DEG C, cultivates in 5%CO2 incubator.
2.2MTT colorimetric method for determining cell viability, above-mentioned three groups of difference logarithmic growth phase SH-SY5Y cell and IMR-32
For cell inoculation in 96 well culture plates, cell density is 1 × 104A/mL, every 100 μ L of hole, 37 DEG C of temperature, under the conditions of 5%CO2
After overnight incubation, two kinds of new alkaloids compound oleraindole A (5-50 μM) of the present invention of various concentration are added in experimental group
Or to H after oleraindole B (5-50 μM), incubation 1h2O2Group and experimental group are separately added into the H of final concentration of 800 μM/L2O2,
It separately sets zeroing group (culture solution of the solvent containing DMSO), every group sets 3 multiple holes, investigates the influence being added after drug to cell.It is above-mentioned
After group of cells culture for 24 hours, the addition 20 μ L of 5mg/mL MTT in each hole cell, 37 DEG C of temperature, 5%CO2Under the conditions of continue to incubate
After educating 4h, culture is terminated, inhales and abandons liquid in hole, 100 μ L dimethyl sulfoxides (DMSO) are added in every hole, vibrate 10min, make intracellular
Crystallization is sufficiently dissolved, each hole light absorption value (A) value of measurement at microplate reader 450nm wavelength, calculating cell survival rate, and cell survival rate=
(AH2O2Damage-A blank)/(A control-A blank).
2.3DCFH-DA method detection SH-SY5Y cell and the intracellular ROS of IMR-32, group of cells are incubated after giving respective substance
It educates for 24 hours, incubation terminates preceding 30min, and each hole is added DCFH-DA, makes final concentration of 10 μm of ol/L, continue to be incubated for 30min in 37 DEG C,
Cell is collected, PBS is washed 2 times, and the cell suspension of same concentrations is made in group of cells by cell count.100 μ L cell suspensions are taken to examine
Survey fluorescence intensity, excitation wavelength 485nm, launch wavelength 538nm.With control group fluorescence intensity be 100%, remaining each group with compare
Group fluorescence intensity compares, and calculates ROS variation intracellular.
2.4INT chromogenic reaction method measures the burst size of LDH, removes above-mentioned control group, H2O2Outside damage model group and experimental group,
It separately sets up blank control group (blank control group not inoculating cell), respective substance culture is added for 24 hours in group of cells, takes each hole supernatant
For 120 μ L into 96 new orifice plates, the LDH detection working solution for adding 60 μ L to prepare is protected from light incubation at room temperature 30min, with more at 490nm
Function microplate reader measures A value, calculates the LDH burst size percentage relative to control tube.LDH release rate=(A administration-A is empty
It is white)/(A control-A blank) 3 experimental results.
Comparative survival rate of cells experimental result is as shown in table 6.
Table 6: influence of the present invention to human neuroblastoma cells' strain SH-SY5Y and IMR-32 comparative survival rate of cells.
Note:*P < 0.05 and H2O2Damage model group compares.
SH-SY5Y cell and the intracellular ROS amount testing result of IMR-32 are as shown in table 7.
Table 7: influence of the present invention to human neuroblastoma cells' strain intracellular ROS amount of SH-SY5Y and IMR-32.
Note:*P < 0.05 compared with the control group,#P < 0.05 and H2O2Damage model group compares
The results are shown in Table 8 for the influence of SH-SY5Y cell and the intracellular LDH release of IMR-32.
Table 8: influence of the present invention to the intracellular LDH release of human neuroblastoma cells' strain SH-SY5Y and IMR-32.
Note:*P < 0.05 compared with the control group,#P < 0.05 and H2O2Damage model group compares
In conclusion the present invention provides two kinds of new indoles alkaloid compounds and its extraction separation method, successively use
Water boiling and extraction, silica gel column chromatography, polyamide column chromatography, compression leg and Sephadex LH-20 purifying, liquid phase separation system in ODS
Standby, successful isolated two kinds of new alkaloids compounds, this method is easy, quickly, environmental protection, and it is isolated through this method
Compound purity is higher, due to two kinds of compound chemical structure uniquenesses of gained, extracts from conventional Chinese medicine purslane, tool
There is anti-inflammatory, antitumor and neuroprotection, therefore two kinds of new alkaloids of the invention and its salt and derivative can be used as naturally
Product develops new Chinese medicine, has broad prospects.
Claims (10)
1. indoles new alkaloids compound oleraindole A in a kind of purslane, which is characterized in that molecular formula are as follows:
C17H13NO4, chemical structural formula is as follows:
。
2. indoles new alkaloids compound oleraindole B in a kind of purslane, which is characterized in that molecular formula are as follows:
C18H15NO5, chemical structural formula is as follows:
。
3. the extraction separation method of the indoles new alkaloids compound as described in claim 1 and 2 is any, which is characterized in that
The following steps are included:
Step 1 takes the dry medicinal material of purslane, and using water boiling and extraction, Aqueous extracts filtering, merging filtrate directly heats concentration, puts
It is cool to room temperature, it is spare to obtain medical fluid；
Step 2, by silicagel column on concentrate in step 1, eluted with ethyl acetate, ethyl acetate be recovered under reduced pressure to medicinal extract, obtains
Ethyl acetate extract；
Step 3 separates ethyl acetate extract in step 2 through polyamide column, using alcohol-water gradient elution, by 70% ethyl alcohol
Upper silicagel column after the coloured moiety merging of elution is evaporated, with ethyl acetate, acetate-methanol gradient elution, through thin-layer chromatography
The coloured moiety of ethyl acetate extract is merged, is concentrated to dryness by detection, colour developing, spare；
Step 4, by gains in step 3 pretreated ODS column (Octadecylsilyl, octadecylsilane bonded silica again
Glue filler) chromatography obtains several elution positions, detected through thin-layer chromatography with methanol-water gradient elution, develops the color, and it will
The elution position of colour developing is concentrated to dryness, and it is spare to obtain concentrate；
Step 5, by gained concentrate pretreated Sephadex LH-20(hydroxypropyl sephadex in step 4), with first
Alcohol elution, is detected through thin-layer chromatography, develops the color, the elution position of colour developing is concentrated to dryness respectively, it is spare to obtain concentrate；
Step 6 carries out HPLC (efficient liquid phase) separation preparation to gained concentrate in step 5, using -0.1% formic acid of acetonitrile as stream
It is dynamic mutually to carry out isocratic elution, two kinds of new indoles alkaloid compounds are prepared.
4. the extraction separation method of indoles new alkaloids compound as claimed in claim 3, which is characterized in that the step 1
Middle water boiling and extraction 2 times decocts 2 hours every time, and water consumption is 8~16 times of medicinal material.
5. the extraction separation method of indoles new alkaloids compound as claimed in claim 3, which is characterized in that the step 2
In ethyl acetate used flowing phase elution program be isocratic elution；Silica gel mesh number 100-200 mesh.
6. the extraction separation method of indoles new alkaloids compound as claimed in claim 3, which is characterized in that the step 3
In second alcohol and water used volume ratio, 0:100,30:70,50:50,70:30 and 100:0；The volume ratio of ethyl acetate and methanol
For 5:1,2:1 and 1:2；Silica gel mesh number 200-300 mesh.
7. the extraction separation method of indoles new alkaloids compound as claimed in claim 3, which is characterized in that the step 4
Preprocessing process with ODS and Sephadex LH-20 gel in step 5 is that methanol impregnated 24 hours, and upper prop is washed with methanol
It balances each other to instilling in water without muddiness, then with initial flow.
8. the extraction separation method of indoles new alkaloids compound as claimed in claim 3, which is characterized in that the step 4
In the volume ratio of first alcohol and water used be 70:30,80:20,85:15,90:10 and 100:0；Filler particle size is 40~70 μm.
9. the extraction separation method of indoles new alkaloids compound as claimed in claim 3, which is characterized in that the step 5
In methanol elution program used be isocratic elution；
- 0.1% formic acid volume ratio of acetonitrile used is 30:70 in the step 6, and two kinds of Compound Retention times are respectively 7.53min
And 8.86min.
10. indoles new alkaloids compound can be used for preparing anti-inflammatory, antitumor and neural as described in claim 1 and 2 is any
The drug of protection.
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