CN116284005B - New alkaloid in herba Portulacae and its extraction and separation method - Google Patents

New alkaloid in herba Portulacae and its extraction and separation method Download PDF

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CN116284005B
CN116284005B CN202310386742.1A CN202310386742A CN116284005B CN 116284005 B CN116284005 B CN 116284005B CN 202310386742 A CN202310386742 A CN 202310386742A CN 116284005 B CN116284005 B CN 116284005B
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英哲铭
谢明
王呈玉
英锡相
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Liaoning University of Traditional Chinese Medicine
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Abstract

The invention relates to the field of traditional Chinese medicine extraction and separation, in particular to novel alkaloid extracted, separated and identified from purslane and an extraction and separation method thereof. The molecular formula of the novel compound is C 7 H 11 N 3 O 2 Designated as 1,5a,6,7,8 a-hexahydropyrroo [3,4-d ]][1,3]diazepine-6,8-diol. Also provides an extraction and separation method of the novel compound, which sequentially adopts alcohol extraction, polyamide column chromatography, silica gel column chromatography, ODS medium-pressure column purification and liquid phase separation. The structure adopts 1 H‑NMR、 13 The method of C-NMR and two-dimensional nuclear magnetic spectroscopy was determined to be a new alkaloid. The compound has potential anti-inflammatory and antioxidant activities, provides a preparation method, and provides a lead and theoretical basis for developing new drugs and developing new components.

Description

New alkaloid in herba Portulacae and its extraction and separation method
Technical Field
The invention relates to the field of traditional Chinese medicine extraction and separation, in particular to alkaloid extracted, separated and identified from purslane medicinal materials and an extraction and separation method thereof.
Background
Herba PortulacaePortulaca oleraceaL.), also known as herba Portulacae, a purslane family plant. The purslane is drought-resistant, waterlogging-resistant, light-resistant, wide in distribution, rich in resources, and attractive as a wild plant for both medicine and food, and the dried overground part of the purslane received in pharmacopoeia of the people's republic of China of 2015 edition is used as a medicine, has the effects of clearing heat and detoxicating, cooling blood and stopping bleeding, stopping diarrhea and the like, and is used for treating heat toxin bloody dysentery, carbuncle and furuncle, eczema, erysipelas, snake and insect bite, hematochezia, hemorrhoidal bleeding, metrorrhagia and the like.
Modern pharmacological researches of purslane show that it has the functions of resisting inflammation, relieving pain, resisting bacteria and viruses, reducing blood pressure, reducing blood fat, resisting oxidation and cancer, relaxing skeletal and smooth muscles, regulating immune function and the like. The researches show that the purslane has a plurality of chemical components which provide a material basis for various pharmacological actions, and the main chemical components of the purslane comprise flavonoids, coumarin, terpenes, steroids, alkaloids, amino acids, various pigments, minerals and the like. Wherein the alkaloid is the main chemical component in purslane, and the alkaloid components reported at present comprise norepinephrine, dopamine, a small amount of dopa, adenosine, uracil, adenine, N-dicyclohexylurea, allantoin and N-trans-feruloyl tyramine; also cyclic dipeptide alkaloids and amide alkaloids: purslane amide a-S.
Most of the chemical components separated from purslane are known at present, and the structural novelty is low, so that development and separation of new compounds in purslane are needed.
Disclosure of Invention
Aiming at the problems, the invention provides a novel compound extracted and separated from purslane, and researches show that the novel compound has anti-inflammatory and antioxidant effects, and simultaneously provides a simple, rapid, environment-friendly and high-purity extraction and separation method aiming at the novel compound.
To achieve the above object, the present invention provides novel compounds of formula C 7 H 11 N 3 O 2 Designated as 1,5a,6,7,8 a-hexahydropyrroo [3,4-d ]][1,3]diazepine-6,8-diol. The chemical structural formula is as follows:
。
in order to achieve the above purpose, the invention also provides a method for extracting and separating the novel alkaloid from the purslane, which comprises the following specific steps:
step 1, taking dry purslane medicinal materials, extracting with alcohol, filtering alcohol extract, mixing filtrates, directly heating and concentrating, and cooling to room temperature to obtain medicinal liquid for later use;
step 2, evaporating the liquid medicine in the step 1, then loading the liquid medicine on a silica gel column, eluting with ethyl acetate, and recovering ethyl acetate under reduced pressure to obtain an ethyl acetate extract;
separating the ethyl acetate extract in the step (2) by a polyamide column, adopting ethanol-water gradient elution to obtain a plurality of elution parts, evaporating 50% ethanol part to dryness, separating by a silica gel column chromatography, wherein silica gel is 200-300 meshes, sequentially carrying out gradient elution by using ethyl acetate, ethyl acetate-methanol and water-injected ethyl acetate-methanol to obtain a plurality of elution parts, detecting by thin layer chromatography, developing, merging and evaporating the developed parts, separating by the silica gel column chromatography, and concentrating to dryness under reduced pressure below 40 ℃ for later use;
step 4, separating the product obtained in the step 3 by chromatography of pretreated ODS column (octocrylylsilly, octadecyl silane bonded silica filler), eluting with methanol-water gradient to obtain a plurality of elution parts, detecting by thin layer chromatography, developing, and concentrating each developed elution part under reduced pressure to dryness to obtain concentrate for later use;
step 5, separating the concentrate obtained in the step 4 through pretreated hydroxypropyl sephadex chromatography, eluting with methanol-water isocratic, detecting through thin layer chromatography, developing color, and concentrating the developed eluting parts under reduced pressure respectively until the developed eluting parts are dry to obtain the concentrate for later use;
and 6, separating and preparing the concentrate obtained in the step 5 through HPLC (high performance liquid phase), and performing isocratic elution by taking methanol-0.1% formic acid water as a mobile phase to finally obtain the compound.
The pretreatment process of the ODS comprises the steps of soaking the ODS in methanol for 24 hours, loading the ODS on a column, washing the ODS with the methanol until the ODS is dripped into water to avoid turbidity, and balancing the ODS by an initial mobile phase.
Compared with the prior art, the invention has the beneficial effects.
The separation and pharmacological activity research of the purslane new alkaloid are not reported by the existing journal of papers; the invention provides an alkaloid from purslane and an extraction and separation method aiming at the novel compound, which adopts alcohol extraction, polyamide column, silica gel column chromatography, ODS medium pressure column and HPLC for separation and purification and preparation, and successfully extracts and separates the novel compound.
Drawings
FIG. 1 shows the novel alkaloids of the present invention 1 H-NMR spectrum.
FIG. 2 shows the novel alkaloids of the present invention 13 C-NMR spectrum.
FIG. 3 is a DEPT135 spectrum of the novel alkaloid of the present invention.
FIG. 4 shows the novel alkaloids of the present invention 1 H- 1 H COSY spectral diagram.
FIG. 5 is a chart of HMBC spectra of the novel alkaloids of the present invention.
FIG. 6 is a HSQC spectrum of the novel alkaloid of the present invention.
FIG. 7 is a ROESY spectrum of the novel alkaloid of the present invention.
Detailed Description
The present invention provides novel compounds of the formula C to achieve the above objects 7 H 11 N 3 O 2 Designated as 1,5a,6,7,8 a-hexahydropyrroo [3,4-d ]][1,3]diazepine-6,8-diol. The chemical structural formula is as follows:
。
the novel compounds are designated as 1,5a,6,7,8 a-hexahydrooxypyrroo [3,4-d ] according to the structure][1,3]Diazepine-6,8-diol, table 1 shows the nuclear magnetic data of the novel compounds: 1 H-NMR 13 C-NMR in DMSO.
Table 1: nuclear magnetic data of novel compounds of the invention
。
Structural identification of the compounds of the present invention is shown in FIGS. 1-7.
1,5a,6,7,8,8a-hexahydropyrrolo[3,4-d][1,3]diazepine-6,8-diol: the light brown paste is easy to dissolve in water, insoluble and slightly soluble in methanol. After spotting on a silica gel thin layer plate, spraying diluted bismuth potassium iodide test solution spots to display orange yellow, and prompting the compound to be a biological alkali component. The molecular weight is 169.0851. Bonding of 1 H-NMR， 13 C-NMR and DEPT data, it is presumed that the compound may have the formula C 7 H 11 N 3 O 2 The unsaturation was 4. 13 C-NMR spectrum and DEPT spectrum show 7 carbon signals, respectively 4 methine carbons [ (]δ:25.31;69.52;67.39;57.41 3 allyl carbons%δ：152.97；139.62；123.75）。
1 H-NMR spectra showed 3 alkene hydrogen signals of delta H 8.39 (1H, s), 5.88 (1H, s), 4.59 (1H, m), 4 methine signals are delta H 2.96 (1H, m)，4.14 (1H，d,J=6.36Hz)，4.34 (1H, d,J=6.06 Hz), 3.55 (1H, m), one active hydrogen δ H 8.62 (1H, s, broad peak), it is presumed that the active hydrogen is linked to N. From the correlation peak of HMBC spectra, it is shown that H-4 (delta H 5.88 And C-2 (delta) C 152.97 Related to H-2 (delta) H 8.39 And C-8a (delta) C 57.41 Related to H-8a (delta) H 3.55 And C-5 (delta) C 123.75 If C-2 is in the low field, C-2 is presumed to be connected to N. According to 1 H- 1 The correlation peak of the H COSY spectrum shows that H-4 (delta) H 5.88 And H-5 (delta) H 4.59 A seven membered N-containing heterocycle, and the heterocycle contains an alkene. H-6 (delta) H 4.14 And C-8 (delta) C 67.39)、C-8a (δ C 57.41 Related to H-8 (delta) H 4.34 And C-5a (delta) C 25.31 If C-6 and C-8 are in the low field, C-6 and C-8 are presumed to be linked to N. According to 1 H- 1 The correlation peak of the H COSY spectrum shows that H-5a (delta) H 2.96 And H-6 (delta) H 4.14 A five membered heterocyclic ring containing N). Furthermore, HMBC spectral phaseThe off-peak indicates H-5 (delta) H 4.59 And C-6 (delta) C 69.52 A) correlation. Thus, based on the above information, the novel compound can be determined to have the above structure.
The invention also provides an extraction and separation method of the novel compound, which comprises the following specific steps of.
Step 1: weighing 80kg of dry purslane, reflux-extracting with 50% ethanol, wherein the ethanol consumption is 8-16 times of that of the purslane, reflux-extracting twice, each time for 2 hours, recovering ethanol under reduced pressure, and cooling to room temperature to obtain a liquid medicine for standby.
Step 2: evaporating part of the liquid medicine obtained in the step 1, separating by silica gel column chromatography, and isocratically eluting with ethyl acetate, wherein silica gel is 100-200 meshes, and recovering ethyl acetate to extract under reduced pressure below 40deg.C to obtain ethyl acetate extract.
Step 3: separating the ethyl acetate extract in the step 2 by a polyamide column, adopting ethanol-water (0:100, 30:70, 50:50, 70:30, 100:0, v/v) gradient elution, evaporating 50% ethanol part to dryness, separating by a silica gel column chromatography, wherein silica gel is 200-300 meshes, sequentially adopting ethyl acetate, ethyl acetate-methanol (5:1, 2:1, 1:2, v:v) and water-injected ethyl acetate-methanol gradient elution to obtain 15 parts (namely 15 bottles, 400mL each), detecting by thin layer chromatography, developing, combining developed 1-3 elution parts, and concentrating the combined 1-3 parts to dryness under reduced pressure below 40 ℃ for later use.
Step 4: separating the product obtained in the step 3 by pretreated ODS medium-pressure column chromatography, wherein the granularity of the filler is 20-40 μm, gradient eluting with methanol-water (84:16, 95:5, 97:3, 100:0, v/v) (pressurizing to make the flow rate be 1mL/min and the temperature be room temperature), obtaining 10 parts (namely, gradient eluting to obtain 10 bottles with 200mL each bottle), detecting by thin layer chromatography, developing, reserving the developed 1-5 parts, and concentrating to dryness under reduced pressure below 50 ℃ for standby. The pretreatment process of the ODS comprises the steps of soaking the ODS in methanol for 24 hours, loading the ODS on a column, washing the ODS with the methanol until the ODS is dripped into water to avoid turbidity, and balancing the ODS with an initial mobile phase.
Step 5: separating the concentrate obtained in step 4 by pre-treated Sephadex LH-20 (hydroxypropyl dextran gel) chromatography, eluting with methanol-water isocratically, detecting by thin layer chromatography, developing, and concentrating the developed elution parts under reduced pressure to dry to obtain concentrate.
Step 6: the novel compounds obtained in step 5 were prepared by HPLC separation with methanol: 0.1% formic acid (95:5) is used as a mobile phase, the detection wavelength is 210 and 254nm, the novel compound is prepared by separation, and the purity measured by a normalization method is 90-99%.
Anti-inflammatory effects of the novel compounds of the present invention.
1. The main material.
1.1 Medicine and reagent: the novel compounds used in the experiments are prepared by the method, the purity is more than 97%, the novel compounds are precisely weighed, and the novel compounds are diluted into the solutions required by the following dosage groups by using DMSO and PBS. Fetal bovine serum (Gibco, USA); CCK-8 kit (Boster Co., U.S.A.); DMSO (Sigma-Aldrich Co., USA); DMEM high sugar culture medium, LPS and IL-1βAnd TNF-αELISA kit (Soy technologies Co., ltd.); penicillin, streptomycin (Hangzhou holly company); PBS (China fir gold bridge biotechnology Co., ltd., beijing).
1.2 Cell lines: RAW264.7 macrophages (american ATCC cell bank).
1.3 Grouping: the control group, the LPS group and the experimental group are divided into one group.
2. Experimental methods.
2.1 Cell culture: DMEM high sugar medium, added with 1.0% fetal bovine serum, and 1% antibiotics (100U/mL penicillin and 100. Mu.g/mL streptomycin), placed at 37℃and 5% CO 2 Culturing in an incubator.
2.2 CCK-8 reagent method for measuring cell viability: each group was inoculated with RAW264.7 macrophages in the logarithmic phase into 96-well plates at a cell density of 1X 10 4 100 mu L per well at 37℃in 5% CO per mL 2 After overnight culture under the condition, adding sample solutions (5 mu M-100 mu M) with different concentrations into the experimental group, incubating for 1h, respectively adding LPS with the final concentration of 1 mu g/mL into the LPS group and the experimental group, additionally arranging zeroing groups (culture solution containing DMSO solvent), arranging 3 multiple holes in each group, and observing the fineness of the mixed solution after adding the medicineInfluence of cells. After culturing the above groups for 24 hours, 10. Mu.L of CCK-8 was added to each well cell at 37℃and 5% CO 2 After incubation for a further 4h under conditions, the absorbance of each well was determined using an enzyme-labeled instrument at a wavelength of 570 nm.
2.3 ELISA method for determining inflammatory factor IL-1β、TNF-α: RAW264.7 macrophages in logarithmic growth phase were inoculated into 96-well culture plates with a cell density of 2X 10 5 1 mL/well at 37℃with 5% CO 2 After overnight incubation under conditions, the experimental groups were incubated with different concentrations of sample solution (1. Mu.M-20. Mu.M) for 1h, LPS (final concentration 1. Mu.g/mL) was added to each well and incubated for 3.25h with 3 duplicate wells per group. ELISA method for measuring IL-1 secreted by RAW264.7 macrophages after treatment of novel compounds derived from purslaneβAnd TNF-αIs contained in the composition.
3. Experimental results.
Experimental results show that the novel compound has no influence on proliferation of macrophage RAW264.7 induced by LPS, and is safe and nontoxic; can effectively inhibit excessive inflammatory cytokine IL-1 generated by macrophage RAW264.7 induced by LPSβAnd TNF-αAnd is concentration dependent.
The results of the cell relative viability experiments are shown in Table 2.
Table 2: the invention affects the relative survival rate of RAW264.7 macrophages
。
Note that: average number ± SD of the average number,n=3， * P<0.05 compared to the control (significant differences in the high concentration group).
ELISA method for determining inflammatory factor IL-1βAnd TNF-αThe results are shown in Table 3.
Table 3: IL-1 secreted by RAW264.7 cells induced by LPSβAnd TNF-αInfluence of the content
。
Note that: * P<0.05 was compared with the control group, # P<0.05 was compared to the LPS group.
Anticholinesterase action of the novel compounds of the invention.
1. The main material.
1.1 Medicine and reagent: the new compound used in the experiment is prepared by the method, and the purity is 90-99%, sodium dihydrogen phosphate, disodium hydrogen phosphate (national pharmaceutical sciences chemical Co., ltd.), physostigmine (Han Xiang Biotechnology), phosphorus 5,5' -dithiobis (2-nitrobenzoic acid) (Dithiobisnitrobenzoic acid, DTNB, shanghai golden ear Biotechnology Co., ltd.), acetylcholinesterase (AChE) and thiocholine iodide (Acetylthiocholine iodide, ATCI, dalian Meen Biotechnology Co., ltd.).
1.2 Grouping: the negative control group, the positive control group and the experimental group are divided into one group.
2. Experimental methods.
2.1 Sample preparation, namely precisely weighing 1mg of the sample and 1mg of physostigmine, and preparing the sample and the physostigmine into five gradient concentrations of lmg/mL, 0.5mg/mL, 0.1mg/mL, 0.05mg/mL and 0.01mg/mL by taking methanol as a solvent. 7.039g of sodium dihydrogen phosphate and 5.996g of disodium hydrogen phosphate are respectively weighed precisely, distilled water is used for constant volume to 50mL, 3.40mL of sodium dihydrogen phosphate and 46.6mL of disodium hydrogen phosphate are taken, and 50mL of PBS (0.1M pH=8.0) is prepared; 0.0588g of DTNB is precisely weighed, 10mL of PBS is added to prepare a DTNB solution (15 mmol/L); precisely weighing 0.01g AChE, adding 10mLPBS, and preparing AChE solution (0.2 u/mL); 0.042g of ATCI was precisely weighed, and distilled water was used to determine the volume to 10mL to prepare an ATCI solution (15 mmol/L).
2.2 Modified Ellman method to determine anticholinesterase activity 140uL PBS (0.1M ph=8.0), 10uL DTNB (15 mmol/L), 15uL AChE (0.2 u/mL), 20 uL sample solution were added sequentially to 96 well elisa plate. The negative control experiments replaced the sample with methanol, and the positive control experiments replaced the sample with physostigmine. After incubation at 37℃for 10min, 10uLATCI (15 mmol/L) was added. After incubation at 20℃for 10min, the absorbance was measured at 410nm using a microplate reader. Inhibition ratio (%) = (blank-sample)/blank×100% was calculated according to the following formula.
3. Experimental results.
Experimental results show that the novel compound has anticholinesterase effect.
The experimental results are shown in table 4.
Table 4: the anticholinesterase activity of the invention
。
In summary, the invention provides a special compound and an extraction and separation method thereof, which sequentially adopts 50% ethanol reflux extraction, polyamide column chromatography, silica gel column chromatography and ODS medium-pressure column separation and purification, and the method is simple, convenient, rapid and environment-friendly, and the compound separated by the method has higher purity, and the special compound, the salt and the derivative thereof can be used as a natural product for developing a novel traditional Chinese medicine because the obtained compound has unique chemical structure and has anti-inflammatory, anti-tumor and anti-oxidation effects.

Claims (6)

1. A novel alkaloid isolated from purslane herb, characterized by the following molecular formula: c (C) 7 H 11 N 3 O 2 Designated as 1,5a,6,7,8 a-hexahydropyrroo [3,4-d ]][1,3]diazepine-6,8-diol, having the chemical formula:
。
2. an extraction and separation method of alkaloid compounds separated from purslane herb according to claim 1, characterized in that the specific steps of the extraction and separation method include:
step 1, taking dry purslane medicinal materials, extracting with alcohol, filtering alcohol extract, mixing filtrates, directly heating and concentrating, and cooling to room temperature to obtain medicinal liquid for later use;
step 2, evaporating the liquid medicine in the step 1, then loading the liquid medicine on a silica gel column, eluting with ethyl acetate, and recovering ethyl acetate under reduced pressure to obtain an ethyl acetate extract;
step 3, separating the ethyl acetate extract in the step 2 by a polyamide column, wherein the volume ratio is 0: 100. 30: 70. 50: 50. 70:30 and 100:0, obtaining a plurality of elution parts, evaporating 50% ethanol part, and separating by silica gel column chromatography, wherein silica gel is 200-300 meshes, ethyl acetate is sequentially used, and the volume ratio is 5: 1. 2:1 and 1:2, gradient eluting with ethyl acetate-methanol and water-injected ethyl acetate-methanol to obtain a plurality of eluting parts, detecting by thin layer chromatography, developing, combining the developed parts, evaporating to dryness, separating by silica gel column chromatography, concentrating under reduced pressure below 40 ℃ until dryness is reserved;
step 4, separating the pretreated ODS column chromatography of the product obtained in the step 3 by using a volume ratio of 84: 16. 95: 5. 97:3 and 100:0, obtaining a plurality of elution parts, detecting by thin layer chromatography, developing, respectively concentrating the developed elution parts under reduced pressure until the elution parts are dried, and obtaining a concentrate for later use;
step 5, separating the concentrate obtained in the step 4 through pretreated hydroxypropyl sephadex chromatography, eluting with methanol-water isocratic, detecting through thin layer chromatography, developing color, and concentrating the developed eluting parts under reduced pressure respectively until the developed eluting parts are dry to obtain the concentrate for later use;
step 6, separating and preparing the concentrate obtained in the step 5 by HPLC, wherein the volume ratio is 95:5 methanol-0.1% formic acid water is used as a mobile phase for isocratic elution, and finally the compound is obtained.
3. The method according to claim 2, wherein the 50% ethanol is extracted under reflux for 2 times each for 2 hours in step 1, and the ethanol consumption is 10 times of that of the medicinal materials.
4. The extraction separation method of claim 2, wherein the mobile phase elution procedure used in step 2 is isocratic elution.
5. The extraction and separation method as claimed in claim 2, wherein the pretreatment process of the ODS and the dextran gel is that the ODS and the dextran gel are soaked in methanol for 24 hours, and then the mixture is put on a column, washed by the methanol until the mixture is dropped into water to have no turbidity, and then the mixture is balanced by an initial mobile phase.
6. Use of an alkaloid compound isolated from a purslane herb of claim 1 in the preparation of an anti-inflammatory, antioxidant drug.
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Title
马齿苋中两种酪胺类生物碱分离及鉴定;蒋明月;英锡相;;辽宁中医药大学学报(09);50-53 *
马齿苋化学成分研究;刘册家;刘佃雨;向兰;周文;邵宁宁;;中药材(11);第1689-1691页 *

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