CN116715708B - Three alkaloid compounds in purslane and extraction and separation method thereof - Google Patents
Three alkaloid compounds in purslane and extraction and separation method thereof Download PDFInfo
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- CN116715708B CN116715708B CN202310774655.3A CN202310774655A CN116715708B CN 116715708 B CN116715708 B CN 116715708B CN 202310774655 A CN202310774655 A CN 202310774655A CN 116715708 B CN116715708 B CN 116715708B
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- oleracrylimide
- ethanol
- extraction
- alkaloid compounds
- eluting
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Polymers & Plastics (AREA)
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- Nutrition Science (AREA)
- Mycology (AREA)
- Botany (AREA)
- Toxicology (AREA)
- Rheumatology (AREA)
- Pain & Pain Management (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the field of traditional Chinese medicine extraction and separation, in particular to three alkaloid compounds extracted, separated and identified from purslane and an extraction and separation method thereof. The molecular formulas of the three alkaloid compounds are C respectively 27 H 33 NO 15 ，C 28 H 35 NO 16 ，C 33 H 43 NO 20 Designated Oleracrylimide D, oleracrylimide E, oleracrylimide F. Also provides an extraction and separation method of the novel compound, which sequentially adopts alcohol extraction, macroporous resin chromatography, polyamide column chromatography, ODS medium-pressure column purification and liquid phase separation. The structure adopts 1 H‑NMR、 13 The C-NMR and two-dimensional nuclear magnetic spectrum analysis methods are determined to be three alkaloid compounds. 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
Technical Field
The invention relates to the field of traditional Chinese medicine extraction and separation, in particular to alkaloid compounds 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 has drought resistance and waterlogging resistance, wide distribution, abundant resources, and is paid attention to as a wild plant for both medicine and food, and the dry overground part of the purslane is loaded in the pharmacopoeia of the people's republic of China of 2020 edition and has the functions of clearing heat and detoxicatingIt has effects of cooling blood, stopping bleeding, and relieving dysentery, and can be used for treating toxic heat, bloody dysentery, carbuncle, furuncle, eczema, erysipelas, snake and insect bite, hematochezia, hemorrhoid, metrorrhagia, and bleeding.
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 research shows that the chemical components in the purslane 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
In order to solve the problems, the invention provides three novel alkaloid compounds extracted and separated from purslane, and researches show that all three novel alkaloid compounds have anti-inflammatory and anti-oxidation effects, and simultaneously provides a simple, rapid, environment-friendly and high-purity extraction and separation method for the novel alkaloid compounds.
To achieve the above object, the present invention provides three novel alkali compounds of the formula C respectively 27 H 33 NO 15 ，C 28 H 35 NO 16 ，C 33 H 43 NO 20 Designated Oleracrylimide D, oleracrylimide E, oleracrylimide F. The chemical structural formula is as follows:
。
in order to achieve the above purpose, the invention also provides three extraction and separation methods of alkaloid compounds in purslane, which specifically comprise the following 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 adding macroporous resin, eluting with ethanol, and recovering ethanol under reduced pressure to obtain an ethanol extract;
step 3, separating the ethanol extract in the step 2 by a polyamide column, adopting ethanol-water gradient elution to obtain a plurality of elution parts, evaporating the ethanol part to dryness, performing chromatography separation by a pretreated ODS column (Octadecylsilly, octadecylsilane chemically bonded silica filler), sequentially eluting by using methanol-water gradient to obtain a plurality of elution parts, detecting by thin layer chromatography, developing, respectively concentrating the developed elution parts to dryness under reduced pressure to obtain a concentrate for later use;
step 4, separating the concentrate obtained in the step 3 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 5, separating and preparing the concentrate obtained in the step 4 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 three alkaloid compounds of purslane are not reported by the existing journal of papers; the invention provides three alkaloid compounds from purslane and an extraction and separation method aiming at the novel compound, which adopts alcohol extraction, macroporous resin chromatography, polyamide column, hydroxypropyl dextran gel chromatography, ODS medium-pressure column and HPLC for separation, purification and preparation, and successfully extracts and separates out the novel compound.
Drawings
FIG. 1 is a UV spectrum of Oleracrylimide D of the present invention.
FIG. 2 is an infrared spectrum of Oleracrylimide D of the present invention.
FIG. 3 is a high resolution mass spectrum of Oleracrylimide D of the present invention.
FIG. 4 is an Oleracrylimide D of the present invention 1 H-NMR spectrum.
FIG. 5 is an Oleracrylimide D of the present invention 13 C-NMR spectrum.
FIG. 6 is a DEPT-135 spectrum of Oleracrylimide D of the present invention.
FIG. 7 is an Oleracrylimide D of the present invention 1 H- 1 H COSY spectral diagram.
FIG. 8 is a chart of HMBC spectra of Oleracrylimide D of the present invention.
FIG. 9 is a HSQC spectrum of Oleracrylimide D of the present invention.
FIG. 10 is a ROESY spectrum of Oleracrylimide D of the present invention.
FIG. 11 is an ultraviolet spectrum of Oleracrylimide E of the present invention.
FIG. 12 is an infrared spectrum of Oleracrylimide E of the present invention.
FIG. 13 is a high resolution mass spectrum of Oleracrylimide E of the present invention.
FIG. 14 shows Oleracrylimide E of the present invention 1 H-NMR spectrum.
FIG. 15 shows Oleracrylimide E of the present invention 13 C-NMR spectrum.
FIG. 16 is a DEPT-135 spectrum of Oleracrylimide E of the present invention.
FIG. 17 shows Oleracrylimide E of the present invention 1 H- 1 H COSY spectral diagram.
FIG. 18 is a chart showing the HMBC spectra of Oleracrylimide E of the present invention.
FIG. 19 is a HSQC spectrum of Oleracrylimide E of the present invention.
FIG. 20 is a ROESY spectrum of Oleracrylimide E of the present invention.
FIG. 21 is a UV spectrum of Oleracrylimide F of the present invention.
FIG. 22 is an infrared spectrum of Oleracrylimide F of the present invention.
FIG. 23 is a high resolution mass spectrum of Oleracrylimide F of the present invention.
FIG. 24 is an Oleracrylimide F of the present invention 1 H-NMR spectrum.
FIG. 25 is an Oleracrylimide F of the present invention 13 C-NMR spectrum.
FIG. 26 is a DEPT-135 spectrum of Oleracrylimide F of the present invention.
FIG. 27 is an Oleracrylimide F of the present invention 1 H- 1 H COSY spectral diagram.
FIG. 28 is a chart showing HMBC spectra of Oleracrylimide F of the present invention.
FIG. 29 is a HSQC spectrum of Oleracrylimide F of the present invention.
FIG. 30 is a ROESY spectrum of Oleracrylimide F of the present invention.
Detailed Description
The invention provides three new compounds with molecular formula of C 27 H 33 NO 15 ，C 28 H 35 NO 16 ，C 33 H 43 NO 20 The chemical structural formula of the compound is named Oleracrylimide D, oleracrylimide E and Oleracrylimide F:
。
the three amide alkaloid compounds are named Oleracrylamide D, oleracrylamide E and Oleracrylamide F according to the structures, and the nuclear magnetic data of the three alkaloid compounds are shown in Table 1: 1 H-NMR 13 C-NMR in DMSO.
Table 1: nuclear magnetic data of three alkaloid compounds of the invention
。
The structural identification of three alkaloid compounds in the invention is shown in figures 1-30.
Olearcrylimide D: yellow powder, easily soluble in water, 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. UV (H) 2 O) λ max 341 nm, IR (KBr)v max : 3477，3081，2933，1722，1639，1585，1247，1174 cm -1 . M/z 270.0771 [ M-C ] from negative ion UHPLC-ESI-QTOF/MS 12 H 21 O 11 ] - Fragment ion peak (calculated as C 15 H 12 NO 4 - 270.0768) deducing that its molecular formula is C 27 H 33 NO 15 From the NMR data, it was deduced that the unsaturation was 12. According to 13 C-NMR and DEPT-135 spectra, there are 27 carbon atoms in Oleracrylimide D, including 2 methylene groups: (delta) C 60.71 68.48), 18 methines: (delta) C 69.76 70.07, 73.27, 73.63, 75.48, 75.75, 76.31, 76.88, 103.37, 104.11, 107.81, 111.68, 115.63, 130.18, 144.01, wherein 115.63 and 130.18 are overlapping), 7 quaternary carbons: (delta) C 126.16 135.22, 143.72, 159.31, 164.31, wherein 126.16 and 143.72 are overlapping). According to 1 As can be seen from the H-NMR spectrum, delta H 7.57（2H，d，J = 8.5 Hz，H-2，H-6）、δ H 6.78 Hydrogen signal and delta of (2 h, d, j=8.5 hz, h-3, h-5) C 130.18 (overlap of C-2 and C-6), delta C 115.63 The carbon signals of (C-3 and C-5 overlap) can determine an AA 'BB' optical rotation system. 13 C-NMR Signal delta C 159 31 (C-4) is located in the low field region, indicating that C-4 is attached to one hydroxyl group. In addition, delta H 6.68 (1H, d, J=15.2 Hz, H-2') and delta H 7.63 The signal of (1H, d, J=15.2 Hz, H-3 ') and their coupling constants indicate the presence of a trans-substituted double bond, H-2' and C-1 (delta) according to HMBC display C 126.16 H-3 'is associated with C-2, C-6, indicating that C-3' is directly linked to C-1. Delta C 164.31 The peak of the (C-1 ') resonance was designated as an aminocarbonyl group, and the correlation of H-3' and HMBC of C-1' revealed that the double bond was linked to the aminocarbonyl group (. Delta.) C 164.31). In addition, protons are at delta H 8.17 (1H, s, H-3 '') and delta H 6.71 (1H, s, H-6 '), in combination with the HMBC correlation of H-3' with C-1' and C-5' and H-6' with C-2' and C-4', a typical tetra-substituted aromatic ring can be deduced. Delta C 135.22 (C-2 '') and delta C 143.72 (C-5 ''), respectively) 13 The C-NMR signals are in the low field region, indicating that C-2'' and C-5'' are attached to the hydroxyl groups. In addition, in 1 In the H-NMR and HMQC spectra, two isomeric proton signals (delta H 4.54, d, j=6.18 hz, h-2' "; 4.20, d, j=7.44 hz, h-2'' '' '') and carbon signal at δ C 104.11 (C-2 '' '), 103.37 (C-2' '' ''), two oxymethylenes (delta) H 3.45, d, J = 12.1 Hz, H-7′′′′a; δ H 3.70, dd, J = 12.2, 4.1 Hz, H-7′′′′b），（δ H 4.20, m, H-7'' '' a; 3.54, m, H-7'' '' b) and carbon signal at delta C 60.71 (C-7 ' ' ' '), 69.76 (C-7 ' ' ' ' '), and 8 oxymethyl hydrogen signals from delta H 3.14 to 3.50, carbon signal delta C 69.76 70.07, 73.27, 73.63, 75.48, 75.75, 76.31, 76.88, coupling constants of binding isomers (j=6.18, 7.44 Hz), indicating the presence of twoβ-D-glucopyranosyl, plus HMBC correlation from H-5 '"to C-2'" indicating twoβ-D-glucopyranosyl is attached at C-5' ". Furthermore, from H-2'' '' to C-4'' (delta) C 143.72 HMBC correlation to show that twoβ-D-glucopyranosyl group is attached to C-4 ''. Furthermore, the ROESY correlation from H-3'' to H-2 'and H-3' shows that the tetra-substituted benzene ring is attached to the acrylamide group and C-1'' is directly attached to the N atom. In summary, oleracrylimide D structure was determined.
Olearcrylimide E: yellow powder, easily soluble in water, 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. UV (H) 2 O) λ max 347 nm, IR (KBr)v max : 3469，2844，2348，1731，1683，1635，1280，1043 cm -1 . M/z 300.0877 [ M-C ] from negative ion UHPLC-ESI-QTOF/MS 12 H 21 O 11 ] - Fragment ion peak of (C as calculated result) 16 H 14 NO 5 - 300.0881) deducing that its molecular formula is C 28 H 35 NO 16 From the NMR data, it was deduced that the unsaturation was 12. According to 1 H-NMR 13 The C-NMR spectrum, oleracrylimide E structure, in addition to having a methoxy group, is closely related to Oleracrylimide D structure. 13 C-NMR and DEPT-135 spectra showed 28 carbon signals in Oleracrylimide E, including 1 methoxy group: (delta) C 55.79 2 methylene groups): (delta) C 60.70 68.28); 17 methine groups: (delta) C 69.84 70.05, 73.28, 73.61, 75.48, 75.75, 76.28, 76.93, 103.39, 104.22, 107.93, 111.66, 112.07, 115.58, 122.18, 143.98, wherein 115.58 is overlapping); 8 quaternary carbons: (delta) C 126.69, 126.71, 135, 32, 143.67, 143.73, 147.75, 148.68, 164.34). Delta in Oleracrylimide E C 55.79 and delta H 3.81 (3H, s) is a typical methoxy signal according to the sequence from delta H Methyl proton signal to C-3 (delta) of 3.81 C 147.75 The HMBC correlation relationship may be linked at the C-3 position. The typical ABX spin system in oleracyl imide E is a 1,3, 4-trisubstituted benzene ring, which can be moved through H-2 (delta H 7.29，d，J = 1.5 Hz）、H-5（δ H 6.78，d，J = 8.1 Hz) and H-6 (delta) H 7.17, dd, j=1.5, 8.1 Hz). In addition, oleracrylimide EβThe type and position of the D-glucopyranosyl group are the same as in Oleracrylimide D. Thus, the Oleracrylimide E structure was determined.
Olearcrylimide F: yellow powder, easily soluble in water, 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. UV (H) 2 O) λ max 336 nm, IR (KBr)v max : 3365，3010，2915，1770，1645，1537，1270，1070 cm -1 . M/z 270.0771 [ M-C ] from negative ion UHPLC-ESI-QTOF/MS 18 H 31 O 16 ] - Fragment ion peak of (C as calculated result) 15 H 12 NO 4 - 270.0773) deducing that its molecular formula is C 33 H 43 NO 20 From the NMR data, it was deduced that the unsaturation was 12. According to 1 H-NMR 13 C-NMR spectrum, oleracrylimide F was very similar in structure to Oleracrylimide D. However, oleracrylimide F contains 3β-D-glucopyranosyl group, delta, according to HMBC spectrum display H The isomer proton signal of 4.26 and C-5' ' ' (delta) C 143.91 For 3, cuesβ-D-glucopyranosyl group is attached to C-5' ″. 13 C-NMR and DEPT-135 spectra showed 33 carbon signals in Oleracrylimide F, including 3 methylene groups: (delta) C 61.01 61.08, 68.53), 23 methines: (delta) C 69.70 69.86, 73.27, 73.38, 74.69, 74.80, 74.84, 75.43, 75.73, 76.47, 76.74, 80.91, 103.06, 103, 43, 104.01, 107.69, 111.69, 115.76, 130.21, 144.01, wherein 115.76 and 130.21 are overlapping), 7 quaternary carbons: (delta) C 124.92, 126.11, 135.19, 143.70, 143.91, 159.32, 164.35). At the position of 1 In the H-NMR and HMQC spectra, three isomeric proton signals (delta H 4.26, d, j=6.18 hz, h-2' "; 4.56, d, j=7.5 hz, h-2' "', 4.27, d, j=6.18 hz, h-2 '" ' ') and carbon signal at δ C 103.06（C-2′' '), 103.43 (C-2 ' ' ' ' '), 104.01 (C-2 ' ' ' ' ' '), three oxymethylenes (delta) H 4.21, d, J = 12.3 Hz, H-7′′′′a; 3.56, m, 7′′′′b），（δ H 3.45,m, H-7′′′′′a; 3.71, m, H-7′′′′′b），（δ H 3.65, m, H-7'' '' a; 3.71, m, H-7'' '' b) and delta C 68.53 (C-7 ' ' ' '), 61.08 (C-7 ' ' ' ' '), 61.01 (C-7 ' ' ' ' ' ') and 12 oxymethyl groups from delta H Hydrogen signal and delta of 3.00 to 3.81 C 69.70 The carbon signals of 69.86, 73.27, 73.38, 74.69, 74.80, 74.84, 75.43, 75.73, 76.47, 76.74, 80.91, combined with the coupling constants of the isomeric proton signals (j=6.18, 7.21 Hz), indicate the presence of threeβ-D-glucopyranosyl group. Furthermore, the HMBC correlation of H-5'' '' to C-2'' '' and H-5'' '' to C-2'' '' indicates threeβ-D-glucopyranosyl groups are attached at C-2'' '' and C-2'' '' positions. Thus, the Oleracrylimide F structure was determined.
The invention also provides an extraction and separation method of the three alkaloid compounds, 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: and (3) evaporating part of the liquid medicine obtained in the step (1), separating by macroporous resin chromatography, and eluting with ethanol isocratically, wherein the macroporous resin is 16-60 meshes, and recovering ethanol to extract under reduced pressure below 40 ℃ to obtain an ethanol extract.
Step 3: separating the ethanol extract in the step 2 by a polyamide column, adopting ethanol-water (0/100, 20/80, 50/50, 80/20, 100/0, v/v) gradient elution, evaporating 20% ethanol partially, separating by pretreated ODS medium-pressure column chromatography, wherein the granularity of the filler is 40-70 μm, adopting methanol-water (84/16, 95/5, 97/3, 100/0, v/v) gradient elution (pressurizing to ensure that the flow rate is 1mL/min, the temperature is room temperature), obtaining 10 parts (namely 10 bottles obtained by gradient elution, each bottle is 200 mL), detecting by thin-layer chromatography, developing color, reserving the developed 1-5 parts, 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 4: separating the concentrate obtained in step 3 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 5: the novel compounds obtained in step 4 were prepared by HPLC separation with methanol: 0.1% formic acid (30/70) is used as a mobile phase, the detection wavelength is 210, 254nm, and the three novel compounds are prepared by separation, and the purity measured by a normalization method is 90-99%.
The three alkaloid compounds have anti-inflammatory effect.
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 incubation, the samples were incubated with Oleracrylamide D, oleracrylamide E, oleracrylamide F in different concentrations (5. Mu.M-50. Mu.M) for 1h, and LPS at a final concentration of 1. Mu.g/mL was added to each of the LPS and experimental groups, and a zeroing group (culture medium containing DMSO medium) was provided, each group was provided with 3 duplicate wells, and the effect on cells after drug addition was examined. 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, the experimental groups were incubated with sample solutions of the three novel base compounds Oleracrylimide D, oleracrylimide E, oleracrylimide F of different concentrations (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, each group was incubated with 3 multiplex wells. 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 three alkaloid compounds have no influence on proliferation of macrophage RAW264.7 induced by LPS, and are 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 influence of three alkaloid compounds on 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 LPS by three alkaloid compoundsβAnd 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.
In summary, the invention provides a special compound and an extraction and separation method thereof, which sequentially adopt 50% ethanol reflux extraction, macroporous resin chromatography, polyamide column chromatography and ODS medium-pressure column separation and purification, and three alkaloid compounds are obtained by successful separation.
Claims (6)
1. Three alkaloid compounds separated from purslane medicinal materials are characterized by respectively having the following molecular formulas: c (C) 27 H 33 NO 15 ，C 28 H 35 NO 16 ，C 33 H 43 NO 20 The chemical structural formula of the compound is named Oleracrylimide D, oleracrylimide E and Oleracrylimide F:
。
2. the method for extracting and separating three alkaloid compounds separated from purslane medicinal materials according to claim 1, which is characterized by comprising 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 adding macroporous resin, eluting with ethanol, and recovering ethanol under reduced pressure to obtain an ethanol extract;
separating the ethanol extract in the step (2) by a polyamide column, eluting by adopting ethanol-water gradient with the volume ratio of 0/100, 20/80, 50/50, 80/20 and 100/0 to obtain a plurality of elution parts, evaporating the ethanol part, separating by pretreated ODS column chromatography, eluting by sequentially using methanol-water gradient with the volume ratio of 84/16, 95/5, 97/3 and 100/0 to obtain a plurality of elution parts, detecting by thin layer chromatography, developing, respectively concentrating the developed elution parts to dryness under reduced pressure to obtain a concentrate for standby;
step 4, separating the concentrate obtained in the step 3 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 5, separating and preparing the concentrate obtained in the step 4 by HPLC, wherein the volume ratio is 30:70 methanol-0.1% formic acid is used as a mobile phase for isocratic elution, and finally the three alkaloid compounds are obtained.
3. The extraction and separation method according to claim 2, wherein in the step 1, 50% ethanol is extracted under reflux for 2 times, each time for 2 hours, the ethanol consumption is 8-16 times of that of the medicinal materials.
4. The extraction separation method as claimed in claim 2, wherein the ethanol 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. An application of three alkaloid compounds separated from herba Portulacae medicinal material in preparation of antiinflammatory and antioxidant medicines is described in claim 1.
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