CN102973619A - Process for extracting indoline amide alkaloid from purslane and detection methods for indoline amide alkaloid - Google Patents
Process for extracting indoline amide alkaloid from purslane and detection methods for indoline amide alkaloid Download PDFInfo
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Abstract
The invention discloses a process for extracting indoline amide alkaloid from purslane. The process comprises the following steps: (1) taking the purslane and carrying out reflux extraction by ethanol; (2) adding an extracting solution onto macroporous resin, eluting by ethanol, decompressing and concentrating; (3) adding a concentrated solution onto a polyamide column, eluting by ammonia water, decompressing, concentrating and drying; and (4) dissolving ammonia water eluate by a solvent, centrifuging, filtering, adding a filtered solution onto sephadex LH-20, eluting by the ethanol, collecting a yellow stripe part, decompressing, concentrating and drying to obtain an indoline amide alkaloid extract, wherein the yield of the extract is 2 mg/g in medicinal materials and the extract mainly contains purslane amides A and B. The invention further discloses a thin-layer chromatography detection method and an HPLC (high performance liquid chromatography) detection method for the indoline amide alkaloid extract.
Description
Technical field
The present invention relates to the alkaloidal technique of enrichment extraction indoline-like amide from the Chinese crude drug Herba Portulacae, and the alkaloidal detection method of this indoline-like amide, pharmaceutical field belonged to.
Background technology
Herba Portulacae (Portulaca oleracea L.) is the annual meat herbaceous plant of Portulacaceae, its aboundresources, be widely distributed in temperate zone, the whole world and torrid areas, its aerial parts uses as the traditional drugs food dual purpose plant in many countries such as China and India, Turkey, Egypt, Greece.How on the books China's successive dynasties books on Chinese herbal medicine medical book is, has the function of heat-clearing and toxic substances removing, cooling blood for hemostasis, is used for that toxic-heat and blood stasis, carbuncle furuncle, eczema, erysipelas, snakeworm are bitten, had blood in stool, hemorrhoidal bleeding and metrostaxis.Modern pharmacological research shows that Herba Portulacae has many-sided effects such as antibiotic, lax skeletal muscle, excited uterus, anti-inflammatory and antalgic, promotion wound healing, atherosclerosis, blood fat reducing, blood sugar lowering, anticancer, antioxidation, defying age and neuroprotective.
Except secondary metabolites such as organic acid, flavone, coumarin and terpenoid, alkaloid is the important chemical constituent of a class in the Herba Portulacae, and its structure type is numerous.Contain norepinephrine, dopamine, DOPA, adenosine, uracil, adenine, 2 such as the Herba Portulacae aerial parts, 5-dicarboxyl pyrroles, 5-hydroxyl-2-carboxyl pyridine, N, N-1,3-Dicyclohexylurea, allantoin; Diketopiperazines amide alkaloid is such as ring (Ile-Phe), ring (phenylalanine-tyrosine), ring (Ala-Leu), ring (alanine-isoleucine), ring (phenylalanine-isoleucine), ring (tyrosine-alanine), ring (Tyr-Leu) and ring (tyrosine-isoleucine); Tetrahydroisoquinolicompounds amide alkaloid such as oleracein E; And indoline-like amide alkaloid oleracein A, B, C, D, F, G etc.In these alkaloids, indoline-like amide alkaloid oleracein A, B, C, D, F, G (Oleracein A-G) are the applicant separates the novel structure that obtains from Herba Portulacae amide alkaloid [Xiang L, Xing DM, Wang W, et al.Alkaloids from Portulaca oleracea L., Phytochemistry, 2005,66 (21): 2595-2601; Liu DY, Shen T, Xiang L.Two antioxidant alkaloids from Portulaca oleracea L., Helvetica Chimica Acta, 2011,94:497-501], its structure is as follows:
At present for the extraction separation method of this indoline-like amide alkaloid monomer chemical compound and utilize the detection method of HPLC/MS/MS to have been reported, but enrichment prepares the technique of total indoline-like amide alkaloid extract and TLC and the HPLC fingerprint atlas detection method of extract there is no report.
Applicant's previous research work shows; this indoline-like amide alkaloid has significant effect of scavenging radical (Yang ZJ; Liu CJ; Xiang L; et al.Phenolic alkaloids as a new class of antioxidants in Portulaca oleracea.Phytotherapy Research; 2009; 23 (7): 1032-1035) and external neuroprotective activity (to orchid; Yang Zijuan. the application .CN of Herba Portulacae amide alkaloid in preparation antioxidation and Neuroprotective Agents, 200810014427.1[P] .2008-03-05).Given this indoline-like amide alkaloid of class formation novelty and derivant thereof have potential researching value aspect pharmacologically active, at Herba Portulacae quality of medicinal material controlling party mask potential using value is arranged, have potential using value aspect the prevention of the neurodegenerative diseases such as AD and PD and treatment, the present invention is intended to report preparation technology and TLC and the HPLC detection method of a large amount of enrichment indoline-like amide alkaloid extracts from Herba Portulacae.
Summary of the invention
For the deficiencies in the prior art, the invention provides the technique of two kinds of enrichment extraction indoline-like amide alkaloid extracts from the Chinese crude drug Herba Portulacae, and the detection method of this indoline-like amide alkaloid extract is provided.
The present invention is achieved by the following technical solutions:
A kind of alkaloidal technique of indoline-like amide of extracting from the Chinese crude drug Herba Portulacae may further comprise the steps:
(1) get dried Herba Portulacae, with 6 times of amount (weight multiple) 60% ethanol (percentage by volume) reflux, extract, 3 times, each 1h; Merge extractive liquid, is concentrated into 0.5g crude drug/ml;
(2) said extracted liquid is added on the macroporous resin of 2 times of volumes, flow velocity by 4BV/h adsorbs, use first the water elution of 10BV, use again 10% ethanol (percentage by volume) eluting of 4.5BV, use again 30% ethanol (percentage by volume) eluting of 4.5BV, collect 30% ethanol elution, concentrating under reduced pressure gets concentrated solution;
(3) above-mentioned concentrated solution is added on the polyamide column of 1 times of volume of the described extracting solution of step (2), use successively the water of 5BV, 70% ethanol elution of 3BV, use again the ammonia eluting (the ammonia mass concentration is 0.25%～25%) of 3BV, collect the ammonia eluent, concentrating under reduced pressure is dry, gets the ammonia eluate;
(4) above-mentioned ammonia eluate is with an amount of ethanol/water ultrasonic dissolution, centrifugal (3000r/min, 10 minutes), supernatant is with 0.45 μ m filtering with microporous membrane, filtrate is added on the polydextran gel Sephadex LH-20 of 50 times of volumes, ethanol elution, collect yellow strip portion, concentrating under reduced pressure is dry, be indoline-like amide alkaloid extract (I), the yield of this extract is the 2mg/g medical material, through and indoline-like amide alkaloid reference substance oleracein A (Oleracein A) and B(Oleracein B) the common thin layer (seeing Fig. 1～3) of mixture, and the HPLC fingerprint map analyzing (is seen Fig. 4, Fig. 5), mainly contain Herba Portulacae amide alkaloid A and B in this extract (I).
Another extracts the alkaloidal technique of indoline-like amide from the Chinese crude drug Herba Portulacae, may further comprise the steps:
(1) get dried Herba Portulacae, with 6 times of amount (weight multiple) 60% ethanol (percentage by volume) reflux, extract, 3 times, each 1h; Merge extractive liquid, is concentrated into 0.5g crude drug/ml;
(2) said extracted liquid is added on the polyamide column of 1.5 times of volumes, use successively the water of 6BV, 10% ethanol elution of 1.5BV, use again 50% ethanol elution of 4.5BV, use again the ammonia eluting (ammonia mass concentration 0.25%～25%) of 3BV, merge 50% ethanol elution and ammonia eluent, concentrating under reduced pressure is dry, gets eluate;
(3) above-mentioned eluate is with an amount of ethanol/water ultrasonic dissolution, centrifugal (3000r/min, 10 minutes), cross 0.45 μ m microporous filter membrane, filtrate is added on the polydextran gel Sephadex LH-20 of 50 times of volumes, ethanol elution, collect orange-yellow strip portion, concentrating under reduced pressure is dry, be indoline-like amide alkaloid extract (II), the yield of this extract is the 3mg/g medical material, through and indoline-like amide alkaloid reference substance oleracein A (Oleracein A) and B(Oleracein B) the common thin layer (seeing Fig. 1～3) of mixture, and the HPLC fingerprint map analyzing (is seen Fig. 6, Fig. 7), mainly contain Herba Portulacae amide alkaloid A and B in this extract (II).
The alkaloidal thin-layered chromatography detection method of a kind of indoline-like amide, step is as follows: get indoline-like amide alkaloid extract, add an amount of methanol/water ultrasonic dissolution, as need testing solution; Other gets the mixture reference substance of oleracein A and B, adds an amount of methanol/water dissolving, in contrast product solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, it is an amount of to draw above-mentioned two kinds of solution, puts respectively on same anti-phase C18 silica gel thin-layer plate, take 20%～40% ethanol as developing solvent, launches, and takes out, and dries UV
254Inspect under the nm, or behind the ammonia cure at UV
365Inspect under the nm, judge testing result: if in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, skin dark stain or the brown color speckle of aobvious same color then contain oleracein A and oleracein B in the indoline-like amide alkaloid extract; Otherwise then without.
In the above-mentioned thin-layered chromatography detection method, developing solvent also can adopt the methanol/water of similar concentration or acetone/water system as developing solvent except the ethanol/water system.
The alkaloidal thin-layered chromatography detection method of another indoline-like amide, step is as follows: get indoline-like amide alkaloid extract, add an amount of methanol/water ultrasonic dissolution, as need testing solution; Other gets the mixture reference substance of oleracein A and B, adds an amount of methanol/water dissolving, in contrast product solution; According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, it is an amount of to draw above-mentioned two kinds of solution, puts respectively on same polyamide film, take 0.01%～0.05% ammonia as developing solvent, launches, and takes out, and dries, at UV
365Inspect under the nm, judge testing result: if in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the brown color speckle of aobvious same color then contains oleracein A and oleracein B in the indoline-like amide alkaloid extract; Otherwise then without.
In the above-mentioned thin-layered chromatography detection method, developing solvent also can be adopted the ethanol that contains ammonia, methanol or the acetone system of similar concentration except the ammonia system.
The alkaloidal HPLC detection method of a kind of indoline-like amide, chromatographic condition is as follows: mobile phase: (the two volume ratio is 20:80 to the aqueous solution of acetonitrile-contain 0.1% formic acid, 0.1% refers to percentage by volume, below with " 0.1% formic acid/water " expression " aqueous solution that contains 0.1% formic acid "); Chromatographic column: Inertsil ODS-3 (4.6 * 250mm, 5 μ m); Guard column: Intersil ODS-SP (4.0 * 10mm, 5 μ m); Flow velocity: 1ml/min; Detect wavelength: 336nm, 240nm; Column temperature: 30 ℃; Sample introduction: 10 μ l; After obtaining finger printing, make comparisons with the collection of illustrative plates of standard substance oleracein A and oleracein B, judge testing result: if in the finger printing, with the corresponding retention time of the collection of illustrative plates of standard substance oleracein A and oleracein B chromatographic peak is being arranged, then proving in the indoline-like amide alkaloid and contain oleracein A and oleracein B.
In the above-mentioned HPLC detection method, mobile phase can also be with acetonitrile-0.1% aqueous formic acid (20:80) similarly other ratio contain the acetonitrile-water system of acid, perhaps methanol-0.1% formic acid/water (35:65), methanol-0.1% glacial acetic acid/water (43:57) or with its similarly other ratio contain the methanol-water system of acid; Detect wavelength and refer to 336nm, perhaps the various wavelength about 336nm; Detected temperatures refers to 30 ℃, or the various temperature between room temperature～45 ℃.
The alkaloidal preparation technology of indoline-like amide macroporous resin, polyamide, polydextran gel Sephadex LH-20 simple, that relate to are extracted in the present invention's enrichment from the Chinese crude drug Herba Portulacae in preparation technology recyclable after alkali treatment, and cost is lower; The qualitative checking method of the indoline-like amide alkaloid extract of setting up is simple.
Description of drawings
Fig. 1: UV
254Inspect anti-phase C-18 silica gel tlc thin layer chromatography under the nm, wherein, 1: indoline-like amide alkaloid extract I, 2: the mixture of oleracein A+B; 3: indoline-like amide alkaloid extract II.
Fig. 2: UV behind the ammonia cure
365Inspect anti-phase C-18 silica gel tlc thin layer chromatography under the nm, wherein, 1: indoline-like amide alkaloid extract I, 2: the mixture of oleracein A+B; 3: indoline-like amide alkaloid extract II.
Fig. 3: UV
365Inspect polyamide film TLC thin layer chromatography under the nm, wherein, 1: indoline-like amide alkaloid extract I, 2: the mixture of oleracein A+B; 3: indoline-like amide alkaloid extract II.
The HPLC finger printing (336nm) of the indoline-like amide alkaloid extract (I) that Fig. 4: embodiment 1 is prepared, wherein, OA: oleracein A, OB: oleracein B.
The HPLC finger printing (240nm) of the indoline-like amide alkaloid extract (I) that Fig. 5: embodiment 1 is prepared, wherein, OA: oleracein A, OB: oleracein B.
The HPLC finger printing (336nm) of the indoline-like amide alkaloid extract (II) that Fig. 6: embodiment 2 is prepared, wherein, OA: oleracein A, OB: oleracein B.
The HPLC finger printing (240nm) of the indoline-like amide alkaloid extract (II) that Fig. 7: embodiment 2 is prepared, wherein, OA: oleracein A, OB: oleracein B.
The specific embodiment
The present invention is further illustrated below in conjunction with embodiment.
1. experiment material and instrument
Experiment material: the Herba Portulacae medical material was purchased from Jinan in 2010 05 month and builds connection pharmacy (lot number 20100502, the Jinan City builds connection Chinese medicine company limited prepared slices of Chinese crude drugs factory and produces), be accredited as Herba Portulacae (Portulaca oleracea L.) by professor Xiang Lan, keep sample (the mark MCX-06-1-4 of this shop) is kept at pharmacognosy teaching and research room of pharmaceutical college of Shandong University.Column chromatography filler: macroporous adsorbent resin (Cangzhou, Hebei province precious grace chemical industry company limited), polyamide (City of Taizhou road and bridge four blue or green biochemical material factory), polydextran gel Sephadex LH-20(Switzerland GE Healthcare Bio-sciences company); Thin layer chromatography: polyamide film (City of Taizhou road and bridge four blue or green biochemical material factory), anti-phase C18 silica gel thin-layer plate (RP-18F
254s) (German Merck company); Conventional analysis pure reagent ethanol, methanol, ammonia and Chromatographic Pure Methanol and acetonitrile are available from Jinan City's sincere opinion on public affairs reagent company limited.
Experimental apparatus: RE52-98 type Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant), the two multiplex vacuum pumps of A circulating water type (Zhengzhou Greatwall Scientific Industrial ﹠ Trading Co., Ltd.) of SHB-IV, LG10-2.4A type centrifuge (Beijing Medical Centrifugal Machine Factory); KQ5200 type ultrasonic cleaner (city of Kunshan instrument plant), AL104 analytical balance (prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit instrument Shanghai company limited), WFH-203 type ultraviolet analysis instrument for three purposed (Industrial Co., Ltd. of upper Nereid section), Shimadzu LC-20AT high performance liquid chromatograph (Japanese Shimadzu company), chromatographic column Inertsil ODS-3 (4.6 * 250mm; 5 μ m) and protection pre-column Intersil ODS-SP (4.0 * 10mm, 5 μ m) all available from Japanese GL Sciences company.
2. experimental procedure
(1) get dried Herba Portulacae, with 6 times of amount (weight multiple) 60% ethanol (percentage by volume) reflux, extract, 3 times, each 1h; Merge extractive liquid, is concentrated into 0.5g crude drug/ml;
(2) said extracted liquid is added on the HPD700 macroporous resin of 2 times of volumes, flow velocity by 4BV/h adsorbs, use first the water elution of 10BV, use again 10% ethanol (percentage by volume) eluting of 4.5BV, use again 30% ethanol (percentage by volume) eluting of 4.5BV, collect 30% ethanol elution, concentrating under reduced pressure gets concentrated solution;
(3) above-mentioned concentrated solution is added on the polyamide column (30～60 order) of 1 times of volume of the described extracting solution of step (2), use successively the water of 5BV, 70% ethanol elution of 3BV, use again the ammonia eluting (ammonia mass concentration 25%) of 3BV, collect the ammonia eluent, concentrating under reduced pressure is dry, gets the ammonia eluate;
(4) above-mentioned ammonia eluate is dissolved with an amount of ethanol/water, centrifugal (3000r/min, 10 minutes), supernatant is added on the polydextran gel Sephadex LH-20 of 50 times of volumes with 0.45 μ m filtering with microporous membrane, filtrate, ethanol elution, collect yellow strip portion, concentrating under reduced pressure is dry, is indoline-like amide alkaloid extract, and the yield of this extract is the 2mg/g medical material.
3. experimental result
Be total to thin layer (seeing Fig. 1～3) through the mixture with indoline-like amide alkaloid reference substance oleracein A (Oleracein A) and B (Oleracein B), and HPLC fingerprint map analyzing (seeing Fig. 4, Fig. 5), mainly contain Herba Portulacae amide alkaloid A and B in this extract (I).
3.1TLC TLC Identification one:
Get total indoline-like amide alkaloid extract (I), add an amount of methanol/water ultrasonic dissolution, as need testing solution.Other gets the mixture reference substance of oleracein A and B, adds an amount of methanol/water dissolving, in contrast product solution.According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, it is an amount of to draw above-mentioned two kinds of solution, puts respectively on same anti-phase C18 silica gel thin-layer plate,, launches as developing solvent with 40% ethanol, takes out, and dries UV
254Inspect (as shown in Figure 1) under the nm, or behind the ammonia cure at UV
365Inspect (as shown in Figure 2) under the nm.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, skin dark stain or the brown color speckle of aobvious same color.
3.2TLC TLC Identification two:
Get total indoline-like amide alkaloid extract (I), add an amount of methanol/water ultrasonic dissolution, as need testing solution.Other gets the mixture reference substance of oleracein A and B, adds an amount of methanol/water dissolving, in contrast product solution.According to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2010) test, it is an amount of to draw above-mentioned two kinds of solution, puts respectively on same polyamide film, take 0.05% ammonia as developing solvent, launches, and takes out, and dries, at UV
365Inspect (as shown in Figure 3) under the nm.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the brown color speckle of aobvious same color.
3.3HPLC finger printing is differentiated
Chromatographic condition: mobile phase: acetonitrile-0.1% formic acid/water (20:80); Chromatographic column: Inertsil ODS-3 (4.6 * 250mm, 5 μ m); Guard column: Intersil ODS-SP (4.0 * 10mm, 5 μ m); Flow velocity: 1ml/min; Detect wavelength: 336nm, 240nm; Column temperature: 30 ℃; Sample introduction: 10 μ l.
The HPLC finger printing of indoline-like amide alkaloid extract (I) such as Fig. 4, shown in Figure 5.
In addition, when mobile phase is methanol-0.1% formic acid/water (35:65), methanol-0.1% glacial acetic acid/water (43:57), OA and OB can reach baseline separation (collection of illustrative plates is not attached) in the extract, above-mentioned each mobile phase but mobile phase acetonitrile-0.1% formic acid/water (20:80) is compared, its peak is narrow, peak shape is good, and separating degree is better, and therefore preferred acetonitrile-0.1% formic acid/water (20:80) is as mobile phase.
1. experiment material and instrument
With embodiment 1.
2. experimental procedure
(1) get dried Herba Portulacae, with 6 times of amount (weight multiple) 60% ethanol (percentage by volume) reflux, extract, 3 times, each 1h; Merge extractive liquid, is concentrated into 0.5g crude drug/ml;
(2) said extracted liquid is added on the polyamide column (30～60 order) of 1.5 times of volumes, use successively the water of 6BV, 10% ethanol elution of 1.5BV, use again 50% ethanol elution of 4.5BV, use again the ammonia eluting (ammonia mass concentration 25%) of 3BV, merge 50% ethanol elution and ammonia eluent, concentrating under reduced pressure is dry, gets eluate;
(3) above-mentioned eluate is dissolved with an amount of ethanol/water, centrifugal (3000r/min, 10 minutes), cross 0.45 μ m microporous filter membrane, filtrate is added on the polydextran gel Sephadex LH-20 of 50 times of volumes, ethanol elution, collect orange-yellow strip portion, concentrating under reduced pressure is dry, is indoline-like amide alkaloid extract II, and the yield of this extract is the 3mg/g medical material.
3. experimental result
Be total to thin layer (seeing Fig. 1～3) through the mixture with indoline-like amide alkaloid reference substance oleracein A (Oleracein A) and B (Oleracein B), and HPLC fingerprint map analyzing (seeing Fig. 6, Fig. 7), mainly contain Herba Portulacae amide alkaloid A and B in this extract (II), but compare the extract I of embodiment 1, impurity is more.
3.1TLC TLC Identification one (with embodiment 1).
3.2TLC TLC Identification two (with embodiment 1).
3.3HPLC finger printing is differentiated
Chromatographic condition: mobile phase: acetonitrile-0.1% formic acid/water (20:80); Chromatographic column: Inertsil ODS-3 (4.6 * 250mm, 5 μ m); Guard column: Intersil ODS-SP (4.0 * 10mm, 5 μ m); Flow velocity: 1ml/min; Detect wavelength: 336nm, 240nm; Column temperature: 30 ℃; Sample introduction: 10 μ l.
The HPLC finger printing of indoline-like amide alkaloid extract (II) such as Fig. 6, shown in Figure 7.
In addition, when mobile phase is methanol-0.1% formic acid/water (35:65), methanol-0.1% glacial acetic acid/water (43:57), OA and OB can reach baseline separation (collection of illustrative plates is not attached) in the extract, above-mentioned each mobile phase but mobile phase acetonitrile-0.1% formic acid/water (20:80) is compared, its peak is narrow, peak shape is good, and separating degree is better, and therefore preferred acetonitrile-0.1% formic acid/water (20:80) is as mobile phase.
Claims (5)
1. one kind is extracted the alkaloidal technique of indoline-like amide from Herba Portulacae, it is characterized in that: may further comprise the steps:
(1) gets dried Herba Portulacae, measure 60% alcohol reflux 3 times, each 1h with 6 times; Merge extractive liquid, is concentrated into 0.5g crude drug/ml;
(2) said extracted liquid is added on the macroporous resin of 2 times of volumes, adsorbs by the flow velocity of 4BV/h, uses first the water elution of 10BV, use again 10% ethanol elution of 4.5BV, use again 30% ethanol elution of 4.5BV, collect 30% ethanol elution, concentrating under reduced pressure gets concentrated solution;
(3) above-mentioned concentrated solution is added on the polyamide column of 1 times of volume of the described extracting solution of step (2), uses successively the water of 5BV, 70% ethanol elution of 3BV, uses the ammonia eluting of 3BV again, collects the ammonia eluent, and concentrating under reduced pressure is dry, gets the ammonia eluate;
(4) above-mentioned ammonia eluate is with an amount of ethanol/water ultrasonic dissolution, centrifugal, supernatant is with 0.45 μ m filtering with microporous membrane, filtrate is added on the polydextran gel Sephadex LH-20 of 50 times of volumes, ethanol elution, collect yellow strip portion, concentrating under reduced pressure is dry, is indoline-like amide alkaloid extract.
2. one kind is extracted the alkaloidal technique of indoline-like amide from Herba Portulacae, it is characterized in that: may further comprise the steps:
(1) gets dried Herba Portulacae, measure 60% alcohol reflux 3 times, each 1h with 6 times; Merge extractive liquid, is concentrated into 0.5g crude drug/ml;
(2) said extracted liquid is added on the polyamide column of 1.5 times of volumes, uses successively the water of 6BV, 10% ethanol elution of 1.5BV, uses 50% ethanol elution of 4.5BV again, use again the ammonia eluting of 3BV, merge 50% ethanol elution and ammonia eluent, concentrating under reduced pressure is dry, gets eluate;
(3) above-mentioned eluate is with an amount of ethanol/water ultrasonic dissolution, and is centrifugal, crosses 0.45 μ m microporous filter membrane, filtrate is added on the polydextran gel Sephadex LH-20 of 50 times of volumes, and ethanol elution is collected orange-yellow strip portion, concentrating under reduced pressure is dry, is indoline-like amide alkaloid extract.
3. alkaloidal thin-layered chromatography detection method of indoline-like amide, it is characterized in that: step is as follows: get indoline-like amide alkaloid extract, add the methanol/water ultrasonic dissolution, as need testing solution; Other gets the mixture reference substance of oleracein A and B, adds the methanol/water dissolving, in contrast product solution; According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put respectively on same anti-phase C18 silica gel thin-layer plate, take 20%～40% ethanol as developing solvent, launch, take out, dry UV
254Inspect under the nm, or behind the ammonia cure at UV
365Inspect under the nm, judge testing result: if in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, skin dark stain or the brown color speckle of aobvious same color then contain oleracein A and oleracein B in the indoline-like amide alkaloid extract; Otherwise then without.
4. alkaloidal thin-layered chromatography detection method of indoline-like amide, it is characterized in that: step is as follows: get indoline-like amide alkaloid extract, add the methanol/water ultrasonic dissolution, as need testing solution; Other gets the mixture reference substance of oleracein A and B, adds the methanol/water dissolving, in contrast product solution; According to the thin layer chromatography test, draw above-mentioned two kinds of solution, put respectively on same polyamide film, take 0.01%～0.05% ammonia as developing solvent, launch, take out, dry, at UV
365Inspect under the nm, judge testing result: if in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, the brown color speckle of aobvious same color then contains oleracein A and oleracein B in the indoline-like amide alkaloid extract; Otherwise then without.
5. alkaloidal HPLC detection method of indoline-like amide, it is characterized in that: chromatographic condition is as follows: mobile phase: acetonitrile-0.1% formic acid/water, the two volume ratio are 20:80; Chromatographic column: Inertsil ODS-3,4.6 * 250mm, 5 μ m; Guard column: Intersil ODS-SP, 4.0 * 10mm, 5 μ m; Flow velocity: 1ml/min; Detect wavelength: 336nm, 240nm; Column temperature: 30 ℃; Sample introduction: 10 μ l; After obtaining finger printing, make comparisons with the collection of illustrative plates of standard substance oleracein A and oleracein B, judge testing result: if in the finger printing, with the corresponding retention time of the collection of illustrative plates of standard substance oleracein A and oleracein B chromatographic peak is being arranged, then proving in the indoline-like amide alkaloid and contain oleracein A and oleracein B.
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| CN103948596A (en) * | 2014-05-04 | 2014-07-30 | 山东大学 | Application for oleracein E in preparation for medicine for resisting dementia neurodegenerative diseases |
| CN106279305A (en) * | 2016-08-15 | 2017-01-04 | 辽宁中医药大学 | Amide alkaloid compound and extraction separation method thereof in Herba Portulacae |
| CN106526058A (en) * | 2016-11-03 | 2017-03-22 | 成都乾坤动物药业股份有限公司 | Thin-layer chromatography-fluorescence analysis method for heat-clearing dysentery-stopping granules |
| CN109824568A (en) * | 2019-04-03 | 2019-05-31 | 辽宁中医药大学 | Two kinds of indoles new alkaloids compounds and its extraction separation method and application in purslane |
| CN110294733A (en) * | 2019-04-03 | 2019-10-01 | 辽宁中医药大学 | One kind Oleracone I of key compound containing peroxide and its extraction separation method and application in purslane |
| CN113912657A (en) * | 2021-11-23 | 2022-01-11 | 辽宁中医药大学 | Three indole alkaloids in purslane, extraction and separation method and application thereof |
| CN115252661A (en) * | 2022-06-13 | 2022-11-01 | 浙江工业大学 | Purslane lactam extract and preparation method and application thereof |
| CN116284005A (en) * | 2023-04-12 | 2023-06-23 | 辽宁中医药大学 | New alkaloid in herba Portulacae and its extraction and separation method |
| CN116621785A (en) * | 2023-04-12 | 2023-08-22 | 辽宁中医药大学 | New alkaloid compound in purslane and extraction and separation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101234121A (en) * | 2008-03-05 | 2008-08-06 | 山东大学 | Application of purslane amide alkaloid in preparing antioxidant and neuroprotective agent |
| CN102389385A (en) * | 2011-05-27 | 2012-03-28 | 上海辉文生物技术有限公司 | Herba portulacae extract and solution, and preparation method and application |
| CN102526138A (en) * | 2012-01-19 | 2012-07-04 | 贾晓斌 | Composition of active components from fresh purslane for decreasing blood sugar, and preparation method thereof |
-
2012
- 2012-12-31 CN CN201210594775.7A patent/CN102973619B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101234121A (en) * | 2008-03-05 | 2008-08-06 | 山东大学 | Application of purslane amide alkaloid in preparing antioxidant and neuroprotective agent |
| CN102389385A (en) * | 2011-05-27 | 2012-03-28 | 上海辉文生物技术有限公司 | Herba portulacae extract and solution, and preparation method and application |
| CN102526138A (en) * | 2012-01-19 | 2012-07-04 | 贾晓斌 | Composition of active components from fresh purslane for decreasing blood sugar, and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| LAN XIANG,DONGMING XING,WEI WANG, RUFENG WANG, YI DING: "Alkaloids from Portulaca oleracea L.", 《PHYTOCHEMISTRY》 * |
| 向兰等: "马齿苋的化学成分研究进展", 《亚太传统医药》 * |
| 曾宪伟等: "马齿苋生物碱对A-549细胞的抑制作用", 《江西食品工业》 * |
| 杨子娟: "马齿苋的化学成分及抗氧化活性研究", 《中国优秀硕士学位论文全文数据库》 * |
| 王仲英等: "马齿苋总生物碱的测定", 《太原理工大学学报》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103948596A (en) * | 2014-05-04 | 2014-07-30 | 山东大学 | Application for oleracein E in preparation for medicine for resisting dementia neurodegenerative diseases |
| CN106279305A (en) * | 2016-08-15 | 2017-01-04 | 辽宁中医药大学 | Amide alkaloid compound and extraction separation method thereof in Herba Portulacae |
| CN106279305B (en) * | 2016-08-15 | 2018-07-27 | 辽宁中医药大学 | Amide alkaloid compound and its extraction separation method in purslane |
| CN106526058A (en) * | 2016-11-03 | 2017-03-22 | 成都乾坤动物药业股份有限公司 | Thin-layer chromatography-fluorescence analysis method for heat-clearing dysentery-stopping granules |
| CN109824568A (en) * | 2019-04-03 | 2019-05-31 | 辽宁中医药大学 | Two kinds of indoles new alkaloids compounds and its extraction separation method and application in purslane |
| CN110294733A (en) * | 2019-04-03 | 2019-10-01 | 辽宁中医药大学 | One kind Oleracone I of key compound containing peroxide and its extraction separation method and application in purslane |
| CN109824568B (en) * | 2019-04-03 | 2021-07-27 | 辽宁中医药大学 | Two indole novel alkaloid compounds in purslane and extraction and separation method and application thereof |
| CN110294733B (en) * | 2019-04-03 | 2021-09-07 | 辽宁中医药大学 | Peroxide bond-containing compound Oleracone I in purslane, and extraction separation method and application thereof |
| CN113912657A (en) * | 2021-11-23 | 2022-01-11 | 辽宁中医药大学 | Three indole alkaloids in purslane, extraction and separation method and application thereof |
| CN113912657B (en) * | 2021-11-23 | 2023-09-19 | 辽宁中医药大学 | Three indole alkaloids in purslane, and extraction and separation method and application thereof |
| CN115252661A (en) * | 2022-06-13 | 2022-11-01 | 浙江工业大学 | Purslane lactam extract and preparation method and application thereof |
| CN115252661B (en) * | 2022-06-13 | 2023-10-20 | 浙江工业大学 | Purslane lactam extract and preparation method and application thereof |
| CN116284005A (en) * | 2023-04-12 | 2023-06-23 | 辽宁中医药大学 | New alkaloid in herba Portulacae and its extraction and separation method |
| CN116621785A (en) * | 2023-04-12 | 2023-08-22 | 辽宁中医药大学 | New alkaloid compound in purslane and extraction and separation method thereof |
| CN116284005B (en) * | 2023-04-12 | 2024-03-01 | 辽宁中医药大学 | New alkaloid in herba Portulacae and its extraction and separation method |
| CN116621785B (en) * | 2023-04-12 | 2024-03-01 | 辽宁中医药大学 | New alkaloid compound in purslane and extraction and separation method thereof |
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