CN101172948B - Method for separation and purification of cajanine and pinostrobin from extract of cajanus cajan branches and leaves - Google Patents
Method for separation and purification of cajanine and pinostrobin from extract of cajanus cajan branches and leaves Download PDFInfo
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- CN101172948B CN101172948B CN2007101445360A CN200710144536A CN101172948B CN 101172948 B CN101172948 B CN 101172948B CN 2007101445360 A CN2007101445360 A CN 2007101445360A CN 200710144536 A CN200710144536 A CN 200710144536A CN 101172948 B CN101172948 B CN 101172948B
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Abstract
The invention relates to a method for separating and purifying functional components of the traditional Chinese medicine, namely, legumin and pinostrobin; aiming at providing a method for easily, safely, economically and effectively extracting and acquiring monomeric compounds of legumin and pinostrobin through separating and purifying from branches and leaves of the dal. The invention has the technical proposal that the method comprises the steps as follows: the extractive ethanol matter is taken from the branches and the leaves of the dal as the raw material, the supersonic oscillating flocculating technology, the negative pressure cavitating and suspending liquid solidifying and extracting technology, the big-pore absorbing resin collecting technology, the positive phase silica gel middle pressure chromatographic separation technology and the recrystalling technology are adopted to produce refined dal element and pinostrobin with the purity being more than 95 percent. The extractive receives simple impurity removal and repurification. The invention which has the advantages of simple method, easy operation, small loss of the target compounds, high yield and high purity of the product is suitable for the industrialized application, and has great importance to the industrialized production.
Description
Technical field
The present invention relates to a kind of method of separation and purification functional ingredient from plant, specifically is separation and purification cajanin and Pinostrobin from pigeonpea branches and leaves ethanol extraction.
Background technology
Pigeonpea [Cajanus cajan (L.) Millsp.] is annual or perennial woody plant, evergreen shrubs for the pulse family pigeonpea belongs to.Having another name called pigeon beans, tree soya bean etc., is a kind of medication among the people.Effects such as refrigerant, the heat that disappears, detoxifcation, analgesic hemostatic, inhibiting bacteria and diminishing inflammation are arranged, (Liu Zhongqiu etc., 1998 such as wound, burn, infection, bedsore and antisepsis and anti-inflammation of being used for the treatment of among the people; Sun Shaomei etc., 1995).In addition, the Semen Cajani extract preparation in national expenditures such as India, Brazil, Cuba, Mexico, South Africa in disease (Abbiw DK., 1990 such as treatment flu, bronchitis, measles, dysentery, hepatitis, tetter, burn, ulcer, tumours; Duke JA.et al, 1994; Milliken W., 1997; Grover JK., 2002), evident in efficacy.The clinical prescription of China is used for the treatment of necrosis of femoral head and circulation system disease (Yuan Hao etc., 2004).
In the present known pigeonpea activeconstituents comprise apigenin, luteolin, Vitexina, Saponaretin, cajanin, Longistylin A, Longistylin C, Pinostrobin, Whitfield's ointment, β-Gu Zaichun, naringenin-4 ', 7-dme, hentriacontane, heneicosane, Cajanol and some volatile oil composition (Lin Li etc., 1999; Christopher JC.et al, 1982; Cheng Zhiqing etc., 1992).
Cajanin in the pigeonpea and Pinostrobin content are higher and active outstanding.Cajanin and other two kinds of stilbene compound prescriptions can suppress bone loss, and it is complete to keep trabecular bone structure; The class estrogen effect of cajanin has good medicinal DEVELOPMENT PROSPECT (Chen Dihua, Sun Lan etc., 2007).And Pinostrobin is a kind of stronger active natural flavone constituents (Liu Xiang etc. that have, 2006), has antibiotic (Bhamarapravati et al, 2006), anti-fragrance, antiproliferative isoreactivity (Jean-Christophe Le Bail, 2000), pharmacological research in recent years finds that it promptly has tangible physiologically active (Pan Yongfeng etc., 2005) in aspects such as (gasteremphraxis) to antiulcer agent, anti-inflammatory, pain relieving, stomach emptying.Although the activity of cajanin that obtains from natural phant and Pinostrobin is outstanding, but up to the present seldom about the report of its production process, have only in the Chinese patent (CN1666745A) " a kind of pharmaceutical composition that is used for the treatment of osteoporosis " and relate to cajanin and Pinostrobin extracting and purifying method, adopt traditional technique means but purify, through silica gel column chromatography repeatedly, process is very long, target compound easily loses, and the bigger organic solvent usage quantity of toxicity is big, is unfavorable for healthy production.In addition, the separation and purification that relates to Pinostrobin is traditional method (Zhou Lidong, 1999; Liu Wenxia, 2006).
In sum, in the process of utilizing plant milk extract separation and purification cajanin and Pinostrobin, main influence factor is ubiquity length consuming time in the sepn process, and the purpose compound runs off big, makes cost raise.Therefore, be necessary to seek the method for a kind of handy and safe economical and efficient ground separation and purification cajanin and Pinostrobin.The present invention is intended to set up a kind of method that combines by various modern separating and purifying technology means, realizes that simply quick, the few separation and purification of loss obtains cajanin and Pinostrobin.
Summary of the invention
The object of the present invention is to provide a kind of ethanol extraction with the pigeonpea branches and leaves is raw material, obtain cajanin and Pinostrobin monomer in a large number, fast by ultra-sonic oscillation flocculation technique, negative pressure-cavitation homogenous solid-liquid phase liquid-solid extraction technology, macroporous adsorbent resin beneficiation technologies, purification on normal-phase silica gel medium pressure column chromatography isolation technique and low temperature crystallization and recrystallization technology, this method is simple, the target compound loss is few.The objective of the invention is to reach by following scheme:
Add 50～60 ℃ of warm water ultra-sonic oscillation, static flocculation in pigeonpea branches and leaves 70～95% ethanol extractions, resultant thickness throw out obtains the product of cajanin and Pinostrobin respectively behind negative pressure-cavitation homogenous solid-liquid phase liquid-solid extraction, Amberlyst process enrichment and purification on normal-phase silica gel medium pressure column chromatography, purity is greater than 80%, obtains purity greater than 95% pure product through low temperature crystallization and recrystallization.
Above-mentioned cajanin and Pinostrobin extracting and purifying method, it is characterized in that, get a certain amount of pigeonpea branches and leaves ethanol extraction, 50～60 ℃ of warm water that add 3～5 times of volumes, vibration flocculation 15～25min under 30～60KHz condition in the ultrasonic oscillation extraction device, static 10～15min, obtain the thickness throw out, with 0.04～0.06MPa negative pressure is that power carries out the negative pressure-cavitation homogenous solid-liquid phase liquid-solid extraction with ethyl acetate, extracting used solvent volume is 0.5～2% (L:g) of extract quality, extraction times is 3～5 times, solvent for use also comprises trichloromethane except that ethyl acetate, methylene dichloride, concentrated extract is to doing, and the solid substance that obtains is with 20-40% ethanol suspendible, being configured to feed concentration is 2～4mg/mL suspendible soup, simultaneously macroporous adsorbent resin is adopted wet method dress post, keep liquid level, the soup of suspendible is passed through adsorption column, applied sample amount 0.5～1BV is 6～8mL/g resin per hour with flow velocity, the PH=4 condition was descended adsorption column.Impurity is washed with 30-40% ethanol 2BV in the absorption back, uses 75-85% ethanol 6～8BV desorb then.Collect desorbed solution, be concentrated into the dried solid substance that obtains.Taking by weighing quality in advance is 2.5～3 times silica gel of extract quality, adopts wet method dress post, and solvent for use also comprises ether, normal hexane except that sherwood oil.With a small amount of organic solvent dissolution of gained solid substance, the chromatographic silica gel of quality such as adding and extract is mixed thoroughly, reducing pressure is spin-dried for, compression leg in the purification on normal-phase silica gel of packing into.Successively with 10-20BV sherwood oil (60-90 ℃), sherwood oil: trichloromethane (10: 1), trichloromethane compress and wash in continuously and take off, and collect elutriant, every part of 1/40～1/20BV.Flow point is collected in the TLC monitoring, merges same section, and developping agent is a sherwood oil: trichloromethane=5: 1～0: 1, ultraviolet wavelength are 254nm and 360nm.Obtain Pinostrobin and cajanin solution successively, the low temperature crystallization obtains product, obtains pure product behind the recrystallization.
The used macroporous adsorbent resin of the extracting and purifying method of cajanin and Pinostrobin is D101, NKA-9, NKA-2, H103 type etc. in the above-mentioned pigeonpea.
The extracting and purifying method used silica gel of cajanin and Pinostrobin is 300～800 orders in the above-mentioned pigeonpea.Silica gel medium pressure post moving phase comprises the different ratios gradient of (sherwood oil, ether, normal hexane)-(methylene dichloride, trichloromethane, ethyl acetate, acetone, methyl alcohol).
The used solvent of recrystallization is sherwood oil, ether, normal hexane, methylene dichloride, trichloromethane, acetone, acetonitrile and methyl alcohol, and recrystallization solvent for use ratio is sherwood oil (or ether, normal hexane): trichloromethane (or methylene dichloride, acetone, acetonitrile, methyl alcohol)=50: 1～1: 1.The low temperature recrystallization temperature is-10 ℃～25 ℃.
Advantage of the present invention:
1. this method is simple, and the cycle is short, can realize large-scale industrialization production.
2. product yield height, purity height.
Description of drawings
Fig. 1 is the structure of cajanin
Fig. 2 is the structure of Pinostrobin
Specific embodiments
Embodiment 1
Take by weighing the ethanol extraction 300g of the dried branches and leaves of pigeonpea, add in 60 ℃ of warm water (1.5L), vibration flocculation 20min under the 30-60KHz condition in the ultrasonic extraction device, static 15min, get the thickness throw out, with the 0.05MPa negative pressure is that power carries out the negative pressure-cavitation homogenous solid-liquid phase liquid-solid extraction with the 4.5L ethyl acetate, concentrated extract, being configured to feed concentration with 30% ethanol is 4mg/mL suspendible soup, applied sample amount 0.8BV, flow velocity is for per hour 8mL/g resin, PH=4 condition descended NKA-9 type macropore to cross adsorption resin column.Impurity is washed with 30% ethanol 2BV in the absorption back, uses 85% ethanol 8BV desorb then.Collect stripping liquid, be concentrated into dried back and dissolve with small amount of methanol, adding 300g order number is that the chromatographic silica gel of 500-800 is mixed thoroughly, 40 ℃ of evaporated under reduced pressure.Take by weighing the 500-800 order silica gel of 2.5～3 times of extract qualities in advance, adopt sherwood oil wet method dress post, during the silica gel of having mixed sample packed in the compression leg, successively with 15BV sherwood oil (60-90 ℃), sherwood oil: trichloromethane (10: 1), trichloromethane compress and wash in continuously and take off, flow point is collected in the TLC monitoring, concentrate and obtain Pinostrobin and cajanin successively, the low temperature crystallization obtains product, obtain compound 1 behind the recrystallization and be Pinostrobin, 33.82mg, purity is 95%, compound 2 is cajanin, 31.36mg, and purity is 96%.
Embodiment 2
Take by weighing the ethanol extraction 300g of the fresh branches and leaves of pigeonpea, add in 55 ℃ of warm water (2L), improve to swing in ultrasonic wave and get in the device vibration flocculation 20min under 30～60KHz condition, static 15min, get the thickness throw out, with the 0.06MPa negative pressure is that power carries out the negative pressure-cavitation homogenous solid-liquid phase liquid-solid extraction with trichloromethane, concentrated extract, being configured to feed concentration with 30% ethanol is 2mg/mL suspendible soup, applied sample amount 0.6BV, flow velocity is for per hour 8mL/g resin, PH=4 condition descended NKA-2 type macropore to cross adsorption resin column.Impurity is washed with 30% ethanol in the absorption back, uses 85% ethanol 7BV desorb afterwards, collects stripping liquid, is concentrated into to do the back with the small amount of methanol dissolving, adds 300g order number for the 300-400 chromatographic silica gel is mixed thoroughly, 40 ℃ of evaporated under reduced pressure.Take by weighing the 300-400 order silica gel of 2.5～3 times of extract qualities in advance, adopt sherwood oil wet method dress post, during the silica gel of having mixed sample packed in the compression leg, successively with 13BV sherwood oil (60-90 ℃), sherwood oil: trichloromethane (10: 1), trichloromethane compress and wash in continuously and take off, and flow point is collected in the TLC monitoring, concentrates and obtains Pinostrobin and cajanin successively, the low temperature crystallization obtains product, obtain compound 1 behind the recrystallization and be Pinostrobin, 30.18mg, purity is 95%; Compound 2 is a cajanin, 28.63mg, and purity is 95%.
Claims (1)
1. the method for separation and purification cajanin and Pinostrobin from a pigeonpea branches and leaves extract, it is characterized in that: the ethanol extraction 300g that takes by weighing the dried branches and leaves of pigeonpea, add in 60 ℃ of warm water of 1.5L, vibration flocculation 20min under the 30-60KHz condition in the ultrasonic extraction device, static 15min, get the thickness throw out, with the 0.05MPa negative pressure is that power carries out the negative pressure-cavitation homogenous solid-liquid phase liquid-solid extraction with the 4.5L ethyl acetate, concentrated extract, being mixed with feed concentration with 30% ethanol is 4mg/mL suspendible soup, applied sample amount 0.8BV, flow velocity is 8mL/g resin per hour, the PH=4 condition descended NKA-9 type macropore to cross adsorption resin column, impurity is washed with 30% ethanol 2BV in the absorption back, use 85% ethanol 8BV desorb then, collect stripping liquid, being concentrated into dried back dissolves with small amount of methanol, adding 300g order number is that the chromatographic silica gel of 500-800 is mixed thoroughly, 40 ℃ of evaporated under reduced pressure, take by weighing the 500-800 order silica gel of 2.5～3 times of extract qualities in advance, adopt sherwood oil wet method dress post, during the silica gel of having mixed sample packed in the compression leg, successively with 15BV 60-90 ℃ sherwood oil, sherwood oil: trichloromethane is 10: 1 the sherwood oil and the mixed solution of trichloromethane, compress and wash during trichloromethane is continuous and take off, flow point is collected in the TLC monitoring, concentrate and obtain Pinostrobin and cajanin successively, the low temperature crystallization obtains product, obtain 33.82mg purity behind the recrystallization and be 95% Pinostrobin, 31.36mg purity is 96% cajanin.
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| CN2007101445360A CN101172948B (en) | 2007-11-01 | 2007-11-01 | Method for separation and purification of cajanine and pinostrobin from extract of cajanus cajan branches and leaves |
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101485714B (en) * | 2009-03-03 | 2010-12-29 | 东北林业大学 | Cajanus cajan leaf extract and application of monomer ingredient thereof in preparing medicament for resisting gram-positive bacteria |
| CN101875648B (en) * | 2010-07-12 | 2012-12-12 | 马灵媛 | Method for extracting and purifying Pinostrobin from medicinal plants, and pharmaceutical preparation and application thereof |
| CN102731460A (en) * | 2011-04-13 | 2012-10-17 | 东北林业大学 | Method for separating and purifying cajanol from pigeon pea rhizomes |
| CN103159621B (en) * | 2011-12-16 | 2015-04-15 | 东北林业大学 | Stilbene active component, preparation method and application thereof |
| CN102703612A (en) * | 2012-06-29 | 2012-10-03 | 东北林业大学 | Method for preparing glucose syrup by performing acidolysis on cellulose biomass by negative pressure mass transfer and diffusion dialysis membrane separation technology |
| CN103073530B (en) * | 2013-02-04 | 2014-07-23 | 河南中医学院 | Method for preparing pinostrobin from lindera reflexa |
| CN105362259B (en) * | 2015-10-20 | 2018-06-15 | 浙江中医药大学 | A kind of Hickory Leaves extract pinostrobin with anti-trioxypurine effect and its preparation method and application |
| CN108096369A (en) * | 2018-01-16 | 2018-06-01 | 河南省中医药研究院 | A kind of trophic nerve cell, the extraction process of anti-apolexis composition |
| CN107879906A (en) * | 2018-01-16 | 2018-04-06 | 河南省中医药研究院 | Extract cajanin, Longistylin A, Longistylin C, Cajanuslactone A technique |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000078324A1 (en) * | 1999-06-18 | 2000-12-28 | Hao Yuan | The leaves of cajanus cajan(l.) millsp and extract, formulation and uses thereof |
| US20040191190A1 (en) * | 2001-08-02 | 2004-09-30 | Gilles Pauly | Cosmetic and/or pharmaceutical preparations containing plant extracts |
| CN1666745A (en) * | 2004-03-12 | 2005-09-14 | 中国医学科学院药用植物研究所 | Drug composition for treating osteoporosis |
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2007
- 2007-11-01 CN CN2007101445360A patent/CN101172948B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000078324A1 (en) * | 1999-06-18 | 2000-12-28 | Hao Yuan | The leaves of cajanus cajan(l.) millsp and extract, formulation and uses thereof |
| US20040191190A1 (en) * | 2001-08-02 | 2004-09-30 | Gilles Pauly | Cosmetic and/or pharmaceutical preparations containing plant extracts |
| CN1666745A (en) * | 2004-03-12 | 2005-09-14 | 中国医学科学院药用植物研究所 | Drug composition for treating osteoporosis |
Non-Patent Citations (2)
| Title |
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| 林励等.木豆黄酮类成分的研究.中国药科大学学报30 1.1999,30(1),21-23. |
| 林励等.木豆黄酮类成分的研究.中国药科大学学报30 1.1999,30(1),21-23. * |
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