CN101898940A - Method for separating and purifying phyllanthin from common leafflower herb - Google Patents
Method for separating and purifying phyllanthin from common leafflower herb Download PDFInfo
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- CN101898940A CN101898940A CN2010101168035A CN201010116803A CN101898940A CN 101898940 A CN101898940 A CN 101898940A CN 2010101168035 A CN2010101168035 A CN 2010101168035A CN 201010116803 A CN201010116803 A CN 201010116803A CN 101898940 A CN101898940 A CN 101898940A
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Abstract
The invention provides a method for separating and purifying phyllanthin from common leafflower herb, which comprises the following steps: airing and crushing a complete stool of the common leafflower herb with water content of about 80%; extracting the stool with ethanol at normal temperature; filtering, repeating filtering, and combining the extracts; carrying out decompression concentration, and loading the concentrated solution to a macroporous resin column with the type of HPD 100, wherein the sample loading flow velocity is 1BV/h, the loading amount is 6.5BV; analyzing the solution with 80% ethanol, wherein the flow velocity is 2BV/h, the dosage of the analytic liquid is 9BV, and the resolution is 76.7%; dissolving extract with ethyl acetate after the phyllanthin analytic liquid is concentrated, adding 5g of dry silica gel into supernate, merging, then carrying out the decompression concentration till dry; loading the extract to a silica gel column to perform gradient elution with petroleum ether: ethyl acetate; combining flows with high content of phyllanthin; concentrating the flows in a refrigerator at the temperature of between 0 and 4 DEG C for refrigeration; and filtering the white flocculent precipitate after 24 hours to obtain crystal of the phyllanthin; continuously putting the filtrate in a refrigerator to refrigerate for 24 hours after the filtrate is decompressed and concentrated, filtering, and combining the crystals; and heating and dissolving the obtained crystal with petroleum ether, putting the crystal in the refrigerator to freeze until white acicular crystal is separated, filtering the crystal, and drying the crystal in vacuum to obtain a phyllanthin sample. The method has the main characteristic of being capable of extracting the high-purity phyllanthin from the common leafflower herb along with simple preparation process and low production cost, and can be applied to industrial production.
Description
Technical field
The present invention relates to the preparation method of a kind of Lignanoids compounds " Common Leafflower Herb element ", specifically relate to from euphorbia plant Common Leafflower Herb herb, extract the method for Common Leafflower Herb element, belong to the comprehensive utilization technique field of medicinal plant.
Background technology
Common Leafflower Herb (Phyllanthus urinaria L.) is the Euphorbiaceae phyllanthus plant, another name Herba Phyllanthi Urinariae, silk tree grass, yin-yang grass, and annual herb, all herbal medicine is traditional herbal medicine.Record the earliest in " the sward property of medicine be equipped with want ", have the effect of calming liver and clearing heat, promoting diuresis to remove toxic substance, China is among the people to be mainly used to treat diseases such as infantile malnutrition, jaundice, hepatitis, enteritis, dysentery, nephritic edema, snakebite
[1]There is kind more than 600 in the phyllanthus plant whole world, and there are 6 subgenus, 33 kinds, 4 mutation in China, and phyllanthus plant can be draft, shrub or arbor, mainly is distributed in the Yangtze valley and southern all provinces
[2], be born in thick grass wetlands such as hillside, roadside, Tian Bian
[1]With Phyllanthusamarus (Phyllanthus amarus L.), treat the hepatitis B virus carriers from India scholar Thyagarajan report, 59% patient's hepatitis B surface antigen (HBsAg) is turned out cloudy
[3], the effect of relevant Common Leafflower Herb has caused extensive concern both domestic and external.
Lignanoids compounds is one of main component of Common Leafflower Herb, and according to the literature, the Common Leafflower Herb element has effect to the glycoprotein of KB cell, thereby the KB cell is had the cytokine activity, and this activity can strengthen because of adding vincaleucoblastine.When adding vincaleucoblastine, inferior Common Leafflower Herb element also has the cytokine activity to the KB cell
[4]And these two lignanoids are to mouse CCl
4With GalN inductive hepatocellular injury significant provide protection is arranged all
[5]Two Jie Ji-butyrolactone diben-zylbutyrolactone has anti-tumor activity
[6]
The Common Leafflower Herb element belongs to the aryl butanes, and this lignans is the simplest lignans of structure.In the plant biological building-up process, this lignans is the foundation stone of synthetic other each lignans, can be converted into other each lignans by it.KadaliS.Sagar etc. have successfully synthesized compound 2-28 by Common Leafflower Herb is plain for precursor, and the HIV (human immunodeficiency virus)-resistant activity of this lignans is studied, and the result shows that compound 2,22,23,28 has showed good HIV (human immunodeficiency virus)-resistant activity, IC
50Value is respectively 0.25,0.07,0.01 and 0.32 μ M, CC
50Value is respectively 74.8,63.5,60.5 and 12.8 μ M, and the inhibition activity of 23 couples of HIV of compound is the strongest, therapeutic index value (CC
50/ IC
50) up to 60000, have the potentiality of the inverase of development of new, be an of great value compound.
Relevant for the plain report that extracts of Common Leafflower Herb, also there is not bibliographical information at present about the plain processing methodes of a large amount of extraction Common Leafflower Herbs.
Summary of the invention
The technical solution used in the present invention comprises: fresh Common Leafflower Herb dries through cleaning impurity elimination, pulverize, and with ethanolic soln extraction, filtration, concentrating under reduced pressure, then with resin absorption, eluent ethyl acetate, elutriant is concentrated, crystallization and make the plain pure product of Common Leafflower Herb.
Therefore, the invention provides a kind of method from Common Leafflower Herb extraction separation Common Leafflower Herb element, step comprises:
1) raw material is handled: the raw material that collects is cleaned, dried to constant weight or dry out of doors 50 ℃ baking oven kind, pulverize, the dry powder particle diameter is between 20 orders～200 orders;
2) extract at room temperature: the Common Leafflower Herb raw material is put into triangular flask, add 95% ethanolic soln of 25 times of raw material weights, need extract at room temperature 2 times, extraction time is about 40min;
3) filter, concentrate: above-mentioned vat liquor is filtered through concentrating under reduced pressure;
4) centrifugation: concentrated solution is carried out centrifugation, take out impurity;
5) macroporous resin adsorption is refining: centrifugal good enriched material is poured into to be equipped with carry out the absorption of post layer in the macroporous resin adsorption resin column of handling well, with 80% ethanolic soln wash-out;
6) concentrate and reclaim ethanol: these solution that elute are concentrating under reduced pressure (ethanol recovery) respectively;
7) silicagel column refining with adsorbents: the mixture acetic acid ethyl dissolution that concentrated, last silicagel column sherwood oil: ethyl acetate gradient elution (volumn concentration of ethyl acetate is 5%, 10%, 15%, 20%, 25%, 30%, 35%), each gradient elution volume is 500mL, about 50mL collects an elutriant, gets the plain crude product of Common Leafflower Herb after elutriant concentrates;
8) recrystallization: use the sherwood oil recrystallization, get highly purified Common Leafflower Herb element.Its recrystallization method is to adopt organic compound process for purification commonly used to operate.
Can reach 98.1% with the Common Leafflower Herb of the said method preparation of the present invention is plain with high performance liquid chromatography (HPLC) mensuration content.
Advantage of the present invention is:
The present invention can extract high purity Common Leafflower Herb element from Common Leafflower Herb, preparation technology is simple, and production cost is low, can be applicable to suitability for industrialized production.
Description of drawings
Fig. 1: the mass spectrum graphic representation of the plain standard substance of Common Leafflower Herb, wherein ordinate zou is represented peak area, X-coordinate is represented disengaging time.
Fig. 2: extract the mass spectrum graphic representation of Common Leafflower Herb element from Common Leafflower Herb, wherein ordinate zou is represented peak area, and X-coordinate is represented disengaging time.
Embodiment:
[0019]Below, the present invention will be further detailed with embodiment, but it is not limited to any or the similar example of these embodiment.
Embodiment 1:
Take by weighing the 50g crude drug, add 6 liters of 95% ethanol, extract at room temperature 60min, filter, the dregs of a decoction repeat once as stated above, united extraction liquid, be evaporated to 100mL, model is the HPD100 macroporous resin column on the concentrated solution, and last sample flow velocity is 1BV/h, applied sample amount is 6.5BV, resolves with 80% ethanol, and flow velocity is 2BV/h, the desorbed solution consumption is 9BV, resolution factor is 76.7%, and after the plain desorbed solution of Common Leafflower Herb concentrated and does, gained medicinal extract was about 1g.Medicinal extract 200mL 98% acetic acid ethyl dissolution, leave standstill and get supernatant liquor, in supernatant liquor, add the dried silica gel of 5g, be evaporated to after the merging do after, on the silicagel column that installs, use sherwood oil: ethyl acetate gradient elution, each elution volume are 500mL, and about 50mL collects an elutriant, merge the high stream part of Common Leafflower Herb cellulose content, put into 0-4 ℃ of refrigerator and cooled after concentrating and hide, occur white flocks behind the 24h, filter the crystal that obtains the Common Leafflower Herb element.Filtrate decompression continues to put into refrigerator cold-storage 24h after concentrating, and filters, and merges crystal.With the crystal that obtains sherwood oil heating for dissolving, put into refrigerator again and be refrigerated to and separate out white, needle-shaped crystals, leach, vacuum-drying 24h, the Common Leafflower Herb element be about 40mg, purity is 98.2%.(sample purity is seen Fig. 2, and Fig. 1 is the standard HPLC collection of illustrative plates of Common Leafflower Herb element)
Embodiment 2:
Take by weighing the 50Kg crude drug, add 5000 liters of 95% ethanol, extract at room temperature 80min, filter, the dregs of a decoction repeat once as stated above, united extraction liquid, be evaporated to 50L, model is the HPD100 macroporous resin column on the concentrated solution, and last sample flow velocity is 1BV/h, applied sample amount is 6.5BV, resolves with 80% ethanol, and flow velocity is 2BV/h, the desorbed solution consumption is 9BV, resolution factor is 75.4%, and after the plain desorbed solution of Common Leafflower Herb concentrated and does, gained medicinal extract was about 0.9Kg.Medicinal extract 200L 98% acetic acid ethyl dissolution, leave standstill and get supernatant liquor, in supernatant liquor, add the dried silica gel of 5kg, be evaporated to after the merging do after, on the silicagel column that installs, use sherwood oil: ethyl acetate gradient elution, each elution volume are 50L, and about 5L collects an elutriant, merge the high stream part of Common Leafflower Herb cellulose content, put into 0-4 ℃ of refrigerator and cooled after concentrating and hide, occur white flocks behind the 24h, filter the crystal that obtains the Common Leafflower Herb element.Filtrate decompression continues to put into refrigerator cold-storage 24h after concentrating, and filters, and merges crystal.With the crystal that obtains sherwood oil heating for dissolving, put into refrigerator again and be refrigerated to and separate out white, needle-shaped crystals, leach, vacuum-drying 24h, the Common Leafflower Herb element be about 30g, purity is 98.8%.
Embodiment 3:
Take by weighing the 20Kg crude drug, add 2000 liters of 95% ethanol, extract at room temperature 50min, filter, the dregs of a decoction repeat once as stated above, united extraction liquid, be evaporated to 20L, model is the HPD100 macroporous resin column on the concentrated solution, and last sample flow velocity is 1BV/h, applied sample amount is 6.5BV, resolves with 80% ethanol, and flow velocity is 2BV/h, the desorbed solution consumption is 9BV, resolution factor is 77.5%, and after the plain desorbed solution of Common Leafflower Herb concentrated and does, gained medicinal extract was about 0.2Kg.Medicinal extract 50L 98% acetic acid ethyl dissolution, leave standstill and get supernatant liquor, in supernatant liquor, add the dried silica gel of 5kg, be evaporated to after the merging do after, on the silicagel column that installs, use sherwood oil: ethyl acetate gradient elution, each elution volume are 10L, and about 1L collects an elutriant, merge the high stream part of Common Leafflower Herb cellulose content, put into 0-4 ℃ of refrigerator and cooled after concentrating and hide, occur white flocks behind the 24h, filter the crystal that obtains the Common Leafflower Herb element.Filtrate decompression continues to put into refrigerator cold-storage 24h after concentrating, and filters, and merges crystal.With the crystal that obtains sherwood oil heating for dissolving, put into refrigerator again and be refrigerated to and separate out white, needle-shaped crystals, leach, vacuum-drying 24h, the Common Leafflower Herb element be about 18g, purity is 99.1%.
Reference
[1] national herbal medicine is write the group volume. national herbal medicine compilation [M] (first volume), Beijing: People's Health Publisher, 1978,263
[2] Li Bingtao. the revision of Chinese phyllanthus plant [J]. Acta Phytotaxonomica Sinica, 1987,25 (5): 371～375
[3]Thyagarajan?S?P，Thiruneelakantan?K，Subramanian?S，et?al.In?vitroinactivation?of?HBsAg?by?Eclipla?alba?Hassk?and?Phllanthus?uiruri?linn[J].Indian?J?Med?Res，1982，76(Suppl)：124～130
[4] Ren Lijuan, Zhang Lan, Zhao Tiande, etc. Common Leafflower Herb is to the provide protection [J] of mouse experiment liver damage. China-Japan Friendship Hospital's journal, 1996,10 (3): 193～196
[5] Zhong Ying, Zuo Chunxu, Li Fengqin, etc. the research of Common Leafflower Herb chemical ingredients and anti-hepatitis B virus activities thereof [J]. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1998,23 (6): 363～364
[6] Zheng Buping, Zheng Feifei, Pu is big. and Dai Nationality's medicine Yexiazhu tablet for treating hepatism is to the influence [J] of Patients with Fatty Liver blood fat. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1998,23 (6): 363～364
Claims (6)
1. preparation method from plant extract Common Leafflower Herb element, it is characterized in that adopting the euphorbia plant Common Leafflower Herb is raw material, extract with 95% alcohol at normal temperature, it is that the HPD100 macroporous resin fully adsorbs with 60% ethanol redissolution back with model that concentrating under reduced pressure is done the back, resolve with 80% ethanol again and collect elutriant, be concentrated into and do with going up silicagel column behind the acetic acid ethyl dissolution, use sherwood oil: the ethyl acetate gradient elution gets the plain crude product of Common Leafflower Herb, and the plain crude product of Common Leafflower Herb obtains highly purified Common Leafflower Herb element with the sherwood oil recrystallization again.
2. according to the preparation method of the said extraction Common Leafflower Herb of claim 1 element, it is characterized in that said raw material is an euphorbia plant Common Leafflower Herb herb, also can be blade, root, stem and fruit.
3. according to the preparation method of the said extraction Common Leafflower Herb of claim 1 element, it is characterized in that said dry powder, extract: make the solvent extract at room temperature 2～3 times with 95% ethanol, about each 40min with alcohol at normal temperature by Common Leafflower Herb.
4. according to the preparation method of the said extraction Common Leafflower Herb of claim 1 element, with behind the extraction using alcohol after 50 ℃ of following concentrating under reduced pressure are done, after ethanolic soln with 60% redissolves, it is the sample solution of 0.149mg/mL that constant volume obtains the plain concentration of Common Leafflower Herb, model is the macroporous resin column of HPD100 on the solution, resolve with 80% ethanol, collect the mixture that is enrichment Common Leafflower Herb element after desorbed solution concentrates.
5. according to the preparation method of the said extraction Common Leafflower Herb of claim 1 element, the preparation that it is characterized in that the plain crude product of said Common Leafflower Herb (3) is that mixture (2) is dissolved in 3～5 times the ethyl acetate solution, last silicagel column sherwood oil: ethyl acetate (volumn concentration of ethyl acetate is 5%, 10%, 15%, 20%, 25%, 30%, 35%) is carried out gradient elution, get the plain crude product of Common Leafflower Herb, the plain crude product of Common Leafflower Herb obtains highly purified Common Leafflower Herb element with the sherwood oil recrystallization again.
6. be 98% according to used sherwood oil concentration in the said recrystallization of claim 5, Tc is 0-4 ℃, and crystallization time is about 24h.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101168035A CN101898940A (en) | 2010-03-03 | 2010-03-03 | Method for separating and purifying phyllanthin from common leafflower herb |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526168A (en) * | 2012-02-21 | 2012-07-04 | 华南理工大学 | Micro-capsules of anti-influenza-virus effective part of phyllanthus urinaria, and preparation method and application thereof |
| CN106572975A (en) * | 2014-06-10 | 2017-04-19 | 阿利奥·马马杜·巴尔德 | Anthostema senegalense-based composition, for use as an anti-aids drug |
-
2010
- 2010-03-03 CN CN2010101168035A patent/CN101898940A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 刘高: "叶下珠中叶下珠素的提取纯化工艺研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526168A (en) * | 2012-02-21 | 2012-07-04 | 华南理工大学 | Micro-capsules of anti-influenza-virus effective part of phyllanthus urinaria, and preparation method and application thereof |
| CN106572975A (en) * | 2014-06-10 | 2017-04-19 | 阿利奥·马马杜·巴尔德 | Anthostema senegalense-based composition, for use as an anti-aids drug |
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Application publication date: 20101201 |