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WO2003080045A1 - Procede permettant d'isoler l'artemisinine d'artemisia annua - Google Patents

Procede permettant d'isoler l'artemisinine d'artemisia annua Download PDF

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Publication number
WO2003080045A1
WO2003080045A1 PCT/IB2002/001204 IB0201204W WO03080045A1 WO 2003080045 A1 WO2003080045 A1 WO 2003080045A1 IB 0201204 W IB0201204 W IB 0201204W WO 03080045 A1 WO03080045 A1 WO 03080045A1
Authority
WO
WIPO (PCT)
Prior art keywords
artemisinin
hexane
extract
aqueous
extraction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2002/001204
Other languages
English (en)
Inventor
Sushil Kumar
Shiv Kumar Gupta
Digvijay Singh
Madan Mohan Gupta
Dharam Chand Jain
Atul Prakash Kahol
Suman Preet Singh Khanuja
Govind Ram
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Council of Scientific and Industrial Research CSIR
Original Assignee
Council of Scientific and Industrial Research CSIR
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Council of Scientific and Industrial Research CSIR filed Critical Council of Scientific and Industrial Research CSIR
Priority to JP2003577873A priority Critical patent/JP4339699B2/ja
Priority to CNA028290003A priority patent/CN1627942A/zh
Priority to PCT/IB2002/001204 priority patent/WO2003080045A1/fr
Priority to AU2002253443A priority patent/AU2002253443A1/en
Priority to US10/112,070 priority patent/US6685972B2/en
Priority claimed from US10/112,070 external-priority patent/US6685972B2/en
Publication of WO2003080045A1 publication Critical patent/WO2003080045A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/282—Artemisia, e.g. wormwood or sagebrush
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials

Definitions

  • the present invention relates to a process for the isolation of artemisinin, an antimalarial agent from the herb of the Artemisia annua plant, comprising of extracting the herb with ethanol, partitioning of the extract between water and hexane, followed by evaporative crystallization of artemisinin from hexane phase to produce substantially pure artemisinin.
  • BACKGROUND ART Artemisinin (Qinghaosu) is a sesquiterpene lactone endoperoxide having potent antimalarial activity (Klayman DL, 1985, Science 228, 1049).
  • Another method of artemisinin extraction involves the extraction of plant material with hexane, followed by partitioning the extract between hexane and acetonitrile followed by chromatographing the acetonitrile phase on silica gel (ElFerali FS, ElSohli HN, 1990, US Patent No. 4,952,603). Slow extraction of artemisnin in hexane needs hot percolation for longer duration, while for partitioning step, acetonitrile being relatively costly relatively costly solvent, increases cost of production for artemisinin.
  • Another disadvantage of this method is that for obtaining artemisinin column chromatography is unavoidable.
  • artemisinic acid being predominant, it tends to elute with artemisinin, thus affecting the purity of artemisinin.
  • the main object of the present invention is to provide a simple, rapid, cost effective and practical method for the isolation of artemisinin from plant Artemisia annua without using chromatography, which yields artemisinin in substantial quantities and purity obviating the drawbacks of the prior art.
  • the present invention provides a process for the preparation of artemisinin from Artemisia annua, said process comprising:
  • step (ii) concentrating the extract of step (i) to recover the polar solvent; (iii) adding water to the aforesaid concentrated extract;
  • step(iv) adding hydrocarbon solvent to partition the aqueous extract of step(iii) into an aqueous layer and an organic layer;
  • step (v) separating the organic layer from the solution of step (iv);
  • step (vi) concentrating the organic layer of step (v) to obtain oily liquid; (vii) adding ethyl acetate to the oily liquid of step (vi);
  • step (viii) treating the solution of step (vii) with a conventional color absorbing substance to remove greenish pigmentation;
  • step (x) evaporating the solution followed by crystallization to obtain pure artemisinin.
  • step (i) dry leaves are taken for extraction.
  • the leaves are powdered before extraction.
  • the non-aqueous polar solvent is selected from the group consisting of ethanol, methanol, acetone, methylisobutyl and hexane.
  • the non-aqueous solvent is ethanol.
  • the ratio of the plant parts to the non- aqueous solvent is in the range of 1:5 to 1:10.
  • the extraction of the plant parts is effected by percolating the herbs with ethanol for four to six hours.
  • percolation of the herb is effected at temperatures between 20 to 50°C.
  • step (ii) wherein in step (ii) the volume of the ethanolic extract is reduced to 1/20* to l/lOO* of its original volume.
  • step (ii) wherein in step (ii) the solvent recovered after concentration is reused in the process step (i).
  • step (iii) water is added four times the quantity of the concentrated ethanolic extract.
  • step (iv) the hydrocarbon solvent used is selected from the group consisting of pentane, hexane and heptane.
  • step (iv) wherein in step (iv) the hydrocarbon solvent used is hexane.
  • step (iv) wherein in step (iv) the aqueous ethanolic extract is partioned in hexane in the ratio of 1 : 1 or 2: 1.
  • step (v) wherein in step (v) the organic layer is concentrated l/20 th to 1/100* of its original volume.
  • step (vii) 10-20% (v/v) ethyl acetate is added to the concentrated hexane solution to induce crystallization of artemisinin from the liquid.
  • the color-absorbing agent is selected from the group consisting of celite, activated charcoal and charcoal.
  • step (x) artemisinin is obtained by slow evaporation followed by crystallization.
  • the process developed in the present invention provides an improved method of isolating the artemisinin from Artemisia annua plant populations.
  • Our method involves cold extraction with ethanol/methanol, which on one hand allows complete extraction of artemisinin with rapidity and saves energy on the other hand in contrast to the methods of extraction using hexane or petroleum ether practiced routinely. Extraction in hexane or ether was very slow and needed hot percolation over several hours.
  • Partitioning of the extract between water and hexane is economical and a completely non-obvious approach to enrich the extract with artemisinin, as 60-70% impurities of extract are left in water layer and complete transfer of artemisinin in to hexane layer.
  • the hexane fraction thus obtained can be used for direct crystallization after a step of de-colorization for obtaining artemisinin without chromatography.
  • invention gives a new approach of isolating the artemisinin from its natural source Artemisia annua plant populations without using chromatography.
  • the mild conditions used for extraction give a clean plant extract and avoid the possible degradation of artemisinin.
  • dried pulverized leaves of Artemisia annua may be extracted by continuous percolation over a period of four to six hours using five to ten fold volume of the non-aqueous solvent selected from ethanol or methanol.
  • the said extraction process may be repeated three to five times using same solvent ratios to ensure maximum extraction of artemisinin from the herb.
  • the resulting extract can be concentrated to 1 to 5% of the original volume by distillation under vacuum.
  • the recovered solvent may be used again in the extraction process.
  • For partitioning the aqueous content and hexane may be used in a ratio of 1 : 1 or 2 : 1 v/v. Partitioning of aqueous content with non-polar solvent may be repeated three to five times using the same solvent ratio in order to ensure maximum transfer of artemisinin to hexane fraction.
  • the combined hexane fractions may be pooled together before they are distilled under vacuum (to recover the solvent for using again) to obtain 1-5% of its original volume.
  • the concentrated liquid may be a light to dark green oily liquid.
  • Ethyl acetate (10- 20% v/v) is added to it. To remove the green pigmentation this liquid is treated with 1- 3%w/v of any colour absorbing substance activated charcoal. The yellowish liquid obtained after removal of activate charcoal (by filtration) may be subjected to the evaporative crystallization yielding substantially pure artemisinin without using chromatography.
  • the extraction of dry, pulverized leaves with non-aqueous ethanol may have several advantages over the previously used hexane or di- ethyl ether solvents. Besides extraction being rapid, ethanol will extract less amount of fatty material, which are considered as an obstacle in the purification and crystallization steps.
  • the extract is partitioned between water and hexane in order to enrich the extract with respect to the artemsinin and remove as many impurities as possible from the concentrate to facilitate the crystallization of artemisinin.
  • partitioning of the ethanol extract is carried out using water and hexane in a ratio ranging from (1 : 1 or 2 : 1), the process of partitioning can be repeated 3 - 5 times to ensure maximum transfer of artemisinin to hexane layer.
  • the partitioning step results in a substantially exclusive transfer of the artemisinin into the hexane layer with concomitant reduction in the amount of material, i.e. only 30 to 35% weight of the original extract is transferred in to hexane layer.
  • hexane fraction can directly be used for crystallization by slow evaporation after concentrating the hexane fraction and mixing 10-20% (v/v) of ethyl acetate.
  • the TLC was carried out in a glass TLC tank saturated with the mobile phase hexane: diethyl ether (1 : 1), plates were developed to a height of 15 cm. Later plates were dried and spots were visualized by immersing the plates in a developing reagent (glacial acetic acid: sulfuric acid: anisaldehyde, 50: 1 : 0.5), followed by heating the plate at 110°C for 15 minutes or until pink spots of artemisinin appeared. For quantification, TLC spots corresponding to artemisinin were scanned at 540 and 610 nm dual wavelength mode of TLC scanner. The results obtained are presented in the form of % artemisinin extracted in to the solvent and are summarized in table - 1.
  • Table 2 Data representing the trend of partitioning of artemisinin between hexane and ethanolic solution with water.
  • artemisisnin can be selectively transferred from ethatnolic extract of Artemisia annua in to hexane by maintaining the above ratios. From hexane fraction, artemisinin can be crystallized directly using known methods of evaporative crystallization.
  • Dry pulverized leaves of Artemisia annua (100 g) was extracted by continuous percolation over a period of four hours using 600 ml of ethanol at 30°C temperature. The process of extraction was repeated four times using same solvent ratios.
  • the combined extract was concentrated under vacuum to reduce the volume to 50 ml, volume was made up to 250 ml by adding water to it. The resulting mixture was partitioned with 250-ml hexane four times.
  • the combined hexane layer was reduced under vacuum (to recover hexane) to 5% of its original volume to result in dark green oily liquid.
  • a 20% v/v ethyl acetate was mixed to this liquid followed by treating this with 1% w/v activated charcoal.
  • Dry pulverized leaves of Artemisia annua (100 g) was extracted by continuous percolation over a period of four hours using 1.0 litre of ethanol at 30°C temperature. The process of extraction was repeated four times using same solvent ratios.
  • the combined extract was concentrated under vacuum to reduce the volume to 80 ml, volume was made up to 400 ml by adding water to it.
  • the resulting mixture was partitioned with 400-ml hexane four times.
  • the combined hexane layer was reduced under vacuum to 50 ml volume to result in dark green oily liquid.
  • a 20% v/v ethyl acetate was mixed to this liquid followed by treating this with 1% w/v activated charcoal.
  • the liquid was filtered as in example 3 and subjected to evaporative crystallization.
  • the artemisinin crystals obtained were weighed to be 605 mg.
  • EXAMPLE 7 Dried pulverized leaves of Artemisia annua (1.0 kg) were extracted by continuous percolation over a period of four hours using 6 litres of ethanol at 30°C temperature. The process of extraction was repeated four times using same solvent ratios. The combined extract was concentrated under vacuum to reduce the volume to 400 ml, volume was made up to 2.0 liters by adding water to it. The resulting mixture was partitioned with 2.0 liters hexane three times.
  • Dried pulverized leaves of Artemisia annua (100 g) were extracted by continuous percolation over a period of four hours using 600 ml of methanol at 30°C temperature. The process of extraction was repeated four times using same solvent ratios.
  • the combined extract was concentrated under vacuum to reduce the volume to 50 ml, volume was made up to 250 ml by adding water to it.
  • the resulting mixture was partitioned with 250-ml hexane three times.
  • the combined hexane layer was reduced 5% of its original volume to result in dark green oily liquid.
  • a 20% v/v ethyl acetate was mixed to this liquid followed by treating this with 1% w/v activated charcoal.

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  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un procédé permettant d'isoler l'artémisinine, un agent antipaludique, de l'herbe de Artemisia annua. Ce procédé consiste à réaliser une extraction à l'éthanol, à séparer l'extrait en une solution biphasique eau/hexane, puis à réaliser une cristallisation par évaporation de l'artémisinine à partir de la phase hexane pour produire de l'artémisinine de grande pureté.
PCT/IB2002/001204 2002-03-25 2002-03-25 Procede permettant d'isoler l'artemisinine d'artemisia annua Ceased WO2003080045A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2003577873A JP4339699B2 (ja) 2002-03-25 2002-03-25 アーテミシア・アニュア（Ａｒｔｅｍｉｓｉａ ａｎｎｕａ）からアーテミシニン（ａｒｔｅｍｉｓｉｎｉｎ）を単離する方法
CNA028290003A CN1627942A (zh) 2002-03-25 2002-03-25 黄花蒿离析苦艾素的方法
PCT/IB2002/001204 WO2003080045A1 (fr) 2002-03-25 2002-03-25 Procede permettant d'isoler l'artemisinine d'artemisia annua
AU2002253443A AU2002253443A1 (en) 2002-03-25 2002-03-25 A process for isolating artemisinin from artemisia annua
US10/112,070 US6685972B2 (en) 2002-03-25 2002-03-27 Process for isolating artemisinin from Artemisia annua

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/IB2002/001204 WO2003080045A1 (fr) 2002-03-25 2002-03-25 Procede permettant d'isoler l'artemisinine d'artemisia annua
US10/112,070 US6685972B2 (en) 2002-03-25 2002-03-27 Process for isolating artemisinin from Artemisia annua

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WO2003080045A1 true WO2003080045A1 (fr) 2003-10-02

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PCT/IB2002/001204 Ceased WO2003080045A1 (fr) 2002-03-25 2002-03-25 Procede permettant d'isoler l'artemisinine d'artemisia annua

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JP (1) JP4339699B2 (fr)
CN (1) CN1627942A (fr)
AU (1) AU2002253443A1 (fr)
WO (1) WO2003080045A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008502712A (ja) * 2004-06-11 2008-01-31 ユニジェン インク． 竹又は竹抽出物を含有してなるアンドロゲン作用組成物
CN100445726C (zh) * 2006-08-09 2008-12-24 重庆医科大学 一种青蒿中青蒿素含量的测定方法
CN103013655A (zh) * 2013-01-09 2013-04-03 新疆大学 苦艾挥发油的制备方法
CN111141846A (zh) * 2019-12-31 2020-05-12 广西仙草堂制药有限责任公司 一种青蒿中青蒿素的含量测定方法
CN114181226A (zh) * 2021-11-04 2022-03-15 河南天源药物研究有限公司 一种青蒿精油的提纯制备工艺
CN116891791A (zh) * 2023-07-12 2023-10-17 苏州满元生物科技有限公司 青蒿油、青蒿油香皂的制备方法及青蒿油香皂

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102890087A (zh) * 2012-11-13 2013-01-23 宁夏医科大学 一种黄花蒿等中药材及含青蒿素成分样品中青蒿素含量的测定方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0908460A1 (fr) * 1997-10-06 1999-04-14 Council of Scientific and Industrial Research Procédé pour la production simultanée d'artemisinine et d'huile essentielle à partir de la plante artemisia annua
US6197767B1 (en) * 1997-04-26 2001-03-06 Paracure, Inc. Arglabin compounds and therapeutic uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197767B1 (en) * 1997-04-26 2001-03-06 Paracure, Inc. Arglabin compounds and therapeutic uses thereof
EP0908460A1 (fr) * 1997-10-06 1999-04-14 Council of Scientific and Industrial Research Procédé pour la production simultanée d'artemisinine et d'huile essentielle à partir de la plante artemisia annua

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
OVEZDURDYEV A ET AL: "Sesquiterpene lactones of two species of artemisia", CHEMISTRY OF NATURAL COMPOUNDS, vol. 23, no. 4, 1987, pages 512 - 513, XP001084753 *
ROTH, RONALD; ACTON, NANCY: "Isolation of arteannuic acid from Artemisia Annua", PLANTA MEDICA, vol. 53, no. 5, 1987, pages 501 - 502, XP001088915 *
SINGH A ET AL: "Evaluation of Artemisia annua Strains for higher Artemisinin Production", PLANTA MEDICA, vol. 54, no. 5, 1988, pages 475 - 476, XP001088281 *
SINGH A K ET AL: "Annphenone, a phenolic acetophenone from artemisia annua", PHYTOCHEMISTRY, PERGAMON PRESS, GB, vol. 44, no. 3, February 1997 (1997-02-01), pages 555 - 557, XP004292837, ISSN: 0031-9422 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008502712A (ja) * 2004-06-11 2008-01-31 ユニジェン インク． 竹又は竹抽出物を含有してなるアンドロゲン作用組成物
CN100445726C (zh) * 2006-08-09 2008-12-24 重庆医科大学 一种青蒿中青蒿素含量的测定方法
CN103013655A (zh) * 2013-01-09 2013-04-03 新疆大学 苦艾挥发油的制备方法
CN111141846A (zh) * 2019-12-31 2020-05-12 广西仙草堂制药有限责任公司 一种青蒿中青蒿素的含量测定方法
CN114181226A (zh) * 2021-11-04 2022-03-15 河南天源药物研究有限公司 一种青蒿精油的提纯制备工艺
CN116891791A (zh) * 2023-07-12 2023-10-17 苏州满元生物科技有限公司 青蒿油、青蒿油香皂的制备方法及青蒿油香皂

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Publication number Publication date
CN1627942A (zh) 2005-06-15
JP2005526096A (ja) 2005-09-02
AU2002253443A1 (en) 2003-10-08
JP4339699B2 (ja) 2009-10-07

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