WO2001064709A1 - Nouveau procede de fabrication de l'acide betulinique a partir de ziziphus jujuba - Google Patents

Nouveau procede de fabrication de l'acide betulinique a partir de ziziphus jujuba Download PDF

Info

Publication number
WO2001064709A1
WO2001064709A1 PCT/IN2000/000019 IN0000019W WO0164709A1 WO 2001064709 A1 WO2001064709 A1 WO 2001064709A1 IN 0000019 W IN0000019 W IN 0000019W WO 0164709 A1 WO0164709 A1 WO 0164709A1
Authority
WO
WIPO (PCT)
Prior art keywords
betulinic acid
crude
solvent
extract
pure
Prior art date
Application number
PCT/IN2000/000019
Other languages
English (en)
Inventor
Sunder Ramadoss
Mohammad Jamshed Ahmed Siddiqui
Original Assignee
Dabur Research Foundation
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 Dabur Research Foundation filed Critical Dabur Research Foundation
Priority to PCT/IN2000/000019 priority Critical patent/WO2001064709A1/fr
Priority to AU2000235744A priority patent/AU2000235744A1/en
Priority to EP00914351A priority patent/EP1259533A1/fr
Publication of WO2001064709A1 publication Critical patent/WO2001064709A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids

Definitions

  • the present invention relates to the field of phytochemistry. More specifically, the invention provides a simple and cost effective method for the isolation of betulinic acid from the bark of Ziz ⁇ hus jujuba.
  • the compound betulinic acid has been isolated mainly from bark, seed, kernels and leaves of various plants.
  • the extraction of biomass with methanol, chloroform, benzene or ethereal solvent yielded an extract which is treated with alkaline solution to selectively extract acidic components, which is subsequently methylated and the product chromatographed to yield methylester of betulinic acid.
  • benzene is highly carcinogenic, this method was abandoned.
  • Filorick (1987), p.58, describes the isolation of betulinic acid from Zizyphus sativa by extraction of the stem bark of the plant with C 6 H 6 :MeOH:NH_ ⁇ OH mixture in the ratio of 100: 1: 1 respectively.
  • the basic compounds were separated from the extract by extraction with aqueous citric acid and benzene fraction left behind is evaporated to dryness and chromatographed on silica gel to yield betulinic acid.
  • Planta Medica, 1988, p.511 relates to the extraction of twig of Agrostistachys hookeri with chloroform, followed by column chromatography of the extract to yield betulinic acid. This involves expensive and tedious steps of chromatography.
  • J.Nat, products. 1994, Nol.57, p.243 relates to the extraction of leaves of Syzigium claviforum with methanol.
  • the methanol extract was partitioned successively with hexane, chloroform, ethyl acetate and n-butanol.
  • Betulinic acid was isolated by repeated column chromatography of chloroform soluble portion.
  • Planta Medica 1997, p.255 describes the isolation of betulinic acid from root bark of Triphyophyllum pelatum and Ancistrocladus heyneanus by extracting with petroleum ether and chromatography of the residue obtained over silica gel.
  • Synthetic communications, 1997, p.1607 describes a method for synthetic conversion of betulin to betulinic acid.
  • US Patent No.5, 804,575 relates to synthetic conversion of betulin (present to the extent of 25%WAV in the bark of white birch, Betula alba) to enantiomerically pure beta—isomer of betulinic acid). Since the above methods relate to synthetic conversion, they are no way related to the applicant's present process.
  • the applicant's recently accepted US Patent Application No.09/040,856 dated March 18, 1998 discloses a process for producing active betulinic acid analogues. This process involves altogether a different procedure.
  • Another feature of the present invention is to employ acylation to derive crude betalinic acid in the process for isolation of pure betulinic acid, which is easy, inexpensive and non-hazardous.
  • the mam objective of the present invention is to provide a simple and cost effective process for the extraction of betulinic acid which forms the basic molecule for the invention of new anticancer drug.
  • Another objective is to provide a process which does not involve tedious step of chromatographic technique at any stage for the isolation of betulinic acid.
  • Yet another objective is to provide a process wherein the solvent used in various steps can be recycled.
  • Still another objective is to provide an ecofriendly process which provides complete isolation of betulinic acid from the raw material used.
  • One more objective relates to a process which results in betulinic acid having purity greater than 98%.
  • the present invention provides a novel process for isolation of betulinic acid from primary extract on a commercial scale from plant extract of Ziziphus jujuba by solvent crystallisation without employing chromatographic separation.
  • the invention provides a novel process for the isolation of betulinic acid on a commercial scale from plant extract of Ziziphus jujuba, said process comprising the steps of preparing an aromatic hydrocarbon solvent extract of bark of Ziziphus jujuba, concentration of extract to a definite volume, chilling, centrifuging or filtering separate the solid (I) and mother liquor.
  • the solid (I) is charcoalised in methanol.
  • methylene chloride is added and chilled. This is then filtered to separate solid (II) and acylated with Py/Ac 0 to get a mixture of acylated product, which is purified by treatment with isopropanol to give pure 3/3- acetoxy betulinic acid.
  • the acetate is treated with methanolic aqueous alkaline solution to yield pure betulinic acid.
  • Fig 1 represents the structure of betulinic acid.
  • the invention provides a process for the isolation of betulinic acid from Ziziphus jujuba, said process comprising the steps of : a) extracting the optionally dried and pulverized parts of Ziziphus jujuba in a solvent, b) preparing a semi-concentrated extract containing betulinic acid, c) chilling the concentrated extract overnight, d) separating the solid from the extract by filtration or centrifugation, e) charcoalising the solid obtained in step (d) with reflux methanol and filtering through celite bed, f) partially concentrating methanolic solution of step (e), adding halogenated hydrocarbon solvent and chilling overnight, g) separation of solid by filtration or centrifugation followed by drying, h) treating the solid of step (g) with Py/Ac 2 0 to yield acetylated product, i) macerating the acetylated product obtained in step (h) with an alcohol to yield pure 3 ⁇ -acetoxy betulinic acid, and (j)
  • the solvent used for the preparation of extract of plants is selected from the group of aromatic hydrocarbons comprising benzene, toluene and xylene.
  • the preferred solvent is toluene.
  • the halogenated hydrocarbon solvent is methylene chloride.
  • the alcohol for washing the acetylated product is selected from methanol, ethanol and isopropanol, preferably isopropanol.
  • the semi-concentrated extract is chilled to yield crude betulinic acid.
  • the preferred alcohol used in step (i) is isopropanol.
  • the chilling in steps (c) and (d) is performed between 0 to 10°C and the partial concentration in step (f) is performed under reduced pressure.
  • the alkali solution in step (j) is prepared in aqueous-alcoholic mixture.
  • the crude betulinic acid is crystallized from the methanol-methylene chloride mixture, preferably in the ratio 1:4.
  • the crude betulinic acid is dissolved in the halogenated solvent containing organic base, preferably pyridine, and treating it with acylating agent, preferably acetic anhydride.
  • the crude 3 ⁇ -acetoxy betulinic acid is purified by maceration with alcoholic solvent, preferably isopropanol.
  • the pure 3 ⁇ -acetoxy betulinic acid is treated with aqueous-alcoholic alkaline solution, preferably aqueous methanolic sodium hydroxide or potassium hydroxide, to give pure betulinic acid ( ⁇ -isomer).
  • aqueous-alcoholic alkaline solution preferably aqueous methanolic sodium hydroxide or potassium hydroxide
  • betulinic acid is isolated by filtration or centrifugation.
  • the preferred part of plant is bark.
  • the present invention provides a novel process for the isolation of betulinic acid having anticancer activity and also a base molecule for the synthesis of other new anticancer analogues produced by the applicants (accepted US application No.09/040,856 filed on March 18. 1998).
  • purification of 3 ⁇ -acetoxy betulinic acid is selected from methanol, ethanol or isopropanol.
  • deacylation of 3 ⁇ -acetoxy betulinic acid is carried out using alcoholic aqueous sodium or potassium hydroxide to get pure betulinic acid.
  • the process illustrated gives quantitative recovery of betulinic acid from the raw material used.
  • the preferred process comprises the following steps: -
  • the bark of Ziziphus jujuba is pulverized and may be optionally dried.
  • Aromatic hydrocarbon solvents selected from benzene, toluene or xylene is added to ground bark and the mixture stirred at approximately 90-95°C for 12 hours.
  • the preferred aromatic hydrocarbon used in the present process is toluene.
  • the semi concentrated extract so obtained contains betulinic acid and other chemical compounds. This is chilled between 0°C-10°C for 16-24 hours. The insoluble material containing betulinic acid is then separated out by filtration or centrifugation. The mother liquor left behind is discarded.
  • step(2) The solid of step(2) is dissolved in hot methanol and refluxed with activated charcoal for an hour and filtered through a celite bed.
  • the clear mother liquor was concentrated to a reduced volume and methylene chloride was added and the solution chilled to 0-10°C for 16-24 hours.
  • the solid is separated by filtration or centrifugation which constituted crude betulinic acid.
  • step(3) Solid of step(3) is dissolved in methylene chloride containing few drops of dry pyridine and treated with acetic anhydride or acetyl chloride. The mixture is stirred at room temperature overnight and worked-up by pouring on to crushed ice with stirring, the organic layer is then separated, washed with dilute acid, aqueous bicarbonate solution, followed by water. The organic layer is dried over anhydrous sodium sulphate, evaporated to dryness to yield crude 3 ⁇ -acetoxy betulinic acid.
  • step (4) The solid of step (4) is washed with isopropanol at room temperature and filtered to separate the solid which constitutes pure 3 ⁇ -acetoxy betulinic acid.
  • step (5) Solid of step (5) is dissolved in methanol and treated with aqueous alkali selected from sodium or potassium hydroxide. The mixture is stirred for 16- 24 hours at room temperature. This is evaporated completely methanol under reduced pressure (100-150 mbar), followed by water, and the pH is adjusted to 7.0 by addition of aqueous dilute hydrochloric acid. The solid obtained is separated by filtration or centrifugation, washed with water to neutrality to yield pure betulinic acid.
  • aqueous alkali selected from sodium or potassium hydroxide
  • EXAMPLE 1 The bark of Ziziphus jujuba optionally dried is finely pulverised (#100 mesh).
  • An aromatic hydrocarbon extract is prepared by stirring 1200 Ltrs. of toluene (in 3 lots) with 100 kgs. of bark (rotation 58 rpm, ca.90-95°C) for 12 hours each time.
  • the toluene solution collected after filtration or centrifugation is concentrated ca. 60-70°C under reduced pressure (150 mbar) to one fiftieth of its original volume, chilled to 0-10°C for a period of 16-24 hours.
  • the solid is separated by filtration or centrifugation, washed with toluene (500 ml) to yield a brown solid (1210 gms).
  • the enriched betulinic acid (720 gms) is dissolved in methylene chloride (7.5 ltrs) containing pyridine (250 ml) and added acetic anhydride (250 ml) and the mixture is stirred at room temperature for 16-24 hours. This is worked-up by washing the reaction mixture with dilute aqueous HCI, aqueous NaHC0 3 followed by water. The organic layer is dried over anhydrous sodium sulphate, filtered and filtrate evaporated to dryness to yield crude 3 ⁇ -acetoxy betulinic acid (810 gms).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Steroid Compounds (AREA)

Abstract

L'invention se rapporte à un nouveau procédé, simple et sans danger pour l'environnement, de fabrication d'acide bétulinique à partir de l'extrait végétal de Ziziphus jujuba (Rhamnaceae) par cristallisation avec solvant sans mise en oeuvre d'une séparation chromatographique.
PCT/IN2000/000019 2000-02-29 2000-02-29 Nouveau procede de fabrication de l'acide betulinique a partir de ziziphus jujuba WO2001064709A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/IN2000/000019 WO2001064709A1 (fr) 2000-02-29 2000-02-29 Nouveau procede de fabrication de l'acide betulinique a partir de ziziphus jujuba
AU2000235744A AU2000235744A1 (en) 2000-02-29 2000-02-29 A novel process for manufacturing betulinic acid from ziziphus jujuba
EP00914351A EP1259533A1 (fr) 2000-02-29 2000-02-29 NOUVEAU PROCEDE DE FABRICATION DE L'ACIDE BETULINIQUE A PARTIR DE i ZIZIPHUS JUJUBA /i

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IN2000/000019 WO2001064709A1 (fr) 2000-02-29 2000-02-29 Nouveau procede de fabrication de l'acide betulinique a partir de ziziphus jujuba

Publications (1)

Publication Number Publication Date
WO2001064709A1 true WO2001064709A1 (fr) 2001-09-07

Family

ID=11076229

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2000/000019 WO2001064709A1 (fr) 2000-02-29 2000-02-29 Nouveau procede de fabrication de l'acide betulinique a partir de ziziphus jujuba

Country Status (3)

Country Link
EP (1) EP1259533A1 (fr)
AU (1) AU2000235744A1 (fr)
WO (1) WO2001064709A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6360949A (ja) * 1986-08-29 1988-03-17 Rikagaku Kenkyusho トリテルペン化合物及び制癌剤
EP0767177A1 (fr) * 1994-06-24 1997-04-09 Nippon Shinyaku Company, Limited Derive du triterpene et preparation medicinale associee
DE19713768A1 (de) * 1997-04-03 1998-10-08 Univ Halle Wittenberg Herstellung von Betulinsäure
EP0943620A2 (fr) * 1998-03-18 1999-09-22 Dabur Research Foundation Dérivés de l'acide bétulinique pour inhibition de la croissance des cancers

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6360949A (ja) * 1986-08-29 1988-03-17 Rikagaku Kenkyusho トリテルペン化合物及び制癌剤
EP0767177A1 (fr) * 1994-06-24 1997-04-09 Nippon Shinyaku Company, Limited Derive du triterpene et preparation medicinale associee
DE19713768A1 (de) * 1997-04-03 1998-10-08 Univ Halle Wittenberg Herstellung von Betulinsäure
EP0943620A2 (fr) * 1998-03-18 1999-09-22 Dabur Research Foundation Dérivés de l'acide bétulinique pour inhibition de la croissance des cancers

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 198817, Derwent World Patents Index; Class B05, AN 1988-115402, XP002155063 *
KUNDU A B ET AL: "ZIZYBERANALIC ACID, A PENTACYCLIC TRITERPENOID OF ZIZYPHUS JUJUBA", PHYTOCHEMISTRY,PERGAMON PRESS,GB, vol. 28, no. 11, 1989, pages 3155 - 3158, XP000964652, ISSN: 0031-9422 *

Also Published As

Publication number Publication date
EP1259533A1 (fr) 2002-11-27
AU2000235744A1 (en) 2001-09-12

Similar Documents

Publication Publication Date Title
Yang et al. Baimantuoluosides DG, four new withanolide glucosides from the flower of Datura metel L.
Siddiqui et al. Kaneric acid, a new triterpene from the leaves of Nerium oleander
US6264998B1 (en) Extracting betulinic acid from Ziziphus jujuba
Lavie et al. Constituents of Citrullus colocynthis (L.) Schrad.
Tupkari et al. Phytochemical study of Solanum xanthocarpum
JP4815041B2 (ja) 酸化窒素合成阻害剤として有用な化合物スコポレチンの単離方法
JPH0248533A (ja) ノブドウ根抽出物よりなる肝疾患治療剤
Horn et al. Transformation of naturally-occurring 1, 9-trans-9, 5-cis sweroside to all trans sweroside during acetylation of sweroside aglycone
Strauss et al. Saponins from root cultures of Phytolacca acinosa
Gupta et al. Two furostanol saponins from Trigonella foenum-graecum
Iwamoto et al. Studies on the Constituents of Actinostemma lobatum MAXIM. I Structures of Actinostemmosides A, B, C and D, Dammarane Triterpene Glycosides Isolated from the Herb
EP1259533A1 (fr) NOUVEAU PROCEDE DE FABRICATION DE L'ACIDE BETULINIQUE A PARTIR DE i ZIZIPHUS JUJUBA /i
US4198344A (en) Process for the preparation of polyhydroxylated steroids, lysergol and ergolinic alkaloids
RU2206572C1 (ru) Способ выделения бетулинола
Sun et al. Four New Cycloartane (= 9, 19‐Cyclolanostane) Saponins from the Aerial Parts of Thalictrum fortunei
Reddy et al. Afromontoside. A new cytotoxic principle from Dracaena afromontana
Djerassi et al. Terpenoids. XXVIII. 1 The Triterpene Composition of the Genus Myrtillocactus2
Atta-Ur-Rahman et al. New steroidal alkaloids from Sarcococca saligna
RU2270202C1 (ru) Способ получения бетулина и лупеола
Alexandre Saponins from Ilex pseudobuxus
Li et al. Further Daphniphyllum Alkaloids from the Bark of Daphniphyllum macropodum Miq.
RU2270201C1 (ru) Способ получения бетулина
Hamed et al. Molluscicidal activity of some constituents isolated from Cestrum purpureum
Li et al. A new diterpenoid alkaloid from Aconitum episcopale
Feng et al. Asparosides A and B, two new steroidal saponins from Asparagus meioclados

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2000914351

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000914351

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2000914351

Country of ref document: EP