Extracting betulinic acid from Ziziphus jujuba

Details Extracting betulinic acid from Ziziphus jujuba Extracting betulinic acid from Ziziphus jujuba

2000-03-01

2001-07-24

Naff, David M. (Department: 1651)

Drug, bio-affecting and body treating compositions

Plant material or plant extract of undetermined constitution...

Containing or obtained from a bark

C424S725000

Reexamination Certificate

active

06264998

ABSTRACT:

FIELD
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
Ziziphus jujuba.
BACKGROUND
Under the auspices of National Cooperative Natural Product Drug Discovery group supported by National Cancer Institute, the potential antitumor activity of approximately 2500 extracts derived from globally collected plants was evaluated. One such extract, prepared from stem bark of
Ziziphus mauritiana
Lam (Rhamnaceae) displayed selective cytotoxicity against cultured human melanoma cells (National Medicine, Vol.1(10), 1995, No.96/29068). The active principle responsible was identified as betulinic acid, whose structure is represented in
FIG. 1
of the accompanying drawings. This prompted the applicants to devise a process for the isolation of betulinic acid from the bark of
Ziziphus jujuba
which is abundantly available in India.
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. Unfortunately, since benzene is highly carcinogenic, this method was abandoned. Some of the common methods employed in the art for the extraction of betulinic acid are given herein below.
Alpin et. al. Chemistry of Titerpenes and related compounds, Part XLIII p3269, relates to extraction of betulinic acid from the bark of
Platanus x hybrida Brot.
In this process, the bark was extracted with methanol and partly evaporated, whereupon betulinic acid was crystallized. The ether-soluble portion of the remaining material was separated into neutral and acidic fractions. From the acidic fraction, some more betulinic acid was isolated as methylester by treatment with diazomethane followed by column chromatography. Unfortunately, this method involves hazardous steps and reagents such as methylating with ethereal diazomethane; accordingly, this process was found to be unfit for commercial exploitation.
Current Science, 1965, p.344, relates to extraction of bark of
ziziphus jujuba
with light petroleum ether and ether solvent; followed by extraction of ether extract with sodium hydroxide solution thereby leading to the isolation of sparingly soluble sodium salt of betulinic acid. In this process extraction of organic layer with alkali solution leads to emulsion problems. Once emulsion is formed no discrete layer separation is obtained. Therefore, it is difficult to implement this process on commercial scale. In addition, this process also leads to wastage of the required compound involving cumbersome operational activities.
Phytochemistry, 1968, Vol.7, p461, relates to the extraction of seed kernels of
Alangium lamarckii
with light petroleum ether, benzene and chloroform respectively. The residue from benzene and chloroform extracts are poured onto ice water to yield a solid. The solid was dissolved in chloroform and extracted with 3% aqueous KOH. The precipitated potassium-salt was filtered, washed with water to free it from alkali. The K-salt was further treated with 2N HCI and extracted with ether. The ethereal solution was charcoalised in MeOH—CHCI
3
and followed by repeated crystallisation of the residue from methanol which finally yielded pure betulinic acid. Even in this process, it is impossible to get discrete layer separation and hence, this process is also unfit for commercial exploitation.
Aus. J. Chem., 1969, 22, p1331, relates to the extraction bark of
Akania lucens
(F. Muell) also known as “turnip wood”, with light petroleum ether and methanol. The ether soluble portions of these extracts were separately worked by extracting with NaHCO
3
, Na
2
CO
3
and NaOH solution, followed by methylation of the residue obtained and purification by column chromatography to yield methylester of betulinic acid. It is apparent that this process is quite cumbersome and unfit for commercial exploitation. This process involves interface formation which is apparent from the citation.
Journal of Indian Chemical Society, 1969, Volume 46, No.4, p.386, relates to preparation of triterpenoids from the bark of
Rhododendron arboreum.
This process is tedious and involves the use of hazardous materials such as ethereal solution of diazomethane. The process is unfit for commercial production of betulinic acid.
Ind. J. of Chemistry (1972), p.152, deals with the isolation of betulinic acid from
Ziziphus rugosa
Lam. by extracting it with alcohol and obtaining concentrated alcoholic extract, which is further extracted with chloroform and filtered to remove the insoluble material. Chloroform soluble portion was evaporated to dryness, defatted with petroleum ether, followed by chromatography of the residue which yielded betulinic acid.
Prot. Nat. Acad. Sci. India, (1975), p.300, describes the extraction of plant
Anemone rivularis
with alcohol, followed by concentration and chilling of extract to give a brown residue which is digested with ether. The ether soluble portion was extracted with potassium hydroxide to yield potassium salt of betulinic acid. The above processes involve usage of ether which is hazardous. In addition, other disadvantages cited earlier such as performing chromatography are also associated with the processes.
Phytochemistry, 1970, Vol.9, p907, relates to the extraction of bark of
Arbutus menziesii
with methanol. The methanolic extract was treated with chloroform and the chloroform solution filtered through alumina and evaporated to yield a residue, which was further treated with methanol and acetic acid to yield a residue. This residue was again chromatographed on silica gel to yield betulinic acid. This process involves expensive and tedious steps of chromatography.
Filoterapia (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
4
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, Vol.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.
Chem. Pharm. Bull. (1996), p.1033, described isolation of betulinic acid from hot chloroform extract of outer bark of
Betula pentayphylla
var
japonica.
The extract is concentrated, added ethanol to crystallise crude betulin. The mother liquor was chromatographed to yield betulinic acid as one of the product.
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 betunic acid.
U.S. Pat. No. 5,804,575 relates to synthetic conversion of betulin (present to the extent of 25% W/W 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 U.S. patent application Ser. No. 09/040,856 dated Mar. 18, 1998 discloses a process for producing active bet

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