Drug – bio-affecting and body treating compositions – Plant material or plant extract of undetermined constitution... – Containing or obtained from artemisia
Reexamination Certificate
2000-03-30
2002-01-08
Weber, Jon P. (Department: 1651)
Drug, bio-affecting and body treating compositions
Plant material or plant extract of undetermined constitution...
Containing or obtained from artemisia
C424S774000, C424S773000, C424S779000, C424S725000
Reexamination Certificate
active
06337095
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a process for the isolation of compound scopoletin useful as a nitric oxide synthesis inhibitor from the plant
Artemisia annua
and other plant families. Scopoletin belongs to the coumarin group of compounds. Scopoletin (7-hydroxy-6-methoxy coumarin is a derivative of benzo-&agr;-pyrone coumarin and is found in plants both in the free state and glycosides.
BACKGROUND OF INVENTION
Natural coumarins exert varied and pronounced effects on living organisms. Coumarins exhibit numerous pharmalogical and physiological activities such as antibacterial, vasoditory and diuretic effects, anticoagulant properties, hepatoprotective and respiratory stimulation. Scopoletin showed very good growth inhibitory properties. (Shilling D G and Yoshikawa F,
Amerchem. Soc. Syinp Series
, 330:335-342). Recently, it was reported that scopoletin showed inhibition of nitric oxide (NO) synthesis in murine macrophages. Macrophages play a major role in last defense against infection and tumor development and this activity is regulated through the production of several mediates. The production of NO by macrophages mediates killing or growth inhibitors of tumor cells, bacteria fungi and parasites. Therefore it will be valuable to develop a potent and economic source of scopoletin, inhibitor of NO for potential therapeutic and commercial use in the future (Tai-Hyun Kang et al: scopoletin: an inducible NO synthesis inhibitory active constituents from
Artemisia feddi, Planta Medica
, 65:400-403, 1999); Invitro inducible NO synthesis inhibitory active constituent from
Ttraxinus rhynchobhyeli
, Plant Medica, 65:656-658, 1999).
Scopoletin is widely distributed in the plant kingdom and is isolated from the different parts (roots, fruits, leaves, stems, etc.) of the plant, but several related members frequently are found together making their isolation difficult. In general isolation of coumarins depends initially upon successive extraction of dried plant with commonly used solvents of increasing polarities (pet ether, benzene, ether, acetone or methanol).
The conventional method used for the isolation of the coumarins used dried powder material extracted with 95% methanol/ethanol, and the concentrated extract was treated with 10% alkali solution. The alkaline solution was extracted with ether to remove the fatty material. The aqueous solution was acidified and extracted with ether or chloroform to obtain crude coumarins. Further, the purification was carried out on column chromatography over silica gel or alumina to get pure coumarins. Certain coumarins (e.g. umbilliferon, scopoletin) can be isolated from plant tissues by sublimation methods (Abu-Mustafa, E. A.; El-Tawil, B. A. H. and M. B. E. Fayez; Phytochemistry, 3, 701 (1964)).
However, the method of isolation of coumarin through acid based treatment is not ideal because it is hardly possible to avoid some degradation of the coumarins which lead to the isolation of artefacts rather than the original compound or the loss of the compounds. In another process purification done by sublimation may involve thermal degradation with formation of artefacts. The overall yield of recovery is reduced. As such no plant with high content of scopoletin for commercial exploitation nor any large scale process for the isolation of scopoletin has been reported.
OBJECTS OF THE INVENTION
The objective of the present invention is to explore new plant/plant part with a high content of scopoletin.
Another objective of the invention is the selection of solvent for the selective extraction of scopoletin.
Still another objective of the invention is to develop an economic process for the isolation of pure scopoletin.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a process for the isolation of nitric oxide synthesis inhibitor compound scopoletin from the plant
Artemisia annua
, and other plant families, which comprises the extracting the powder plant parts with an aqueous acetonitrile solvent, partitioning with a chlorinated solvent, evaporating the solvent, crystallizing a residue from the solvent and employing chromatography to yield pure scopoletin.
Novelty of the Process
1. Selection of the plant part for the extraction of scopoletin improved the yield and reduced the processing cost.
2. The extraction of the plant part with aqueous acetonitrile is more economic with a better yield of scopoletin as compared to the use of other polar solvents like methanol or ethanol.
3. In this process, by avoiding any acid base treatment for the isolation of scopoletin the yield was improved.
4. The selective transfer of scopoletin from the aqueous extract to the halogentaed non polar phase resulted in easy purfication and isolation of pure scopoletin.
5. The purificaton of the crude extract resulting in crystallisation of 50% of scopoletin before the chromatography.
6. The process allows the reuse of solvents and silica gel.
7. These advantages are of significant economic values for large scale production of scopoletin.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for the isolation of compound scopoletin which is used as nitric oxide synthesis inhibitor from
Artemisia annua
and other plant families, said process comprising:
a) extraction of dried powdered material of different plant parts with an aqueous acetonitrile solvent in the ratio of 1:5 for 6 to 8 hrs.,
b) concentration of the extracted solvent upto 30% of its original extract under vacuum,
c) partitioning the concentrated extract with halogenated solvent to transfer scopoletin in the non-polar halogenated solvent,
d) drying halogenated solvent over anhydrous sodium sulphate and evaporating the solvent,
e) crystallizing the residues in methanol and filtering the crystals,
f) concentrating the filtrate and chromatographed on silica gel,
g) eluting scopoletin in chloroform methanol mixture; and crystallization of the fractions containing the scopoletin to get the pure scopoletin compound.
In one embodiment of the invention,
Artemisia annua
plant was selected for the isolation of scopoletin.
Artemisia annua
ariel parts from Spain have reported 0.02% scopoletin and England (cult) 0.034%. China has also reported the presence of scopoletin from the whole plant (Brown, G. D.: Two new compounds from
Artemisia annua
: J.Nat. Prod. 58, 300 (1992); Marco, J. A.; Sawz, J. F.; Bea, J. F.; Barber, O.: Phenolic constituents from
Artemisia annua
: Pharmazie 45, 382-383 (1990); Liu, H. M.; Li, G. L., Wu, H. Z.: Studies on the constituents of Quinghao:Yao Hsuch Hsuch Pao, 37, 129-143 (1979)).
In India CIMAP, Lucknow, has developed a new variety of
Artemisia annua
“Jeevan raksha” producing high content of artemisinin and biomass (stems & leaves). Artemisinin and its derivative are reported as potent against chloroquine resistant multi drug resistant and severe complicated malaria.
Artemisia annua
plant is the only source of artemisinin.
Screening of all the three major parts of the
A. annua
plant of the new variety for scopoletin was carried out by HPLC. The content of scopoletin obtained in different plant parts are as follows; leaves 0.2%, Stems 0.3% and roots 0.004%. The yield of the scopoletin is very high as compared to other reported plants. The stem part of the plant
Artemisia annua
is a waste material as no artemisinin is present in the stems. The biomass of the stem portion of the plant is five times more than of the leaves. Therefore, we have selected the stem part of the plant
Artemisia annua
for the isolation of scopoletin. Also the stem portion of the stems was found to contain less colouring and fatty material which eases the isolation and purification of scopoletin.
In the prior art, it has been observed that non polar solvents for extraction of the plant materials were employed, resulting in less recovery of coumarins. Still, polar solvents (methanol and ethanol) used for the extraction of coumarins resulted in a higher amount of total extract having more colouring and fatty material. The separation of fatty and col
Bhakuni Rajendra Singh
Gupta Madan Mohan
Gupta Shiv Kumar
Jain Dharam Chand
Kahol Atul Prakash
Council of Scientific and Industrial Research
Merchant & Gould P.C.
Patten Patricia
Weber Jon P.
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