Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing a...
Reexamination Certificate
2002-01-15
2003-03-04
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing a...
C435S052000
Reexamination Certificate
active
06528280
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a process for the preparation of derivatives of
Ruscus Aculeatus
steroid glycosides (ruscosaponins).
More particularly, the invention relates to the preparation of desglucodesrhamnoruscin by hydrolysis of ruscoside and/or desglucoruscoside through the fermentation route.
BACKGROUND OF THE INVENTION
Desglucorhamnoruscin and the corresponding free aglycons, ruscogenin and neoruscogenin, which can be easily obtained from desglucodesrhamnoruscin by acid hydrolysis, are valuable pharmaceutical active principals having antiinflammatory and connective-protecting activities.
The chemical preparation of said active principles starting from ruscoside or desglucoruscoside is however problematic, since it requires drastic conditions, such as hydrolysis with strong acids, and complex operative steps, which yield a very heterogeneous mixture of intermediates and products.
It would therefore be highly desirable to provide a process for the preparation of desglucodesrhamnoruscin, which overcomes the drawbacks mentioned above connected with the known chemical processes.
SUMMARY OF THE INVENTION
The present invention meets such a need, by providing a process for the preparation of desglucodesrhamnoruscin which comprises the hydrolysis of
Ruscus aculeatus
steroid glycosides (ruscosaponins) through fermentation of a substrate containing said glycosides by means of fungi of the
aspergillus niger
species.
A culture broth is typically used as a nutrient complex substrate.
Fermentation is generally carried out at a temperature of 25-30° C., preferably 27° C., under stirring and aeration so as to attain a pO2 higher than or equal to 50%.
The concentration of the starting steroid glycosides usually ranges from 5 to 15% w/v, preferably from 8 to 10% w/v and the pH of the culture broth ranges from 4 to 6, preferably 4.5-5.5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The biotechnological process of the present invention allows to carry out the whole reactions sequence in a single fermentation step, in that the microorganism, selected with suitable microbiological techniques, is capable of expressing the necessary enzymatic activities for operating all the transformation sequential reactions, from the starting complex heteroglycoside to the monoglycoside or the final aglycone. Said hydrolase transformations comprise in fact a sequence of &bgr;-glucosidase, &agr;-rhamnosidase reactions on the intermediates which are successively released during the process. A further &agr;-arabinosidase reaction allows to obtain the free aglycone (ruscogenin).
Said approach is quite novel, in that no applicative examples of said procedure for the preparation of said products can be found in literature.
The microorganisms suitable for carrying out the transformations involved are obtained by selection on synthetic or semi-synthetic media, added with the same substrates to be transformed, in addition to or in place of the conventional carbon sources (glucose, saccharose, and the like). The concerned substrates (ruscoside, desglucoruscoside) can be added in this case in even high concentrations, e.g. 90-100 g/L. The agarized isolation media comprise the usual formulations for microbiology, such as Malt Agar and Czapek Agar, or similar formulations, wherein the nitrogen source is represented by peptones, urea, ammonium nitrate, and the like, whereas the conventional carbon source (glucose, saccharose) has been substituted or supplemented by ruscoside or desglucoruscoside. Said media can be further added with mineral salts of potassium, magnesium, manganese, zinc etc., such as phosphates, sulfates and/or chlorides. The pH of the isolation media can range from 4 to 6, preferably from 4.5 to 5.5.
The microorganisms suitable to the required biotrasformations are recovered by scalar dilution and plating of aqueous suspensions of samples of soil, humus, vegetable extracts and other similar organic sources.
The microbial cultures selected as described above are isolated in microbiology test-tubes containing the same culture media and used for the biotransformation of ruscoside and desglucoruscoside, added in high concentrations (to 100 g/L) to liquid culture media containing the same nitrogen sources as used in the isolation media, such as urea or peptone, with the addition of phosphates and other mineral salts, as described above, at pH ranging from 4 to 6, preferably 4.5÷5.5.
Following the procedures described it has been found that selected cultures of
Aspergillus niger
are capable of transforming ruscoside and desglucoruscoside into desglucodesrhamnoruscin, a direct precursor of ruscogenins, by a sequence of enzymatic &bgr;-glucosidase and &agr;-rhamnosidase reactions.
A subsequent &agr;-arabinosidase reaction provides the saponin in the aglyconic form (ruscogenin-neoruscogenin).
The selected culture is capable of operating said transformations growing in controlled (thermostatic) conditions, at optimal temperatures ranging from 25° C. to 30° C., under stirring on a rotatory shaker (200÷300 rpm). Said fermentation can also be carried out in a suitable bioreactor, at different scale levels, for the industrial production of the desired saponin derivatives.
The microorganisms used for said biotransformation are capable of steadily maintaining the catalytic activity, even for repeated fermentation cycles, in batch or continuous processes.
The present process provides important advantages, such as less complex the steps for the separation and recovery of the product, and is also easy to carry out as well as cost-saving.
The selected microorganisms can be frozen for the to long-term storage, in suspensions enriched with cryopreservatives, such as glycerol, peptone and the like, at temperatures ranging from −80° C. to −196° C. (in liquid nitrogen), or subjected to freeze-drying treatments.
The progress of the bioconversion can be monitored by TLC and HPLC analysis on the culture broth, using the following analytical methods:
TLC Analysis
Silica gel plates 60 F250 Merck
Eluents:
A) Ethyl acetate-Methanol 9:1
B) Ethyl acetate-Methanol-Water 100:15:10.
Detection: reaction with 10% sulfuric acid and heating to 120° C. for 5 minutes, then visible and UV detection.
HPLC Analysis
Column: Supelcosil LC18, 250×4,6 mm, 5 &mgr;m
Eluent: acetonitrile-water 60:40
Wavelength: 200 nm
Injection volume: 10 &mgr;l
Flow: 1 mL/min.
The final biotransformation products, such as desglucodesrhamnoruscin, can be recovered by extraction of the culture broth with n-butanol, subsequent purification steps with chlorinated solvents (such as trichloroethane) and silica gel chromatography. Finally, the product can be crystallized from different solvents, such as isopropanol, ethyl acetate, chloroform, acetone, methanol. In addition to the main product, desglucodesrhamnoruscins esterified at C-2′, for example with 2-hydroxy-3-methylpentanoic acid, can be obtained.
The saponins in the aglyconic form (ruscogenins) are obtained by acid hydrolysis of the fermentation products described above.
The following examples disclose the invention in greater detail.
REFERENCES:
patent: 2 104 911 (1972-04-01), None
patent: 1 380 253 (1975-01-01), None
Computer Caplus Abstract 1976:87880 Perepelitsa et al Prikl.Biokhim Mik (1975)11 (6) 901-5.*
Computer Caplus Abstract 1975:528650 Perepelitsa et al Khim Prir.Soedin (1975) 11 (2) 260-1.*
ATCC Catalogue Bacteria & Bacteriophages 18thEd 1993 p. 544.*
Computer Pascal Abstract 1994-0473378 Ray et al Jour Microbial Biotech (1993) 8(2) 84-95.*
Computer Pascal Abstract 1985-0261580 Okada Agric. & Biol. Chem. (1985) 49 (5) 1257-1265.
Acquati Walter
Ponzone Cesare
Indena S.p.A.
Lilling Herbert J.
Pennie & Edmonds LLP
LandOfFree
Process for the preparation of derivatives of ruscus... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for the preparation of derivatives of ruscus..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for the preparation of derivatives of ruscus... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3074738