Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2001-01-12
2003-04-08
Wilson, James O. (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S025000, C536S004100, C536S116000, C536S128000, C435S072000, C435S074000
Reexamination Certificate
active
06544965
ABSTRACT:
The present invention relates to compounds having the formula
wherein R can be H or CH
3
.
Hereinafter in the present description, compound (I) wherein R═H is called FPA.
Compound (I) wherein R═CH
3
, is called FPB.
FPA (phenylpropanoid glucoside) has been previously described and called teupolioside (Chem. Nat. Compd. 1991, 27:5 556-559), as secondary metabolite, having an antimicrobial activity, present in the plant
Teucrium polium
. The presence of FPA however has never been described in other different plants or in “in vitro” cell cultures of any plant.
FPB, on the other hand, is a compound which has never been described in literature and as such is therefore an object of the present invention. According to a first aspect of the present invention, a production process of FPA and FPB compounds (jointly called FPs, or raw FPs), is proposed.
This consists in cultivating cells of a cellular line taken from a plant of
Ajuga reptans
. For this vegetable species, bibliographical reference can be made to Cantino-Sanders, Syst. Bot. Vol. 11 (1986), pages 163-185.
According to the invention, parts of this plant (leaves, shoots and roots) are sterilized by means of sequential washings with ethanol at 70% for 5 minutes, sodium hypochlorite at 2% for two minutes and mercuric chloride at 0.2% for 45 seconds. After each washing with the sterilizing agents, the parts treated are rinsed with sterile distilled water.
The leaves, shoots and roots are cut into portions, sterilely planted in Gamborg B5 medium [O. L. Gamborg et al. Exp. Cell. Res. 50, (1968), page 151] containing 1 mg/L of naphthalene-acetic acid, 1 mg/L of kinetin, 0.2 g/L of 2, 4-dichlorophenoxyacetic acid (G5 medium) and 7 g/L of agar and are kept in the dark at 28° C.
After 10-15 days an undifferentiated tissue (callus) develops which, after a further 20-30 days, is transferred onto agarized slants of the same medium. 20 days at 28° C. in the dark are normally required for obtaining well grown cultures. After 2 or 3 transfers, stabilized cultures are obtained which are used as inoculum for cultures in suspension.
The cultures grown on solid medium (undifferentiated callus cultures) consist of small masses of colorless and easily disagreeable cells. The elliptic-spherical shaped cells have a diameter of 50-100 &mgr;m. Under the above conditions, the calluses undergoing cultivation do not show any signs of organogenesis or any differentiation process. When exposed to light with an intensity equal to at least 2000 lux, the calluses become green due to the biosynthesis of the chlorophyll. Exposure to light, however, does not influence the biosynthesis of the phenylpropanoids.
When cultivated in liquid mediums, the undifferentiated callus cultures of
Ajuga reptans
grow in small aggregrates made up of 5-50 cells. The cells have the same shape and size as those grown on solid mediums.
About 1-2 grams of callus (fresh weight) can therefore be transferred to a 300 ml Erlenmeyer flask containing 50 ml of liquid G5 medium. After 28 days of incubation at 28° C. in the dark on a trolley with orbital stirring rotating at 120 revs/minute, the dry weight of the culture is about 15 mg/mL, and 5 mL of vegetative culture are inoculated into Erlenmeyer flasks each containing 50 mL of liquid G5 medium. These cultures are incubated at 23° C. in the dark on a trolley with orbital stirring at 120 revs/minute for 10-14 days.
According to the process of the present invention, the cells are cultivated in a liquid medium. Flasks or fermenters made of glass or other materials generally used, such as, for example, stainless steel, can be adopted. The liquid medium can be a nutritive solution containing an assimilable carbon source, an assimilable source of organic or inorganic nitrogen, inorganic salts and, optionally vegetable hormones and/or vitamins. The assimilable carbon source may consist of carbohydrates such as sucrose, fructose, glucose, starch, dextrin, glycerol, mannitol and mannose.
The assimilable source of organic or inorganic nitrogen can consist of aminoacids or their mixtures, peptides or proteins or their hydrolyzate, casein hydrolyzate, a hydrosoluble fraction of cereals such as the distillation residue of maize or wheat in the production of alcohol, or yeast and also inorganic nitrates and inorganic ammonium salts.
The process of the invention is typically carried out in a culture in suspension, for example in a flask under stirring or in an aerated fermenter with a pH ranging from 5 to 7, preferably 6.5 and at temperatures ranging from 18 to 36° C., preferably 23° C.
The best culture conditions are generally in the dark at a pH of 5.5 to 6.5 and at temperatures ranging from 18 to 32° C., for a duration of 8 to 16 days. The production of raw FPs begins after 2-3 days of growth and reaches its maximum after 10-14 days.
The extraction of the raw FPs can be effected starting either from cells or from the culture medium or from the culture in toto.
The raw FPs are extracted from the filtered cells or from the culture in toto with a solvent miscible with water, such as methanol, ethanol or acetone. The raw FPs are extracted from the culture medium separated from the cells, by means of extraction in solid phase.
REFERENCES:
patent: 0 466 375 (1992-01-01), None
patent: 0 471 081 (1992-02-01), None
patent: WO 92/16544 (1992-01-01), None
patent: WO 99/45919 (1999-09-01), None
Gao et al, “Radical Scavenging Activity of Phenylpropanoid Glycosides in Caryopteris incana”, Biosci. Biotechnol. Biochem., vol. 63, No. 6, issued 1999, pp. 983-988.*
Chemical Abstarct, vol. 117, No. 17, Oct. 26, 1992 Columbus, Ohio, US; Oganesyan, G.B. et al.: “Phenylpropanoid glycosides of Teucrium polium” p. 428; col. r; *abstract* & KHIM. PRIR. SOEDIN., 1991, pp. 630-634 XP002166801.
Malakov, P.Y. and Papanov, G.Y.: “Areptins A and B two new neo-clerodane diterpenoids from ajuga reptans” PHYTOCHEM., vol. 49, No. 8, 1998, pp. 2443-2447. XP000986295.
Terahara, N. et al.: “Triacylated anthocyanins from ajuga retans flowers and cell cultures” PHYTOCHEM., vol. 42, No. 1, 1996, pp. 199-203. XP000986294.
Calcagno, M.P. et al.: “New phytoecdysteroids from roots of ajuga retans varieties” TETRAHEDRON vol. 52, No. 30, 1996 pp. 10137-10146, XP004104054.
Danieli Bruno
Frigerio Giuliano
Marchioretto Danila Ingrid
Minghetti Anacleto
Perellino Nicoletta Crespi
Giuliani S.p.A.
Hedman & Costigan ,P.C.
White Everett
Wilson James O.
LandOfFree
Compounds with an antioxidant activity, compositions useful... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Compounds with an antioxidant activity, compositions useful..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Compounds with an antioxidant activity, compositions useful... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3019243