BG110966A - A method for isolating a mixture of terpenoids and flavonoids from stuff of ethereal oils - Google Patents

Abstract

The invention refers to a method for obtaining phytoproducts from waste, as well as from fresh pine loppings with applications in pharmaceutical and food industry, as well as a nicotine antidote. Themethod involves an extraction by means of an alkaline solution of chloroform:ethanol, distillation of the solvent and an extraction by means of 5% NaHCO3. The composition of the bicarbonate extract reveals the presence of diterpene acids: calitrisoena, dehydrobietine, abietine, pymaric and triterpene acids: oleanolic and ursolic. Through LC/MS analyses the following flavonoids were identified: prodelfinidin, dimer of antocyanines, procyanidin. The content of procyanidin and of antocyanidin helps to elucidate the cardiotonic effect of coniferous extracts.

Description

Description of the invention

1. Name of the invention Method for isolating a mixture of terpenoids and flavonoids from coniferous essential oil raw materials

2. FIELD OF THE INVENTION The invention relates to the preparation of phyto-products for use in pharmaceutical compositions and in the food industry, as well as as an antidote for nicotine in the manufacture of cigarettes, after pharmacological and toxicological studies have been carried out.

Literature data on methods used so far to isolate biologically active extracts from coniferous raw materials, composition and fields of application:

There is a known method for the isolation of diterpenic acids by dehydroabietic and pimaric boron resins by repeated extraction with hexane, for which anti-nicotinic action has been established Azano, C.Ohkubo, S.Tamura;

Agr.Biol.Chem., 1972, Vol.36, Noll, 1051-2053).

By treating pine needles with water, organic solvents and mixtures with water at a temperature of 40-110 °, an extract was used as a nutritional supplement and a dosage form for lowering blood pressure / US 2007/148266 A 1 /

Pine needle extracts have been described as useful for specific beverages in JP 08107778 A, JP 07059538 and for the preparation of JP 01218562 A. rice cakes.

U. S. Pat. No. 6,329,000 approves the use of boron extracts obtained by extraction with water and alcohol for the treatment of myocarditis, angina, arrhythmia, dementia and high blood pressure.

U. S. Pat. No. No. 5,690,984 describes a beverage made from pine needles by boiling with water useful for high blood pressure.

U.S. Pat. No. No. 5,607,971 uses Pinus ponderosa extracts with methanol, diethyl ether and methylene chloride for vasoactive lipids. Procyanidins and prodelfinidins are known flavonoid components of boron, showing anti-inflammatory, anti-arthritic, anti-allergic activity, protecting the skin from aging, suppressing TPA tumors / Carcinogenesis, 1999 Sep. USA /, as well as antiviral and immunological protective action.

It is known that the distillation of essential oil of pine / needles, twigs, resin / water vapor leaves a large amount of waste - about 20,000 tonnes per year, containing valuable bioactive substances.

In addition, fresh pine twigs and needles remain as a waste in the production of pinewood in sawmills.

4.3 It is an object of the invention to provide a method for the maximum isolation of a mixture of mono-, ses-, di- and triterpenoids and flavonoids from freshly cut off and terpentine-derived essential oil waste, in particular pine.

According to the invention, a method has been devised for isolating a mixture of terpenoids and flavonoids from waste essential oil, in particular pine, after the distillation of the essential oil, and also from fresh pine nuts, waste from the production of pine wood.

♦ · ♦ ♦ ...

· I: -. ·: *: Ί. : ·: ::: ·: · ··· ...... ....:.

The method consists of extraction with an alkaline (5% KOH) solution of chloroform: ethanol = 1: 1. To isolate the biologically active substances, the extract, after distilling off the solvent, is acidified with dilute acid (HCl: H2O = 1: 1) by litmus, extracted with an organic solvent (chloroform) and extracted with 5% NaHCCh / or a saturated solution. The composition of this bicarbonate extract, after acidification, extraction with organic solvent (chloroform) and solvent distillation shows the presence of a mixture of di- and triterpenoic acids and unsaturated fatty acids, which are analyzed by thin layer chromatography / TLC. -, UV and mass of the spa ect, gas chromatographic mass spectral analysis.

The extract obtained showed enhanced antinicotinic action due to the presence, in addition to diterpenic acids, 4 epidehydroabietic and pimaric and triterpenic acids ursolic and hydroxyoleanolic.

The pharmacological and toxicological studies performed at NIHFI at SOFARMA show an effect lowering blood pressure and pulse rate when smoking, normalizing blood flow to the aorta. The extract can be administered in an amount of about 10mg per cigarette or tablets and candy can be prepared filler.

The advantage of the patent is the creation of a method for the complete extraction of mono-, sesame-, di- and triterpenoids, flavonoids and unsaturated fatty acids from the waste oil after the distillation of the essential oil, essential oil or freshly collected, in particular pine needles, branches and other .The method allows the use of complex essential oily raw material, obtaining biologically active substances for preparations with curative prophylactic action, antitumor, antinicotinic action.

The invention is illustrated by the following examples:

1,200g boron needles, waste after boron distillation, oil, extracted three times with an alkaline solution (5% KOH) of chloroform: ethanol = 1: 1.The extract obtained is removed by distillation. A yield of 7% of boron is obtained. This residue was acidified with dilute HCl to acidic pH in litmus, extracted with chloroform and extracted with 5% HCO3. The bicarbonate extract was acidified and extracted with chloroform. The solvent was removed in vacuo. A) containing mainly di- and triterpene vi and fatty acids, 16.6% by extract, were analyzed by preparative thin layer chromatography on silica gel in mobile phase: chloroform: ethyl acetate: ethanol = 100: 9: 1, at infinite front for 21 hours and by gas-liquid chromatography spectrometry.

Identify:

Triterpenic acids:

2-hydroxyoleanolic / cratogolic / acid, isolated for the first time from boron raw material, yield 0.63% extract extract and 0.04% dry drug, Rf = 0.18.Mac spectrum: M 472, characteristic fragment ions: m / d 248/100% / , 203/90% /, 223/15% /, 133/34% /, 189/32% /, ursolic acid - isolated for the first time from boron raw material, yield 0.34% by extract and 0.025 by dry drug.1H = 0.31. The mass spectrum is identical to that of ursolic acid: Molecular ion at m / z 456/15 / and characteristic ions: m / z: 248/100 /, 207, 203/20 /, 133/10 /, 189. The structure was also confirmed by IR data.

Diterpenic acids:

4-epi dehydroabietic acid / cratoleic acid, first isolated from boron raw material. Mass spectrum shows molecular ion at m / z 300/10 /, more important fragment ions: 285/90% /, 239/100 /, 267 / Dehydroabietic acid - molecular ion m / z 300/10 /, (M-CH 3) at m / z 285, ions at m / z 239/100 / and 197/41 /. these two epimeric acids is based on the difference in ion intensities (M-CH 3) at m / z 285. Gas methyl chromatographic mass analysis (GC / MS) of the methylated acid fraction showed the presence of seven more isomeric abietic acids - meth esters with a molecular peak m / z 316, pimaric and dihydropimaric acids.

The total amount of di- and triterpenic acids was 16.6% of the extract extract. The remaining neutral fraction (Boron pinol K) was analyzed by TLC hGC-MS. The sterol-β sitosterol and campesterol and triterpene alcohols α-amyrin and β-amyrin were detected.

The isolated acidic and neutral fractions of di- and triterpenoids were standardized by industry norms, analyzed for lead and arsenic content, and obtained permission from the MHC for use in the food and pharmaceutical industries. The anti-tumor activity of the extracts obtained, as well as from boron and juniper extract / “Tumor screening of conifers, E. Voinova, P. Hadzhieva, M. Ancheva, D. Popov; C.R.Acad.Bulg. Sci, 44, 3, 117-126 (Amy's 1991J / .C method was found to have no genotoxic effect.

Example 2. An extract of terpenoids and flavonoids from fresh pine was obtained by the same method.

The acid fraction (Boron pinol A) isolated with 5% NaHCO3 as well as the neutral (Boron pinol K) fractions were analyzed by combined GC / MS and Liquid Chromatographic Mass Spectral (LC / MS) analyzes. Therefore, in this case, unlike Example 1, boron oil is not pre-distilled with water vapor, these extracts contain, in addition to iterpenoids, also mono, sesame terpenoids and flavonoids.

The following flavonoids were identified by LC / MS negative ion mode assays: prodelfinidine, M 593; anthocyanins dimer - glycosides of peonidine and dolphinidin-M 928; procyanidin - an anthocyanidin trimer with M 864; epicatechin oligomer with M 290 on the monomer. The content of procyanidins and anthocyanins contributes to clarify the cardiotonic action of coniferous extracts / ”Discover the Amazing Power of Pine Needle Oil, http: //www.nanume.net/wp / category / health / alternative medicines /

GC / MS analysis of the isolated extracts / methylated acid fraction / showed also the content of the following mono- and sesquio-terpenoid components: borneol, cs-muurol, cadin, kuben, kubenol, α-cadinol, cedrol borneolacetate. as well as citric, p-coumaric and labd-8 (20) -en 15,18-diarbosilic acid.

Claims (1)

Author's claim: Method for the isolation of terpenoids and flavonoids from essential oily raw materials by extraction with chloroform and ethanol, characterized in that the raw material is extracted with an alkaline solution of chloroform: ethanol = 1: 1 and the extract obtained after removal of the solvent by 5 After acidification of the bicarbonate extract, it is extracted with an organic solvent and the solvent is distilled off. Author's claim: Method for the isolation of terpenoids and flavonoids from essential oils by extraction with chloroform and ethanol, characterized in that the raw material is extracted with an alkaline solution of chloroform: ethanol = 1: 1 and the resulting extract after solvent distillation / extraction is extracted with 5 % o NaHCO3 ^ After acidification of the bicarbonate extract, it is extracted with an organic solvent and the solvent is distilled off.