Unsaponifiable Residue Research Articles

LIPOPHILIC SUBSTANCES OF THE ROOTS AND RHIZOMES OF LEUZEA SAFFLOWER (RHAPONTICUM CARTHAMOIDES (WILLD.) ILJIN)

The qualitative composition of lipophilic secondary metabolites in the medicinal plant leuzea safflower (Rhaponticum carthamoides (Willd.) Iljin) has been studied by gas chromatography - mass spectrometry. The roots and rhizomes of this plant species were used as the raw material. Hexane and methyl tert-butyl ether (MTBE) were used as extractants. Two extraction schemes were used (exhaustive and sequential), which allowed us to obtain more detailed information on the composition of components of the raw material. The obtained lipophilic extracts were separated into acid and neutral components by treating with NaOH and KOH solutions. The acid components were transformed into methyl ethers by treatment with diazomethane. The neutral components of the unsaponifiable residue were analysed without derivatisation. The fractions enriched with hydrocarbons, ketones, sterols, aliphatic and triterpene alcohols were obtained by chromatographic separation through a column with silica gel. A series of low-polar compounds were identified that have not been detected previously in this kind of raw material. By comparing the mass spectra obtained in the work with the available databases, the following compounds were identified: aliphatic acids with chain length 10-30 carbon atoms (including unsaturated and dicarboxylic), the compounds of cinnamic series, as well as benzoic, salicylic, sterculic, 9,10-octadecadienic and phenylpropanoic acids. More than 90 triterpene and aliphatic compounds of the unsaponifiable residue and 33 components of free and bound acids were identified. In addition to n-alkanes, the hydrocarbon fraction contains also squalene, as well as saturated and unsaturated hydrocarbons with shorter chains. Aliphatic and terpene aldehydes and ketones were detected. Highly active triterpene alcohols and ketones are represented mainly by ursane and oleanane derivatives. The major sterol component (β-sitosterol) is accompanied by cholesterol, campesterol, stigmasterol, stigmast-7-en-3-β-ol and stigmastan-3-β-ol.

TERPENE AND ALIPHATIC PRODUCTS OF SAPONIFICATION OF THE ETHER EXTRACT OF EUPHORBIA VIRGATA WALDST. ET KIT

The composition of the lipophilic components of Euphorbia virgata Waldst. et Kit. has been studied. Acidic and neutral components have been identified using gas-liquid chromatography with mass spectrometric detection (GC-MS) following alkaline hydrolysis of the whole extract. The plant material was extracted with methyl tert-butyl ether (MTBE). Total acids isolated during sample preparation were analysed after methylation with diazomethane. The composition of the neutral components of unsaponifiable residue was studied without derivatisation, but after separation by column chromatography on silica gel into the groups of substances of different polarity. As a result, aliphatic acids with the chain length 10-30 carbon atoms, including unsaturated ones, were identified by comparison with databases. In addition, two compounds rarely occurring in plant materials were detected: ricinoleic and octadeca-9-en-12-ynic acids. More than 100 triterpene and aliphatic compounds of the unsaponifiable residue and 25 components of acidic fractions were identified. The hydrocarbon fraction was determined to contain, in addition to n-alkanes, also substantial amounts of unsaturated branched hydrocarbon squalene. Highly active triterpene alcohols and ketones are mainly represented by ursane, lupane and cycloartane derivatives. The main sterol component (β-sitosterol) is accompanied by cholesterol, campesterol, stigmasterol, stigmast-7-en-3-β-ol and stigmastan-3-β-ol. A substantial contribution from euphanic compounds characteristic of the plants of the Euphorbiaceae family has been revealed.

COMPOSITION OF LIPOPHILIC COMPONENTS OF RHODODENDRON ADAMSAY ETHER EXTRACT AND AC-TIVITY AGAINST SARS-COV-2 MAIN PROTEASE

The composition of the lipophilic components of the Adams rhododendron RhododendronadamsiiRehd. Acid and neutral components were identified using gas-liquid chromatography with mass spectrometric detection. Methyl tert-butyl ether (MTBE), which has all the advantages of diethyl ether, but is devoid of its disadvantages, was used as the raw material extractant. It does not form peroxides and does not create increased gas contamination due to its higher boiling point. As a result, comparison with databases identified triterpene, phenolcarboxylic and aliphatic acids with a chain length of 8 to 30 carbon atoms, including saturated, unsaturated and dibasic acids. More than 150 triterpene and aliphatic components of the unsaponifiable residue and acid fractions have been identified. Previously, unsaponifiable residues of lipophilic extracts were practically not studied. Only components of Rh.adamsiiand other species of the genus rhododendron essential oils have been studied in detail. For bioactivity testing, samples of the whole extract and its fractionation products were prepared. Some of the studied samples show inhibitory activity against the main protease of SARS-CoV-2.

EFFECT OF AN EXTRAGENT ON THE COMPOSITION OF THE LIPOPHILIC CONSTITUENTS OF THE EXTRACTS OF RHODIOLA ROSEA L. AND ON THE EXTRACTS ACTIVITY

The composition of the plant Rhodiola rosea L. lipophylic substances was studied. Acidic and neutral components were identified by gas-chromatography-mass-spectrometry. With methyl-tert-butyl ether (MTBE) as an extractant instead of the volatile solvent diethyl ether, lipophylic extract was obtained. Methyl-tert-butyl ether used as an extraction solvent for raw materials has all the advantages of diethyl ether, being free of its disadvantages. It does not form peroxides or produce elevated partial gas pressure due to its higher boiling point. As a result, comparison with databases identified some triterpene, phenolic and aliphatic acids with chain lengths 12 to 30 carbon atoms, including saturated, unsaturated, and dibasic acids. In addition to the components known from the literature, more than 50 triterpene and aliphatic compounds were detected in the unsaponifiable residue and acidic fractions for the first time. The hexane extract and the product obtained by the stepwise extraction of MTBE after the extraction of low-polarity compounds with hexane were investigated in a similar way. In the case of an extract obtained using MTBE after the extraction of low-polarity components with hexane, there was shown a more efficient extraction of benzoic and cinnamic acids compared to the exhaustive extraction of MTBE. These acids are absent in the hexane extract. Ethanol extraction was also carried out to test bioactivity: exhaustive and after hexane and MTBE extraction. Extracts obtained using MTBE and ethanol showed anti-virus activity against Ebola psevdovirus.

Evaluation of the Qualities of Camelina sativa Oil as Raw Material for Biokerosene

The advanced biofuels are an important target within EU considering the 2030 Energy View. There is a rising demand in finding alternative second generation bio-fuels. With the aim of describing the characteristics of the oil obtained from crops grown in Transylvania and to compare them with values reported from other countries, the following chemical analyses were performed: free fatty acids, fatty acid composition, water content, nitrogen, sulfur, aluminum, calcium, iron, phosphorus, magnesium, sodium, potassium and unsaponifiable residue. The obtained results were compared with the results found in the literature and a comparison was made between the Camelina sativa oil and Brassica napus L. oil regarding fatty acid content. No significant difference were found between the results from the literature and results obtained by us, regarding fatty acid content. The most abundant fatty acid found was α-linolenic acid in concentration of 32.79% mg and 36.96% mg. The sulfur content was between 22-33 ppm (m/m). Calcium was found in the highest concentration of 1630-1670 ppm (m/m).

Effect of avocado and soybean unsaponifiables on gelatinase A (MMP-2), stromelysin 1 (MMP-3), and tissue inhibitors of matrix metalloproteinase (TIMP- 1 and TIMP-2) secretion by human fibroblasts in culture.

In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). They can also respond to growth factors and cytokines. In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable [ASF] or soy unsaponifiable [SSF]) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts. Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0. 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta). MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis. TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis. In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF. The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml). A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations. Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF. As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen. ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml. These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.

Effects of three avocado/soybean unsaponifiable mixtures on metalloproteinases, cytokines and prostaglandin E2 production by human articular chondrocytes.

The in-vitro effects of avocado and soybean unsaponifiable residues on neutral metalloproteinase activity, cytokines and prostaglandin E2 (PGE2) production by human articular chondrocytes were investigated. Avocado and soybean unsaponifiable residues were mixed in three ratios: 1:2 (A1S2), 2:1 (A2S1) or 1:1 (A2S2). Freshly isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1beta, (IL-1beta) (17 ng/ml), with or without unsaponifiable residue mixtures at a concentration of 10 microg/ml. A/S unsaponifiable residues were also tested separately at concentrations of 3.3, 6.6 and 10 microg/ml. All A/S unsaponifiable mixtures reduced the spontaneous production of stromelysin, interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) by chrondrocytes. At concentrations of 3.3 and 6.6 microg/ml, A/S residues, tested separately, were potent inhibitors of the production of IL-8 and PGE2. Nevertheless, only avocado residue inhibited IL-6 production at these concentrations. A/S unsaponifiable mixtures had a more pronounced inhibitory effect on cytokine production than avocado or soybean residues added alone. As anticipated, IL-1beta induced a marked release of collagenase, stromelysin, IL-6, IL-8 and PGE2. A/S unsaponifiable mixtures partially reversed the IL-1 effects on chrondrocytes. These findings suggest a potential role for A/S unsaponifiable extracts in mitigating the deleterious effects of IL-1beta: on cartilage.

Sterols ofArtemia salina

In order to study the sterols, a mixture of 10.35 g of dewatered shrimps and 41.20 g of a 10% solution of potassium hydroxide in ethanol was boiled under reflux for 1 h and was extracted with diethyl ether (3 × i00 ml). The combined ethereal extracts were washed with saturated aqueous sodium chloride solution and dried with anhydrous sodium sulfate. After the solvent had been distilled off, 0.240 g of unsaponifiable residue was obtained. This 0.240 g of unsaponifiable residue was chromatographed on a column (700 × i0 mm) of silica gel (KSK, grain size 0.140-0.315 mm) to give 0.096 g of a white solid product.

Light microscopy investigation of the action of the unsaponifiable residue of vegetable oils on rat periodontal disease induced by a high carbohydrate diet.

Rats, submitted to a sucrose diet for 12 months, were normally fed for another 6 months either with or without addition of the unsaponifiable residue of vegetable oils. It appears that unsaponifiable substances reinforce the collagen fiber network of the periodontium. The presence of a dense supracristal barrier accounts for the lack of an infraosseous pocket and for the limitation of bone resorption despite the high level of the sucrose diet.

Analysis of Fat-Soluble Vitamins. XXVI. High Performance Liquid Chromatographic Determination of Vitamin D in Fortified Milk and Milkpowder

Vitamin D is determined by high performance liquid chromatography (HPLC) in samples containing other fat-soluble vitamins. The vitamin D in the unsaponifiable residue is extracted and separated from interferences by straight phase chromatography, and the fraction corresponding to vitamin D3 is collected and quantitated using the AOAC official final action HPLC method for vitamin D3. Analysis of a synthetic mixture gave reasonable recoveries. The method measures potential vitamin D3 content in milkpowder samples containing 2 IU vitamin D/g in the presence of all known vitamin D3 isomers, vitamin A, and vitamin E.

Analysis of Fat-Soluble Vitamins. XXIII. High Performance Liquid Chromatographic Assay for Vitamin D in Vitamin D3 and Multivitamin Preparations1

Vitamin D is determined in preparations containing other fat-soluble vitamins by high performance liquid chromatography (HPLC). The unsaponifiable residue is extracted and separated from interferences by reverse phase chromatography; the fraction corresponding to vitamin D3 is collected and quantitated using normal phase chromatography (amylalcohol-n-hexane as mobile phase) by measuring the vitamin D3 and pre-vitamin D3 peaks at 254 nm. Previtamin D3 content is calculated as vitamin D3 with a conversion factor (determined on the equipment used). Application of the method to vitamin AD3 mixtures in oils gives 98-102% recovery. The reproducibility, using an external standard, is 2-3%, calculated as the coefficient of variation; with an internal standard, the coefficient of variation is 1-1.5%. The method measures potential vitamin D3 content in preparations containing greater than or equal to 200 IU/g in the presence of all known vitamin D3 isomers, vitamin A, and vitamin E.

Determination of Unsaponifiable Residues in Milk

Abstract Unsaponifiable residues are determined in milk samples by the following procedure: oily drug bases and fats are extracted with iso-octane (rather than the usual mixed ether solvents) in an alkaline alcohol-acetone mixture; this extract is concentrated and the fatty constituents are saponified with KOH. The unsaponifiable matter is then re-extracted with iso-octane and the residual soaps and extraneous constituents are removed by water washing and column chromatography. Mineral oil added at levels of 12–404 ppm to milk samples was quantitatively recovered, with an average value of 98%. Unsaponifiable matter in market milk was also determined; results demonstrated good precision between duplicate analyses. Milk-out studies were performed to determine the rate of diffusion and dissipation of infused formulations.

Determination of Unsaponifiable Residue in Candy

Determination of Unsaponifiable Residue in Candy

γ線照射の卵黄脂質への影響

殻付鶏卵にCo60を用いて照射線量1×105および5×1O5rでγ線照射を行ない, -5°～-8°において卵黄の1.6%NaCl3倍稀釈液より遠心分離によりリポ蛋白質を分離し,またこの稀釈液のエーテル抽出部を遊離脂質とし,さらに遠心分離とエーテル抽出をくりかえし段階的にリポ蛋白質と遊離脂質を分離した.このリポ蛋白質およびこれに結合する脂質ならびに遊離脂質の収量および化学的性質を検討した.その結果,酸価,鹸化価,ヨウ素価および不鹸化物含有量より見た卵黄脂質自体の化学的性質はγ線照射によりあまり変化しないが,脂質の卵黄中における結合状態には照射により変化があるものと推定された.

Spectrophotometric assay of α‐tocopherol

AbstractThe benzene washing procedure for the isolation of unsaponifiable residues has been applied to vitamin E preparations containing α‐tocopheryl esters only.The isolation procedure is carried out once (after saponification) or twice (before and after saponification). In the solution thus obtained, β‐tocopherol is determined spectrophotometrically; the absorbance at the maximum is corrected for by‐absorption.By avoiding quantitative extractions and transfers, contact with air is reduced to a minimum; ascorbic acid is used as an antioxidant.

Analysis of Poison Ivy Fruit Fat

The whole fat of poison ivy fruit was analyzed and its composition was determined. The fatty acids isolated included myristic, palmitic, stearic, linoleic, and oleic. The study of the glyceride composition disclosed the presence of fully saturated glycerides, disaturated-mono-unsaturated glycerides and monosaturated-diunsaturated glycerides. β-Sitosterol was isolated and identified. The presence of unsaturated hydrocarbons and pigments in the unsaponifiable residue was indicated. The pericarp fat and the seed oil were examined individually and their partial analysis is reported.

The antivitamin D factor in roughages.

Vitamin D was separated from the rachitogenic substance in dry grass and hay by chromatography [no details]. Fractions of the unsaponifiable residue of the grass fat, with addition of fixed amounts of vitamin D, were tested on rachitic rats, with radiography of the bones as criterion, to ascertain the content of rachitogenic substance. A fraction was obtained which contained all the vitamin D but not the rachitogenic substance. The last was present chiefly in the carotene fraction; the fraction containing xanthophylls was inactive. The method was applied to routine estimation of roughages. In hay dried in windrows or cocks or on tripods the vitamin D content ranged from 1005 to 1375 I.U. per kg., and there was little increase after chromatography, showing the absence of rachitogenic substance. In artificially dried grass the values ranged from 270 to 1150 before chromatography, and from 595 to 1790 after it; the amount of rachitogenic substance was, therefore, considerable. The results were in accordance with the finding of Grant (Abst. 1730, Vol. 24), that carotene is the rachitogenic substance. The rachitogenic substance was not a phytosterol precipitable with digitonin.-E. M. Hume. (Abstract retrieved from CAB Abstracts by CABI’s permission)

399. The estimation of vitamin A in butter: A critical study of methods

1. The Lovibond Tintometer has been found more reliable than either the Pulfrich Photometer or the Spekker Absorptiometer for the assessment of the blue colour from the Carr-Price test on butterfat unsaponifiable residues.2. Using a Beckman Spectrophotometer, vitamin A in butterfat unsaponifiable residues has been estimated also by the intensity of absorption at 325 mµ. From spectrophotometric readings at this wavelength and at 450 mµ, the irrelevant absorption, due both to carotenoid and non-carotenoid compounds, may be calculated as follows:Corrected density reading at 325 mµ = 9/10 (A – 12/100B),where A = density reading of the unsaponifiable residue in petrol ether at 325 mµ, B = density reading of the same solution at 450 mµ.3. The Morton & Stubbs equation for irrelevant absorption in fish-liver oils is not generally applicable to butterfat unsaponifiable residues.4. A comparison of the vitamin A content of butterfat samples by the Carr-Price test and by the direct spectrophotometric method has shown a reasonably good agreement between the two values.

RESIN-RUBBER FROM CANADIAN GROWN PLANTS: IV. ANALYTICAL STUDY OF MILKWEED POD GUM

Fresh pod gum has an acetone-soluble content (resin) of 83.4%, and a benzene-soluble content of 7.3%. Steam distillation of the resin furnishes a small amount (0.1%) of an essential oil. Acidification of the saponifiable portion (55%) of the resin yields an oil consisting of a mixture of fatty acids. Palmitic, stearic, oleic, linoleic, and linolenic acids were identified. Resin acids are absent. The unsaponifiable portion (45%) contains a large amount of α-amyrin, and extremely small amounts of sterol (probably heterogeneous) and resene. Polyisoprene, an oil containing free acids, and an unsaponifiable residue make up the benzene extract. Water-solubles include calcium tartrate, soaps, and a material yielding a mixture of sugars on hydrolysis. The residue comprises lignin, cellulose, protein, and inorganic materials. From these data the dry gum contains approximately 50% of fatty acid esters, 26% of α-amyrin, 6% of polyisoprene, 4% of ash, 3% of lignin, 2% of cellulose, and small amounts of protein, sterol, resene, essential oil, and calcium tartrate. Unidentified substances comprise 7.7%.

The Unsaponifiable Residue of Human Liver. I. Preparation and Primary Chromatographic Fractionation1

The Unsaponifiable Residue of Human Liver. I. Preparation and Primary Chromatographic Fractionation¹