Acid hydrolysis of saponins extracted in tincture

Background Saponins are secondary metabolites from plants added to shampoos and beverages to make them foam, and the sapogenins released from them upon acid hydrolysis are commonly used as starting materials for steroidal drugs. However, current methods embed the saponin in a thick “gum” material consisting of multiple impurities. This gum limits access to the saponin, reducing the efficiency of hydrolysis and requiring large amounts of heat, organic solvents and effort to recover the sapogenin. For centuries, herbalists have been making tinctures by soaking plant materials at room temperature, in mixtures of alcohol and water. Many herbal tinctures contain saponins floating freely in solution, gum free. The saponin from sarsaparilla (Smilax spp), sarsasaponin, yields the sapogenin, sarsasapogenin, upon acid hydrolysis. The retail price of sarsasapogenin is very high but would be lower if the “gum problem” could be avoided. Materials and methods We incubated sarsaparilla tincture under different conditions of temperature, acidity and duration then used quantitative nuclear magnetic resonance (qNMR) to measure the amount of sarsasapogenin produced by hydrolysis as well as the amount of its epimer, smilagenin. Results and discussion Most, if not all the sarsasaponin in sarsaparilla root powder is extracted into a solution of 45% ethanol (55% water) at room temperature and stays suspended without formation of any particles (gum). Acid hydrolysis of the saponin in this solution is very efficient, approaching 100%. The sarsasapogenin released by hydrolysis and the smilagenin produced by its epimerisation, migrate into the chloroform phase. Conclusion Sarsaparilla saponin diffuses into and disperses in a solution of alcohol:water (45:55) at room temperature. Hydrolysis of saponins in tincture provides a simple, inexpensive and environmentally friendly alternative.

Activity of Piperaceae extracts and fractions in the control of Phytomonas serpens

ABSTRACT: Protozoa of the genus Phytomonas are harmful parasites to several agricultural crops of economic importance. Due to their recognized biological activity, crude extracts of Piper aduncum, P. crassinervium, P. hispidum, and P. amalago leaves, were tested using the microdilution plate technique to assess the antiparasitic potential against Phytomonas serpens. Results showed that the ethanolic crude extract of P. crassinervium and P. amalago presented the best inhibitory concentration for 50% of the cells (IC50), 16.5 µg mL-1 in chloroform phase, and 18 µg mL-1 in aqueous phase, respectively, after 48 h treatment. Cytotoxicity analyses were performed using the colorimetric method of sulforhodamine-B in LLCMK2 mammalian cells. The chloroform phase of P. crassinervium was subjected to the fractionation process, in which the ethyl acetate and dichloromethane fractions obtained better IC50 values. Scanning electron microscopy (SEM) images showed alterations in the cell membrane of the treated parasites. The data obtained indicate a potential antiparasitic effect of the Piper species analyzed against P. serpens, being considered promising candidates for formulations of bioproducts to control the parasite.

MIKROBA ENDOFIT DARI TANAMAN SRIKAYA (Annona squamosa L.) SEBAGAI PENGHASIL ANTIMIKROBA Staphylococcus aureus DAN Candida albicans

Various studies indicated that endophytic microbes lived in the plant tissues and produced antimicrobial compounds. Sugar-apple plant (Annona squamosa L) contained alkaloids, cyanogenic glycosides, and flavonoids. The purpose of this reasearch were (1) to determine the endophytic microbes isolated from sugar-apple plant (2) to study inhibiting capabillity of endophytic isolate against Staphylococcus aureus and Candida albicans, (3) to analyze antimicrobial compounds produced by the potential endophytic isolate. Diffusion agar plate methode was used to assessed antimicrobial activity. Antimicrobial compounds were analyzed by Thin Layer Chormatography (TLC) and High Performance Liquid Chormatography (HPLC), compared with erythromycin, metronidazole and tetracycline. Twelve bacterial isolates and 24 fungus were isolated. Selected bacteria, BMC 1.1, showed the biggest clear zone on C. albicans culture on agar medium, meanwhile selected fungi, BTCK 1.1T, formed the biggest colony on S. aureus culture on agar medium. TLC and HPLC analysis showed that the Rf value of BMC 1.1 and BTCK 1.1T chloroform phase fractions was similiar to metronidazole. Metronidazole concentration in C1, C2, Ck1 and Ck2 fraction were 170.98 ppm, 18.27 ppm, 1.51 ppm and 4.14 ppm respectively.

Allelopathy and Preliminary Separation of Allelopathic Substance in the Extracts of Wheat Seed Germination

One variety of durum wheat (Triticum durum L.), ‘Longfu 30’, was choosen to study the allelopathic effects of seed germination extracts on other plants. The allelopathic effects of different polarity extracts were measured in terms of germination rate and germination speed on a test cucumber variety ‘Jinyou No.2’ (Cucumis sativus L.), and the allelopathic substance in chloroform extract was preliminarily separated with HSCCC and determined by GC-MS. The results showed that the allelopathy of wheat seed germination extracts varied with the polarity of solvent, and the substance in chloroform phase was the most active and showed a significantly inhibitory effect on seed germination of cucumber by 80.7% under Petri dish culture. The major allelopathic substance in wheat seed germination extracts were phenol, amine, piperazine, alcohols, esters and ketones.

Absolute Configuration of Antifi brotic (+)-Episesamin Isolated from Lindera obtusiloba BLUME

Fractionation of a 70% ethanolic extract from twigs of Lindera obtusiloba BLUME (Japanese spicebush, Tohaku) yielded fi ve fractions of different polarity. The antifi brotic activity within the chloroform phase was best assessed by an in vitro bioassay using rat hepatic stellate cell (HSC) proliferation and their autocrine transforming growth factor beta (TGF-β) expression as sensitive fi brosis-associated read out. Chromatography of the chloroform extract on Sephadex LH-20 or liquid-liquid extractions yielded a crystalline compound as an active principle, which was identifi ed from NMR and ESI-MS analyses, its melting point, and its optical rotation as (7S,7´R,8R,8´R)-3,4:3´,4´-bis(methylenedioxy)-7,9´:7´,9-diepoxy-lignane [(+)-episesamin]. X-Ray diffraction confi rmed the structure and provided, for the fi rst time, directly its absolute confi guration. (+)-Episesamin blocked proliferation and the profi brotic autocrine TGF-β expression HSC without signifi cant cytotoxicity.

Antiphospholipid antibodies and atherosclerosis

β2-Glycoprotein I (β2-GPI) is a major antigen for anticardiolipin antibodies (aCL) induced in patients with antiphospholipid syndrome and their antigenic epitopes are cryptic. The epitopes appear on the surface of β2-GPI molecule only when β2-GPI interacts with lipid membranes containing negatively charged phospholipids or polyoxygenated polystyrene surface. Our data also indicated that CuSO4-oxidized low density lipoproteins (oxLDL) are subsequently targeted by β2-GPI and aCL; however, malonedialdehyde (MDA)-modified LDL were recognized neither by β2-GPI nor aCL. β2-GPI binding to LDL was rapidly increased by incubation with CuSO4. Oxidation of lipoproteins was accompanied with the increment of thiobarbituric acid-reactive substances (TBARS) and denature of apolipoprotein. Ligands on LDL for β2-GPI seemed to be intermediate oxidative derivatives which were extractable into the chloroform phase by Bligh and Dyer's extraction, but not MDA. Further, immune responses against β2-GPI, as an anti-atherogenic protein, were demonstrated to induce atherogenic effect in in vitro oxLDL uptake by macrophages.

Spectrophotometric determination of sulfanilic acid and sulfonamides in pharmaceutical samples with potassium 1,2-naphthoquinone-4-sulfonate

An accurate and simple method is proposed for the determination of sulfanilic acid in the presence of sulfonamides. This method is based on measuring the intensity of the red colour that develops when sulfanilic acid is allowed to react with potassium 1,2-naphthoquinone-4-sulphonate (NS) in a chloroaceticchloroacetate buffer at pH 3,4. Colour development reaches completion after 2 h, allowing sulfanilic acid to be quantified spectrophotometrically at 470 nm (ε = 4.7 × 103 L mol−1 cm−1). The main product causing colour formation, potassium 1,2-naphthoquinone-4-(N-aminophenylen-4-sulphonate) (NSSA), was isolated and characterized. When samples also contain sulfonamides an extraction into chloroform must be performed. Sulfanilic acid in binary mixtures with sulfanilamide, sulfacetamide, or sulfathiazole can be determined either by direct measurement of the aqueous phase after extraction at 470 nm or by subtracting from the absorbance of the aqueous phase before extraction the absorbance of sulfonamide as determined by measuring the extracted chloroform phase at 345 nm. Sulfadiazine, sulfamethoxipyridazine, and sulfamethoxazole interferences are prevented by their extraction into chloroform at pH 7.2; these species cannot be determined. The effects of pH, reagent concentration, time, and temperature on colour formation were investigated. In all cases the standard addition method gave more accurate results. The method was applied to several pharmaceutical samples. Keywords: spectrophotometry, sulfanilic acid, sulfonamides, potassium 1,2-napthoquinone-4-sulphonate.

Extraction-photometric determination of indium with 8-quinolinol in layered crystals of the type A2VB3VI

A method for determining indium in milligram samples of indium-doped crystals of bismuth telluride is described. The pH effect of the aqueous phase (Vaq = 40 ml) on the extraction yield is examined, also by considering the results computed with the data so far published. In a single extraction at pH 3·6-4·2 (acetate buffer) the tris(8-quinolinolato)indium(III) chelate is quantitatively transferred to the chloroform phase (Vorg = 10 ml). Bismuth present in excess is masked with thiosulfate. The absorbance of the chloroform extract is measured at 400 nm in a glass cell of 10-mm path-length (or in a 50-mm cell when m(In) < 20 μg). A blank extraction is carried out simultaneously with the use of a solution of pure Bi2Te3 and taken as reference. The calibration within the range of 10 - 130 μg of In(III) is performed by adding a standard indium(III) solution to the matrix solution obtained by dissolving pure Bi2Te3. The accuracy of the method has been checked by analysis of polycrystalline reference materials of known composition. The method is applicable for the analysis of milligram samples containing 0·6 - 5% of indium, with a relative standard deviation of 1 - 4%.