Purification Of Crude Extract Research Articles

Chemical composition and in vitro chemoprevention assessment of Eugenia jambolana Lam. (Myrtaceae) fruits and leaves

Eugenia jambolana has been used as an antioxidant, anti-inflammatory and antidiabetic. This study performed a chemical and chemopreventive screening of extracts and fractions from leaves and fruits from Eugenia jambolana. The purification of the crude extract from leaves afforded two known flavonoids, tricetin-4′-O-α-l-rhamnopyranoside (1), and myricitrin (2), whereas the purification of crude extract from fruits afforded one anthocyanin, malvidin-3-O-gentibioside (3). Additionally, seven anthocyanins were tentatively identified. The antigenotoxicity results showed that post-treatment with extracts and fractions mitigated the damage induced by hydrogen peroxide. In assessing the antimutagenicity, the samples were able to reduce the frequency of micronucleus formation for both pre-treatment and post-treatment. Also, extracts and fractions promoted the induction of quinone reductase activity. Such extracts and fractions were able to induce detoxifying enzymes and reverse DNA damage, an essential balance for its applicability as a chemopreventive agent.

In - vitro and in - vivo antiproliferative activity of Byrsocarpus coccineus (Connaraceae)

The incidence of cancer is on the increase worldwide, and there are concerns of selectivity of action and significant deleterious effects associated with existing anticancer drugs. Based on these facts, tremendous effort is being made worldwide to discover and develop new anticancer agents with better efficacy and safety profile. In this study, various extracts of Byrsocarpus coccineus, a Nigerian medicinal plant, were evaluated for antiproliferative activity in in-vitro and in-vivo assays. The sulforhodamine B (SRB) (in-vitro) using A549 (lung), HCT-116 (colon), PC3 (prostate), A431 (skin), HeLa (cervix), THP-1 (leukemia), NCI-H322 (lung), and COLO-205 (colon) human cancer cell lines, Sarcoma-180 (S-180) ascites and L1210 lymphoid leukemia (in-vivo) antiproliferative assays were used in this study. Based on the standard of 30 µg/mL as the upper IC50 limit for purification of crude extracts, BC-A001 (ethanolic extract; IC50 12 µg/ml with A431); BC-A002 (hydroethanolic extract; IC50 125% considered to depict activity). The results obtained in this study suggest that the extracts of B. coccineus possess significant antitumor activity in-vitro against skin, lung, colon and prostate human cancer cell lines with BC-A002 demonstrating moderate activity in the S-180 ascites model with no activity in the L1210 lymphoid leukemia test.

요소분해효소 기반 식물추출액을 이용한 광산폐기물 내 중금속 오염 저감

ABSTRACT Acid mine drainage occurrence is a serious environmental problem by mining industry, it usually contains high levels ofmetal ions, such as iron, copper, zinc, aluminum, and manganese, as well as metalloids of which arsenic is generally of thegreatest concern. An indigenous plant extract was used to produce calcium carbonate from Canavalia ensiformis aseffective biomaterial, and its ability to form the calcium carbonate under stable conditions was compared to that ofpurified urease. X-ray diffraction and scanning electron microscopy were employed to elucidate the mechanism of calciumcarbonate formation from the crude plant extracts. The results revealed that urease in the plant extracts catalyzed thehydrolysis of urea in liquid state cultures and decreased heavy metal amounts in the contaminated soil. The heavy metalamounts were decreased in the leachate from the treated mine soil; 31.7% of As, 65.8% of Mn, 50.6% of Zn, 51.6% of Pb,45.1% of Cr, and 49.7% of Cu, respectively. The procedure described herein is a simple and beneficial method of calciumcarbonate biomineralization without cultivation of microorganisms or further purification of crude extracts. This studysuggests that crude plant extracts of Canavalia ensiformis have the potential to be used in place of purified forms of theenzyme during remediation of heavy metal contaminated soil.Key words :Urease, Plant extract, CaCO

Calcite precipitation by ureolytic plant (Canavalia ensiformis) extracts as effective biomaterials

An extract of the indigenous plant jack bean (Canavalia ensiformis) was used to produce calcite, an effective biomaterial. The formation of calcite using this extract was compared, under stable conditions, to that using commercially available purified urease. Xray diffraction and scanning electron microscopy were employed to elucidate the mechanism of calcite formation from the crude plant extracts. The results revealed that urease in the jack bean crude extracts catalyzed the hydrolysis of urea in liquid-state cultures. The procedure described herein is a simple and useful method of calcite mineral precipitation that does not require cultivation of microorganisms or further purification of crude extracts. This study suggests that crude extracts of Canavalia ensiformis have the potential to be used in place of purified forms of urease during remediation of cracks and to increase the strength of materials.

Extraction, Purification and Characterization of Apricot Seed β-Galactosidase for Producing Free Lactose Cheese

β-Galactosidase was extracted (with acetate buffer pH 5) from apricot seeds, partially purified by ammonium sulphate (30-70%) and dialyzed for 24 hr. The crude extract had a higher specific activity (9.93 U/mg proteins) which indicates a high enzyme yield. Purification of crude extract with ammonium sulphate (30-70%) increased the specific activity and purification fold to 29.07 U/mg proteins and 2.92, respectively which was improved by dialysis to 32.68 U/mg proteins and 3.29, respectively. The extracted enzyme exhibits an optimum temperature at 70°C and 5 pH optima. The enzyme activity was almost stable between pH 5.0 and 6.5, where more than 90% of its activity was remained with incubation at the previous pH for 30 min. Further more; it was thermostable when incubated for 30 min. at temperature ranges of 55-70°C with a complete loss of activity at 80°C. The activity of β-galactosidase was enhanced by different concentration of Ca+2. There were no significant (P>0.05) changes in flavor, body texture, and overall acceptability between free lactose white cheese treated by apricot seeds β –galactosidase (9865 U/ 500 ml milk) and untreated cheese.

SEPARATION AND PURIFICATION OF THREE FLAVONOIDS FROM DAPHNE GENKWA SIEB. ET ZUCC.: COMPARISON IN PERFORMANCE BETWEEN MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY AND HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

The two methods of medium-pressure liquid chromatography (MPLC) and high-speed countercurrent chromatography (HSCCC) are compared in the separation and purification of crude extract from Daphne genkwa Sieb. et Zucc. The MPLC method was performed with the mobile phase composed of methanol-0.1% aqueous acetic acid in the gradient elution which yielded apigenin, 3′-hydroxygenkwanin, and genkwanin at high purities of 95.3%, 94.0%, and 91.5%, respectively, for 230 min. The same three components were isolated by HSCCC also at high purities of 93.4%, 94.3%, and 92.8%, respectively, using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:7:5:5, v/v) for 400 min. Although both methods produced effective separations of Daphne genkwa Sieb. et Zucc., MPLC gave much shorter separation time, whereas HSCCC yielded substantially greater amounts of target compounds due to avoiding irreversible sample loss onto the solid support with less consumption of the solvent. The chemical structures of purified three compounds were identified by HPLC, ESI-MS, and 1H NMR.

Simultaneous isolation of artemisinin and its precursors from Artemisia annua L. by preparative RP‐HPLC

It is still a major challenge to simultaneously isolate artemisinin and its precursors, especially dihydroartemisinic acid and artemisinic acid, from herbal Artemisia annua. A rapid, economical and automatical chromatographic separation process to isolate and purify artemisinin, dihydroartemisinic acid and artemisinic acid at the same time on a preparative scale was developed. The procedure included solvent extraction of ground Artemisia annua leaves by refluxing and purification of crude extract by preparative reverse-phase high-performance liquid chromatography (RP-HPLC). Fractions containing artemisinin and its precursors were collected and identified by gas chromatography and mass spectrometry. High purity of artemisinin, dihydroartemisinic acid and artemisinic acid was obtained by preparative HPLC with a C(18) column and 60% acetonitrile in water as the mobile phase. The techniques described here are useful tools for the preparative-scale isolation of artemisinin and its precursors in a fast, cost-effective and environmental friendly manner.

PLE and GC–MS Determination of Polybrominated Diphenyl Ethers in Soils

We describe a pressurized liquid extraction (PLE) method, followed by gas chromatography-mass spectrometry, for the determination of polybrominated diphenyl ethers (PBDEs) in soil samples. Different extraction solvents (isohexane, dichloromethane, acetone and methanol), either alone or in combination, were used to extract these target compounds from spiked soil samples. Variables affecting the PLE extraction efficiency, such as pressure, temperature, number of extraction cycles and static extraction time were studied; the optimum parameters were 1,500 psi, 100 °C, 1 cycle and 5 min, respectively. Purification of crude extracts from the PLE system was achieved by adsorption chromatography using different proportions of acid silica and activated silica; the most effective mixture being either 10 g acid modified silica : 5 g silica or 15 g acid modified silica : 0 g silica. The PLE based method was compared with more traditional Soxhlet and ultrasonic extraction methods; overall the mean percentage recoveries using PLE were 95 ± 9%, and compared favourably with the Soxhlet 94 ± 4% and were better than the ultrasonic method 108 ± 36%. The limits of detection and quantification of the proposed method were 11–54 and 38–180 pg g−1, respectively, for the different PBDE studied. The developed method was linear over the range from 0.05 to 30 ng g−1 except for PBDE 183 of 0.10–30 ng g−1. The established method was successfully applied to soil samples from Scotland, UK, for the determination of the target PBDEs.

Advanced mass spectrometry techniques for identification, characterisation and structural elucidation of natural products

Mass-based structural identification of natural products frequently employs combinations of different mass spectrometers, including triple quadrupole and ion trap instruments. These methods often provide adequate information for tentatively identifying structures of analogues of previously characterized compounds. For completely unknown compound, tentatively identified structures are often further examined by high-resolution MS, from which elemental compositions can be obtained. Here it is required to measure the mass-to-charge ratios with the smallest mass uncertainties possible. Ideally, routines with sufficient resolution and mass accuracy yield single empirical formulae for every compound analysed. In the absence of a unique empirical formula, analysis can be highly ambiguous. In most studies, quadrupole TOF and Fourier-transform MS (orbitrap, ICR) instruments are used. This presentation illustrates state-of-the-art techniques for screening and identifying natural products. (1) Characterization of hyperforin in extracts of St. John's wort (Hypericum perforatum) is illustrated. The strategy used was a combination of MS2 and MS3 experiments, performed on QqLIT and high-resolution FTICR instruments. This combination allowed fragmentation schemes and chemical structures to be proposed. (2) Analysis of Boswellia from the Boswellia serrata tree is demonstrated. Specific active anti-inflammatory ingredients in this herb have been identified, boswellic acids. In animal studies, these acids have been shown to significantly reduce inflammation. We are interested in the metabolism, as the clinical efficiency appears high despite the low observed plasma levels, possibly involving unknown metabolites. (3) Characterization of active species in marine toxin extracts is shown. Analysis of phytoplankton allowed for detection of several previously characterized toxins as well as new unexpected compounds. Purification of crude extracts into several fractions using mass-triggered preparative chromatography permitted enrichment of different species, resulting in high quality mass spectra for all known and unknown species

Pressurized liquid extraction in determination of polychlorinated biphenyls and organochlorine pesticides in fish samples

Pressurized liquid extraction (PLE) is a relatively new technique applicable for the extraction of persistent organic pollutants from various matrices. The main advantages of this method are short time and low consumption of extraction solvent. The effects of various operational parameters (i.e. temperature of extraction, number of static cycles and extraction solvent mixtures) on the PLE efficiency were investigated in this study. Fish muscle tissue containing 3.2% (w/w) lipids and native polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and other related compounds was used for testing. Purification of crude extracts was carried out by gel permeation chromatography employing Bio-Beads S-X3. Identification and quantitation of target indicator PCBs and OCPs was performed by high-resolution gas chromatography (HRGC) with two parallel electron-capture detectors (ECDs). Results obtained by the optimized PLE procedure were compared with conventional Soxhlet extraction (the same extraction solvent mixtures hexane–dichloromethane (1:1 v/v) and hexane–acetone (4:1 v/v) were used). The recoveries obtained by PLE operated at 90–120 °C were either comparable to “classic” Soxhlet extraction (for higher-chlorinated PCB congeners and DDT group) or even better (for lower chlorinated analytes). The highest recoveries were obtained for three static 5 min extraction cycles.

Production, extraction and purification of phycobiliproteins from Nostoc sp.

One strain of nitrogen-fixing cyanobacterium ( Nostoc sp. PCC 9202) was grown indoors in 1 litre glass air-lift reactors as well as 17 litre polyethylene bags. Temperature dependence of growth-kinetic parameters, as well as of biomass and phycobiliprotein productivities, were determined. Harvesting and phycobiliprotein extraction methodologies are presented. The purification of crude extracts was performed by means of ultrafiltration or (NH 4) 2SO 4 precipitation followed by gel filtration and ion-exchange chromatography. Final phycocyanin and phycoerythrin preparations were characterized by purity ratios above 4 and 5, respectively. Phycobiliprotein production cost determination for each purity grade and cumulative phycobiliprotein weight losses along purification steps are shown. Production cost for high purity phycoerythrin (30 US$ per g) seems to be far below the market price.

Separation and Identification of Phenolic Acids from Some Species of the Asteraceae Family Using HPLC with a Diode Array Detector†

The qualitative and quantitative analysis of phenolic acids was carried out using methods of HPLC with the diode array detector (DAD). The quantitative analysis of chosen phenolic acids was carried out using the Multicomponent Analysis method (MCA). Solid-phase extraction was applied for the purification of crude extract from aerial part of three species of fam. Asteraceae (Chrysanthemum maximum Ram.(DC), Chrysanthemum segetum L., Rudbeckia laciniata L.). The choice of optimal conditions of analysis was made using DryLab+ software. The part of this work was presented at the 3rd European Congress of Pharmaceutical Sciences, Edinburgh, 15–17 September 1996.

Saponin‐like in vitro characteristics of extracts from selected non‐nutrient wild plant food additives used by Maasai in meat and milk based soups

Twelve plant additives used by the Batemi and Maasai of East Africa in meat and milk based soups were investigated for saponin‐like characteristics and in vitro interactions with cholesterol and cholesterol analogues. Purification of crude extracts by n‐butanol extraction revealed that Albizia anthelmintica, Acacia goetzii, and Myrsine africana had frothing ability, molluscicidal activity, haemolytic activity and TLC activities indicating saponins. They also had significant (p < 0.05) haemolytic activity that was affected by the presence of cholesterol, cholesterol analogues, conjugated and non‐conjugated bile salts (p < 0.05). Methanol, ethyl acetate, aqueous and n‐butanol extracts of A. anthelmintica and methanol, ethyl acetate and aqueous extracts of A. goetzii bound significant amounts of cholesterol solubilized in ethanol (p < 0.05). Thus, saponin‐like compounds are a detectable component of the Maasai diet and extracts likely containing saponins do interact with cholesterol and analogues involved in lipid metabolism that have also been proposed to be involved in in vivo mechanisms of saponin induced hypocholesterolemia. These results suggest that investigation of the use of A. anthelmintica and A. goetzii as dietary additives, the use of herbs in the Maasai diet and the contribution of natural products to the health of people maintaining indigenous diets is worth further investigation.

Sample preparation for taxol and cephalomannine determination in various organs of Taxus sp.

Solid-phase extraction and preparative thin-layer chromatography were applied as sample preparation techniques for the purification of crude extracts from twigs and needles of various Taxus species as well as for the isolation of taxol and cephalomannine for further reversed phase high performance liquid chromatography analysis. Significant differences in the contents of taxanes examined were found. The preparative chromatographic methods used were compared and evaluated as routine and reproducible procedures for the rapid isolation and determination of taxol and cephalomannine in plant extracts.

Assessment of Various Techniques for the Quantitative Extraction of Lysophospholipids from Myocardial Tissues

Lipid extraction methods were evaluated for their effectiveness in extracting lysophosphatidyicholines and lysophosphatidylethanolamines from tissues and for subsequent recoveries during purification of crude extracts. The acid-butanol technique, although effective in complete extraction, resulted in partial hydrolysis (2-10%) of phospholipids containing 1-alk-1′-enyl-2-acylglycerophospholipids (plasmalogens) to produce artifactual lysophospholipids. This problem was avoided using a neutral butanol extraction or Bligh and Dyer techniques, but these resulted in only partial recoveries (60-72 and 75-80%, respectively) of these lipids. Tissue extracted with neutral chloroform-methanol mixtures provided virtually complete extraction (97-100%), but subsequent losses (up to 15%) occurred during purification of crude extracts with Folch synthetic upper phases. These losses could be circumvented by purification of the crude extract on Sephadex G-25 column. As an alternative, the Folch extraction technique was modified to achieve complete recoveries of lysophospholipids. This involved extraction of the tissue with a chloroform-methanol-saline biphasic system. After removal of the lower lipid phase, the upper phase containing residual tissue was reextracted twice more with Folch lower phase and once with lower phase containing HCl. This last extract was neutralized with NH3 vapor before pooling with the preceding extracts. This method (i) circumvents plasmalogenic hydrolysis, (ii) avoids use of time-consuming column chromatography, (iii) eliminates the losses of lipids during purification, and (iv) allows highly reproducible quantitative analyses of all lipid fractions including lysophospholipids and nonesterified fatty acids from myocardial tissue.

The identification of microbial metabolites of sulfolithocholic acid.

Sulfolithocholate a major detoxified form of lithocholic acid, is biotransformed by human intestinal microflora to four major metabolites. Purification of crude extracts by Sephadex LH-20 chromatography gave three major radioactive peaks, after incubation studies, which were characterized by thin-layer and gas--liquid chromatography, as well as combined gas--liquid chromatography--mass spectrometry. Peak I has a polarity similar to that for 3 beta-palmitoyl-isolithocholic acid and transesterification of the metabolite showed that it was a mixture of palmitoyl, palmitoleyl, stearyl, and oleyl esters of isolithocholate. Peak II was identified as chol-3-en-24-oic acid and Peak III was a mixture of lithocholic (mainly) and isolithocholic acids. The fatty acyl derivatives of isolithocholate and delta 3-cholenate are new microbial metabolites of sulfolithocholate and represent unique classes of bile acids that should be included in routine fecal analysis of bile acids.

The role of molybdenum in the synthesis of nitrate reductase in cauliflower ( Brassica oleracea L. var. Botrytis L.) and spinach ( Spinacea oleracea L.)

1. 1.The 185W analogue of nitrate reductase was produced in molybdenum-deficient cauliflower ( Brassica oleracea L. var. Botrytis L.) leaves following petiolar uptake of 185W from a molybdenum-free medium. l-Azetidine-2-carboxylic acid in the nutrient solution had no effect on analogue formation, but puromycin decreased by 75% 185W incorporation into protein in the fraction normally associated with nitrate reductase. The analogue co-purified with spinach ( Spinacea oleracea L.) nitrate reductase. 2. 2.An antiserum prepared against partially purified spinach nitrate reductase inhibited enzyme activity. 3. 3.Using the antiserum a method was developed, based on 50% inactivation of a “standard” nitrate reductase preparation, to measure the amount of material in solution cross-reacting as nitrate reductase protein but having no enzyme activity. Crude extracts and solutions resulting after purification with respect to nitrate reductase were obtained from molybdenum-deficient spinach plants grown with nitrate either as the sole nitrogen source of supplemented with ammonium salt. These were assayed for their content of cross-reacting material. 4. 4.The results indicate that approximately 30% of the normal complement of nitrate reductase protein found in extracts of healthy spinach plants was present in extracts of molybdenum-deficient spinach plants either as aponitrate reductase orits immunochemically cross-reacting sub-units. Purification of crude extracts with respect to nitrate reductase resulted in a preferential and substantial loss of cross-reacting material relative to the enzyme. It is suggested that breakdown of the apoprotein is rapid in vivo compared with that of the holoenzyme. Continued net increase of nitrate reductase resulting from metal incorporation into apoprotein is dependent on protein synthesis.