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|Substrate For (Enzyme):||α-Galactosidase|
High purity Verbascose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.Hide Abstract
McCleary, B. V. & Matheson, N. K. (1974). Phytochemistry, 13(9), 1747-1757.
Germinating seeds of lucerne, guar, carob and soybean initially depleted raffinose series oligosaccharides and then galactomannan. This depletion was accompanied by a rapid increase and then a decrease in α-galactosidase levels. Lucerne and guar contained two α-galactosidase activities, carob three and soybean four. One of these in each plant, from its location in the endosperm, time of appearance and kinetic behaviour, appeared to be primarily involved in galactomannan hydrolysis. This enzyme in lucerne had MW of 23 000 and could not be separated from β-mannanase by (NH4)2SO4 fractionation, DEAE, CM or SE-cellulose chromatography or gel filtration, but only by polyacrylamide gel electrophoresis. In guar, carob and soybean, it could be separated by ion-exchange chromatography and gel filtration. In lucerne, carob and guar most of the total increase in activity was due to this enzyme. The other α-galactosidases had MWs of about 35 000 and could be separated from β-mannanase by dissection, ion exchange cellulose chromatography and gel filtration. They were located in the cotyledon-embryo and appeared to be primarily involved in galactosylsucrose oligosaccharide hydrolysis.Hide Abstract
Monitoring of early‐stage water uptake by hyperspectral imaging and evaluation of nutritional and technological functionality of germinated faba bean (Vicia faba L.) var. minor and var. major as food ingredients.
Holopainen‐Mantila, U., Sarlin, T., Mäkinen, O., Laitila, A. & Sozer, N. (2021). Legume Science, e124.
Faba bean (Vicia faba L.) is a potential, sustainable protein alternative. Germination behavior of Vicia faba L. var. minor and Vicia faba L. var. major needs further studies in order to enable larger scale bioprocessing. In this study, early-stage water uptake of two distinct faba bean varieties was assessed by hyperspectral imaging. Nutritional and technological functionality of germinated faba bean ingredients as such and in combination with fermentation were evaluated. Hyperspectral imaging revealed that early-stage water uptake in faba beans occurred evenly from the different sides of the beans. Germination on petri dishes and in pilot-scale showed that smaller faba beans moistened and germinated significantly (p < 0.05) faster and retained water better through germination than larger faba beans. Germinated faba flour of minor-type variety resulted in 72% higher dextran production in Weissella confusa VTT E-143403 fermentation than respective native faba flour. With both types of faba bean varieties, germination as such, and with minor-type variety, germination as combined with fermentation decreased notably the content of raffinose family oligosaccharides. These bioprocessing methods also improved functionality of faba bean ingredients by increasing protein separation efficiency in air classification, protein solubility, foaming capacity, and foam stability. Based on this study, minor- and major-type or small- and large-seeded faba bean varieties set different requirements to the germination process. In addition, germination alone or as combined with fermentation was proved to improve the nutritional and technological quality of faba bean material promoting its use in several food applications including also gluten-free products.Hide Abstract
Multiple reaction monitoring for identification and quantification of oligosaccharides in legumes using a triple quadrupole mass spectrometer.
Prieto-Santiago, V., del Mar Cavia, M., Barba, F. J., Alonso-Torre, S. R. & Carrillo, C. (2022). Food Chemistry, 368, 130761.
Raffinose family oligosaccharides are non-digestible compounds considered as dietary prebiotics with health-related properties. Hence, it is important to develop highly specific methods for their determination. An analytical method is developed in this study for oligosaccharide identification and quantification using liquid chromatography-tandem mass spectrometry equipped with a triple quadrupole analyser operating in Multiple Reaction Monitoring mode. Raffinose, stachyose and verbascose are separated in a 10-minute run and the method is validated over a broad concentration range, showing good linearity, accuracy, precision and high sensitivity. A low-cost, short eco-friendly procedure for oligosaccharide extraction from legumes, with a high recovery rate extraction, good repeatability and reproducibility is also proposed. No plant-matrix effects were demonstrated. The method applied to the screening of 28 different legumes revealed species-related traits for oligosaccharide distribution, highlighting Pisum sativum (9.22 g/100 g) as the richest source of these prebiotics and its suitability as a functional food ingredient.Hide Abstract
Enhanced nutritional value of chickpea protein concentrate by dry separation and solid state fermentation.
Xing, Q., Dekker, S., Kyriakopoulou, K., Boom, R. M., Smid, E. J. & Schutyser, M. A. (2020). Innovative Food Science & Emerging Technologies, 59, 102269.
A sustainable dry processing method to obtain nutritional and functional chickpea products was developed, yielding protein concentrates suited to prepare products without additives. Chickpeas were milled and air-classified into protein and starch-enriched concentrates. Subsequently, spontaneous solid state fermentation (SSF) with daily back-slopping was performed at 37°C. The dominant autochthonous lactic acid bacteria (LAB) strains in chickpea flour and enriched fractions were Pediococcus pentosaceus and Pediococcus acidilactici. Strains were selected on their ability to metabolise flatulence-causing α-galactosides. SSF reduced the pH of the doughs in 24h from 6.6 to 4.2. After 72 h, concentrations of raffinose and stachyose were reduced by 88.3-99.1%, while verbascose became undetectable. Moreover, phytic acid reduced with 17% while total phenolic contents increased with 119%. Besides the observed differences in smell, texture and colour, the sourdoughs showed 67% higher water-holding capacity. This natural route to produce chickpea concentrates thus increases both the nutritive and technical functionality.Hide Abstract
Xu, Y., Wang, Y., Coda, R., Säde, E., Tuomainen, P., Tenkanen, M. & Katina, K. (2017). International Journal of Food Microbiology, 248, 63-71.
Fava bean flour is regarded as a potential plant-based protein source, but the addition of it at high concentration is restricted by its poor texture-improving ability and by anti-nutritional factors (ANF). Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) are regarded as good texture modifiers. In this study, fava bean flour was fermented with Leuconostoc spp. and Weissella spp. with or without sucrose addition, in order to evaluate their potential in EPS production. The contents of free sugars, organic acids, mannitol and EPS in all fermented fava bean doughs were measured. Rheological properties of sucrose-enriched doughs, including viscosity flow curves, hysteresis loop and dynamic oscillatory sweep curves, were measured after fermentation. As one of the ANF, the degradation of raffinose family oligosaccharides (RFO) was also studied by analyzing RFO profiles of different doughs. Quantification of EPS revealed the potential of Leuconostoc pseudomesenteroides DSM 20193 in EPS production, and the rheological analysis showed that the polymers produced by this strain has the highest thickening and gelling capability. Furthermore, the viscous fava bean doughs containing plant proteins and synthesized in situ EPS may have a potential application in the food industry and fulfill consumers' increasing demands for “clean labels” and plant-originated food materials.Hide Abstract
Raffinose family oligosaccharides in seeds of Pisum wild taxa, type lines for seed genes, domesticated and advanced breeding materials.
Gawłowska, M., Święcicki, W., Lahuta, L. & Kaczmarek, Z. (2016). Genetic Resources and Crop Evolution, 64(3), 1-10.
The content of raffinose family oligosaccharides (RFOs) in pea seeds constrains their usage in feeding humans and animals. In our research, the content of soluble carbohydrates—particularly α-D-galactosides of sucrose (RFOs)—was analyzed. The materials were as follows: 248 accessions from the Polish Pisum Genebank including representatives of taxa (from species to convarietas), type lines for genes controlling seed characters, and breeding materials and cultivars. Accessions were divided into homogeneous groups considering content of total soluble carbohydrates, total RFOs and individual RFOs: raffinose, stachyose and verbascose. The highest content of total soluble carbohydrates and total RFOs were stated for accessions with wrinkled seeds (r and rb genes) and the lowest content for seeds of the wild species P. fulvum Sibth. et Sm. Accessions valuable for breeding (for further decreasing of anti-nutritional compounds) were found among domesticated taxa (P. sativum subsp. sativum convar. vulgare Alef. And speciosum (Dierb.) Alef.), breeding lines, and some wild taxa. Accessions with decreased content of a total RFOs and verbascose are particularly valuable. It was found that the content of total RFOs was the most highly, frequently, and positively correlated with a stachyose and verbascose content. However, in P. fulvum seeds with the lowest content of RFOs and verbascose, a high correlation between the content of total RFOs and stachyose was revealed. Contents of all oligosaccharides were substantially lower in lines with dominant alleles of main pea seed genes (R, A, and I). It can be assumed that wild, primitive peas were characterized by not-all-to-high (rather not high) content of oligosaccharides; then recessive mutations in mentioned genes resulted in an increased content of RFOs. It seems to be an interesting observation from an evolutionary point of view.Hide Abstract
Condezo-Hoyos, L., Pérez-López, E. & Rupérez, P. (2015). Food Chemistry, 180, 265-271.
Optimization and validation of evaporative light scattering detector (ELSD), aided by response surface methodology (RSM), has been developed for the liquid chromatography analysis of a wide molecular weight (MW) range of carbohydrates, including polysaccharides and oligosaccharides. Optimal experimental parameters for the ELSD detection were: 88.8°C evaporator temperature, 77.9°C nebulizer temperature and 1.1 standard litres per minute nitrogen flow rate. Optimal ELSD detection, used together with high performance size exclusion chromatography (HPSEC) of carbohydrates, gave a linear range from 250 to 1000 mg L-1 (R2 > 0.998), with limits of detection and quantitation of 4.83–11.67 and 16.11–38.91 mg L-1, respectively. Relative standard deviation was lower than 1.8% for intra-day and inter-day repeatability for apple pectin, inulin, verbascose, stachyose and raffinose. Recovery ranged from 103.7% to 118.3% for fructo-oligosaccharides, α-galacto-oligosaccharides and disaccharides. Optimized and validated ELSD detection is proposed for the analysis of high- to low-MW carbohydrates with high sensitivity, precision and accuracy.Hide Abstract
Popova, A. V., Rausch, S., Hundertmark, M., Gibon, Y. & Hincha, D. K. (2015). Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1854(10), 1517-1525.
The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses.Hide Abstract
Slupski, J. & Gebczynski, P. (2014). International Journal of Food Sciences and Nutrition, 65(4), 419-425.
Immature seeds of five bean cultivars (flageolet-type and those intended for dry-seed production) were assessed for changes in water-soluble carbohydrates including raffinose family oligosaccharides (RFOs) due to boiling, sterilization, and storage of the sterilized product. About 100 g fresh weight of edible portion of fresh bean seeds contained 2449.3–3182.6 mg total soluble sugars, of which RFOs comprised 44–49%. The highest amounts of these compounds were found in the seeds of the cultivars Laponia and Mona. The dominant oligosaccharide was stachyose. Boiling fresh seeds to consumption consistency reduced total soluble sugars and RFOs: average values were 57% and 55%, respectively. Sterilization in cans resulted in 65% reductions of both total soluble sugars and RFOs. In general, there were no changes in the content of soluble sugars in canned and sterilized products stored for 12 months.Hide Abstract
Słupski, J., & Korus, A. (2014). International Journal of Refrigeration. 43, 187-193.
Immature seeds of five bean cultivars (flageolet-type and those intended for dry-seed production) were evaluated for changes in the content of water soluble carbohydrates due to freezing, storage and preparation for consumption. Analyses were conducted in two differently treated products: 1. blanched, frozen, stored for 0–12 months, then boiled, 2. boiled, frozen, stored for 0–12 months, then microwave heated. Fresh bean seeds contained 2449.3–3182.6 mg total soluble sugars per 100 g of edible portion, of which raffinose family oligosaccharides (RFOs) comprised 43.8–49.3%. The dominant raffinose family oligosaccharide was stachyose. In seeds having undergone either of the above processing methods and then prepared for consumption, the content of total soluble sugars decreased by 51–64% and RFOs by 40–54%, depending on the cultivar. In general, there were no differences in the content of soluble sugars in products prepared for consumption in different way both immediately after freezing and after 12-month storage.Hide Abstract
Lahuta, L. B. & Górecki, R. J. (2011). Acta Physiologiae Plantarum, 33(3), 725-733.
During germination of winter vetch (Vicia villosa Roth.) seeds, the degradation of raffinose family oligosaccharides and galactosyl pinitols occurred faster in axis than in cotyledons. After 7 days of germination, all α-D-galactosides disappeared and the soluble carbohydrates in seedling tissues consisted of D-pinitol, sucrose, fructose, glucose and myo-inositol. Osmotic stress caused by incubation of seedlings in PEG 8000 solution (−0.5, −1.0, and −1.5 MPa) for 48 h induced the activity of crucial enzymes of the RFOs pathway, i.e. galactinol synthase and raffinose synthase, in both the root and epicotyl but not in cotyledons. The root and epicotyl accumulated elevated amounts of galactinol and raffinose as the osmotic potential was lowered. This process was transient because when PEG solution was replaced with water, galactinol and raffinose were degraded, thus confirming their direct involvement in the response of tissues to osmotic stress. Among other soluble carbohydrates, only sucrose accumulated in response to stress. The results did not show potential role of D-pinitol in the adjustment of winter vetch seedlings to osmotic stress.Hide Abstract
Lahuta, L. B., Górecki, R. J., Zalewski, K. & Hedley, C. L. (2007). Acta Physiologiae Plantarum, 29(6), 527-534.
The aim of the present study was to determine the effect of accelerated ageing on the composition and content of the soluble carbohydrates in pea seeds of six genotypes differing in the composition of raffinose family oligosaccharides. A gradual decrease in the concentration of higher homologues of raffinose was observed along with seed ageing. At the same time the seeds lost vigor, viability and germination capacity. No increase in the concentration of reducing sugars was recorded, but sorbitol accumulated in pea embryos. Sorbitol accumulation may indicate seed quality deterioration during storage.Hide Abstract
Kochhar, A., Rees, T. A. & Pollock, C. J. (2003). Brazilian Journal of Botany, 26(4), 525-531.
Lamium album accumulates starch, sucrose and raffinose-family oligosaccharides (RFO) as the major products of photosynthesis. These products were measured in leaves throughout a sixteen-hour photoperiod and under various irradiance conditions. There was continuous accumulation of sucrose and starch. The rate of gas exchange was higher at 500 µEm2 s-1 and 900 µEm2 s-1 than at 300 µEm2 s-1. The rate of photosynthesis did not decline over the sixteen-hour photoperiod, which suggested that there was no short-term feed back inhibition due to sucrose accumulation in this plant. When the products of photosynthesis were compared at the end of the photoperiod, only sucrose increased in abundance at high irradiance. The RFO pool in leaves was shown to contain raffinose, stachyose and verbascose; galactinol was also present. 14CO2 feeding demonstrated that roots and flowers were the major sinks. The middle leaves were major source leaves whilst young leaves acted as both sources and sinks.Hide Abstract
Smiricky, M. R., Grieshop, C. M., Albin, D. M., Wubben, J. E., Gabert, V. M. & Fahey, G. C. (2002). Journal of Animal Science, 80(9), 2433-2441.
Fourteen ileally cannulated pigs (BW = 35 ± 2 kg) were randomly allotted to a replicated 7 × 7 Latin square design experiment to evaluate the influence of the soybean oligosaccharides (OS), raffinose and stachyose, on ileal nutrient digestibility and fecal consistency. Semipurified diets containing soy protein concentrate (SPC) or soybean meal (SBM) as the sole protein sources were fed. Soy solubles (SS), a by-product of SBM processing containing 3.5% raffinose and 11.5% stachyose, were used to increase dietary raffinose and stachyose concentrations. The seven dietary treatments were SPC, SPC + 9% SS, SBM, SBM + 9% SS, SBM + 18% SS, SBM + 24,000 U α-galactosidase enzyme preparation/kg diet, and a low-protein casein (LPC) diet used to calculate true digestibility. Diets, with the exception of the LPC diet, were formulated to contain 17% CP. All diets contained 0.5% chromic oxide as a marker for ileal digestibility determination. The experimental periods were divided into a 5-d diet adaptation followed by 2-d of ileal digesta collection. Diets and digesta were analyzed for DM, N, Cr, amino acids (AA), raffinose, and stachyose. Fecal consistency was determined on d 6 and 7 of each experimental period. The apparent and true ileal AA digestibilities were not different (P< 0.05) for the SPC and SBM control diets. When SS was added to the SPC diet, apparent and true N and AA digestibilities were depressed (P< 0.05) with the exception of Trp and Pro. The apparent and true ileal N and AA digestibilities were not different (P> 0.05) between the SBM control and SBM + 9% SS diets with the exception of Glu. There was a linear decrease (P< 0.05) in apparent and true DM, Val, Gly, and Tyr digestibilities when increasing levels of SS were added to the SBM diet. The addition of α-galactosidase did not improve apparent or true ileal N or AA digestibilities except for apparent and true Val and Tyr. Ileal raffinose digestibility was improved (P< 0.05) by addition of α-galactosidase, but was not affected by any other dietary treatment. Ileal stachyose digestibility was not affected (P> 0.58) by treatment. Fecal consistency likewise was not affected (P> 0.36) by dietary treatment. In conclusion, soy OS reduced nutrient digestibilities, but the reductions were small, ranging from approximately 1.1 to 7.4 percentage units. This suggests that other factors may be negatively impacting SBM digestibility.Hide Abstract
Schrier, A. A., Hoffmann-Thoma, G. & van Bel, A. J. (2000). Functional Plant Biology, 27(9), 769-778.
Given the fact that plant species with a type 1 (symplasmic) minor vein ultrastructure seem to dominate in the tropics and subtropics, and species with a type 2 (apoplasmic) minor vein ultrastructure in the temperate and boreal climate zones, a cold sensitivity of symplasmic phloem loading was postulated. Electron microscopic observations were taken as support for this proposal. The objective of the present work was to test this postulate by measuring physiological parameters correlated to phloem loading. Carbohydrate levels in the leaf, minor vein loading of 14CO2-derived assimilates in leaf segments and exudation of sugars and 14C-labelled compounds in several species from families with known phloem-loading pathways were compared in 10 and 20°C-adapted plants at both 10 and 20°C. No essential differences in response to temperature between symplasmically and apoplasmically phloem-loading species were observed. Carbohydrate availability for export was essentially similar, phloem loading was fully operative at 10°C, and exudation of sugars equally reacted to low temperature in symplasmic and apoplasmic species. Apparently, the geographical distribution of type 1 and 2 species is not explained by a difference in temperature sensitivity of the phloem-loading mode.Hide Abstract
Chabrillange, N., Dussert, S., Englemann, F., Doulbeau, S. & Hamon, S. (2000). Seed Science Research, 10(3), 393-396.
Large differences in seed desiccation sensitivity have been observed previously among ten coffee species (Coffea arabica, C. brevipes, C. canephora, C. eugenioides, C. humilis, C. liberica, C. pocsii, C. pseudo-zanguebariae, C. sessiliflora and C.stenophylla). Of these species, C. liberica and C. humilis were the most sensitive to desiccation and C. pseudozanguebariae the most tolerant. A study was carried out using the same seed lots to investigate if these differences in desiccation tolerance could be correlated with differences in soluble sugar content. Soluble sugars were extracted from dry seeds and analysed using high performance liquid chromatography. The seed monosaccharide (glucose and fructose) content was very low (1.5 to 2 mg g-1 dry weight [dw]) in all species studied. The sucrose content ranged from 33 mg g-1 dw in C. liberica seeds to 89 mg g-1 dw in seeds of C. pocsii. Raffinose was detected in the seeds of only five species (C.arabica, C.brevipes, C.humilis, C.sessiliflora, C.stenophylla), among which only three species (C.arabica, C.sessiliflora and C.brevipes) also contained stachyose. Both raffinose and stachyose were present in very low quantities (0.3–1.4 mg g-1 dw and 0.1–0.7 mg g-1 dw, respectively). Verbascose was never detected. No significant relationship was found between seed desiccation sensitivity and: (i) the sugar content; (ii) the presence/absence of oligosaccharides; and (iii) the oligosaccharide:sucrose ratio.Hide Abstract
Rodríguez-Bürger, A. P., Mason, A. & Nielsen, S. S. (1998). Journal of Agricultural and Food Chemistry, 46(12), 4806-4813.
Common beans are an important source of energy and nutrients, but have significant amounts of antinutritional factors and a limited digestibility. A nutritious weaning food was developed by combining fermented black beans and rice. Raw beans were coarsely ground, soaked, cooked, fermented with Rhizopus oligosporus for 15, 20, or 25 h, and then homogenized to obtain a supernatant and a precipitate. Raw, cooked, and fermented beans, and the precipitates were chemically characterized and the data statistically analyzed to choose an optimum fermentation time to develop the weaning food product. Ash and mineral contents of the beans decreased after soaking and in the precipitates. Cooking improved protein digestibility and decreased the levels of lectin and trypsin inhibitor. The oligosaccharide content of beans fermented 25 h was lower than in the other treatments. The weaning food product (27% 25 h fermented beans, dry weight/73% cooked rice, dry weight) had an in vitro protein digestibility of 86% and a very low content of oligosaccharides.Hide Abstract
Johansen, H. N., Glitsø, V. & Bach Knudsen, K. E. (1996). Journal of Agricultural and Food Chemistry, 44(6), 1470-1474.
The effect of extraction solvents and temperatures on extraction yields of monosaccharides, sucrose, and raffinose oligosaccharides from plant materials was investigated. Toasted soybean meal, cotton seed meal, field peas, and a feed mixture were extracted in either water, 50% (v/v), or 80% (v/v) aqueous methanol or ethanol at 20 or 50°C or at the boiling point of the solvent. Extraction in 80% (v/v) alcohol was strongly influenced by the extraction temperature and maximum extraction was only achieved at the boiling point. Extraction in water and 50% (v/v) methanol or ethanol was less heat sensitive and gave comparable results. Aqueous ethanol (50%, v/v) was as effective as 50% (v/v) methanol, whereas lower yields were seen at higher alcohol strength. There was no consistent difference in the extraction yield when comparing reflux with constant stirring and water bath with occasional mixing for any of the extraction solvents used.Hide Abstract