Ultrasound pretreatment of wheat dried distiller’s grain (DDG) for extraction of phenolic compounds

Wheat Dried distiller’s grain (DDG), a coproduct from the ethanol production process, is rich in potentially health-promoting phenolic compounds. In the extraction of phenolic compounds from DDG, the DDG cell wall is an important barrier for mass transfer from the inside to the outside of the cell. The effect of high-power ultrasound pretreatment on destruction of DDG cell walls and extraction yield and rate was investigated. Direct sonication by an ultrasound probe horn at 24 kHz was applied and factors such as ultrasound power and treatment time were investigated. The method of nitrogen (N₂) adsorption at 77 K was used as a means to determine and compare the changes in physical properties (specific surface area, pore volume and pore size) of the treated samples at different levels of ultrasound power and treatment time. Increasing specific surface area, pore volume and pore size caused by ultrasonic treatment implied development of new or larger pores and damaged cell walls. Also, it was observed that the ultrasound pretreatment of DDG particles increased the extraction yield and rate of phenolic compounds from DDG by 14.29%. Among tested ultrasound conditions, 100% ultrasound power for 30 s was evaluated as the best pretreatment condition.     

The development and evaluation of ultrasound for the treatment of bacterial suspensions. A study of frequency,power and sonication time on cultured Bacillus species

Some species of bacteria produce colonies and spores which agglomerate in spherical clusters (Bacillus subtilis) and this serves as a protection for the organisms inside against biocidal attack. Flocs of fine particles e.g. clay can entrap bacteria which can also protect them against the biocides. It is because of problems such as these that alternative methods of disinfecting water are under active investigation. One such method is the use of power ultrasound, either alone or in combination with other methods. Ultrasound is able to inactivate bacteria and deagglomerate bacterial clusters or flocs through a number of physical, mechanical and chemical effects arising from acoustic cavitation. The aim of this study was to investigate the effect of power ultrasound at different powers and frequencies on Bacillus subtilis. Viable plate count techniques were used as a measure of microbial activity. Results showed a significant increase in percent kill for Bacillus species with increasing duration of exposure and intensity of ultrasound in the low-kilohertz range (20 and 38 kHz). Results obtained at two higher frequencies (512 and 850 kHz) indicated a significant increase in bacteria count suggesting declumping. In assessing the bacterial kill with time under different sonication regimes three types of behaviour were characterized:

• High power ultrasound (lower frequencies) in low volumes of bacterial suspension results in a continuous reduction in bacterial cell numbers i.e. the kill rate predominates.

• High power ultrasound (lower frequencies) in larger volumes results in an initial rise in cell numbers suggesting declumping of the bacteria but this initial rise then falls as the declumping finishes and the kill rate becomes more important.

• Low intensity ultrasound (higher frequencies) gives an initial rise in cell numbers as a result of declumping. The kill rate is low and so there is no significant subsequent decrease in bacterial cell numbers.

     

Ultrasound-assisted rapid extraction and kinetic modelling of influential factors: Extraction of camptothecin from Nothapodytes nimmoniana plant

Ultrasound-assisted extraction (UAE) of commercially important natural product camptothecin (CPT) from Nothapodytes nimmoniana plant has been investigated. The influences of process factors such as electric acoustic intensity, solid to liquid ratio, duty cycle, temperature and particle size on the maximum extraction yield and kinetic mechanisms of the entire extraction process have been investigated. The kinetics results showed that increasing the intensity, duty cycle, solid to liquid ratio and decreasing the particle size lead to substantial increase in extraction yields compared to classical stirring extraction. Different kinetic models were applied to fit the experimental data. The second order rate model appears to be the best. The extraction rate constant, initial extraction rate and the equilibrium concentration for all experimental conditions have been calculated. SEM analysis of spent plant material clearly showed hollow openings on cell structure, which could be directly correlated to explosive disruption by the action of ultrasound waves. Overall 1.7-fold increase in extraction yields of CPT (0.32% w/w) and decrease in time from 6 h to 18 min was observed over the stirring method.     

Ultrasound-assisted extraction of anthraquinones from roots of Morinda citrifolia

This study investigated the use of ultrasound-assisted extraction (UAE) to improve the extraction efficiency of the classical solvent extraction techniques such as maceration and soxhlet extraction to extract anti-oxidant activity compounds, anthraquinones, from the root of Morinda citrifolia. The effects of different extraction conditions were determined, i.e., temperature of (25, 45, 60 °C), ultrasonic power, solvent types, and compositions of ethanol in ethanol–water mixtures. The results show that the yield increases with increasing extraction times and extraction temperatures. The percent recovery of anthraquinones using ultrasound was found to be highly dependent on the type of solvents (acetone > acetonitrile > methanol > ethanol). Furthermore, the use of ethanol–water solution as extraction solvent increased the yield of anthraquinones due to the relative polarity, the swelling effect of plant tissue matrix by water, and increased sound absorption. To achieve the same recovery as that achieved by UAE, soxhlet extraction and maceration required much longer time.     

Study of cell killing effect on S180 by ultrasound activating protoporphyrin IX

The present study was initiated to investigate the potential biological mechanism of cell killing effect on isolate sarcoma 180 (S180) cells induced by ultrasound activating protoporphyrin IX (PPIX). S180 cells were exposed to ultrasound for 30 s duration, at a frequency of 2.2 MHz and an acoustic power of 3 W/cm² in the presence of 120 μM PPIX. The viability of cells was evaluated using trypan blue staining. The generation of oxygen free radicals in cell suspensions was detected immediately after treatment using a reactive oxygen detection kit. A copper reagent colorimetry method was used to measure the level of FFAs released into cell suspensions by the process of cell damage induced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (i.e., SOD, CAT, GSH-PX) in S180 tumor cells. Treatment with ultrasound and PPIX together increased the cell damage rate to 50.91%, while treatment with ultrasound alone gave a cell damage rate to 24.24%, and PPIX alone kept this rate unchanged. Colorimetry and enzymatic chemical methods showed that the level of FFAs in cell suspension increased significantly after the treatment, while the activity of all the above enzymes decreased in tumor cells at different levels, and were associated with the generation of oxygen free radicals in cell suspension after treatment. The results indicate that oxygen free radicals may play an important role in improving the membrane lipid peroxidation, degrading membrane phospholipids to release FFAs, and decreasing the activities of the key antioxidant enzymes in cells. This biological mechanism might be involved in mediating the effects on S180 cells and resulting in the cell damage seen with SDT.     

Ultrasonic assisted far infrared drying characteristics and energy consumption of ginger slices

Three drying methods, including far infrared drying, infrared convection drying, and ultrasonic pretreatment assisted far infrared drying, were adopted in the drying of ginger slices. The effects of main parameters (ultrasonic pretreatment power and time, far infrared temperature and power, sample thickness, infrared convection temperature) on the drying kinetics, energy consumption, and color change were investigated and discussed in detail. The results showed that the drying process of ginger slices was controlled by falling rate period. For far infrared drying, the drying rate increased with the increase of infrared temperature and decrease of sample thickness, while the infrared power had no obvious effect on the drying process. The infrared convection drying showed the fastest drying rate and the smallest color change, however, the energy consumption was the highest. For ultrasonic pretreatment assisted far infrared drying, an appropriate ultrasonic pretreatment time and power would promote the far infrared drying process and the energy consumption was only slightly increased. However, the color change was relatively large. The ultrasound technology showed its greatest potential to enhance the drying rate at the early stage of drying and increasing ultrasonic power was more effective than prolonging the pretreatment time in promoting far infrared drying.     

Optimization of ultrasound assisted extraction of abalone viscera protein and its effect on the iron-chelating activity

This study aims to investigate effects of ultrasound assisted extraction on the abalone viscera protein extraction rate and iron-chelating activity of peptides. The optimal conditions for ultrasound assisted extraction by response surface methodology was at sodium hydroxide concentration 14 g/kg, ultrasonic power 428 W and extraction time 52 min. Under the optimal conditions, protein extraction rate was 64.89%, compared with alkaline extraction of 55.67%. The iron-chelating activity of peptides affected by ultrasound technology was further evaluated by iron-chelating rate, FTIR spectroscopy and LC-HRMS/MS. Alcalase was the suitable enzyme for the preparation of iron-chelating peptides from two abalone viscera proteins, showing no significant difference between their iron-chelating rate of 16.24% (ultrasound assisted extraction) and 16.60% (alkaline extraction). Iron binding sites from the two hydrolysates include amino and carboxylate terminal groups and peptide bond of the peptide backbone as well as amino, imine and carboxylate from side chain groups. Moreover, 24 iron-chelating peptides were identified from hydrolysate (alcalase, ultrasound assisted extraction), which were different from the 27 iron-chelating peptides from hydrolysate (alcalase, alkaline extraction). This study suggests the application of ultrasound technology in the generation of abalone viscera-derived iron-chelating peptides which have the ability to combat iron deficiency.     

Fragmentation of chitosan by ultrasonic irradiation

Kinetics of chitosan fragmentation by ultrasonic irradiation at frequency of 20 kHz, and the effects of experimental variables (power of ultrasound, chitosan concentration and solution temperature) on fragmentation were investigated. The kinetics studies were followed by measuring solution viscosity of the original and its fragments, and determining average number of chain scission of the fragments. The effects of ultrasonic power, chitosan concentration and solution temperature on fragmentation process were followed by viscometry and size exclusion chromatography. The chemical structure of the original chitosan and its fragments were examined by (1)H NMR spectroscopy and elemental analysis. The experimental results showed that the rate of fragmentation increased with an increase in power of ultrasound. Chain scission increased with an increase in power of ultrasound; and solution temperature, but a decrease in chitosan concentration. The chemical structure and polydispersity of the original and the fragments were nearly identical. A model based on experimental data to describe the relationship between chain scission and experimental variables (power of ultrasound; irradiation time; reduced concentration, c[eta]; and solution temperature) was proposed. It was concluded that ultrasonic irradiation is a suitable method to perform partial depolymerization and to obtain moderate macromolecules from large ones.     

Ultrasound assisted enhancement in natural dye extraction from beetroot for industrial applications and natural dyeing of leather

There is a growing demand for eco-friendly/non-toxic colorants, specifically for health sensitive applications such as coloration of food and dyeing of child textile/leather garments. Recently, dyes derived from natural sources for these applications have emerged as an important alternative to potentially harmful synthetic dyes and pose need for suitable effective extraction methodologies. The present paper focus on the influence of process parameters for ultrasound assisted leaching of coloring matter from plant materials. In the present work, extraction of natural dye from beetroot using ultrasound has been studied and compared with static/magnetic stirring as a control process at 45 °C. The influence of process parameters on the extraction efficiency such as ultrasonic output power, time, pulse mode, effect of solvent system and amount of beetroot has been studied. The use of ultrasound is found to have significant improvement in the extraction efficiency of colorant obtained from beetroot. Based on the experiments it has been found that a mixture of 1:1 ethanol–water with 80 W ultrasonic power for 3 h contact time provided better yield and extraction efficiency. Pulse mode operation may be useful in reducing electrical energy consumption in the extraction process. The effect of the amount of beetroot used in relation to extraction efficiency has also been studied. Two-stage extraction has been studied and found to be beneficial for improving the yield for higher amounts of beetroot. Significant 8% enhancement in % yield of colorant has been achieved with ultrasound, 80 W as compared to MS process both using 1:1 ethanol–water. The coloring ability of extracted beet dye has been tested on substrates such as leather and paper and found to be suitable for dyeing. Ultrasound is also found to be beneficial in natural dyeing of leather with improved rate of exhaustion. Both the dyed substrates have better color values for ultrasonic beet extract as inferred from reflectance measurement. Therefore, the present study clearly offers efficient extraction methodology from natural dye resources such as beetroot with ultrasound even dispensing with external heating. Thereby, also making eco-friendly non-toxic dyeing of fibrous substances a potential viable option.     

Spatial-temporal dynamics of cavitation bubble clouds in 1.2 MHz focused ultrasound field

Cavitation bubbles have been recognized as being essential to many applications of ultrasound. Temporal evolution and spatial distribution of cavitation bubble clouds induced by a focused ultrasound transducer of 1.2 MHz center frequency are investigated by high-speed photography. It is revealed that at a total acoustic power of 72 W the cavitation bubble cloud first emerges in the focal region where cavitation bubbles are observed to generate, grow, merge and collapse during the initial 600 μs. The bubble cloud then grows upward to the post-focal region, and finally becomes visible in the pre-focal region. The structure of the final bubble cloud is characterized by regional distribution of cavitation bubbles in the ultrasound field. The cavitation bubble cloud structure remains stable when the acoustic power is increased from 25 W to 107 W, but it changes to a more violent form when the acoustic power is further increased to 175 W.     

Application of ultrasound as pretreatment for extraction of podophyllotoxin from rhizomes of Podophyllum peltatum

The effect of high-power ultrasound pretreatment on the extraction of podophyllotoxin from Podophyllum peltatum was investigated. Direct sonication by an ultrasound probe horn was applied at 24 kHz and a number of factors were investigated: particle size (0.18-0.6 mm), type of solvent (0-100% aqueous ethanol), ultrasonic treatment time (2-40 min), and power of ultrasound (0-100% power intensity, maximum power: 78 W). The optimal condition of ultrasound was achieved with 0.425-0.6 mm particle size, 10 min sonication time, 35 W ultrasound power, and water as the medium. There was no obvious degradation of podophyllotoxin with ultrasound under the applied conditions, and an improvement in extractability was observed. The SEM microscopic structure change of treated samples disclosed the effect of ultrasound on the tissue cells. The increased pore volume and surface area after ultrasonic treatment also confirmed the positive effect of ultrasound pretreatment on the extraction yield of podophyllotoxin from the plant cells.     

Ultrasound assisted supercritical fluid extraction of oil and coixenolide from adlay seed

Oil and coixenolide are important components of adlay seed (Coix lachrymal-jobi L. var. Adlay) with many beneficial functions to human health. In this work, a novel extraction technique--ultrasound assisted supercritical fluid extraction (USFE)--was studied. Effects of operating conditions on the extraction, including extraction temperature (T), pressure (P), time (t), CO(2) flow rate (F) and ultrasonic power (I) were investigated. There are optimum temperatures which gives the maximum extraction yields (EYs) for the supercritical fluid extractions with and without ultrasound. The effect of pressure on EYs for is similar to that of pressure on CO(2) density. Based on the yield of extraction, the favorable conditions for supercritical fluid extraction (SFE) were: T at 45 degrees C, P at 25 MPa, t at 4.0 h and F at 3.5L/h. While ultrasound was applied as in USFE, the following parameters were preferred: T at 40 degrees C, P at 20 MPa, t at 3.5h and F at 3.0 L/h, respectively. The results show that supercritical fluid extraction with the assistance of ultrasound could reduce the temperature, pressure, CO(2) flow rate, as well as time used in the process. Compared with SFE, USFE could give a 14% increase in the yield for extracting oil and coixenolide from adlay seed with less severe operating conditions.     

Mass transfer enhancement in supercritical fluids extraction by means of power ultrasound

The use of high-intensity ultrasound represents an efficient manner of producing small scale agitation, enhancing mass transfer on supercritical fluids (SF) extraction processes. In this way, a supercritical CO(2) extraction of oil from particulate almonds using power ultrasound was studied. To examine the effect of the acoustic waves all experiments were performed with and without ultrasound. A power ultrasonic transducer for a working frequency of about 20 kHz was constructed and installed inside a high-pressure 5 l SF extractor. The experimental tests were carried out with CO(2) at 280 bar and 55 degrees C. Grounded almonds with an oil content of about 55%, in an amount of 1500 g were deposited inside the SF reactor where the solvent was introduced at a flow rate of 20 kg/h. The results show that the kinetics and the extraction yield of the oil were enhanced by 30% and 20% respectively, when a power of about 50 W was applied to the transducer. The average time of each extraction process was of about 8 h and 30 min. In addition, the transducer was also used as a sensitive probe capable to detect the phase behavior of supercritical fluids when it was driven with low power signals.     

Using low intensity ultrasound to improve the efficiency of biological phosphorus removal

Low intensity ultrasound can produce various effects on biological materials, such as stimulating enzyme activity, cell growth, biosynthesis, etc., which may improve the efficiency of enhanced biological phosphorus removal (EBPR). We adopt total phosphorus (TP) and dehydrogenase activity (DHA) as indicators to confirm the feasibility of applying low intensity ultrasound in EBPR. Single-factor experiments and orthogonal test were conducted in batch anaerobic/oxic (A/O) process simulation to study the influence of ultrasonic intensity and exposure time in the EBPR process. The results showed that the optimal ultrasonic parameters were 0.2 W/cm² and 10 min under which condition the TP concentration in the effluent was 35–50% lower than that of the control (without ultrasonic irradiation). Changes of sludge activities after ultrasonic irradiation were examined. The improvement of sludge activity by ultrasound took 4 h after irradiation to reach the peak level, when an increase above 50% of DHA has been achieved by ultrasonic irradiation, and the enhancing effects induced by ultrasound disappeared in 16 h after irradiation. A tentative mechanism of biological phosphorus removal enhancement stimulated by ultrasound was discussed based on these phenomena.     

A kinetic study of the effect of ultrasound power on the sonohydrolysis of tetraethyl orthosilicate

A kinetic study of the ultrasound-stimulated and acid-catalyzed sonohydrolysis of tetraethyl orthosilicate (TEOS) in solventless TEOS–water heterogeneous mixtures was carried out by means of a calorimetric method as a function of the ultrasound power. The hydrolysis reaction starts in acidulated heterogeneous water–TEOS mixtures after an induction period under ultrasonic stimulation. The ultrasound power seems to play a role on the dynamical coupling of the system originating a continuum upward shifting of the base line during the induction period of sonication. The rate in which the base line is upward shifted diminishes with the power. The best coupling between the ultrasound and the reactant heterogeneous mixtures for this experimental setup was found to occur at 50 W, for which the gelation time was found to be a minimum. The kinetics of the heterogeneous TEOS sonohydrolysis was studied on the basis of a dissolution and reaction modeling. The heterogeneous reaction pathway as deduced from the kinetic study was drawn in a ternary diagram as a function of the ultrasound power.     

Ultrasonic dyeing of cationized cotton fabric with natural dye. Part 1: cationization of cotton using Solfix E

The dyeing of cationized cotton fabric with Solfix E using colouring matter extracted from Cochineal dye has been studied using both conventional and ultrasonic techniques. Factors affecting dye extraction such as ultrasound power, particle size, extraction temperature and time were studied. The results indicated that the extraction by ultrasound at 300 W was more effective at lower temperature and time than conventional extraction. The effect of various factors of dye bath such as pH, salt concentration, ultrasound power, dyeing time and temperature were investigated. The colour strength values obtained were found to be higher with ultrasound than with conventional techniques. The results of fastness properties of the dyed fabrics were fair to good. The scanning electron microscope (SEM) images of the morphological and X-ray analyzes were measured for cationized cotton fabrics dyed with both conventional and ultrasound methods, thus showing the sonicator efficiency.     

An investigation of the effect of ultrasonic waves on the efficiency of silicon extraction from coal fly ash

Burning of coal accounts for an enormous proportion of the current energy supply, especially in developing countries. Burning of coal produces large amounts of coal fly ash, which causes serious environmental problems unless it is managed properly. Using chemical analysis, we found that coal fly ash could be a promising source of Si, Al, Ca and some rare earth elements, especially with the assistance of some measures such as ultrasound. In this study, we extracted silicon from coal fly ash using an alkaline dissolution strategy and investigated the effects of temperature and ultrasonic power on the efficiency of silicon extraction. During a 70 min reaction, the efficiency of silicon extraction increased markedly, from 9.41% to 34.96%, as the reaction temperature increased from 70 °C to 110 °C. With ultrasound assistance, ultrasonic waves enhanced the extraction of silicon at both 80 °C and 110 °C at 720 W ultrasound, increasing the efficiency of silicon extraction from 6.01% to 15.36% and from 34.96% to 54.42%, respectively. However, at 900 W ultrasonic power, extraction was slightly inhibited at both temperatures, causing a little decrease in efficiency.     

Ultrasound-assisted extraction of rare-earth elements from carbonatite rocks

In view of the increasing demand for rare-earth elements (REE) in many areas of high technology, alternative methods for the extraction of these elements have been developed. In this work, a process based on the use of ultrasound for the extraction of REE from carbonatite (an igneous rock) is proposed to avoid the use of concentrated reagents, high temperature and excessive extraction time. In this pioneer work for REE extraction from carbonatite rocks in a preliminary investigation, ultrasonic baths, cup horn systems or ultrasound probes operating at different frequencies and power were evaluated. In addition, the power released to the extraction medium and the ultrasound amplitude were also investigated and the temperature and carbonatite mass/volume of extraction solution ratio were optimized to 70 °C and 20 mg/mL, respectively. Better extraction efficiencies (82%) were obtained employing an ultrasound probe operating at 20 kHz for 15 min, ultrasound amplitude of 40% (692 W dm⁻³) and using a diluted extraction solution (3% v/v HNO₃ + 2% v/v HCl). It is important to mention that high extraction efficiency was obtained even using a diluted acid mixture and relatively low temperature in comparison to conventional extraction methods for REE. A comparison of results with those obtained by mechanical stirring (500 rpm) using the same conditions (time, temperature and extraction solution) was carried out, showing that the use of ultrasound increased the extraction efficiency up to 35%. Therefore, the proposed ultrasound-assisted procedure can be considered as a suitable alternative for high efficiency extraction of REE from carbonatite rocks.     

Effects of processing parameters in the sonic assisted water extraction (SAWE) of 6-gingerol

The use of water in subcritical conditions for extraction has several drawbacks. These include the safety features, higher production costs and possible degradation of the bioactive compounds. To overcome these problems, sonic energy and an entrainer were used as external interventions to decrease the polarity of water at milder operating conditions. The effect of low (28 kHz) and high (800 kHz) frequencies of sonication in the extraction of the main ginger bioactive compound (6-gingerol) were compared. Six parameters were studied: mean particle size (MPS, mm), time of extraction, applied power, sample to solvent ratio (w/v), temperature of extraction, and the percentage of entrainer. The optimum conditions for high frequency SAWE prototype were MPS 0.89–1.77 mm, 45 min, 40 W applied power, 1:30 (w/v), 45 °C, and 15% of ethanol as entrainer. Two-way analysis of variance (ANOVA) gave the most significant parameter, which was power with F (1, 45.07), p < 2.50 × 10⁻⁹. Although the effect of low frequency was stronger than high frequency, at the optimum conditions of the sample to solvent ratio 1:30 (w/v) with 700 mL solvent and temperature 45 °C, the concentration and recovery of 6-gingerol from high frequency of SAWE prototype was 2.69 times higher than at low frequency of SAWE. It was found that although the effects of high frequency (800 kHz) were negligible in other studies, it could extract suitable compounds, such as 6-gingerol, at lower temperature. Therefore, the effects of sonication, which cause an enlargement in the cell wall of the ginger plant matrix, were observed using a Scanning Electron Microscope (SEM). It was found that the applied power of sonication was the most significant parameter compared to the other parameters.     

Optimization of ultrasound-assisted extraction of anthocyanins in red raspberries and identification of anthocyanins in extract using high-performance liquid chromatography-mass spectrometry

Anthocyanins (Acys) are naturally occurring compounds that impart color to fruit, vegetables and plants. The extraction of Acys from red raspberry (Rubus idaeus L. var. Heritage) by ultrasound-assisted process (UAP) was studied. A central composite rotate design (CCRD) was used to obtain the optimal conditions of ultrasound-assisted extraction (UAE), and the effects of operating conditions, such as the ratio of solvents to materials, ultrasonic power and extraction time, on the extraction yield of Acys were studied through response surface methodology (RSM). The optimized conditions of UAE were as follows: ratio of solvents to materials was 4:1 (ml/g), extraction time was 200 s, and ultrasonic power was 400 W. Under these conditions 34.5 mg of Acys from 100 g of fresh fruits (T_Acy, expressed as cyanidin-3-glucoside), approximately 78.13% of the total red pigments, could be obtained by UAE. The Acys compositions of extracts were identified by high-performance liquid chromatography–mass spectrometry (HPLC–MS), 12 kinds of Acys had been detected and eight kinds of Acys were characterized. Result indicated that cyanidin-3-sophoroside, cyanidin-3-(2^G-glucosylrutinoside), cyanidin-3-sambubioside, cyanidin-3-rutinoside, cyanidin-3-xylosylrutinoside, cyanidin-3-(2^G-glucosylrutinoside), and cyanidin-3-rutinoside were main components in extracts. In addition, in comparison with the conventional solvent extraction, UAE is more efficient and rapid to extract Acys from red raspberry, due to the strong disruption of fruit tissue structure under ultrasonic acoustic cavitation, which had been observed with the scanning electron microscopy (SEM). However, the Acys compositions in extracts by both methods were similar, which were investigated using HPLC profile.