medium ceramsite powder grinding mill in republic of korea

medium ceramsite powder grinding mill in republic of korea

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Purple glutinous rice bran (Kum Doi Saket rice (KUM)) contains high content of edible polysaccharides and anthocyanins and has an excellent antioxidant activity. This research aimed to optimize the extraction of crude polysaccharides from defatted purple glutinous rice bran using an ultrasonic-assisted extraction (UAE) and compared with a hot water extraction (HWE). Results showed that optimal extraction condition was as follows: a defatted rice bran to water ratio of 1:20 w/v, extraction temperature and time of 70 °C for 20 min. Under the optimal extraction condition, the yield of polysaccharide of UAE (4%) was significantly higher than that obtained from the HWE (0.8%). Additionally, antioxidant activities of extracted polysaccharide including IC50 value DPPH, IC50 value ABTS, and FRAP value were 1.09 mg/mL, 2.80 mg/mL and 197 µM Fe2+/g, respectively. It is suggested that the UAE process is promising method to decrease the processing time and to enhance extracted polysaccharide yields by 4 times

Polysaccharides have been studied for their various biological activities such as inhibiting free radicals, and antimicrobial, antitumor, anticancer, antiviral, anticoagulant and immunological activities. The biological effects of polysaccharides depend on their chemical composition, molecular weight and structure1. The polysaccharides extracted from edible and medicinal plants, e.g., Pteridium aquilinum, Ficus carica L., Lycium barbarum L., Portulaca oleracea L. and Arachis hypogaea seeds, were reported to be good sources of antioxidants2. Polysaccharides can inhibit free radicals generated in living organisms and protect the body tissue, thereby helping to prevent various diseases caused by tissue damage3. Many studies emphasized on the water-soluble polysaccharides, which are negatively charged such as with hydroxyl groups and oxygen atoms, and they can be free radical scavengers and metal chelators, leading to inhibition of lipid peroxidation4

optimization of ultrasonic-assisted extraction of

The KUM is a purple pigmented cultivar belonging to the species Oryza sativa L. It is widely grown in the north and northeast of Thailand and has high antioxidant activities because of its anthocyanin component5. The extracted polysaccharides from the defatted rice bran showed anti-cancer and/or anti-tumor properties6. Our previous reports showed a high potential of polysaccharides from the KUM defatted bran (KUM-DB) with antioxidant and antimicrobial activities7. Moreover, these polysaccharides were also modified using sulphation. The sulphated polysaccharides significantly increased immunomodulatory activity8

Extraction method is important to provide biologically active compounds. The highest active compounds and low undesired materials were target in extraction. The liquid-solid extraction is commonly used to extract the required compounds. Several factors such as type of extraction, temperature, time, solvent and extracted material are key parameters. The low extraction temperature is mostly considerable because the biologically active compounds can be degraded or lost during extraction. The time-consuming of extraction is also concerned9. HWE is the common boiling or refluxing method using for the extraction of botanical polysaccharides. Although, HWE is a low-cost process, it uses high temperatures (95–100 °C), long extraction times and generally has a low yield10. The three variables significantly affected the HWE yield of Epimedium acuminatum. The yield was increased with an increasing ratio of water to raw material and extraction temperature from 70 to 83.5 °C, but beyond 83.5 °C, the yield was decreased with increasing extraction temperature. When the extraction time increased from 3.5 to 4 h, the yield increased significantly11

The UAE has applied compared with other techniques for the extraction of bioactive compounds such as microwave-assisted extraction (MAE), supercritical fluid extraction, and enzyme-assisted extraction12. UAE is often inexpensive and simple. It could increase the yield of extracted components, decrease the extraction time and use lower temperatures. Therefore, it is usually used for extraction of thermolabile and unstable compounds13. For example, the UAE extracted polysaccharides from white button mushroom (Agaricus bisporus) showed that the UAE gave a higher yield than the HWE and MAE, with the UAE showing relative increases of 155 and 28%, respectively10. Ultrasonic has also been used to degrade polysaccharides to enhance antioxidant properties. The degraded polysaccharides did not have a different main structure but did have a reduced molecular weight14 and also it is very useful for extraction of polysaccharides, essential oils, proteins, peptide and pigment15

Although some studies showed improvement of the extract’s properties including antioxidant activity using UAE, there is little information focusing on the crude polysaccharide extract from rice bran, especially the purple glutinous rice. Therefore, this study aimed to investigate and optimize the UAE of polysaccharide from purple glutinous rice bran cv. Kum Doi Saket (colored rice) using three independent variables including the ratio of defatted purple glutinous rice bran to water (1:1–1:3 (w/v), extraction temperature (30–70 °C) and extraction time (20–60 min) through a response surface methodology (RSM). Furthermore, it was also attempted to compare the physicochemical characteristics and antioxidant activities of polysaccharides extracted by UAE and HWE. The aim was to optimize extraction conditions while obtaining high antioxidant activities of obtained polysaccharides

optimization of ultrasonic-assisted extraction of

Kum Doi Saket rice bran was purchased from a local grinding mill company (Organic Germinated Brown Rice Community Enterprise, Chiang Mai, Thailand) and defatted using hexane7,8. Chemicals used in the experiments, including 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), sulfur trioxide–trimethylamine (STMA), anhydrous dimethyl sulfoxide (DMSO), D-glucose and ascorbic acid (vitamin C), were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Other reagents in this experiment were analytical grade purchased from Union Science Co., Ltd. (Chiang Mai, Thailand)

The optimal extraction condition for crude polysaccharides from KUM-DB using UAE with a static power of 150 W ultrasonic bath (S100 H, Elma Schmidbauer GmbH, Singen, Germany) was determined using RSM with Design-Expert software (version 7.0, Stat-Ease, Inc., Minneapolis, MN, USA). A Box Behnken Design was selected due to high efficient in an interation and applied to optimize the extraction variables, including the ratio of KUM-DB to water (X1) from 1:10-1:30 (w/v), extraction temperature (X2) from 30–70 °C and extraction time (X3) from 20–60 min. A total of 15 treatments were used (Table 1)

For each treatment, 30 g of KUM-DB was put into a 3 L stainless steel bath and water was added according to the design (Table 1). After extraction, crude polysaccharide was centrifuged and evaporated to obtain concentrated supernatants. Next, the starch and protein in the supernatants were removed, and then, three volumes of 95% ethanol were added to precipitate polysaccharide. Subsequently, the precipitate was collected by centrifugation and washed with absolute ethanol. Afterwards, the precipitate was dialyzed in tap water and distilled water, respectively. Finally, the ultrasonicated crude polysaccharide (KUM-U) was dried in a vacuum oven and kept in an aluminum foil laminated polyethylene pouch at 4 °C for further analysis7,8

optimization of ultrasonic-assisted extraction of

The HWE was extracted using a sample to water ratio of 1:15 (w/v) at 90 °C for 2 h done twice following the condition as described by Surin et al.7,8. In comparison, the UAE was extracted using ultrasonic extractor at KUM-DB to water ratio of 1:20 (w/v) at 70 °C for 20 min. Further processing of the extract was as stated above to obtain the hot water extract (KUM-H). The KUM-H polysaccharide was used as control and compared with optimal extraction condition of KUM-U polysaccharide

The physiochemical properties were evaluated as described by Surin et al.7. The yield percentage of crude polysaccharide was calculated as the ratio of crude polysaccharide per KUM-DB. The carbohydrate and protein content were evaluated using the phenol–sulfuric acid colorimetric method (D-glucose as the standard) and the Coomassie Brilliant Blue reaction method (BSA as the standard), respectively. While the starch content was measured using a total starch assay kit (AA/AMG method, Megazyme, Wicklow, Ireland)

Anthocyanin content of crude polysaccharide was measured by following the method of Settapramote et al.16 with slight modification. Briefly, an HPLC (Agilent series 1100, Waldbronn, Germany) coupled with a diode array detector was used to evaluate anthocyanin contents. The samples were dissolved in HPLC grade water (RCI-Labscan, Thailand), afterwards they were filtered through a 0.45 μm membrane disc. The prepared samples, 20 μL, were injected into a Pursuit XRs5 C18 column (250 × 4.6 mm, Agilent, USA), ambient temperature and the wavelength was 520 nm. The samples were performed with a mixture of A: 4% phosphoric acid and B: 10% acetic acid/5% acetonitrile/1% phosphoric acid in water. A gradient elution of 0–20–40% A by linear increase from 0–20–25 min

optimization of ultrasonic-assisted extraction of

The sample solutions were prepared by dissolving the dried powder in distilled water at final concentrations of 0.312 to 10 mg/mL. Seven methods of in vitro antioxidant activity were evaluated using the methods as described previously8. The result of DPPH radical scavenging activity, ABTS radical scavenging activity, superoxide anion scavenging activity, hydroxyl radical scavenging activity and metal chelating assay were expressed as IC50 values, which referred to the concentration of polysaccharide required to scavenge 50% of the radicals. Reducing power and Ferric reducing antioxidant power (FRAP) assay were also performed

The sample solution (2 mL) was mixed with 2 mL of 0.2 mmol/L DPPH solution. The mixed solution was kept in the dark at 30 °C for 30 min. The absorbance of the mixed solution was measured at 517 nm using a spectrophotometer (model UV-2101PC, Shimudzu, Kyoto, Japan) against a blank (distilled water)

The sample solution was added to the ABTS radical solution with a ratio of 1:20. The mixed solution was kept at 30 °C for 6 min. The absorbance was measured at 734 nm against a blank (distilled water instead of sample solution)

optimization of ultrasonic-assisted extraction of

The polysaccharide solution (1 mL) was added to 2.0 of mL Tris–HCl buffer (16 mM, pH 8.0) containing 76 µM NBT and 394 µM NADH. Subsequently, 0.4 mL of PMS was added, and the mixed solution was incubated at 30 °C for 5 min. The absorbance was measured at 560 nm against a blank (distilled water instead of sample solution)

The polysaccharide solution (1 mL) was mixed with 2 mL of 9 mmol/L FeSO4 solution, 2 mL of 9 mmol/L salicylic acid in 96% ethanol, and 2 mL of 8.8 mmol/L H2O2 solution. The solution was incubated at 25 °C for 60 min. The absorbance of the mixed solution was measured at 510 nm against a blank (distilled water instead of sample solution)

The chelating effect of different polysaccharides on ferrous ion was measured by mixing 1 mL of samples with 0.1 mL of 2 mM FeCl2 and 0.2 mL of 5 mM ferrozine. The solution was kept at 30 °C for 10 min. The absorbance was measured at 562 nm against a blank (distilled water instead of sample solution). The inhibition percentage of ferrozine–Fe2+ complex formation was determined

optimization of ultrasonic-assisted extraction of

The different concentrations of samples were mixed with 2.5 mL of 0.2 M sodium phosphate buffer at pH 6.6 and 2.5 mL of 1% potassium ferricyanide [K3Fe(CN)6] solution. The mixed solution was kept at 50 °C for 20 min. Then 2.5 mL of 10% trichloroacetic acid solution was added. Then 2.5 mL of the upper layer of solution was removed and mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl3. The absorbance of the mixed solution was measured at 700 nm

The sample solution (80 µL) was added to 2 mL of FRAP reagent, and the mixed solution was incubated at 37 °C for 8 min. The absorbance of the mixed solution was measured at 593 nm. A standard curve was prepared using FeSO4•7H2O solution from 100 to 1000 µM. The results were expressed as the concentrations of FeSO4•7H2O with equivalent antioxidant activity

Response surface methodology was applied to optimize the experimental data using Design-Expert (version 7.0, Stat-Ease, Inc., Minneapolis, MN, USA). Data were expressed as mean ± standard deviation from duplicates. A polynomial equation was fitted to the data to obtain a regression equation. And the comparison of the physicochemical properties and antioxidant activity of KUM-H and KUM-U are reported as mean ± standard deviation from triplicates. The statistical significance was evaluated using one-way analysis of variance (ANOVA) at 95% confidence

optimization of ultrasonic-assisted extraction of

The ratio of KUM-DB to water, extraction temperature and extraction time significantly (p ≤ 0.05) affected the yield of the crude polysaccharides (Table 1). The regression equation of yield was a good fitting with a coefficient of determination (R2) of 0.905. The value was close to 1 indicating that the experimental and predicted values had a high degree of correlation17. The yields of the crude polysaccharides extracted using UAE were in the range of 1.15–4.31% (Table 1)

According to the tri-dimensional response surface contour plots in Fig. 1a, the yield showed a positive correlation with ratio of KUM-DB to water and extraction temperature. At all extraction temperatures, increasing the ratio of KUM-DB to water had a positive impact in the range of 1:10 to 1:25 w/v but it gave slightly lower yield, when the ratio of KUM-DB to water was greater than 1:25 w/v. The result is in agreement with the study previously of Cheung and Wu18. They reported that the ratio of material to water and extraction temperature (70–80 °C) affected the polysaccharide yield from Zizyphus jujuba cv. jinsixiaozao extracted by UAE improving the mass transfer rates. In addition, the yield of crude polysaccharides was rapidly increased when increasing the extraction temperature and the highest yield was observed at 60–70 °C. Using lower extraction temperatures (below 40 °C) could not disrupt the cell wall causing to obtain a low yield of polysaccharides from rice bran

Tri-dimensional response surface contour plots showing the experimental factors: (X1) ratio KUM-DB to water, (X2) extraction temperature and (X3) extraction time on yield (a–c), IC50 of DPPH assay (d–f), IC50 of ABTS assay (g–i) and FRAP assay (j–l)

optimization of ultrasonic-assisted extraction of

The tri-dimensional response surface contour plots of the ratio of KUM-DB to water (X1) with the extraction time (X3) on the yield of crude polysaccharides are shown in Fig. 1b. The yield of crude polysaccharides rapidly increased with increasing the ratio of KUM-DB to water from 1:15 to 1:20 w/v but the yields were slightly decreased when the ratio of KUM-DB to water was higher than 1:20 w/v. For the extraction time, the yield increased with increasing extraction time. Overall, the UAE increase the yield of polysaccharides because it can disrupt the cell wall of plant tissues, resulting in the polysaccharides easily released into the water at the early period of extraction19

Figure 1c shows the effect of extraction temperature and extraction time on the crude polysaccharides yield at a fixed the ratio of KUM-DB to water of 1:20 w/v. For the extraction temperature, the crude polysaccharides yield was slightly increased with increasing temperature and had the highest yield at 70 °C. For extraction time, the crude polysaccharides yield decreased with increased extraction time. The longer extraction time led to degrade the polysaccharides into free sugars, resulting in a decrease of polysaccharides yield12,20

The DPPH assay has been widely used as a tool for evaluating the free radical scavenging ability of antioxidant agents10. Crude polysaccharides could inhibit free radicals to become a stable molecule by donating electrons or hydrogen atom to the free radical21. The regression equation of DPPH radical scavenging activity was obtained by fitting the quadratic polynomial equation with the data. The effect of extraction on the IC50 DPPH scavenging activity is shown in Table 1. It was found that only extraction time (X3) had significantly (p ≤ 0.05) affected the IC50 value of DPPH scavenging activity. The interaction effects of extraction time (X3) with ratio of KUM-DB to water (X1) and with extraction temperature (X2) on the IC50 DPPH scavenging activity are also shown in Fig. 1e,f, respectively. As shown in Fig. 1f, the extraction time had a positive effect on the IC50 DPPH scavenging activity. These results showed that the lower IC50 DPPH value was observed when extraction time was shorter than 30 min. The lower IC50 DPPH value corresponds to a higher potential to inhibit free radicals22. When extracting for long time, the IC50 DPPH value increased. However, the ratio of KUM-DB to water and extraction temperature had no significant (p > 0.05) effect on the IC50 DPPH value (Fig. 1d)

optimization of ultrasonic-assisted extraction of

The ABTS assay is often used to estimating total antioxidant power7. From the regression equation, it was found that the extraction time (X3) was the most significant factor affecting the IC50 value of ABTS as shown in Table 2. The extraction time had a negative effect on the antioxidant activity. The IC50 value of ABTS increased when extracted for longer times. The higher value of IC50 ABTS corresponds to lower activity. Therefore, the greatest IC50 value of ABTS scavenging activity of crude polysaccharides was found when the extraction time was shorter than 30 min (Fig. 1h,i). The mechanism to explain this, is that the plant cell wall was disrupted by the collapse of cavitation bubbles, which were near a cell walls which increased the release of the crude polysaccharide. With longer extraction times, the polysaccharides were noticeably degraded to shorter chains and some functional groups were cut23. Therefore, using the shorter extraction time is potentially beneficial for the inhibition of free radicals. The ratio of KUM-DB to water and extraction temperature had no significant (p > 0.05) effect on the IC50 value for ABTS radical scavenging activity (Fig. 1g)

The ferric-reducing activity power (FRAP) assay is another assay to evaluate the antioxidant activity of crude polysaccharides. This method measures the ability of a sample to reduce the TPTZ-Fe(III) complex to the TPTZ-Fe(II) complex24. The crude polysaccharides were in the range of 166–200 µM Fe2+/g (Table 1). The regression equations with FRAP were obtained by fitting the quadratic polynomial equation with the data, as shown in Table 2. The results showed that extraction time was a negative factor on the FRAP of crude polysaccharides. The higher values of FRAP correspond to higher activity. The tri-dimensional plot of the interaction between extraction time (X3) with the ratio of KUM-DB to water (X1) and with extraction temperature (X2) showed the quadratic effect on FRAP of crude polysaccharides are shown in Fig. 1j–1, respectively. Figure 1k shows that the FRAP value increased with shorter extraction times. The similar results of the interaction between extraction time (X3) and extraction temperature (X2) are presented in Fig. 1

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