Separation Science and Technology ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lsst20 Subcritical Ethanol-Water and ionic liquid extraction systems coupled with multi-frequency ultrasound in the extraction and purification of polysaccharides Otu Phyllis Naa Yarley, Azumah Bright Kojo, Telfer Felix Adom, Cunshan Zhou, Xiaojie Yu, Agyapong Henrietta, Oklu Matthew Makafui, Arhin Reuben Essel & Osae Richard To cite this article: Otu Phyllis Naa Yarley, Azumah Bright Kojo, Telfer Felix Adom, Cunshan Zhou, Xiaojie Yu, Agyapong Henrietta, Oklu Matthew Makafui, Arhin Reuben Essel & Osae Richard (2021): Subcritical Ethanol-Water and ionic liquid extraction systems coupled with multi- frequency ultrasound in the extraction and purification of polysaccharides, Separation Science and Technology, DOI: 10.1080/01496395.2021.1987470 To link to this article: https://doi.org/10.1080/01496395.2021.1987470 Published online: 12 Oct 2021. Submit your article to this journal Article views: 48 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=lsst20 SEPARATION SCIENCE AND TECHNOLOGY https://doi.org/10.1080/01496395.2021.1987470 Subcritical Ethanol-Water and ionic liquid extraction systems coupled with multi-frequency ultrasound in the extraction and purification of polysaccharides Otu Phyllis Naa Yarley a,b, Azumah Bright Kojob, Telfer Felix Adomc, Cunshan Zhou a, Xiaojie Yua, Agyapong Henriettab, Oklu Matthew Makafuib, Arhin Reuben Esselb, and Osae Richardd aSchool of Food and Biological Engineering, Jiangsu University, Zhenjiang, People’s Republic of China; bFaculty of Applied Sciences, Department of Science Laboratory Technology, Accra Technical University, Accra, Ghana; cSchool of Engineering, Department of Food Process Engineering, University of Ghana, Legon, Ghana; dSchool of Applied Science and Technology, Department of Food and Postharvest Technology, Cape Coast Technical University, Cape Coast, Ghana ABSTRACT ARTICLE HISTORY This study obtained crude sorghum leaf sheath polysaccharide (39.99% wet matter (wm)) by Received 7 July 2021 subcritical ethanol-water (40% v/v) extraction (180°C, 40 min). The subcritical extraction solution Accepted 24 September 2021 was transformed into an ionic liquid aqueous two-phase extraction system and subsequently KEYWORDS coupled with ultrasound extraction to obtain partially purified polysaccharides (PPP). PPP yields Sorghum bicolor (L.); of 20.89%, 27.38%, and 36.49% (wm) were obtained using 60 kHz, 20/60 kHz, and 20/40/60 kHz viscoelasticity; particle size ultrasound frequencies, respectively. Polysaccharide functional groups such as hydroxyl, aldehyde, distribution; molecular and amide were detected using Fourier Transform Infrared Spectroscopy (FT-IR). Amylose contents weight; scanning electron of 15%, 18%, and 25% were obtained for PPP under single, dual, and tri-frequencies, respectively. microscopy Amylose contents were associated with aggregation of PPP particles sizes after heat exposure (70°C for 1 h 50 min). Triple-frequency extracted polysaccharides with the highest uronic acid (1.51%) and polyphenolic (27.79%) contents had an IC50 of 1.37 mg/mL in an in-vitro hydroxyl scavenging activity assay. Three interesting co-extracted bioactive phytochemicals; 2-amino-5[(2-carboxy) vinyl]-Imidazole, N-[4-bromo-n butyl]-2-Piperidinone, and 3-Trifluoroacetyl Pentadecane were detected. The PPP extract showed antioxidant activity and contained phytochemicals with poten- tial antimicrobial and antiviral activities, and thus may be useful in food, nutraceutical, and pharmaceutical applications. Introduction temperature[6]. This is due to the reduction in strength Sorghum bicolor (sorghum) is a grass species that origi- of its intermolecular forces allowing better matrix parti- nates from Northeastern Africa. It is mainly cultivated to cle penetration. Also, at elevated temperatures, surface obtain its grain for food, animal feed, and ethanol tension decreases allowing an excellent coat of feedstock production.[1] Nutritional analysis of the alcohol extract by solvent. This leads to an increase in the rate of of forage sorghum or leaf sheath showed the presence of extraction. Fortunately, subcritical water extraction carbohydrates (75.39 g), proteins (4.87 g) and dietary operates at such elevated temperatures (100–374°C) fiber (50.30 g).[2] Research has also shown that con- and pressure, high enough to maintain the liquid [7] sumption of diets prepared with the sorghum leaf sheath state. However, water used as a solvent in this form provides natural antioxidants and essential fatty acids of extraction has high polarity. The ethyl (C2H5) group that can fight cardiovascular-related diseases.[3] of ethanol is nonpolar and can therefore serve as an Bioactive polysaccharides present in such natural adjunct in water to achieve a less polar medium. This sources are responsible for antioxidation and other bio- can facilitate the easy dissolution of nonpolar substances logical activities.[4] Thus, novel extraction techniques to (water-insoluble polysaccharides). However, polysac- increase the extraction yield and purity of such bioactive charides solubility in ethanol-water solutions was compounds have become a paramount interest to found to decrease at 80% ethanol content. [8] In previous researchers. work, a ratio of 40%: 60% (v/v) ethanol-water used in the For a good extraction yield of moderately polar and formation of an ionic liquid aqueous two-phase system nonpolar targets, a less polar medium driven by elevated (ILATPS) was established to be the most appropriate temperature is required.[5] The viscosity of the extrac- volume ratio for remarkable extraction yield of sorghum tion solvents decreases with an increase in leaf sheath polysaccharides. [9] Thus, this work employed CONTACT Cunshan Zhou cunshanzhou@163.com School of Food and Biological Engineering, Jiangsu University, Zhenjiang, People’s Republic of China © 2021 Taylor & Francis Group, LLC 2 O. P. NAA YARLEY ET AL. 40% ethanol as an adjunct in the water for subcritical rheometer, respectively; and 4) investigate the antioxi- extraction of crude polysaccharides from sorghum leaf dant activity and phytochemical constituents of the sheath. polysaccharides extract. Ultrasound sonication can disrupt cell wall structure via cavitaton and thus accelerate the diffusion of cell content through membranes.[10] Ultrasound generates Materials and methods cavitation bubbles through the propagation and interac- Sample preparation tion of ultrasound pressure. When cavitation bubbles collapse, microturbulence, perturbation, liquid circula- Dried S. bicolor leaf sheath was collected from Ghana. The tion, and rotational flow structure occur and it is known leaf sheath was pulverized into powder, sieved, and as eddy.[11] Interparticle collision together with eddies defatted using the method described by Otu et al., [17] ensures a dramatic eddy diffusion and internal diffusion (2018). and also speeds up mass transfer of solvent from con- tinuous phase into plant cells. Furthermore, a quick Chemicals and reagents moving stream of liquid passes through the cavity at the surface whenever there is a collapse of the bubble Ionic liquid, 1-octyl-3-methylimidazolium chloride [C8 near the liquid-solid interface. Particles of the surfaces mim]Cl, potassium carbonate (K2CO3), ethanol, metha- are impacted and cell walls are disrupted which pro- nol, sulfuric acid (H2SO4), phenol, glucose, coomassie motes the release of intracellular components into G-250, bovine serum albumin (BSA), phosphoric acid solvents.[12–15] Ultrasound extraction has been estab- (H3PO4), potassium bromide (KBr), gold palladium, lished to be an alternative to conventional extraction Fe(II) sulfate (FeSO4), salicyclic acid, hydrogen peroxide methods, and is effective even at low temperatures. It (H2O2), ABTS [2,2ʹ-azino-bis (3-ethylbenzothiazoline- has the advantage of avoiding the use of organic solvent 6-sulfonic acid)], potassium peroxidate, and carbazole. and has reduced extraction time[16] All chemicals were purchased from Sigma (St. Louis, As described by Otu et al. (2018), [17] crude polysac- MO, USA). charides water extract was separated, freeze dried and subsequently incorporated into an Ionic Liquid Aqueous Subcritical ethanol-water extraction (SEWE) Two-Phase System (ILATPS) coupled with ultrasound treatment to partially purify extract within a short time. Research has demonstrated that ethanol combined with The novelty of this study was the use of ethanol-water in subcritical water treatment can improve the recovery a subcritical extraction and shortening the processing efficiency of by-products from raw sample material.[19] time by skipping protocol for the precipitation of crude Therefore, 40% (v/v) of the ethanol-water solution was polysaccharides extract. Ionic liquid and salt were then prepared. Subcritical ethanol-water extraction was simi- introduced into the extraction solution to produce an larly carried out as described by Yabalak and Ahmet ethanol-adjunct ionic liquid aqueous-two phase system. (2012) with little modification.[20] To a 25 mL Teflon This system with its crude polysaccharides content was cup, 120 mg of dried sample powder and 14.4 mL of the coupled with ultrasound treatment to produce partially prepared ethanol-water solution were added (Fig. 1). purified polysaccharides with much higher yield within The Teflon cup was then fitted into a pressure cell, a short time. screwed tight, and placed into a hot-air oven. Crude Research has established that the method of extrac- polysaccharides from sample powder were then tion has a direct influence on the amount and type of extracted using designed single factors; temperature biomolecules obtained.[18] Therefore, the specific objec- (100, 120, 140, 160,180, 200°C) and time (10, 20, 30, tives of this work were: 1) determine the effect of sub- 40, 50, 60 min). The polysaccharide content was critical ethanol-water extraction system on yield of measured using the phenol-sulfuric method. polysaccharides; 2) determine the effect of subcritical and frequency-varied ultrasound-assisted extraction sys- tem on yield of partially purified polysaccharides; 3) Ionic liquid aqueous two-phase system (ILATPS) characterize the particle size, morphological structure, Three (3) sets of sorghum leaf sheath crude polysacchar- primary structural changes, molecular weight, and rheo- ides solutions were obtained using subcritical ethanol- logical properties of partially purified polysaccharides water extraction methodology as described above (sec- using dynamic light scattering, scanning electron micro- tion 2.2). The polysaccharide solutions were poured scopy, Fourier transform-infrared spectroscopy, high- from the Teflon cups into 50 mL plastic tubes. Ionic performance size-exclusion chromatography and liquid, [C8mim]Cl (10.5 g), K2CO3 salt (5.1 g) were SEPARATION SCIENCE AND TECHNOLOGY 3 Figure 1. Schematic diagram of subcritical ethanol-water & biphasic ultrasound extraction of sorghum leaf sheath polysaccharides. Experimental conditions; 40% (v/v) ethanol solution at 180 °C for 40 mins, and ultrasound treatment with 60 or 20/60 or 20/40/60 KHz, 120 W/L for 30 min at 35 °C. added and vortexed to complete a 30 mL ILATPS Characterization formation. Each sample set was exposed to a different ultrasound frequency and therefore assigned the Preliminary characterization names; subcritical ethanol-water extraction – mono The three sets of partially purified samples (SEWE-M, (SEWE-M), subcritical ethanol-water – dual (SEWE- SEWE-D, and SEWE-T) were desalinated using D), and subcritical ethanol-water – triple (SEWE-T). a dialysis membrane and thereafter freeze-dried. The Partially purified polysaccharides were finally carbohydrate and protein content was determined obtained by treating the formed ILATPS with ultra- using the phenol-sulfuric [22] and Bradford method .[23] sound waves. Purifed samples were desalinated using The apparent amylose content was measured using the a recyclable dialysis membrane (D44 mm, MWCO iodometric method. [24] The phenolic contents were [25] 8000–14000) enabling filtration of the salt and IL. It measured as described by Kumar et al. (2008). The was then freeze dried for further analysis. Carbazole-sulfuric acid method was adopted for the measurement of uronic acid in the samples.[26] All experiments were conducted in triplicate. Single-factor design for ultrasound-assisted purification Particle size (dynamic light scattering, DLS) The hydrodynamic diameters of the three polysacchar- As described by Yan et al. (2018), [21] a triple-band ultra- ides samples (SEWE-M, SEWE-D, and SEWE-T) were sound water bath (KQ-300 DE, Kunshan Ultrasonic measured. Using the dynamic light scattering method as Instruments Co., Ltd., China) was used in this work. described by Ren et al. (2015).[27] Briefly, the hydrody- Three major parameters, extraction frequencies namic diameters of the partially purified samples in (Mono – 20, 40, 60 kHz, Dual – 20/40, 40/60, 20/ water (2 mg/mL) were examined using dynamic scatter- 60 KHz, and Tri – 20/40/60 kHz), extraction temperature ing on a Litesizer 500 (Anton Paar, UK). The solutions (25, 30, 35, 40, and 45°C), and extraction time (15, 20, 25, containing the samples were filtered through a 0.45 μm 30, 35, and 40 min) were explored. A power of 240 W and syringe filter. The solutions were measured initially at power density of 120 W/L was used in this work. room temperature (25°C) and subsequently heated at 4 O. P. NAA YARLEY ET AL. � � 70°C for 1 h 50 min and then cooled down to a tempera- A1 A2 ture of 25°C and again measured. Each sample was ScavengingRate ¼ 1 x100% (1) A0 measured 10 times under each condition. Where A0 is the absorbance of the control group (without polysaccharides), A1 is the absorbance of the test group, Scanning electron microscopy (SEM) A2 is the absorbance of the background group. The Scanning Electron Microscope (S-3400 N, Tokyo, Japan) was used to observe the morphological charac- teristics of the three polysaccharide samples. Each sam- IC50 ¼ %Max:inhibition ple was coated with a conductive layer of gold- 50%xð%Max:inhibition %Min:inhibitionÞ palladium. (2) Fourier transform-infrared (FT-IR) spectroscopy ABTS radical scavenging assay. The ABTS radical The infra-red spectra of the samples were determined scavenging assay was measured using the method using the FT-IR spectrophotometer (Nicolet, described by Zhou et al. (2011)[29] with slight modifica- Nexusn670) as described by Otu et al. (2018).[17] tion. Briefly, ABTS (50 mL, 7 mM) was mixed with Briefly, IR spectra were determined between an absor- 140 mM potassium peroxydisulfate (890 μL), then kept bance mode of (4000– 400 cm−1) and a resolution of in the dark at room temperature for 12–16 h before use. (4 cm−1). The samples were prepared in a variety of concentra- tions (5–30 mg/mL). The polysaccharides solution Molecular weight (HPSEC) (0.2 mL) was added to the ABTS·+ solution (5 mL). High-performance size-exclusion chromatography was The absorbance of the mixture was measured at used to determine the molecular weight of samples as 734 nm after holding at room temperature for 6 min. described by Otu et al. (2018).[17] All measurements were made in triplicate. The ABTS radical scavenging rate was calculated using the following formula in Eq. (3). Again, the IC50 Rheological properties was calculated using Eq. (2). The steady shear and oscillatory tests of samples at � � a concentration of 5 mg/mL were conducted with A AScavengingRate ¼ 1 1 2 x100% (3) a DHR-1 rheometer (TA Instruments, Surrey, UK) A0 using a cone – and – plate geometry. Conditions used Where A0 is the absorbance of the control group (without were as described by Otu et al. (2018).[17] polysaccharides), A1 is the absorbance of the test group, A2 is the absorbance of the background group. Antioxidant activity in-vitro Hydroxyl radical scavenging assay. The hydroxyl radi- Phytochemical constituent analysis of extracted cal assay was performed as described by Jen et al. polysaccharides (GC-MS/MS) (1998).[28] Briefly, samples were dissolved in distilled Polysaccharides extract was dissolved in methanol water (5– 30 mg/mL). The sample solution (1.0 mL) (1 mL) and filtered through a 0.2 µm syringe filter and was mixed with FeSO4 (9 mM, 1.0 mL) and 9 mM carefully kept at 4°C for 24 h before analysis using the salicylic acid solution (1 mL, 50% ethanol). Then, GC-MS/MS .[30] To identify the compounds observed, 8.8 mM H2O2 (1.0 mL) was added to start the reaction. the mass spectra of the analytes were screened against The mixture was kept in a water bath at 37°C for 1 h. The the NIST mass spectral database. The retention indices background was mixed as described above except 50% were also compared either with literature values or ethanol (1.0 mL) was used in place of the salicylic acid authentic compounds.[31,32] solution. For the control, distilled water (1.0 mL) was Phytochemical constituents were identified using substituted for the polysaccharide solution. After warm- GC-MS/MS (Agilent 789A) equipped with a DB-5 MS ing in a water bath, the absorbance of the mixture was column (30 m × 0.25 mm i.d., 0.25 um film thickness, measured at 510 nm. All measurement was done in J&W Scientific, Folsom, CA).[33] Helium was used as the triplicate. carrier gas at the rate of 1.0 mL/min. The effluent of the The hydroxyl radical scavenging rate was calculated GC column was introduced directly into the source of using the following formula in Eq. (1). The half-maximal the MS via a transfer line (250°C). The ionization voltage inhibitory concentration, IC50, was calculated using was 70 eV and the ion source temperature was 230°C. Eq. (2). Scan range was 41– 450 amu. SEPARATION SCIENCE AND TECHNOLOGY 5 Statistical analysis (60 kHz), dual-frequency (20/60 kHz), and tri-frequency All experiments were conducted in triplicate and means (20/40/60 kHz). Different ultrasound temperatures (25, determined. The statistical difference in means was 30, 35, 40, and 45°C) were used for the extraction of determined using one-way ANOVA (OriginPro 2015). partially purified polysaccharides (PPP) under each Statistical difference between means was considered sig- ultrasound extraction frequency (Table 1). The highest nificant if (p ˂ 0.05). polysaccharide extraction yields of 27.19%, 30.29%, and 42.69%, (p < .05) were recorded at 35°C temperature for Results and discussion mono, dual, and tri extraction frequency, respectively. The highest extraction yield for Aloe vera polysaccharide Single-factor design for subcritical adjunct water was attained at room temperature (25°C) using extraction ILATPS[37] . It was explained that at a certain extraction Effect of temperature on polysaccharides extraction temperature, water is transferred from the top-to- At a fixed extraction time of 30 min and different extrac- bottom phase of ILATPS. The transferred water tion temperatures of 100, 120, 140, 160,180, and 200°C, decreases the salting-out effect at the bottom phase and polysaccharides from dried sample powder were subsequently decreases polysaccharides yield. Again, extracted (Fig. 2a). Extraction at 180°C gave the highest transient cavitation filled bubbles undergoes irregular yield of polysaccharides (44.96% wet matter basis, p < oscillation and implodes, producing high local increase .05). A significant decrease (p < .05) was recorded at of temperature and pressure that in turn disintegrate the [38] 200°C. Similarly, the optimum temperature of 140°C in cells for mass transfer. Ultrasound temperature a subcritical extraction attained a 12.28% (dry matter directly impacts the formation of transient cavitaional basis) pectic polysaccharides in a 10%(v/v) ethanol- bubbles. Nagalingam and Yeo (2018) [39] showed that at water.[34] Though higher yield can be obtained at 10°C, bubble population under the horn of an ultra- a much higher temperature, thermal degradation of sound was very low. Hence, very less mass loss values polysaccharides has been found to occur at the third were recorded. At 30°C and 50°C, high bubble popula- stage of a mass loss experiment.[35] The temperature tion was observed, which in turn increased the mass loss 180°C was therefore selected for this work. during machining. At 70°C and 90°C, very higher bubble population was observed, but it was noted that the Effect of time on polysaccharides extraction bubbles did not implode but instead settled near the The extraction temperature was adjusted to 180°C and horn and on the sides of the workpiece and the test polysaccharides were extracted at different times (10, 20, chamber. This may be the reason for the observed 30, 40, 50, 60 min) (Fig. 2b). The extraction time of reduction in polysaccharides yield for experimental tem- 40 min gave the highest polysaccharides yield of 42.60% peratures beyond 35°C. Thus, the temperature 35°C was (wet matter basis) (p < .05). A significant decrease (p < selected for this work. .05) in polysaccharides yield was recorded at 50 and 60 min. Within 43.65 min at 210°C, 25.1% (wet matter Effect of ultrasound frequencies on polysaccharides basis) of Grifola frondosa polysaccharides were obtained extraction by subcritical water extraction .[36] The much higher The ultrasound irradiation temperature and time were extraction yield in this work within a similar extraction adjusted to 35°C and 30 mins, respectively. Variation of time may be attributed to the nonpolar effect of ethanol frequencies included: mono-frequency (20, 40, 60 kHz) on the extraction water medium and the ultrasound and dual-frequency (20/60, 20/40, 40/60 kHz) and triple- treatment. An extraction time of 40 min was thus selected frequency (20/40/60 kHz). Triple frequency gave the best for this work. The yield of crude polysaccharides extract results. The highest polysaccharide extraction yields of obtained based on selected temperature (180°C) and time 27.71%, 29.39%, and 37.52% (p < .05) were recorded for (40 min) has been presented in (Fig. 3). mono – 60 kHz, dual – 20/60 KHz, and triple – 20/40/ 60 kHz ultrasound frequencies, respectively (Table 1). The Single-factor design for ultrasound-assisted results agree with the report that multi-frequency gives a much higher extraction yield than single polysaccharides purification frequency.[40,41] In an experimental report, Guo and Zhu Effect of ultrasound temperature on polysaccharides (2017)[42] observed that, as ultrasound frequency extraction increased, maximum radius (Rmax) of cavitation bubble Ultrasound time was fixed at 30 min throughout the was found to have slightly reduced and oscillation interval experiment. In a stepwise manner, ultrasound extraction was shortened. This implied that completion of bubble frequencies were adjusted to obtain a mono-frequency growth was made difficult whilst bubble collapse became 6 O. P. NAA YARLEY ET AL. Figure 2. Effect of temperature a) for 30 min under (100, 120, 140, 160,180, and 200 °C) and time b) at 180 °C under (10, 20, 30, 40, 50, 60 min) on the yield (Mean ± SEM) of sorghum leaf sheath crude polysaccharides under subcritical ethanol-water extraction. Means with different letters are significantly different (Turkey’s HSD, p < .05). an easy occurance at a high frequency. Therefore, Effect of ultrasound time on polysaccharides frequent cavitation bubble collapse, possibly aided by extraction multi-frequency obsviously increased the rate of mass Adjustment of ultrasound operational parameters to transfer. Thus, the selection of highest frequencies in obtain a temperature of 30°C was made. Secondly, step- this work (60 kHz, 20/60 KHz, and 20/40/60 KHz) for wise adjustments to obtain (i) 60 KHz mono frequency, mono, dual, and triple ultrasound frequencies, (ii) 20/60 KHz dual-frequency, and (iii) 20/40/60 kHz respectively. triple frequency were made. Varying ultrasound time SEPARATION SCIENCE AND TECHNOLOGY 7 Figure 3. Yield (mean ± SEM) of sorghum leaf sheath polysaccharides (crude and partially purified) under selected best conditions for subcritical (180 °C and 40 min) and ultrasound extractions (60 kHz or 20/60 kHz or 20/40/60 kHz, 35 °C, time 30 min, 120 W/L) respectively. Means with different letters are significantly different (Turkey’s HSD, p < .05). (15, 20, 25, 30, 35, and 40 min) extraction of the purified ultrasound extraction frequency (Table 1). In a work polysaccharides under each ultrasound frequency were by Otu et al. (2018), [17] a notable yield of sorghum leaf carried out (Table 1). The highest polysaccharide extrac- sheath polysaccharides was similarly achieved by cou- tion yield of 26.03%, 29.13%, and 41.52% (p < .05) were pling dual-frequency ultrasound and ILATPS within recorded at 30 min time for mono, dual, and tri 30 min. Therefore, an extraction time of 30 min was selected for this work. The yield of purified polysaccharides extracted based Table 1. Effect of single factors temperature, time, and frequency on selected ultrasound frequencies (mono - 60 kHz, dual on yield of Sorghum bicolor leaf polysaccharides under ultra- - 20/60 kHz, and triple - 20/40/60 kHz), temperature sound extractions under constant experimental conditions of (35°C), and time (30 min) under this part of work has (30 min, 30°C, and 60 or 20/60 or 20/40/60 KHz). also been presented in (Fig. 3). Polysaccharides Yield (%) Temperature (oC) Mono frequency Dual frequency Triple frequency 25 20.99 ± 0.30 22.28 ± 0.22 31.19 ± 0.26 Preliminary characterization 30 23.19 ± 0.21 22.54 ± 0.22 31.97 ± 0.26 35 27.19 ± 0.23 30.29 ± 0.26 42.69 ± 0.27 40 20.86 ± 0.25 25.00 ± 0.23 38.42 ± 0.31 The total carbohydrates, proteins, apparent amylose, 45 18.02 ± 0.21 22.54 ± 0.21 38.17 ± 0.30 uronic acid, and polyphenolic content of the lyophilized Polysaccharides Yield (%) polysaccharides have been presented in Table 2. Time (mins) Mono frequency Dual frequency Tri frequency 15 20.73 ± 0.23 17.76 ± 0.30 31.06 ± 0.26 Swamy and Narayana (2001)[43] discovered that the 20 24.09 ± 0.24 19.18 ± 0.21 36.23 ± 0.26 mechanism of combining two frequencies of ultrasound 25 24.74 ± 0.23 20.60 ± 0.23 37.91 ± 0.27 30 26.03 ±0.25 29.13 ± 0.26 41.52 ± 0.31 gave better efficiency when compared to single- 35 24.35 ± 0.21 27.58 ± 0.26 32.61 ± 0.30 frequency ultrasound sonication. Following this discov- 40 23.70 ± 0.22 24.09 ± 0.28 30.55 ± 0.21 [44] Polysaccharides Yield (%) ery, Manickam et al. (2014) found that cavitation Frequency (KHz) Mono frequency Dual frequency Tri frequency effects were higher for the triple frequency operation 20 17.63 ± 0.25 - - 40 20.09 ± 0.21 - - than with dual and single frequency. The mode of opera- 60 27.71 ± 0.22 - - tion of the mono frequency in this work allowed cavita- 20/40 - 19.05 ± 0.30 - tion bubble formation, growth, and collapse at 60 kHz 40/60 - 17.63 ± 0.21 - 20/60 - 29.39 ± 0.23 - frequency continuously without pulse time. The dual 20/40/60 - - 37.52 ± 0.21 and the triple frequency on the hand started with the 8 O. P. NAA YARLEY ET AL. Table 2. Preliminary characterization of SEWE-M, SEWE-D, and carbohydrate content increased, the associated proteins SEWE-T polysaccharides. were effectively transferred into the top phase. This Composition SEWE-M SEWE-D SEWE-T explains why the protein content of extracted polysac- Carbohydrate (%) 60.17 ± 0.15 69.21 ± 0.17 80.83 ± 0.16 Protein (%) 0.085 ± 0.16 0.050 ± 0.14 0.034 ± 0.14 charides reduced with an increase in carbohydrate Uronic acid (%) 1.04 ± 0.12 1.12 ± 0.20 1.51 ± 0.22 content. Amylose (%) 15.35 ± 0.16 18.67 ± 0.29 25.27 ± 0.19 Oxidation at the carbon-6 of sugars produce uronic Polyphenol (%) 24.23 ± 0.12 26.22 ± 0.06 27.79 ± 0.08 acids.[46] This may explain the observed increase in the uronic acid content of samples as the total carbohydrates lowest frequency (20 kHz) and switched to a much higher increased. That is, as the availability of carbohydrates frequency in the next cycle without pulse time. This rise, possible oxidation of carbohydrate into uronic acid effected continuous production of low to high level tur- also increases. Similarly, apparent amylose content was bulent cavitation throughout the cycles and may have found to have increased with an increase in carbohy- caused better disruption of plant cells for mass transfer. drate content. A report by Kumari et al. (2007), [47] This mechanism explains the observed pattern of increase indicated an increase in apparent amylose content with in total carbohydrates and polyphenolic recorded values increased resistant starch content. The higher cavita- for single, dual, and triple frequency ultrasound-extracted tional effect may have aided in the extraction of resistant polysaccharides in this work (Table 2). starch and therefore the increase in amylose content. There was a close content correlation observed between the primary metabolite (carbohydrates) and sec- Fourier transform-infrared (FT-IR) ondary metabolites (polyphenolic). Primary and second- ary metabolites are usually not thoroughly separated The FT-IR analysis of all samples in this work has been during extraction. This may explain the observed close presented in (Fig. 4). The objective was to investigate content correlation. Total proteins associated with any possible primary structural changes. A peak was extracted polysaccharides were lowest with dual and triple observed at 3,196 cm−1 which is known to be the hydro- frequency ultrasound extraction. Sugar molecules have xyl stretching vibration.[48] This first peak is usually been found to possess OH groups that can destroy the associated with carbohydrate compounds confirming natural hydrogen bond network of water. Eventually, new the extracted samples to be carbohydrate. A second hydrogen bond networks between water molecules and peak was also observed at 2989 cm−1 which is the alkyl sugars are formed. This reduces “free” water molecules in C-H vibration. Its intensity relative to other peaks indi- the bottom phase of ILATPS, and therefore forces pro- cates the size of the alkyl group. teins to be transferred to the top phase[45] . As the Figure 4. FT-IR spectra of SEWE-M, SEWE-D, and SEWE-T polysaccharides. SEPARATION SCIENCE AND TECHNOLOGY 9 C-O stretching vibration[50] was observed. A fifth peak was found at the band 882 cm−1 also known as β anomer[51] denotes the presence of a dehydrated form of a salt or organic derivative. Finally, a weak absorption band between 600 and 703 cm−1 was attributed to O-H stretching vibrations.[52,53] Particle size (dynamic light scattering, DLS) The particle size distribution of the three polysacchar- ides SEWE-M, SEWE-D, and SEWE-T in aqueous solu- tions has been displayed in (Fig. 5). When the ultrasound single frequency extracted polysaccharide SEWE-M was dispersed in water, the particle size dis- tribution with diameters that ranged (94.30– 1754.44 nm) and main peak diameter at (919.99 nm) was observed (Fig. 5a). Holding the aqueous solution under a temperature of 70°C for 1 h 50 min, the particle size distribution increased to (233.92– 4535.64 nm) and displayed peak diameter at (2329.46 nm). A similar trend of particle size distribution was observed for the multi-frequency extracted polysac- charides SEWE-D and SEWE-T. Dispersing these two samples in water, SEWE-D recorded a particle size distribution with diameters in the range of 106.34– 1585.78 nm and a main peak diameter at 625.02 nm (Fig. 5b). The sample SEWE-T recorded a particle size distribution with diameters in the range 167.54– 4946.67 nm and peaked at 2936.99 nm (Fig. 5c). Application of temperature of 70°C for 1 h 50 min similarly increased particle size distribution of SEWE- D and SEWE-T. The sample SEWE-D recorded a particle size distribution with diameters in the range 362.67– 5806.10 nm and a main peak diameter at 2611.65 nm. The sample SEWE-T recorded a parti- cle size distribution with diameters in the range 319.29– 6552.50 nm and peaked at 3379.21 nm. The aggregation of particles observed after heat expo- sure can be attributed to retrogradation which refers to the reformation of a more ordered structure.[54] It is affected by the presence of sugars or other hydroxyl-containing molecules.[55] It is known to mostly favor amylose chains.[56] The percentage of amylose in polysaccharides increased with an increase in carbohydrate content (Table 2). Therefore, more intense retrogradation or aggregation was observed for multi-frequency extracted samples. Figure 5. Particle size distribution of polysaccharides: a) SEWE-M, b) SEWE-D c) SEWE-T. SEM and molecular weight of polysaccharides The third peak observed at 1662 cm−1 is referred to as The morphological structure of polysaccharides SEWE-M, Amide I stretching which depicts the presence of some SEWE-D, and SEWE-T have been presented in (Fig. 6). soluble proteins in polysaccharides.[49] Another peak The sample SEWE-M presented a grain morphological ranged at the band 1,042– 1,064 cm−1 known to be structure that was round and loosely packed. The multi- 10 O. P. NAA YARLEY ET AL. Figure 6. SEM of extracted polysaccharides under different ultrasound frequencies. Table 3. Molecular weight of Sorghum bicolor leaf However, there were clear differences in viscosity. The polysaccharides. sample SEWE-M with smaller particle size distribution Molecular Weight (g/mol) recorded the highest G” values (Fig. 7b). The samples, Samples Mn Mp Mw Mz Mw/Mn SEWE-D and SEWE-T with much larger particle size SEWE- M 1,160,000 1,285,000 1,202,000 1,243,000 1.037 SEWE- D 48,500 39,720 55,140 70,860 1.137 distributions, recorded lowest G” values. This indicates SEWE- T 2,688 2,090 3,133 4,671 1.166 that SEWE-M was the most viscous. In a similar report by Afoakwa et al. (2008), [57] an increase in particle size distribution gave rise to a significant reduction in viscosity. frequency extracted samples SEWE-D and SEWE-T pre- All three polysaccharide samples, however, showed sented a densely packed morphology. The multi- dominance in elasticity since tan δ was (< 1) throughout frequency ultrasound extracted polysaccharides showed the experiment (Fig. 7c). The samples SEWE-M, SEWE- a dramatic deformation of cells compared to the mono- D, and SEWE-T displayed a near-Newtonian flow beha- frequency extract (Fig. 6). The triple-frequency ultrasound vior, with constant records throughout the shear rate treatment showed the most deformed and compact cells. experiment (Fig. 7d). Molecular weight reduced with the use of multiple frequencies of ultrasound extraction (Table 3). This can be ascribed to the vigorous cavitational effect of multiple Antioxidant activity ultrasound frequencies on the sizes of extracted poly- saccharides. The mono-frequency ultrasound extracts Scavenging effect on hydroxyl radical presented the highest molecular weight (1,202,000 g/ The hydroxyl scavenging activity of ascorbic acid and mol) due to the use of cavitation with lowest intensity. the three polysaccharides samples within the final calcu- lated concentration range 1.25–7.5 mg/mL have been Rheological properties displayed in (Fig. 8). The sample SAW-T displayed a very close scavenging rate as ascorbic acid and Viscoelasticity and steady shear flow properties of all recorded the same value (79%) at the final concentration three polysaccharides have been displayed (Fig. 7). The of 7.5 mg/mL. The IC50 of SEWE-M, SEWE-D, SEWE- three polysaccharides samples proved to be elastic in T, and ascorbic acid were found to be 2.25, 1.87, 1.37, nature. As frequency increased from (1–10 Hz), G’ and 1.25 mg/mL, respectively. An interesting report by values increased consistently (Fig. 7a). Noda et al. (1997)[58] revealed a reduction in hydroxyl SEPARATION SCIENCE AND TECHNOLOGY 11 Figure 7. Rheological properties of SEWE- M, D and T polysaccharides: a) storage modulus G’ b) loss modulus G” c) tangent tan δ in dynamic frequency test d) steady shear flow curves. Figure 8. Scavenging effects of ascorbic acid and polysaccharides: a) Hydroxyl radical and b) ABTS radical. scavenging inhibition of various plant extracts after efficiency of triple frequency ultrasound gave rise to ascorbate oxidase treatment. The extraction technique low molecular weight polysaccharides. Low molecular used to obtain polysaccharides may therefore have weight polysaccharides are known to give rise to high a direct impact on the inhibition ability. The high Table 4. Phytochemical Constituents of Sorghum bicolor Leaf Sheath (SEWE- T). No. RT Name of Compound Molecular Molecular Weight Area Sum (%) 1 5.89 Hexadecane, 1-chloro C16H33Cl 260 3.83 2 6.82 Imidazole, 2-amino-5-[(2- carboxy)vinyl]- C6H7N3O2 153 10.87 3 7.97 3- Trifluoroacetoxypentadecane C17H31F3O2 324 5.57 4 8.62 3-Butoxy-1,1,1,7,7,7- hexamethyl-3,5,5- tris(trimethylsiloxy)tetrasiloxa C19H54O7Si7 591 7.05 5 11.28 2-Piperidinone, N-[4-bromo-nbutyl]- C9H16BrNO 234 9.49 6 13.51 Oxalic acid, allyl pentadecyl ester C20H36O4 340 9.95 7 29.60 Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13, 13-tetradecamethyl C14H44O6Si7 505 5.74 8 30.77 Tris(tertbutyldimethylsilyloxy)arsane C18H45AsO3Si3 468 7.06 12 O. P. NAA YARLEY ET AL. hydroxyl activity. Furthermore, phenolic content of the heat exposure. The triple frequency extracted polysaccharides polysaccharides in this work was observed to increase presented the most deformed and compact cells. Though the with increase in inhibition activity (Table 1) (Fig. 8a). polysaccharides displayed strong elasticity, the viscosity decreased with an increase in particle size distribution. The Scavenging effect on ABTS radical hydroxyl and ABTS radical scavenging ability of triple- The ABTS scavenging activity of ascorbic acid and the frequency ultrasound extracted polysaccharides was as strong three polysaccharides samples within the final calculated as ascorbic acid. Under ABTS radical scavenging rate analysis, concentration range of 0.19– 1.14 mg/mL have been both triple-frequency ultrasound extracted sample and ascorbic displayed in (Fig. 8b). Similar to the ascorbic acid acid recorded an IC50 of 0.19 mg/ml. Some of the co-extracted scavenging rate (99–100%), polysaccharides extracted phytochemical constituents, Imidazole 2-amino-5-[(2- car- in this work displayed 100% scavenging rate at all boxy)vinyl]-, 2-Piperidinone N-[4-bromo-n butyl]- and experimental concentrations. An IC50 of 0.19 mg/mL 3-Trifluoroacetyl Pentadecane are potentially bioactive. The was therefore recorded for ascorbic acid, SEWE-M, combined thermal and non-thermal extraction method, there- SEWE-D, and SEWE-T. Similarly, analysis of some fore, gave a high yield of bioactive polysaccharides that can be plant ethanolic extracts revealed higher antiradical and useful for the food, nutraceutical, and pharmaceutical industry. antioxidant activity with ABTS scavenging activity among other scavenging activities conducted. This was attributed to a greater quantity of phycobilin pigments Disclosure statement and phenolics extracted.[59] The high ABTS activity observed may be further attributed to the appreciable No conflict of interest. amount of uronic acid content of all samples. Higher uronic acid content has also been discovered to give rise to high ABTS activity .[60] Funding The authors are grateful for the support provided by the National Natural Science Foundation of China (32072174) Phytochemical constituents of extracted and Social Development of Jiangsu Province – General Polysaccharides (GC-MS/MS) Project (BE2020776). A total of 8 different phytochemical compounds were co- extracted with the triple frequency extracted polysaccharides and identified based on peak area, molecular weight, and ORCID molecular formula (Table 4). Bioactive compounds, 2-amino- Otu Phyllis Naa Yarley http://orcid.org/0000-0001-6042- 5-[(2- carboxy)vinyl]-Imidazole and N-[4-bromo-n butyl]- 7952 2-Piperidinone with peak areas of 10.87% and 9.49% at RT of Cunshan Zhou http://orcid.org/0000-0001-9119-3941 6.82 and 11.28, respectively, have antimicrobial potentials.[61,62] Again, 3-Trifluoroacetyl Pentadecane with a peak area of 5.57% at RT of 7.97 has been reported to have an anti-viral ability.[63] References Other phytochemicals identified were Hexadecane 1-chloro, [1] Harlan, J. R.; De Wet, J. M. A Simplified Classification of 3-Butoxy-1,1,1,7,7,7- hexamethyl-3,5,5- tris(trimethylsiloxy)tet- Cultivated Sorghum 1. Crop Sci. 1972, 12(2), 172–176. DOI: rasiloxane, Oxalic acid allyl pentadecyl ester, Heptasiloxane 10.2135/cropsci1972.0011183X001200020005x. 1,1,3,3,5,5,7,7,9,9,11,11,13, 13-tetradecamethyl, and Tris(tert [2] Okubena, O.; Makanjuola, S.; Ajonuma, L.; Dosunmu, A.; butyldimethylsilyloxy) arsane. Umukoro, S.; Erah, P. 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