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1. Weekly Report Muhammad Imran Prof. Yao-Tung Lin National Chung Hsing University, Department of Soil and Environmental Sciences April 30 , 2024, Taiwan 1

2. Preparing Blank and Standard Solutions Blank Solution The blank solution is prepared by mixing 1 mL of distilled water, 1 mL of 5% phenol, and 5 mL of concentrated sulfuric acid. Standard Solution A stock glucose solution of 100 μg/mL is diluted to create standard solutions in the range of 20, 40, 60, 80, and 100 μg/ mL. 1mL of each standard is combined with 1 mL of 5% phenol and 5 mL of sulfuric acid. Chemicals and Materials Glucose Phenol Sulfuric acid DI water Pipette Beakers Tubes (10mL) Spectrophotometer Lab Note 1 Page No # 321 Date: 2024/04/92

3. Spectrophotometric Measurement 1 Solution Preparation The blank and standard solutions are prepared according to the instructions. 2 Absorbance Measurement The absorbance of the standard solutions is measured at 490 nm using the spectrophotometer, with the blank solution as the reference. 3 Calibration Curve The absorbance values of the standards are plotted against their corresponding glucose concentrations to create the calibration curve.

4. Calibration Curve Blank 20 μg /mL 40 μg /mL 60 μg /mL 80 μg /mL 100 μg /mL Lab Note 1 Page No # 321 Date: 2024/04/92 4

5. Project Report Introduction Okra is a vegetable rich in bioactive compounds like polysaccharides, known for its medicinal properties and diverse applications in food and pharmaceutical industries. Okra polysaccharides, primarily composed of galactose, rhamnose, and galacturonic acid, exhibit antioxidant, lipid-lowering, and immune-modulating effects. These polysaccharides serve as natural stabilizers in food production and substitutes for traditional ingredients in fat-free biscuits. Polysaccharides, including those from okra, possess a range of beneficial properties like antioxidant, antitumor, and immune-stimulating activities, potentially preventing oxidative damage. 1.1 Okra, Polysaccharides, importance, extraction methods 1.2 Extraction methods, UAE based extraction. 1.3 ILs, DESs: Discusses the properties, applications 1.4 DES based plants extracts 1.5 DESs in Polysaccharide Extraction: Explores DESs' effectiveness in polysaccharide extraction 5

6. Project Report Introduction Polysaccharides, vital biomolecules, are found in various organisms and play crucial roles in cellular structures. Traditional polysaccharide extraction methods, like hot water extraction (HWE), are being enhanced by techniques such as ultrasonic and microwave assistance for improved efficiency. New extraction technologies offer alternatives to traditional methods, addressing issues like energy consumption and purification drawbacks. Ecologically friendly approaches, like enzyme-assisted extraction, are gaining attention for their potential to increase polysaccharide extraction rates and sustainability. 1.1 Okra, Polysaccharides, importance, extraction methods 1.2 Extraction methods, UAE based extraction. 1.3 ILs, DESs: Discusses the properties, applications 1.4 DES based plants extracts 1.5 DESs in Polysaccharide Extraction: Explores DESs' effectiveness in polysaccharide extraction 6

7. Project Report Introduction Deep eutectic solvents (DESs) are eco-friendly alternatives to ionic liquids, offering low vapor pressure and high stability. They are cost-effective and versatile, widely used for extracting biologically active compounds from natural sources. DESs show promise in green technologies, serving as efficient solvents for various applications including synthesis, catalysis, and phytochemical extraction. 1.1 Okra, Polysaccharides, importance, extraction methods 1.2 Extraction methods, UAE based extraction. 1.3 ILs, DESs: Discusses the properties, applications 1.4 DES based plants extracts 1.5 DESs in Polysaccharide Extraction: Explores DESs' effectiveness in polysaccharide extraction 7

8. Project Report Introduction Deep eutectic solvents (DESs) show efficiency in breaking down cellulose and improving extraction of plant-derived active ingredients, but their use in polysaccharide extraction is limited by high viscosity. DESs offer versatility and potential across various applications due to their ease of preparation, cost-effectiveness, non-toxicity, and recyclability. Different DES-based extraction methods, including ultrasonic-assisted extraction (UAE), have been developed for polysaccharide extraction, offering simplicity, low cost, and high efficiency. A study developed an extraction process for okra polysaccharides using DESs and ultrasonic-assisted extraction, aiming to optimize extraction parameters, characterize the extracted polysaccharides, and assess their antioxidant properties for potential industrial applications. 1.1 Okra, Polysaccharides, importance, extraction methods 1.2 Extraction methods, UAE based extraction. 1.3 ILs, DESs: Discusses the properties, applications 1.4 DES based plants extracts 1.5 DESs in Polysaccharide Extraction: Explores DESs' effectiveness in polysaccharide extraction, Objectives 8

9. Project Report Materials and methods Fresh Okra were purchased from Taichung vegetable market (Taichung, ROC, Taiwan). Choline chloride ≥ 98 %, Citric acid ≥ 98 %, Urea ≥ 99 %, 1,4-Butanediol > 99 %, Glycerol 99 %, Citric acid≥98 %, Malic acid >98 %, Ethylene glycol>99.5 %, D-glucose ( Glc ), D-glucuronic acid ( GlcA ), D- galactose (Gal), D-glucuronic acid ( GlcA ), L-rhamnose ( Rha ), D-mannose (Man), D-arabinose (Ara), 1-phenyl-3-methyl-5-pyrazolone (PMP) and inulin were bought XX Biochemical co., LTD (XX). Other chemical reagents and solvents were purchased from XX Chemical Reagents Co., Ltd (XX, XX). All other chemicals and reagents were of analytical grade. Deionized water was used throughout the experiments. 2. Materials and methods 2.1. Materials and Chemicals 9

10. Project Report Materials and methods The okra was washed and cleaned by vegetables detergent. After the Okra were driend , cut and remove the seed and head while using pericarp for the powder. The Okra were packed into Zip bag no.4 around 40-50gm in each bag. Kept into the freezer at -20 o C before going for freeze drying. After freeze drying stored at -20 o C , and then grind with the help of grinder. Thereafter, the obtained sample was sieved through a .22mm mesh sifter and preserved at 4 o C for use. DESs were synthesized via heating and stirring. Briefly, HBA and HBD were mixed using a magnetic stirring bar and heated at 80 ◦C for 3 h, or until a homogeneous and stable liquid was formed, confirming successful preparation of DES. During the synthesis process, a certain amount of water was required to reduce the viscosity of DES. For the initial screening, 30 % (w/w) of water was added to each NADES sample. In this study, CC was used as the HBA, and one component were used as the HBD, as listed in Table XX. Six CC-based DESs were successfully synthesized. The tailor-made DES for OPs extraction was selected based on the polysaccharide yield. DES was prepared by the following flow chart method. 2.2. Okra sample collection and pretreatment 2.3.1.1 OKRA dried, ground, and filtered. 2.3. Preparation and optimization of DESs 10

11. Project Report Materials and methods The okra was washed and cleaned by vegetables detergent. After the Okra were driend , cut and remove the seed and head while using pericarp for the powder. The Okra were packed into Zip bag no.4 around 40-50gm in each bag. Kept into the freezer at -20 o C before going for freeze drying. After freeze drying stored at -20 o C , and then grind with the help of grinder. Thereafter, the obtained sample was sieved through a .22mm mesh sifter and preserved at 4 o C for use. DESs were synthesized via heating and stirring. Briefly, HBA and HBD were mixed using a magnetic stirring bar and heated at 80 ◦C for 3 h, or until a homogeneous and stable liquid was formed, confirming successful preparation of DES. During the synthesis process, a certain amount of water was required to reduce the viscosity of DES. For the initial screening, 30 % (w/w) of water was added to each NADES sample. In this study, CC was used as the HBA, and one component were used as the HBD, as listed in Table XX. Six CC-based DESs were successfully synthesized. The tailor-made DES for OPs extraction was selected based on the polysaccharide yield. DES was prepared by the following flow chart method. 2.2. Okra sample collection and pretreatment 2.3.1.1 OKRA dried, ground, and filtered. 11

12. Project Report Results and Discussion 3.2.1. Effect of liquid-solid ratio on the extraction yield The extraction yield of six OPs components at varying liquid-material ratios was assessed under specific conditions: ultrasonic power of 100 W, 4 hours duration, temperature of 80 ◦C, and 30% water content in the DESs . Hence, XX:X should be chosen as the optimal extraction liquid-material ratio for OPs. 3.2.2. Effect of extraction temperature on the extraction yield The extraction yields of OPs components at various ultrasonic temperatures were conducted with an ultrasonic power of 100 W, an extraction duration of 4 hour, a 30 % water content of DESs, and a liquid-material ratio of 30:1. Consequently, XX ◦C was identified as the optimal extraction temperature for OPs. 3.2.3. Effect of extraction time on the extraction yield The extraction yields of OP components at various ultrasonic durations were conducted under the following conditions: ultrasonic power of 100 W, temperature of 80 ◦C, 30 % water content of DESs, and a liquid-material ratio of 30:1. Based on these findings, an extraction time of XX min is recognized as optimal. 3.2.4. Effect of Ultrasound power on the extraction yield The extraction yields of OP components using various ultrasonic powers were conducted under the following conditions: 4 hours of ultrasonic time, a temperature of 80 ◦C, 30 % water content of DESs, and a liquid-material ratio of 30:1. Consequently, XX ◦C was identified as the optimal extraction temperature for OPs. 3.2. Single Factor Experimental Analysis 12

13. Project Report Results and Discussion 3.2.1. Effect of liquid-solid ratio on the extraction yield The extraction yield of six OPs components at varying liquid-material ratios was assessed under specific conditions: ultrasonic power of 100 W, 4 hours duration, temperature of 80 ◦C, and 30% water content in the DESs . Hence, XX:X should be chosen as the optimal extraction liquid-material ratio for OPs. 3.2.2. Effect of extraction temperature on the extraction yield The extraction yields of OPs components at various ultrasonic temperatures were conducted with an ultrasonic power of 100 W, an extraction duration of 4 hour, a 30 % water content of DESs, and a liquid-material ratio of 30:1. Consequently, XX ◦C was identified as the optimal extraction temperature for OPs. 3.2.3. Effect of extraction time on the extraction yield The extraction yields of OP components at various ultrasonic durations were conducted under the following conditions: ultrasonic power of 100 W, temperature of 80 ◦C, 30 % water content of DESs, and a liquid-material ratio of 30:1. Based on these findings, an extraction time of XX min is recognized as optimal. 3.2.4. Effect of Ultrasound power on the extraction yield The extraction yields of OP components using various ultrasonic powers were conducted under the following conditions: 4 hours of ultrasonic time, a temperature of 80 ◦C, 30 % water content of DESs, and a liquid-material ratio of 30:1. Consequently, XX ◦C was identified as the optimal extraction temperature for OPs. 3.2. Single Factor Experimental Analysis 13

14. Light intensity and ambient temperature Sensor No data for the temperature and humidity, Will update tomorrow and focus on these four steps Will update the company tomorrow. 14

15.  S.N  Title Apr May Jun Jul Aug Se; Oct Nov Dec 2.1 Materials and Chemicals 2.2 Okra sample collection and pretreatment 2.3 Preparation and optimization of DESs 2.4 Deep eutectic solvent-based ultrasound-assisted extraction of OPs 2.4.1 Deep eutectic solvent-based ultrasound-assisted extraction procedure 2.4.2 Combined extracts concentrated and precipitated with anhydrous ethanol. 2.4.3 Determination of TSC using phenol-sulfuric acid method. 2.5 Single-factor experiment design 2.5.1 liquid-solid ratio 2.5.2 extraction temperature 2.5.3 extraction time 2.5.4 extraction power 2.6 BBD and statistical analysis 2.6.1 BBD with RSM applied to design experimental project. 2.6.2 Three major experimental factors and extraction yield of OPs as response variables. Research project schedule

16.  S.N  Title Apr May Jun Jul Aug Se; Oct Nov Dec 2.7 Determination of the contents and components of OPs 2.7.1 Total sugar contents 2.7.2 Monosaccharides composition and contents 2.7.3 Pectin determination 2.7.4 Dextran content 2.7.5 Uronic acid contents 2.8 Analysis of molecular structure of OPs 2.8.1 Functional Groups (FTIR) 2.8.2 Molecular Composition (GC) 2.8.3 Structural morphology (SEM) 2.8.4 Surface bonding (NMR) 2.8.5 Crystallographic configuration (XRD) 2.8.6 Specific surface area (BET) 2.9 In-vitro bioactivity evaluation of Okra PS 2.9.1 Antioxidant capacity assessment Research Manuscript submission

17. Thanks 17