Green Synthesis and Characterization of Selenium Nanoparticles and Its Agricultural Application

THESIS

GREEN SYNTHESIS AND CHARACTERIZATION OF SELENIUM NANOPARTICLES AND ITS AGRICULTURAL APPLICATION

WARINDA FUANGCHOONUCH

GRADUATE SCHOOL, KASETSART UNIVERSITY Academic Year 2019

THESIS APPROVAL GRADUATE SCHOOL, KASETSART UNIVERSITY

DEGREE: Master of Science (Chemistry) MAJOR FIELD: Chemistry DEPARTMENT: Science

TITLE: Green Synthesis and Characterization of Selenium Nanoparticles and Its Agricultural Application

NAME: Miss Warinda Fuangchoonuch

THIS THESIS HAS BEEN ACCEPTED BY

THESIS ADVISOR (Mrs. Nongpanga Jarussophon, Ph.D.)

DEPARTMENT HEAD (Assistant Professor Taeng-On Prommi, Ph.D.)

APPROVED BY THE GRADUATE SCHOOL ON

ACTING DEAN (Associate Professor Ladawan Puangchit, D.Sc.)

THESIS

GREEN SYNTHESIS AND CHARACTERIZATION OF SELENIUM NANOPARTICLES AND ITS AGRICULTURAL APPLICATION

WARINDA FUANGCHOONUCH

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science (Chemistry) Graduate School, Kasetsart University Academic Year 2019

ABST RACT Warinda Fuangchoonuch : Green Synthesis and Characterization of Selenium Nanoparticles and Its Agricultural Application. Master of Science (Chemistry), Major Field: Chemistry, Department of Science. Thesis Advisor: Mrs. Nongpanga Jarussophon, Ph.D. Academic Year 2019

Selenium nanoparticles (SeNPs) were synthesized by the reduction of sodium selenite using ascorbic acid as a reducing agent in the presence of chitosan (Cs) and poly-γ-glutamic acid (PGA) as the stabilizer and capping reagent to provide chitosan-selenium nanoparticle (Cs-SeNPs) and poly-γ-glutamic acid selenium nanoparticle (PGA-SeNPs). Their size and morphology were characterized by dynamic light scattering (DLS), UV-Vis spectroscopy, fourier transform infrared spectrometer (FT-IR) and transmission electron microscopy (TEM). The result showed that both spherical Cs-SeNPs and PGA-SeNPs have a diameter of about 100 nm while the naked SeNPs have particle size about 150 nm. The presence of selenium nanoparticles was confirmed by UV-visible spectroscopy for surface plasmon resonance (265 nm). The highest selenium uptake in sunflower sprout was observed on SeNPs while both Cs-SeNPs and PGA-SeNPs displayed higher seed germination. Moreover, the effect of selenium accumulation related to its antioxidant activity was also studied by DPPH assay and ABTS assay. The result showed that the ethanolic extract of sunflower sprout with accumulated PGA- SeNPs has the highest antioxidant activity.

______/ ____ / ____ Student's signature Thesis Advisor's signature

ACKNOWLEDGEMENTS

First, I would like to thank my advisors, Dr. Nongpanga Jarussophon for her suggestion, encouragement, support and constant inspiration throughout the duration of my graduate study and research. She was always there to listen and showed me in different ways to approach a research problem and the need to be persistent to accomplish any goals. Without her, this thesis would not have been possible. I would like to thank Dr. Sagaw Prateepchinda, Miss Kanjanaporn Chompoonuch for antioxidant activity, Asst. Prof. Dr. Orawan Chunhachart for PGA, Dr. Kanok-on Amprayn for ICP, Dr. Suwatchai Jarussophon for DLS, Asst. Prof. Dr. Sasiwadee Boonya-uthayan, and Dr. Wanpen Laosripaiboon for always listen and give valuable advice on my thesis problems. Financial support for this research was granted through a scholarship of the Graduate School, Kasetsart University, and the authors would like to acknowledge the National Research Council of Thailand (NRCT), National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), and Thailand Institute of Scientific and Technological Research (TISTR). Special thanks to the Chemistry Laboratory Kasetsart University Kamphaeng Sean Campus for Laboratory. I especially thank my friend for helping me during my graduate study. Finally, I would like to thank my lovely parents and my sister, who love me and support me through everything I do in my life.

Warinda Fuangchoonuch

TABLE OF CONTENTS

Page

ABSTRACT ...... C

ACKNOWLEDGEMENTS ...... D

TABLE OF CONTENTS ...... E

LIST OF TABLES ...... F

LIST OF FIGURES ...... H

LIST OF APPENDIX TABLES ...... K

LIST OF APPENDIX FIGURES...... M

INTRODUCTION ...... 1

OBJECTIVES ...... 4

LITERATURE REVIEWS ...... 5

MATERIALS AND METHODS ...... 25

RESULTS AND DISCUSSION ...... 31

CONCLUSIONS...... 68

LITERATURE CITED ...... 70

APPENDICES ...... 76 CURRICULUM VITAE ...... 103

LIST OF TABLES

Page

Table 1 Some examples of size controlled synthesis of selenium nanostructures. (Asere, 2013)...... 20 Table 2 Sizes of synthesized selenium nanoparticle using different volume of sodium selenate to ascorbic acid...... 31 Table 3 Sizes of selenium nanoparticle synthesized on the basis of different ratios of sodium selenite to ascorbic acid...... 32 Table 4 Sizes of selenium nanoparticle synthesized on the basis of the ratios of sodium selenite to ascorbic acid with different concentrations when being kept for 7 days...... 34 Table 5 Sizes of selenium nanoparticle synthesized on the basis of the ratios of sodium selenite to ascorbic acid with different concentrations when being kept for 22 days...... 34 Table 6 Sizes of selenium nanoparticle synthesized on the basis of the ratios of sodium selenite to ascorbic acid with different concentrations when being kept for 1 month...... 35 Table 7 Sizes of selenium nanoparticles synthesized on the basis of the ratios of different volume of sodium selenite to ascorbic acid...... 36

Table 8 Sizes of selenium nanoparticle synthesized at different reaction times...... 38 Table 9 Sizes of selenium nanoparticle synthesized at Different volumes of water 10, 25, 50, 100 mL ...... 39 Table 10 Selenium particle sizes synthesized by adding water for stopping reaction as per scheduled times 2, 4, 6, 8, and 10 days...... 40

Table 11 Selenium nanoparticle sizes at 40, 60, and 80 ºC...... 41

Table 12 Chitosan-coated selenium nanoparticle sizes...... 43

Table 13 Sizes of chitosan - coated selenium nanoparticles after 7 days passed...... 44

Table 14 Sizes of chitosan - coated selenium nanoparticles after 22 days passed...... 44

Table 15 Sizes of chitosan - coated selenium nanoparticles after 1 month passed...... 45

Table 16 Short chain chitosan-coated selenium nanoparticle sizes...... 46

Table 17 Sizes of short chain chitosan-coated selenium nanoparticles after 7 days passed...... 47 Table 18 Sizes of short chain chitosan-coated selenium nanoparticles after 22 days passed...... 47 Table 19 Sizes of short chain chitosan-coated selenium nanoparticles after 1 month passed...... 48

Table 20 PGA-coated selenium nanoparticle sizes...... 49

Table 21 Sizes of PGA-coated selenium nanoparticles after 7 days passed...... 50

Table 22 Sizes of PGA-coated selenium nanoparticles after 22 days passed...... 50

Table 23 Sizes of PGA-coated selenium nanoparticles after 1 month passed...... 51 Table 24 Long chain chitosan-coated selenium nanoparticle sizes that are synthesized by using different water volume ...... 52 Table 25 PGA-coated selenium nanoparticle sizes that are synthesized by using different water volume...... 52

Table 26 Total selenium concentration in sunflower sprouts after 7 days...... 59 Table 27 The percentage seed germination of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS...... 61 Table 28 The shoot lengths sunflower sprout of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS...... 62 Table 29 The root lengths sunflower sprout of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS...... 64 Table 30 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C- PGA and C-CS towards DPPH radicals...... 66 Table 31 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C- PGA and C-CS towards ABTS radicals...... 67

LIST OF FIGURES

Page

Figure 1 Se8 rings ...... 5

Figure 2 Scopus-indexed articles for polysaccharide based metallic nanoparticles ...... 7 Figure 3 Overall approaches for the synthesis of polysaccharide-based metallic nanoparticles...... 8

Figure 4 Structure of a. chitin and b. chitosan ...... 8

Figure 5 Chitosan production process...... 9

Figure 6 PGA structure ...... 13 Figure 7 Pictorial representation of the interface of Se with soil, plant and atmosphere...... 15

Figure 8 The probable mechanism involved in the formation of nanoparticles (Se0) . 26 Figure 9 Sizes of selenium nanoparticle synthesized on the basis of different ratios of sodium selenate to ascorbic acid...... 32 Figure 10 Sizes of selenium nanoparticle synthesized on the basis of different ratios of sodium selenite to ascorbic acid...... 33 Figure 11 Sizes of selenium nanoparticle synthesized of the ratios of sodium selenite to ascorbic acid with different concentrations when being kept for 7, 22 days and 1 months...... 35 Figure 12 Solution colors of solution after selenite interacted with ascorbic acid after 30 minutes a. 1:1, b. 1:2, c. 1:3, d. 1:4, e. 1:5 ...... 36 Figure 13 Sizes of selenium nanoparticles synthesized on the basis of the ratios of different volume of sodium selenite to ascorbic acid...... 37 Figure 14 Colors of solution after selenite interacted with ascorbic acid at different times a. 15 minutes, b. 30 minutes, c. 1 hour, d 90 minutes. e. 2 hours...... 37

Figure 15 Sizes of selenium nanoparticle synthesized at different reaction times...... 38 Figure 16 Sizes of selenium nanoparticle synthesized at different volumes of water 10, 25, 50, 100 mL ...... 39 Figure 17 Selenium particle sizes synthesized by adding water for stopping reaction as per scheduled times 2, 4, 6, 8, and 10 days...... 40

Figure 18 Selenium nanoparticle sizes at 40, 60, and 80 ºC...... 41

Figure 19 Shows 100 nm selenium nanoparticle measured at 80 ºC from DLS...... 42 Figure 20 Shows 100nm selenium nanoparticle measured at a. 40 ºC and b. 60 ºC from DLS...... 42 Figure 21 Chitosan-coated selenium nanoparticles with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 µM ...... 43 Figure 22 Chitosan-coated selenium nanoparticles with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 µM after being kept for 7, 22 days and 30 days...... 45

Figure 23 Short chain chitosan-coated selenium nanoparticle sizes...... 46 Figure 24 Short chain chitosan-coated selenium nanoparticles with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 µM after being kept for 7, 22 days and 30 days...... 48

Figure 25 PGA-coated selenium nanoparticle sizes...... 49 Figure 26 PGA-coated selenium nanoparticles with different concentrations at 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 mM after being kept for 7, 22 days and 30 days...... 51 Figure 27 Chitosan-coated and PGA-coated selenium nanoparticle sizes using different water volume...... 52

Figure 28 UV-Vis spectrum of SeNPs, CS-SeNPs and PGA-SeNPs ...... 54

Figure 29 (a.) The FTIR spectra of SeNPs, Cs-SeNPs, PGA-SeNPs, Cs and PGA .... 55 Figure 30 Morphology and formation of SeNPs freshly synthesized within CS solution and PGA solution. (a), (b) TEM image of SeNPs. (c), (d) TEM image of CS-SeNPs. (e), (f ) TEM image of PGA-SeNPs...... 56 Figure 31 Morphology and formation of SeNPs freshly synthesized within CS solution and PGA solution. (a.) The size distribution of SeNPs measured DLS, (b.) The size distribution of CS-SeNPs measured DLS, (c.) The size distribution of PGA- SeNPs measured DLS...... 57

Figure 32 Representative samples of SeNPs with chitosan and PGA ...... 58

Figure 33 Total selenium concentration in sunflower sprouts after 7 days...... 59 Figure 34 The percentage seed germination of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-Cs...... 61 Figure 35 The shoot lengths sunflower sprout of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS...... 63 Figure 36 The root lengths sunflower sprout of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS...... 65

Figure 37 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS towards DPPH radicals...... 66 Figure 38 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS towards ABTS radicals...... 67

LIST OF APPENDIX TABLES

Page

Appendix Table A1 The percentage seed germination of SeNPs, Cs-SeNPs, PGA- SeNPs, control, C-selenite, C-PGA and C-CS..…...…………...... 81

Appendix Table A2 The effect of SeNPs uptake in sunflower sprout……………….83

Appendix Table A3 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C- selenite, C-PGA and C-CS towards DPPH radicals………………………………….85

Appendix Table A4 DPPH assay showing enhanced antioxidant activity of C-PGA...... 86 Appendix Table A5 DPPH assay showing enhanced antioxidant activity of control...... 87

Appendix Table A6 DPPH assay showing enhanced antioxidant activity of Cs-SeNPs ………………………………………………………………………………..88 Appendix Table A7 DPPH assay showing enhanced antioxidant activity of PGA- SeNPs. ……………………………………………………………...... 89

Appendix Table A8 DPPH assay showing enhanced antioxidant activity of SeNPs…………………………………………………….…………………………..90 Appendix Table A9 DPPH assay showing enhanced antioxidant activity of C-Se….91

Appendix Table A10. DPPH assay showing enhanced antioxidant activity of C-Cs ……………………………………………………………...... 92

Appendix Table A11 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS towards ABTS radicals……………………………….93 Appendix Table A12 ABTS assay showing enhanced antioxidant activity of C-PGA ………………………………………………………...…………….….....94

Appendix Table A13 ABTS assay showing enhanced antioxidant activity of Control.…...…………...... 95

Appendix Table A14. ABTS assay showing enhanced antioxidant activity of Cs- SeNPs………………………………………………………………………………...96

Appendix Table A15 ABTS assay showing enhanced antioxidant activity of PGA- SeNPs.. ……………………..………………………………………………………..97 L

Appendix Table A16 ABTS assay showing enhanced antioxidant activity of SeNPs………………………………………………………...……………………....98

Appendix Table A17 ABTS assay showing enhanced antioxidant activity of C- Cs...... 99 Appendix Table A18. ABTS assay showing enhanced antioxidant activity of C- Se……………………………………………………………………………………100

Appendix Table B1 Data from Laboratory in reaction of ethyl acetate synthesis. ...100

LIST OF APPENDIX FIGURES

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Appendix Figure A1 UV–vis spectrum of (a) SeNPs, (b). Cs-SeNPs and (c) PGA- SeNPs.………………………………………………………………………………..78 Appendix Figure A2 The FTIR spectra of (a) SeNPs, (b) Cs-SeNPs, (c) PGA-SeNPs, (d)Cs and (e) PGA……………………………………………………...…………….79

Appendix Figure A3 DPPH assay showing enhanced antioxidant activity of C-PGA ………………………………………………………………….…………………….87

Appendix Figure A4. DPPH assay showing enhanced antioxidant activity of control………………………………………………………………….……………..88

Appendix Figure A5 DPPH assay showing enhanced antioxidant activity of Cs-SeNPs. …………………………….……………………………………………...89

Appendix Figure A6. DPPH assay showing enhanced antioxidant activity of PGA- SeNPs… ……………………………………………………………………………..90

Appendix Figure A7 DPPH assay showing enhanced antioxidant activity of SeNPs………………………………………………………………………………...91

Appendix Figure A8 DPPH assay showing enhanced antioxidant activity of C-Se. ..92 Appendix Figure A9 DPPH assay showing enhanced antioxidant activity of C-Cs. …………………………….……………………………………………...... 93

Appendix Figure A10 ABTS assay showing enhanced antioxidant activity of C- PGA…………………………………………………………………………………..94 Appendix Figure A11 ABTS assay showing enhanced antioxidant activity of Control………………………………………………………………………………..95 Appendix Figure A12 ABTS assay showing enhanced antioxidant activity of Cs- SeNPs………………………………………………………………………………...96 Appendix Figure A13 ABTS assay showing enhanced antioxidant activity of PGA- SeNPs………………………………………………………………………………...97 Appendix Figure A14. ABTS assay showing enhanced antioxidant activity of SeNPs………………………………………………………………………………...98

Appendix Figure A15 ABTS assay showing enhanced antioxidant activity of C- Cs……………………………………………………………………………………99

Appendix Figure A16 ABTS assay showing enhanced antioxidant activity of C- SeNPs………………………………………………………………………………100

GREEN SYNTHESIS AND CHARACTERIZATION OF SELENIUM NANOPARTICLES AND ITS AGRICULTURAL APPLICATION

INTRODUCTION

Selenium (Se) is an essential element, and necessary for a healthy liver. It has high potential to defense the cell damage and other organs from oxidation. There are several researches shown that the properly supplied selenium can increase the level of enzyme such as glutathione peroxidase (GPx), prevent the antioxidant accumulation, and decrease the cell damage (Zhai et al., 2017). Only a little amount of selenium is necessary for the operation of human and animal cell significantly. However, high amount of selenium that exceed the maximum requirement of body can be severely toxic to human and animal, but its deficiency can be illness too such as Keshan type of heart disease. It can be cause the rapid symptoms and also a chronic symptoms including cardiomegaly, arrhythmia, heart attack, Kashin back, deterioration, dead of muscle and the illness with joint stiffness and pain and cancer. The National Academy of Science in U.S. reported that the recommended daily allowance (RDA) of selenium in adult is 55 microgram and the maximum should not be exceed 400 microgram (Saha, 2017). Therefore, the selenium was brought to be selenium metal nanoparticles which has the oxidation number equal to zero (Se 0). The selenium in Se (0) form has been attracted considerably since it has lower toxicity, be widely used in all areas of life due to its high biological activity and can be absorbed well in the body compared to other forms of selenium. Nanomaterials have become the attractiveness of several research works. Various kinds of nanoparticles such as titanium oxide, silver, gold, and cadmium selenide nanoparticles were used as catalysts, stain-resistant clothing, sunscreens, cosmetics and electronics, including drug delivery. Selenium nanoparticles display an important role on the biology and medicine, in virtue of their excellent biological activities and low toxicity. Recently, selenium nanoparticles have been used as additives in growth of corn and cereals as well as in vitamins replacing organic selenium compound to replenish the essential trace selenium in human’s body. The application of nanoparticles can be associated with many fields like medical, food industries, environmental studies, electronics production, energy generation and agriculture. However, its application has the limitation in facts: (1) economic and environment-friendly ways to synthesize small SeNPs are needed and (2) SeNPs are usually enlarge, aggregate and unstable form (gray/black analog) that is thermodynamically more stable, leading to a lower 2

bioactivity and bioavailability. Natural polysaccharides have potential ability to disperse and stabilize SeNPs, which can interact with nano-sized materials and prevent them from aggregation. Besides, polysaccharides exhibit excellent bioactivity such as antioxidant, anti-inflammation, and so forth, resulting in SeNPs encapsulated by polysaccharides may have more activities than naked SeNPs. The reason is that polysaccharides own the hydrophilic groups such as hydroxyl, which can interact with nano-sized materials and prevent them from aggregation. Besides, polysaccharides exhibit excellent bioactivity such as antioxidant, anti-inflammation, and so forth, resulting in SeNPs encapsulated by polysaccharides may have more activities than naked SeNPs. Chitosan (Cs) is an amino-polysaccharides. The process to produce in large scale of chitosan start from chitin, which can be extracted from the Crustaceans by- products such as shrimp, clam, and crab. Chitin then react with the alkali through the deacetylation (N.V. and Kumar, 2000).The positive traits of chitosan give to this polymer numerous and unique physiological and biological properties with great potential in a wide range of industries such as pharmacology, medicine, and agriculture .One of the most important bioactivity of chitosan on plants is stimulation of seed germination. This water-soluble chitosan can be easily further modified in order to fine tune the chemical and physical properties (Ali and Ahmed, 2018) for controlling and releasing of drugs used in various medical applications (Islam et al., 2015). Moreover, it was interested to be a biodegradation, biocompatibility, hydrophilic properties, low cost and non-toxic properties of chitosan. Besides, chitosan is known to exhibit great bioactivities, such as antitumor, antibacterial, hypocholesterolemic, and antihypertensive activities. In addition, it has also been confirmed that chitosan could be a good stabilizer for SeNPs. Poly-glutamic acid (PGA) is possibly produced by some microorganism and also be naturally decomposed, and safe for human and environment. From the structure of PGA, it is found that the structure is long chain substance and there are carboxylate groups. The large amount of carboxylate results in PGA being able to hold water and retain moisture well in the soil. There is a study of stability in natural degradation and water adsorption capacity of poly-glutamic acid by the occurrence of create 3D bonds between molecules It was found that the hydrogel of the polyglutamic acid substance absorbs water well and the polyglutamic acid is a source of carbon, nitrogen and microbial growth minerals. From the property of natural self- degradation, water absorption, and naturally decompose make the PGA have a lot of application for several industries.(Khalil et al., 2017) In food industry, it was used as a moisturizer or emulsion stabilizer. In the cosmetic industry, it was used together with other ester components to increase the flexibility and the strength of thermoplastic (Najar and Das, 2015) Furthermore, there was a report revealed the 3

PGA was applied to use as a targeting nano capsule drug carrier, which can protect the drug from the stomach enzyme damaging. Due to necessity and requirement of selenium (Se); therefore, instead of directly buying organic selenium supplements in yeast form that are sold in markets, receiving Se indirectly by eating plants absorbing and accumulating Se from soil would be easier, cheaper, and safer compared to a chance to have excessive or insufficient selenium from taking directly selenium supplements. In Thailand health problems caused by Se deficiency or excessive selenium supplementation or consumption have not been reported and there has not been a report on selenium nutritional status in Thai people. A report showed that areas with soils containing Se from 0.125-0.175 mg/kg would not have problems related to the deficiency of Se in population but areas with soils containing Se more than 3mg/kg are considered soils with excessive levels of Se which can cause selenosis, a population health problem, due to receiving too much Se. From this part of review, these result can be hypothesized that the se concentration in the soil is related to human or animal health.

Production of selenium in plants is focused on growing plants and vegetables for use as human foods such as spring onion, onion, fruit, rice, etc. In case growing areas contain low levels of Se, increasing the amount of Se in plant foods can be done by giving soils instant bio-fertilizer mixed with Se or by spraying method. However, Se fertilizers sold in markets are quite expensive since they are imported from foreign countries as sodium selenite, sodium selenate, selenium amino acid chelate whose prices range from 1,700 – 1,900 US$ or about 54,000 – 60,000 baht per ton. Therefore, the synthesis of selenium fertilizers and producing fertilizer formulas with selenium supplementation domestically would help to reduce cost of production and farmers who would like to produce high selenium plants can reduce Se fertilizer costs accordingly.

This work, presented herein, focuses on the preparation of SeNPs in one-step by the reduction of sodium selenite by ascorbic acid. To prevent aggregation of the synthesized particles and to improve the stability, chitosan and PGA has been introduced into the redox system. The aim of this work was to optimize the synthesis conditions of synthesized SeNPs, Cs-SeNPs and PGA-SeNPs productions. The physical properties of selenium nanoparticles and its encapsulated form are characterized and their plant uptakes are also be evaluated.

OBJECTIVES

1. To synthesize the selenium nanoparticles (SeNPs), chitosan-selenium nanoparticles (Cs-SeNPs) and poly-glutamic acid-selenium nanoparticles (PGA-SeNPs)

2. To encapsulate selenium nanoparticles by using chitosan and poly-glutamic acid.

3. To investigate the physical properties of SeNPs, Cs-SeNPs and PGA-SeNPs and their plant uptake.

LITERATURE REVIEWS

Selenium (Se), as the essential micronutrient, was discovered by Jons Jacob Berzilus. Selenium is a non-metallic element that is belong to the 16th group between sulphur and tellurium. Its atomic number is 34. It has 6 stable isotopes called as nuclear isomers. It exists in three well defined allotropic forms such as amorphous selenium which is red, crystalline trigonal which have helical chain in black, and the crystalline monoclinic (α, β, γ) having Se8 rings Figure 1 which also in red color. (Chhabria and Desai, 2016) Selenium is a semi metal and stable stable at ordinary temperatures. When it burns, it produces a blue flame and selenium dioxide. Selenium can be combined with many elements (hydrogen, fluorine, chlorine, bromine, phosphorus, etc.). Selenium usually presents in nature and in organism as an organic or inorganic form. The most organic form are in the selenomethionine (Se-Met) and selenocrysteine (Se-Cys). On the other hands, the inorganic form are in the selenite -2 2- -2 (SeO3 ), selenide (Se ), selenate (SeO4 ) and the selenium element (Se).

Figure 1 Se8 rings

There are various reports that selenium is involved in the antioxidant defense system of liver and has been responsible for protecting against oxidative stress in human body. Selenium is present in at least 25 human selenoproteins and enzymes as selenocysteine, which plays an essential role in preventing various diseases such as cardiovascular disease, hypercholesterolemia and certain cancers. (Preparation and antioxidant) Many studies demonstrated that the supplementation of Se can increase the enzyme level such as GPx etc., prevent the accumulation of free radical species, and reduce the cellular damage. (Skalickova et al., 2017) However, the narrow margin between the effectiveness and toxic cause the limitation to apply this substance. Selenium was also considered as owning the multiple valence states (+6, +4, +2, 0, -1, -2) and more complex antioxidant activities. The Se (0) has thus gained more attention because of its low toxicity and good bioavailability compared with Se (IV) and Se (VI), since both of them having an ability to capture free radicals. Nevertheless, poor water solubility and ability to transform into the analogue, make Se (0) difficult to be used for food and medicine fields. The solubility in water of this substance can be improve by reducing size into nano-scaled particles and increase the specific surface with convenient nanotechnology. 6

Selenium nanoparticles has also been provided better biocompatibility, bio- efficiency, and lower toxicity as compared to various organic and inorganic forms of selenium. (Wen Liu et al., 2012) used SeNPs tagged with 5-flurouracill which can enhances the cellular uptake of SeNPs through endocytosis which enhanced its anticancer ability. SeNPs exhibit much lower execution of toxicity while increase the activity of the selenoenzymes. (Bai et al., 2017) present the result that selenite for 11.0 mg Se/kg by weight can kill all mice. However, SeNPs-M did not cause any mortality at 14.6 mg Se/kg by weight. The LD50 of selenite and SeNPs-M was 3.4 and 6.23 mg Se/kg by weight, respectively. Indicating that the acute of toxicity of SeNPs- M was only 1/18 of the selenite based on Se dose. SeNPs in the form of SeNPs-M was much safer than selenite. However, the application of SeNPs is restricted by the economic and environment-friendly ways to synthesise small amount of SeNPs and SeNPs can be enlarged, aggregated and transformed into gray/black analog that is thermodynamiccally stable, but biologically inert. These problems are related to the final size and stability of SeNPs on which activities of SeNPs are dependent. It seems only SeNPs in smaller size with good stability can guarantee high bioactivities. To solve these problems, some compounds including polysaccharides, monosaccharides, proteins, amino acids and even the culture of microorganism, have been utilized to stabilize SeNPs. Among them, polysaccharides are considered as a suitable material for fabricating SeNPs for the energy efficiency and eco-friendliness. (Zhai et al., 2017)

Bio-polymer is the eco-friendly natural polymer that was widely used in the medical, agricultural, environmental activities since it is renewable, sustainable, and nontoxic when compared with the polymer that made from petroleum. Natural polysaccharide has great property because their several chemical property and structure of them. The difference ranging in charge, chain lengths, monosaccharide’s sequences, and stereochemistry give high capacity for the development in advanced functionalized materials and biomedicines. Especially, the hydrophilic groups of polysaccharides can form non-covalent. Moreover, polysaccharides can be naturally decomposed which is stable and biologically appropriated. Polysaccharides can be reducing agents as a good stabilizer to control the physical properties of nanoparticles. (Wang et al., 2017) Over the last decades, remarkable progress has been achieved in the studies of polysaccharide-based metallic nanoparticles through polysaccharides encapsulation Figure 2. The previous research also rarely studies in selenium nanoparticles. 7

Figure 2 Scopus-indexed articles for polysaccharide based metallic nanoparticles

Nanoparticles can be green synthesized using starting materials via physical and chemical processes, called that top-down and bottom-up synthesis. The nanoparticles that produced in these syntheses have differenced in elemental composition, shape, size, and chemical or physical properties. The top-down synthesis is a size reduction of a large solid material into smaller size. The size reduction may involve physical and chemical treatments, such as mechanical milling, chemical etching, thermal ablation, laser ablation, and explosion process. (Asere, 2013) With this top-down synthesis after preliminary treatment, the metallic nanoparticles surface will be changed and the high temperature and pressure during the size reduction may induce the oxidation of the nanoparticles. This can cause the physical properties and surface chemistry changing. On the other hand, bottom-up synthesis (self-assembly) is decomposing or reducing valent state of metallic precursor to zero-valent state to form the building blocks (smaller entities) followed by the nucleation and nanocrystals growth. During this process, the metallic ions can be encapsulated by polysaccharide through non-covalent bonding, and then the order of free energy is altered to realize the stabilization, morphological control and kinetic growth of the metallic nanoparticles. The stereogenic centers will also help to anchor the metallic nanoparticles. In contrast of the harsh reaction condition of top-down approach, the bottom-up synthesis process can occur in the bulk solution or in droplets, which is easy to regulate through controlling the process conditions. Therefore, polysaccharide-based metallic nanoparticles synthesized are relatively homogeneous compared to those synthesized through top-down approach as Figure 3. (Wang et al., 2017) 8

Figure 3 Overall approaches for the synthesis of polysaccharide-based metallic nanoparticles.

Chitosan Chitosan is the long-chain polysaccharide which connect together with the β- (1→4) bond between D-glucosamine (deacetylate unit) and N-acetyl-D-glucosamine (acetylated unit) as show in Figure 4. In the chitosan production process, it comes from chitin which normally has been found in the hard case of sea organism such as shrimps, clam, and crab, react with the alkali trough the deacetylation (N.V. and Kumar, 2000) as show in Figure 4.

Figure 4 Structure of a. chitin and b. chitosan 9

Figure 5 Chitosan production process.

Chitosan is a natural polymer useful for medical and pharmaceutical applications. Among biocompatible and biodegradable natural polymers it displays interesting biological activities. Recent studies prove that chitosan has mucoadhesive properties (Lehr et al, 1992) and therefore it seems particularly useful to formulate bio-adhesive dosage forms for mucosal administration. Furthermore, chitosan can be used as the wall material of the capsule. Chitosan capsules can be applied in food, drug, and biochemical areas (Chen and Tsaih, 1997). Controlled release by using chitosan method is often applied to drug controlled release studies. The release behaviors of drug that coated by chitosan- collagen matrices is pH dependent. Chitosan controlled release drug. microspheres cross-linked with glutaraldehyde were used Drug delivery was affected by the extent of cross-linking.

The property of chitosan at low pH, amino group will has protonation and cause the chitosan to be polycationic. It affects to the increasing of solubility. In this acid state, the polycationic of chitosan can do the sulfonation and became negative. Then, it cause more solubility to chitosan. (Liu, W. et al., 2012) With the property that can transform to be chelate film with the light structure of metal ion and can be decomposed naturally (optical structural characteristics), it was applied to agricultural filed such as bio-pesticide. Moreover, it can be applied as the mold prevention in plant in medical field such as becoming a bandages to stop bleeding and drug carrier to increase the efficiency and effectiveness of drug as well. (Jayakumar et al., 2011)

Applications of Chitosan

Chitosan has been reported to have useful applications in the medical, cosmetic and pharmaceutical area, which are described below,

Medical Application Chitosan has been used as blood coagulant, a wound healing accelerator, soft and hard contact lens and artificial kidney membrane.

Cosmetic Applications The main application areas in cosmetic are in hair care and skin care consumer products.

Pharmaceutical Application Chitosan has many applications in pharmaceutical, examples of reports of its uses in this area are: - As a direct compression diluent. - As a vehicle for sustained release. - Enhancement of dissolution rate of insoluble drug. - As a tablet binder. - As disintegrant in pharmaceutical tablet - Formulation of film dosage form. - Gel preparation - Microencapsulation for anticancer, cell culture and other drugs. (Chen and Tsaih, 1997) studied the effect of preparation method and characteristics of chitosan on the mechanical and release properties of the prepared capsule. They found axial ratio and hemoglobin release percent of the capsule increased with the increase of the chain flexibility parameter, but decreased with the increased of the chitosan molecular weight or decreased degree deacetylation of chitosan.

(Mi et al., 1997) studied the effect of viscosity and swelling ability on the release rates of drugs, theophyline. Chitosan tablets containing theophyline were prepared by directly compressing the wet or dry blended polymer-drug powders. The results showed the release of theophyline increased with a decrease in the viscosity of the blending chitosan solution. On the other hand, the swelling ability of the polymer greatly influences the release kinetics of the theophyline tablets.

(Thawatchai, 1994) studied effect of variables in chitosan film formulations on propranolol hydrochloride tablets. The results showed that the plasticized coated tablets with the higher M.W. of chitosan exhibited slower drug release than those 11

coated with the plasticized lower M.W. of chitosan. For plasticized coated tablets with chitosan as the amount of propylene glycol increased, the drug release was faster.

(Gasser et al., 1998) studied the reaction of the microcapsule, alginate- chitosan-I by using sodium alginate solution drop in chitosan solution, or using calcium alginate beads incubated with chitosan solution. They found that the sodium alginate that were dropped in chitosan solution provided the chitosan 0.015 mg per mm2 of the capsule surface while the calcium alginate beads that were incubated with the chitosan solution provide more than 2 mg per mm2. Moreover, the decreasing of molecular weight of chitosan to be lower than 20,000 Da and increasing the porosity of alginate can affect to the accumulation of alginate-chitosan to be noticeably increased.

(Yadav et al., 2012) reported the curcumin encapsulated in chitosan nanoparticles to apply as a stable detoxifying agent for arsenic poisoning. The results showed that chitosan nanoparticles of less than 50 nm in diameter. Sodium arsenite (2 mg/kg) and ECNPs (1.5 or 15 mg/kg) were orally administered to male Winstar rats for 4 weeks to evaluate the therapeutic potential of ECNPs in blood and soft tissues. Arsenic significantly decreased blood α-aminolaevulinic acid dehydratase (α-ALAD) activity, reduced glutathione (GSH) and increased blood reactive oxygen species (ROS) while only arsenic exposure resulted in significant decreased in hepatic GSH, superoxide dismutase

(Chen et al., 2015) reported the synthesis of selenium nanoparticles by using chitosan (CS) and carboxymethyl chitosan (CCS) as capping agents with a simple synthesis using ascorbic acid as a reducer. They investigated by using TEM, UV-vis spectroscopy (UV-vis), Dynamic Light Scattering (DLS), Fourier transform infrared (FTIR), and Thermogravimetric analysis (TGA), and analyzed the antioxidant activity by DPPH and ABTS method. It was found that SeNPs were monodisperse (average size 50 nm) and are ligated by CS and CCS to produce nanocomposites. When the time increase to be 120 days, the average size of nano particle will be increased to 180 nanometers or more. The antioxidant activity was higher than the CCS-SeNPs about 93.5 %, at the concentration 0.6 mmol/L. Moreover, SeNP, CS-SeNPs, and CCS- SeNPs increased an ability to prohibit the antioxidant than the Na2SeO3.

(Pavlina Jelinkova et al., 2016) studied the selenium nanoparticles by complexing them with chitosan and nano selenium particle. The chitosan was synthesized with Schiff base. They tested the catalyzed particles as the bacteria preventer and the result found that the selenium nanoparticles was about 29.4 nanometers with Zeta potential of 44.3 mV. They investigated the speed of selenium nanoparticles with chitosan and derivate in Staphylococcus aureus, Escherichia coli and Staphylococcus aureus (MRSA). The testing result showed that the nano particle 12

can prohibit only the negative gram bacteria Escherichia coli, but chitosan Schiff base can prohibit bacterial cell growth. Therefore, it seem to provide a good solution when use the nano selenium particles with chitosan Schiff bases to prohibit the bacteria.

(Zhai et al., 2017) reported the stability of CS-SeNPs and their antioxidant property with different molecular weight of chitosan.They found that the CS-SeNPs which have different molecular weight can produce the round particles at 103 nm within 30 days. The antioxidant testing in the pattern of DPPH, ABTS and lipid peroxide found that the CS-SeNPs can the antioxidant in many levels. The ability to prohibit the antioxidant from ABTS will be increased up to 87.45 ± 7.63% and 89.44 ± 5.03% of Cs(l) – SeNPs and CS(h) – SeNPs, respectively. Moreover, they can protect the activity of glutathione peroxidase (GPx) efficiently and protect the production of lipofuscin which is from the UV-ray of D-galactose in rat, respectively. Additionally, the rapid toxicity of CS(l)-SeNPs was lower than H2SeO3 about 10 times. (Bai et al., 2017) studied the synthesis of selenium nanoparticles by capping with chitosan and drying using spray-dryer. They found that the particles distribution was good, and the particles were trigonal. The particles was about 35 nm and the results from rapid toxicity testing shown that the SeNPs-M was safer than the selenite for 18 times. Moreover, SeNPs-M can be the antioxidant. The antioxidant testing was found the increasing of glutathione peroxidase, superoxide dismutase, and catalase. SeNPs level can increase the biological efficiency and safety. Therefore, the SeNPs- M might be considered as the potential additional foods with the property of antioxidant that can be applied to both humans and animals.

(Zeng et al., 2018) reported the synthesis of selenium nanoparticles with chitosan (CTS-SeNPs) by using selenite and ascorbic acid as reducers and measured the size of crystalline and stability of CTS-SeNPs by using transmission electron microscopy (TEM), Dynamic Light Scattering (DLS), and scanning electron microscope (SEM). The results shown that the size of CTS-SeNPs has diameter around 54 nm under the appropriated state (temperature 24 °C, reaction time 2 hours, concentration of ascorbic acid 0.04 M, and concentration of chitosan 1.0 mg/mL). CTS-SeNPs were in round shape and have a good distribution. They can be stable for 60 days at the temperature 4 °C. Moreover, the diabetic prohibit ability of CTS-SeNPs in diabetic rats which obtained streptozotocin (STZ) was evaluated, it was found that the CTS-SeNPs at 2.0 mg Se/kg bw can decrease the blood sugar more than any other amount of CTS-SeNPs and any other selenium matter which have the same selenium amount.

Poly-γ -Glutamic Acid Poly-γ-glutamic acid (PGA) is the natural polymer which can be synthesized by several microorganism especially Bacillus (B. licheniformis and B. subtilis). PGA is a homo-polyamide which consist of linked glutamic monomer that chained amide between α-amino and γ-amino carboxyl PGA. All of the monomer can be only L- glutamic acid, or only D-glutamic, or both D-glutamic and L-glutamic in the constant ratio depending on the concentration of bacteria or the material for producing poly-γ- glutamic acid. This type of polymer can be saluted (free acid from) or completely saluted in salted form which have various cation (Na+, Mg2+, K+, NH4+, or Ca2+). (Khalil et al., 2017) PGA synthesized from microorganism can be easily decomposed naturally and non-toxic to human and environment. The more molecular weight of PGA chain which consist a lot of large carboxyl unit can provide high efficiency such as high moisture absorbability. There is a report show that the absorbability can be compared with hyaluronic acid (HA) which can be found in connecting tissue, epidemic tissue, and nervous system. Moreover, γ-PGA has been reported to promote the growth and yield of wheat plants (Z. Xu et al., 2013), and increase dry weights of stems and roots of cucumber young plants (Qiu et al., 2018). In addition, (Chunhachart et al., 2014) found that γ-PGA can relieve lead poisoning in Chinese flowering cabbage, a cause that inhibits the length of roots and the growth of young plants. Moreover, the advantages of the PGA can be applied to several industries such as: - The water absorber in food industry - The carrier to carry the drug to target organs (Najar and Das, 2015) - The moisturizer and emulsion stabilizer in cosmetic industry

Figure 6 PGA structure

(Chen et al., 2008) studied the covering of small gold nanoparticles by using polystyrene and poly(glutamic acid) copolymer through the heating at 100 °C for 2 hours, and then slowly cooled down by using water/dimethyl formamide (H2O/DMF) as a stabilizer of AuNPs during the cooling down process. The polymer can be self- assembly. From the study, it found that the reaction in covering of small gold nanoparticles provide the red dregs. It can be indicated that there are no dregs of gold which is in purple. The measurement of the nano aurum particles size by using TEM 14

was found to be only 5 nm when compared with polystyrene and poly (acrylic acid) copolymer provided coated gold nanoparticles about 32 nm. Then, they studied the bioconjugation of antibody molecule of the nano aurum particles, which covered by polystyrene and poly (glutamic acid) with IgG and A33 type in rate. The study found that the bioconjugation with the gold nanoparticles coated by polystyrene and poly (glutamic acid) provide purple solution and effect to IgG and A33 denaturing too.

(Shima et al., 2013) reported the effect of nano poly(-glutamic acid)-graft-L- phenylalanine ethyl ester [(g-PGA-Phe) NPs] size against the dendritic cell (DCs). They found that g-PGA-PheNps having 40 nm can move to lymph nodes in vivo system faster than the size 100 and 200 nm and can be pass through many dendritic cells. On the other hand, the amount of g-PGA-PheNps per 1 dendritic cell at the size 100 and 200 nm are more than the size of 40 nm. From the growth of dendritic cell testing, it found that g-PGA-Phe NPs at the size 40 nm can help the growth of cells more efficient than the other sides.

(Chen et al., 2012) studied the medical effect of camptothecin which was encapsulated by PGG supramolecular nanoparticle (CPT-PGA SNPs) with the different size at 37 and 104 nm in PBS solution. They found that CPT-PGA SNPs at 37 nm can prohibit growing of tumor cells in rat within 6 days and more effective within 12 days. Moreover, they found that the rats that obtained the CPT-PGA SNPs at 37 nm were not lose their weight. It can be indicated that CPT-PGA SNPs at 37 nm has not serious toxicity.

(Seephinta et al., 2019) studied the effects of γ-polyglutamic acid (γ-PGA) and Bacillus subtilis cells on germination, growth and biochemical contents in Khao Dawk Mali 105 rice seedling were determined in laboratory scale. The results showed that the germination percentage, germination energy and germination speed were not affected by γ-PGA and B. subtilis cells. For the seedling vigor index, B. subtilis cells tend to reduce the seedling vigor compared with the control. Considering the 21-day- old rice seedlings, the results showed that γ-PGA at the concentration of 50 mg.kg- 1sand gave the significantly highest shoot length. Whereas, the root length was gradually reduced with increasing the γ-PGA and B. subtilis cells concentration. However, the addition of γ-PGA and B. subtilis cells significantly increased both shoot and root in term of fresh and dry weight. Moreover, γ-PGA and B. subtilis cells significantly influenced the increase in sugar content, total free amino acid, total chlorophyll and carotenoids content when compared with the control.

Selenium uptake in plants

Plants uptake selenium in the form of Se (IV), Se (VI) and organic Selenium (selenocysteine and selenomethionine). Plants cannot directly take up metallic selenide and elemental Se because these forms of Se are water insoluble. Selenium is chemically similar to sulfur (S). Selenite is transported probably via phosphate transporters, whereas selenate can pass through sulfate transporters and channels in the plasma membrane of root cells as Figure 7. The transportation selectivity S and Se depends on the plant species and amount of the element presenting in soil. However, the increased transfer of Se from root to aerial parts greatly depends on plant species, type and form of Se species present in soil/growth medium. Selenite is less accumulated in shoot compared to Se (VI) and most of Se is transported to shoot in Se (VI) form. Selenate could be transferred to shoot from root by xylem and then to reproductive organs by phloem. Selenate ions are readily absorbed into xylem and hence transported via xylem sap. The uptake, translocation and distribution of Se depend upon plant species, phases of development, form and concentration of Se, physiological conditions (salinity and soil pH) and presence of other substances, activity of membrane transporters, translocation mechanisms of plant. (Gupta and Gupta, 2016) (Saha, 2017)

Figure 7 Pictorial representation of the interface of Se with soil, plant and atmosphere.

(Mahdavi and Rahimi, 2013) studies a role of chitosan for modulating the plant response to several abiotic stresses including salt and water stress. Ajowan almost cultivated in arid and semi-arid land in Iran, which is often confront to drought and salinity stress. This study would like to find out how chitosan seed pretreatment 16

could alleviate the salt stress condition on germination and growth of ajowan. The results show that all of chitosan concentrations increased germination percentage, germination rate, seedling vigour index, length and dry weight of hypocotyl and radicle compared to control although, 0.2% chitosan concentration was more effective than other treatments. Salinity caused a significant reduction in germination percentage, shoot length, root length, shoot and root dry weight and relative water content, while chitosan adjusted the salt toxicity.

(Yookate et al., 2014) reported the sunflower sprout was the useful food material for good health because of their containing with high vitamin and enzyme. Sunflower sprouts were produced by showing of sunflower seed in the suitable seedling media for 7 days. Seeding media and soaking treatment of seed were the important factor for the production of sunflower sprouts. Comparison of various seedling media and the process of seed soaking before sowing were studied. There were 2 experiments. Experiment 1: seeds were sowed in 13 seedling media for 7 days. Sowed seeds in burnt rice husk had the highest percentage of germination, seedling high and fresh weight per 100 seeds. Experiment 2: Seeds were subjected to soaking treatments by soaking in the water at 27 oC and 50 oC for 4, 8, 12, and 16 hr. There was an significant interaction (p <0 .0 1 ) between water temperature and duration of soaking on percentage of seed germination, seedling heigh and hypocotyl width. Soaking of seeds in warm water (50 oC) for 16 hr was the best treatment for production of sunflower sprout.

(Chimsonthorn et al., 2018) studied the the effect of pre-harvest chitosan treatments on appearance and bioactive compounds enhancement of sesame sprout during storage. The sesame sprouts were sprayed with chitosan at the concentration of 0, 0.1, 0.5 and 1 % (w/v) before harvested 24 hours. After harvest, the sesame sprouts were held at 5 ± 1 oC for 6 days. The appearance, weight loss, antioxidant activity and bioactive compounds such as total phenol, flavonoids and ascorbic acid contents of the sprouts were determined. The results indicated that the sesame sprouts treated with 0.1 % chitosan showed the best result for maintaining the appearance. The increase in weight loss and stalk water-soak of all the treatments were delayed over the storage. Antioxidant activity and total phenol contents were enhanced by chitosan treatments at the concentration of 0.1 or 0.5 % compared to the control. Both chitosan treatments also maintained high level of total flavonoid content compared to the control. In conclusion, chitosan treatments at the concentration of 0.1 or 0.5 % could enhance nutritional value in sesame sprouts during cold storage by inducing bioactive compounds contents.

(Supapvanich et al., 2018) studied the effects of pre-harvest chitosan treatments on antioxidant activities and bioactive compounds of sunflower sprouts during cold storage were investigated. The sprouts were watered with 0, 0.1, 0.5 and 17

1 .0 % (w/v) chitosan solutions prior harvest 24 h. After harvest, the sprouts were stored at 4 ±1 °C for 9 d. The investigated parameters were visual appearance, antioxidant activities such as ferric reducing antioxidant potential (FRAP), DPPH radical scavenging (DFRS) activity and bioactive compounds including total phenols, flavonoids and ascorbic acid concentrations. The visual appearance of the sprouts of all treatments was maintained during storage at 4 ± 1 °C for 9 d. Pre-harvest chitosan treatment enhanced antioxidant and DFRS activities, total phenols, flavonoids and ascorbic acid contents of the sprouts, especially at 0.1 % chitosan. During the storage, 1.0 % chitosan treatment induced FRAP, DFRS activity and all bioactive compounds rather than others treatments. These suggest that pre-harvest chitosan treatment is an effective alternative improving nutritional quality of the sunflower sprouts during cold storage.

(Junmatong, 2017) studied the effects of salicylic acid (SA) soaking on seed germination, growth and antioxidant capacity of pea seedlings (Pisum sativum L.) were investigated. Pea seeds were soaked in SA aqueous solution at 0 (control), 250, 500 and 1000 μM for 8 hours. The results showed that SA soaking at 500 μM before cultivation was shown to promote growth of pea seedlings. Percentage of seed germination, shoot length and seedling fresh weight of the treatment were significantly (p < 0 .0 5 ) higher than those of the control seedlings throughout the germination period. The treatments of SA at 250, 500 and 1000 μM significantly (p < 0.05) improved total antioxidant capacity when compared with the controls throughout germination time. However, the 1000 μM SA was the most effective in enhancing the total antioxidant capacity. Thus, SA soaking could be applied for enhancing seedling growth, seedling production and antioxidant content of pea seedlings.

(Jirapong et al., 2018) found that the fungi are an important problem in seed cultivation. Aspergillus sp. was isolated from red cabbage sprouts by tissue transplanting method. This fungus was cultured and tested inhibition on Potato Dextrose Agar (PDA) mixed with chitosan, in the concentration of 0, 100, 200, 300, 400 and 500 ppm. All chitosan mixed in Potato Dextrose Agar (PDA) was most effective to inhibition Aspergillus sp. than control (0 ppm). Therefore, Chitosan used in 0, 100 and 200 ppm to germinate red cabbage sprout. After 2 days planting, treatment used chitosan will increase the percentage of seed germination, height of sprouts and reduce the percentage of red cabbage. However, the fungus was found after 8 day of planting. In addition, this research not found infected Salmonella spp. and Escherichia coli in red cabbage sprouts.

(Suthamwong et al., 2010) found that selenium is an essential mineral for humans and animals. Receiving a small amount of selenium is beneficial to health as it plays an important role in reducing and preventing the incidence of cancer, AIDS and has antioxidant properties. Selenium exists in two forms as organic and inorganic. 18

Inorganic selenium can be converted to organic forms e.g., selenomethionine, seleno- cystine and selenocysteine by bacteria and plants. In the experiment, selenate concentration at 0, 30, 60, 90, 120, 150, and 180 µg/ml (Na2SeO4) were given to mung beans. The experimental result indicated that mung beans accumulated the highest selenium of 1,031 mg/kg when using the selenate concentration at 90 µg/ml. The total of selenium accumulation in mung beans had increased and had the highest amount after being treated for 168 hours. Based on the analysis of protein pattern with SDS-PAGE technique, it was found that the mung beans that were treated with selenate expressed more the 48.6 kDa and 27.3 kDa than the control group. It could be concluded that these 2 proteins are probably related to the accumulation ability of selenium in mung beans.

(Chantiratikul et al., 2018) conducted a study on the suitability of selenium concentration and cultivation period for producing high selenium leaf mustard and Hairy basil sprouts under hydroponic system. Experiment 1: Leaf mustard and Hairy basil were cultivated in Hoagland’s solution supplemented 0, 5, 10, 20, and 30 mgSe /L from sodium selenite for 10 days in a completely randomized design. The findings from the experiment revealed that leaf mustard died when cultivated in the solution contained 20-3 0 mgSe/L. Yield of leaf mustard sprouts cultivated in the solution contained 10 mgSe/L was not different from that of the control group. Survival rate of Hairy basil sprouts decreased when cultivated in the solution contained 3 0 mgSe/L. The height of Hairy basil sprouts cultivated in the solution contained 5 -10 mgSe/L was greater than Hairy basil sprouts cultivated in the solution contained 20-30 mgSe/ L. Yield of Hairy basil sprouts decreased when cultivated in the solution contained 10-30 mgSe/l. Selenium concentration in leaf mustard and Hairy basil sprouts increased according to selenium levels in the solution. Experiment 2 : Leaf mustard and Hairy basil were cultivated in Hoagland’s solution supplemented 10 mgSe/L for 0, 2, 4, 6, 8, and 10 days in a completely randomized design. The experimental result showed that survival rate of leaf mustard and Hairy basil sprouts decreased when cultivation period increased. Yield of leaf mustard sprouts increased when they were cultivated for 6 days or longer. Fresh yield of Hairy basil sprouts extremely increased on the day 8 of cultivation period while dry yield on the days 6-10 was not different. Selenium concentration in leaf mustard sprouts and Hairy basil sprouts increased in accordance with cultivation period. It can be concluded that the suitable selenium and cultivation period for producing high selenium leaf mustard and hairy basis sprouts are 10 mgSe/L and 10 days respectively.

The Synthesis of Selenium nanoparticles

Nano form of selenium can be used for various applications due to its advantages over the bulk form such as low toxicity, better reactivity, low dosage, etc. Hence, it is very important to choose a suitable synthetic protocol to maintain its properties forming a better nano product. To synthesize the Selenium nanoparticles, several factors need to be controlled in order to define the properties of nanoparticles such as size, shape, surface, and component. The factors are the concentration of the substrate, temperature, time of the reaction, and pH. There are several reports for the synthesis of selenium nanoparticles such as chemical reduction, biological synthesis, solvothermal route, hydrothermal route, microwave assisted synthesis, green synthesis, electrodeposition method, pulsed laser and ablation method. (Hosnedlova et al., 2018)

Biological synthesis

Recently, researchers focused more on biosynthesis due to the fact that it can reduce the chemical wastes, and to be more eco-friendly by using bacteria, mold, yeast, and plant extraction. The main components for the green synthesis of nanoparticles are a middle solvent for the synthesis, the eco-friendly reducer, non- toxic stabilizer. The advantages of the biological synthesis is the reduction of chemical using and the particles obtained from the microorganism and the plant extract can be both reducer and stabilizer. Hence, they can reduce the production cost (Ramamurthy et al., 2013). The disadvantage is that the difficulty to control size, shape, and crystalline of nanoparticles.

Chemical reduction Chemical synthesis is the most famous way to produce the nanoparticle since it can synthesize in high volume, and easily to control size and shape. The chemical synthesis involved the substances in the reaction i.e. inorganic selenium, reducing agent such as NaBH4, ascorbic acid, and gallic acid, the stabilizer or capping agent which most of them are polymers such as polyvinyl alcohol (PVA), sodium dodesyl sulfate (SDS). These stabilizers become the inhibitor of the oxidation reaction and prevention of the particles accumulation. Moreover, they also control size of nano selenium.

Table 1 Some examples of size controlled synthesis of selenium nanostructures. (Asere, 2013)

Structure Reagents Stabilizer Advantage

Simple and large- Ultra-long wire SeO and ammonia No template 2 scale production

Trigonal Se Uniform t-Se Na SeSO PVA nanorod bundles 2 3 nanorods

Spherical Se Na SeSO and 2 3 PVA Ambient conditions nanoparticles organic acids

Trigonal Se Simple and no Na SeSO and H N Hydrazine nanorods 2 3 2 4 stabilizer

H2SeO3 and Polysacchar Very stable in Se nanoparticles ascorbic acid ides solution

Na2SeO3 and Good stability of Se Se nanoparticles L-cysteine L-cysteine nanoparticles

Amorphous Se SeO2 and . SDS Room temperature nanoparticles Na2S2O3 5H2O

Trigonal Se Glucose and Large scale and nanowires and No template environment- nanotubes Na2SeO3 friendly

Single- crystalline Se SeO2 and NaBH4 CMC Room temperature nanorods

(NH ) S O and High yield and low Se nanowires 4 2 2 3 SDS Na2SeO3 reaction temperature

Table 1 (Continued)

Se nanoparticles Na2SeO3 and CNXL CNXL Mild condition

Food surfactants Se nanoparticles Rapid and mild SeO and KBH (e.g.; and nanorods 2 4 condition potassium sorbate)

Easily scalable and Se nanoparticles H SeO and TSA TSA 2 3 long-term stability

(Gao, X. et al., 2002) used the beta-mercaptoethanol as a reducing agent to synthesize the selenium nanoparticles (HSSN) at the size 32 nm. The synthesis used the mixed stabilizer by Li et al. (2009). It was discovered that using hydrochloride with sodium dodecyl sulfate and poly vinyl alcohol to stabilize the system can produce the selenium nanoparticles at 30 nm.

(Zhang et al., 2004) reported the synthesis of selenium nanoparticles by using seleneous acid as the substrate and reacted with an ascorbic acid used as a reducer in many stabilizers such as polysaccharide like chitosan (CTS), Konyag Glucomanman (KGM), acasia (ACG), and carboxymethyl cellulose (CMC). They found that the stability of nano-selenium in different solution were varied.

(Lin et al., 2004) revealed the synthesis of selenium nanoparticles by using chemical reduction. They use the selenous acid as a substrate which can be prepared from selenium dioxide, and use SDS as the anionic surfactant with temperature control to be at 80 °C. The reaction was set at 30 s, 60 s, 90 s, 150 s, and 210 s. The reactions were quenched by freezing at the iced pool and characterized by TEM, X- ray (EDS). The diameter of nano particles produced are at 32.0 ± 8.4 nm (30 s), 39.6 ± 10.1 nm (60 s), 73.6 ± 12.6 nm (90 s), 93.1 ± 6.8 nm (150 s), and 188.8 ± 12.7 nm (210 s), respectively. Moreover, The investigation found that they are pure Selenium.

(Shin et al., 2007) prepared the selenium nanoparticles with the dimeter 10- 20 nm by using cellulose nanocrystal (CNXL) as a reducer and used sodium selenite as the substance. The reaction was carried out under hydrothermal condition (120-160 °C). The temperature of the reaction affected to the size of selenium nanoparticles. This was confirmed by physical property analysis using X-ray, TEM and FESEM to identify the character of the selenium nanoparticles on the surface of CNXL. 22

(Li et al., 2010) reported the synthesis of selenium nanoparticle starting from sodium selenite as the starting material and use L-Cysteine as the surfactant reducer in the 1:4 ratio for synthesis the selenium nanoparticles in round shape with the size 100 nm.

(Barnaby et al., 2013) synthesized the round shape nano selenium at the size more than 500 nm by employing dithiothreitol and gallic acid as a reducer and used sodium selenite as the substance. The transformation was controlled in the pH range of 5-7. With this method, nano fabric at the size of 50 - 57 nm was obtained. At pH 5, the color of gallic acid was changed to be yellow and orange, and change to dark brown at pH 7. This phenomenon can indicate the changing of shape and size of the selenium nanoparticles.

(Dwivedi et al., 2011) developed the synthesis based on chemical reduction to synthesize selenium nanoparticles (size 40-100 nm) by using sodium seleno sulphate as the substance in organic solvent. Poly vinyl alcohol has been used as the stabilizer. The color of nanoparticles were changed to orange-red in a few minutes. However, in case of poly vinyl alcohol, the dark red crystalline of selenium was obtained, which can be measured by UV-visible, X-ray, and TEM. This method was simple and convenience.

(Avendano et al., 2016) synthesized the nano particle of selenium from selenite by using Pseudomonas putida KT2440. The result shown that P. putida can reduce selenite but cannot reduce selenate. Moreover, the measurement by the TEM showed that the size of selenium nanoparticles were in range of 100 – 500 nm. With DLS technique confirmed, the size of selenium nanoparticles were in range of 70-300 nm. From this study, it can be stated that the Pseudomonas putida KT2440 can be used to synthesize the selenium nanoparticles.

(Li et al., 2010) studied the synthesis of selenium nanoparticles by using selenite glycerin as the substance and glucose as the reducing agent. The particles were characterized by X-ray diffraction (XRD), UV-Vis absorption spectroscopy, and High-Resolution Transmission Electron Microscope (HRTEM). Glycerin can help to control the size of the particles and cause the particles become more stable. From this experiment, the selenium nanoparticles 2-6 nm were obtained, which is smaller than the best value (more than 20 nm) can be produced. This size range (2-6 nm) was the best report ever.

(Ashwini et al., 2019) studied the synthesis of nano-selenium was achieved from sodium selenite by a simple precipitation method using the reducing power of ascorbic acid. The high-speed centrifuge was used to separate selenium nanoparticles from aqueous solution. Some of the important human pathogens like Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were used for examining the 23

antibacterial response of selenium nanoparticles. Results of this study demon- strate that synthesized selenium nanoparticles exhibit a spherical shape with average diameter range between 15 and 18 nm. They can be used as an antibacterial agent and also in medicinal applications for the treatment of humans with certain bacterial diseases.

(Gangadoo et al., 2017) studied simple and reproducible solution phase synthesis approach for selenium nanoparticles by reducing selenium tetrachloride in the presence of ascorbic acid. An optimized condition with poly (sodium 4- styrenesulfonate) produced stable and spherical narrowly size distributed nanoparticles (46 nm), which are considered highly monodisperse. The presence of selenium nanoparticles was confirmed by UV-visible spectroscopy for surface plasmon resonance (262 nm), elemental dispersive spectroscopy (11 KeV and 12.5 KeV) and size ranges characterized by dynamic light scattering (PDI D 0.04, size range of optimized nanoparticles D 35 nm to 75 nm), and visualized using scanning and transmission electron microscopy.

(Jadhav and Khanna, 2015) studied one-pot microwave assisted synthesis of selenium nanoparticles by decomposition of cycloocteno-1,2,3-selenadiazole in the presence of oleic acid and diphenyl ether. Three different forms of Se nanoparticles i.e. red, black amorphous and crystalline red were synthesized by varying the reaction time with microwave irradiation. The formation of Se nanoparticles from such a microwave reaction was monitored by UV-Visible spectroscopy at specific intervals. X-ray diffraction (XRD) measurement and Raman spectroscopy. They confirmed that trigonal crystal structure of the Se nanoparticles so-isolated. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed the globular morphology of the red and black Se particles. The particle size distribution was found to be between 1–20 nm for red Se. Thermogravimetric analysis (TGA) revealed a decomposition range between 180–240 ºC leading to the possibility of interconversion of the crystal structure. A %wt loss between 94-99% was observed for various forms of Se.

(Srivastava and Mukhopadhyay, 2013) synthesized the selenium nanoparticles with the size range of 30 nm to 150nm. The formation of selenium nanorods (trigonal) was also observed. XRD and SAED analysis revealed that synthesized selenium nanoparticles were selenium crystal of the pure hexagonal phase. A hypothetical mechanism for biological synthesis of selenium nanoparticles was also proposed.

(Kokila et al., 2017) synthesized the selenium nanoparticles (Se-NPs) was done by a simple precipitation method using the aqueous extract of Diospyros montana. The leaf extract to be worked as good capping and stabilizing agent with the 24

formation of stable nanoparticles. TEM and DLS analysis was determined by the size of Se-NPs ranges from 4 to 16 nm. The zeta potential was obtained at -22.3 mV. DPPH and reducing power activity showed the potential antioxidant property of the biosynthesized Se-NPs. These nanoparticles suspension exhibit more significant antimicrobial activity against both microorganisms such as Gram (+) Staphylococcus aureus, Gram (-) Escherichia coli (bacteria) and Aspergillus niger (fungai). The cytotoxicity of Se-NPs was assayed against human breast cancer cells (MCF-7). The anti cancer activity was found that Se-NPs are able to inhibit the cell growth by dose- dependent manner.

(Kannan et al., 2014) synthesized nanorods of one-dimensional (1D) trigonal selenium (t-Se) using biomolecule substances for five different aging times (1 h, 2 h, 3 h, 1 day and 4 days) by precipitation method. XRD analysis indicates a shift of the (1 0 1) plane towards higher diffraction angle for 1 day aging time. It is observed that the crystallite size decreases with increase in aging time except for an aging period of 4 days. FTIR analysis confirmed that the presence of stretching and bending vibrations of Se–O in both synthesized and commercial selenium samples are the same at 465, 668 and 1118 cm−1. The FESEM micrographs are evident for the changes of rod size as a function of aging time. It is observed that the optical band gap energy is increased with aging time up to 1 day, whereas it decreases in 4 days aging time.

MATERIALS AND METHODS

Materials

1. UV-VIS Spectrophotometer (T80 UV/Vis Spectrometer, Japan) 2. Oven (ED53 WTB Binder, USA) 3. Analytical Balance (Mettler Toledo Newclassic MF, Switzerland) 4. Rotary shaker GFL 3005 (Lab Unlimited Carl Stuart Group. USA) 5. Micropipette (PIPETMAN® neo) (GILSON, USA) 6. Fourier transform Infrared Spectrometer (FT-IR) from Perkin Elmer 7. Block digestion 8. Dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS.) 9. Transmission electron microscopy (TEM) (JEM-2100 Plus; JEOL, Tokyo, Japan) 10. Graphite Atomic Absorption Spectrometer (GFAAS) (Varian, SpectraAA) 11. Rotary evaporator (Buchi, Switzerland) 12. Freeze Dryer Coolsafe (100-90 Pro, Denmark) 13. Microplate reader (Biotek Synergy H1, VT, USA)

Chemicals 1. Sodium selenite from sigma Aldrich, Inc. (St. Louis, MO, USA) 2. Ascorbic acid from Ajax Finechem Pty Ltd., Australia. 3. Selenium dioxide from sigma Aldrich, Inc. (St. Louis, MO, USA) 4. Sodium dodecyl sulfate from Ajex Finechem Pty Ltd., Australia. 5. Hydrochloric from Ajex Finechem Pty Ltd., Australia. 6. Sodium-metabisulfite 7. Chitosan from Ajax Finechem Pty Ltd., Australia. 8. Poly-γ-glutamic Acid 9. Nitric acid from Ajex Finechem Pty Ltd., Australia. 10. Hyperchloric acid from Ajex Finechem Pty Ltd., Australia. 11. Sodium selenite from sigma Aldrich, Inc. (St. Louis, MO, USA) 12. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) from Sigma-Aldrich, Missouri, USA 13. 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) from Sigma-Aldrich, Missouri, USA 14. Potassium persulfate from Sigma-Aldrich, Missouri, USA 15. (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) from Sigma-Aldrich, Missouri, US

METHODS

1. The synthesis of selenium nanoparticles by using sodium selenite as a starting material. The synthesis of selenium nanoparticles (Li et al., 2010)

1.1. Sodium selenite (Na2SeO3) was used for the synthesis of Se nanoparticles where ascorbic acid (C6H8O6) acted as a reducing operator. A stock of aqueous solution of (25mM : 100 mM, 50 mM : 100mM, 100 : 50mM, 200mM : 50mM) ascorbic acid was prepared in 1 : 4 ratio. The ascorbic acid was dropwisely added to the sodium selenite solution. The reaction mixture was kept under stirring and at ambient temperature for 30 min. The mixture was allowed to react in the concentrated form until a color change from colorless to red color. After completion, the mixture was diluted to 25 mL by MilliQ water then the stability of SeNPs was studied by aside at room temperature at 1, 7, 22 and 30 days.

Figure 8 The probable mechanism involved in the formation of nanoparticles (Se0)

1.2. The best ratio of sodium selenite per ascorbic acid providing the highest size of SeNPs was selected to further study the effect of the volume portion of Sodium Selenite per Ascorbic acid, the volume of water to stop the reaction, and the reaction time that effect the size of the selenium nanoparticles.

2. The synthesis of selenium nanoparticles. The synthesis of selenium nanoparticles (Lin et al., 2004) was achieved by using selenous acid as the substance with 5.4 mM concentration, which could be prepared from the selenium dioxide dissolving in water.

SeO2(s) + H2O(l) → H2SeO3(aq) To the selenous acid solution (10 mL), sodium dodecyl sulfate (SDS) was added drop wisely and stirred. The final concentration of SDS was kept to 10 mM. Then, sulfur dioxide (SO2), the reducing agent, which can be prepared from sodium metabisulfite (Na2S2O5)was added. The pH of the solution was adjusted to be 3.2 by 27

adding conc. hydrochloric acid approximately 40 µL. the final concentration of sodium-metabisulfite was maintained at 5.4 mM.

Na2S2O5(s) + H2O → 2NaHSO3(aq) + + NaHSO3(aq) + H → Na + H2O(l) + SO2(aq) The reaction was kept at 40, 60, 80 °C for 30 s and then was quenched by cooling the reaction with iced water since the redox reaction are slowly occurred at ambient temperature. Reaction equation

2- + H2SeO3(aq) + 2SO2(aq) + H2O(l) → Se(s) + 2SO4 (aq) + 4H Half reaction + H2SeO3 +4H +4e → Se+3H2O 2- SO2 +2H2O → SO4 +4H+ +2e

3. The synthesis of selenium nanoparticles encapsulated with chitosan

Cs-SeNPs were synthesized by the reduction of selenite according to (Bai et al., 2017). 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 µM of chitosan and 0.4 g of ascorbic acid were dissolved in 25 mL of 1% (w/w) acetic acid. Next, selenite solution (0.4g in 10 mL of water) was added into the mixture of chitosan and ascorbic acid solution. After the reaction completed, the mixture was diluted to 25 mL using MilliQ water. The resulting solution was then mixed to obtain the colloidal solution of Cs-SeNPs. This nano-particle can be kept at room temperature then study the stability of CS-SeNPs by aside at room temperature at 1, 7, 22 and 30 days.

4. The synthesis of selenium nanoparticles encapsulated with PGA

PGA-SeNPs were synthesized by the reduction of selenite. 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 mM of PGA and 0.4 g of ascorbic acid was dissolved in MilliQ water. Then, selenite solution (0.4g in 10 mL of water) was added into the mixture of PGA and ascorbic acid solution. After the reaction completed, the mixture was diluted to 25 mL using MilliQ water. The resulting solution was then mixed to obtain the colloidal solution of PGA-SeNPs. This nanoparticle can be kept at room temperature. This nano-particle can be kept at room temperature then study the stability of SeNPs by aside at room temperature at 1, 7, 22 and 30 days.

The analysis of the nanoparticles Characteristics The characterization of the synthesized selenium nanoparticles was achieved by several techniques such as UV-Vis spectrophotometer, Dynamic light scattering (DLS), Fourier transform Infrared Spectrometer (FT-IR) and Transmission electron microscopy (TEM) 28

TEM TEM (JEM-2100 Plus; JEOL, Tokyo, Japan) was applied to study the morphological characteristics of SeNPs, Cs-SeNPs and PGA-SeNPs. Briefly, diluted SeNPs, Cs-SeNPs and PGA-SeNPs solutions were dropped onto copper grids and dried in clean air. TEM observation was carried out at an accelerating voltage of 200 kV.

UV-Visible analysis

The UV–vis spectroscopy was performed on the UV-vis spectrometer (T80 UV/Vis Spectrometer, Japan) in the spectral region of 200–400 nm, using aqueous samples in 1 cm width quartz cuvettes.

FTIR

In the FT-IR spectrum, the samples were determined by using the model of Perkin-Elmer in the transmittable mode at the ranges from 4000–400 cm−1

DLS

The particle size and size distribution of the selenium nanoparticle sample was analysed by dynamic light scattering using the Malvern Zetasizer Nano ZS. A volume of 1 mL of the SeNP solution in 2 mL of Milli-Q water was placed in a polystyrene cuvette and measured at 25 ºC. Measurements of size and polydispersity index (PDI) were obtained for both freshly synthesized SeNPs and washed nanoparticles in triplicate. The particle size was given as mean and standard error.

5. The effect of SeNPs, Cs-SeNPs and PGA-SeNPs uptake in sunflower sprout

Plan the Completely Randomized Design (CRD) by selecting sunflower seeds from the farm. Pathum Thani province that is completely disease free and insect divides the seeds into 7 groups then seed is soaked in the solution SeNPs, CS-SeNPs, PGA-SeNPs, Control, Control PGA (C-PGA), Control Chitosan (C-Cs), Control Selenite (C-Se) at concentration 1000 ppm

5.1 Germination and sample preparation

Each 4 g of sunflower sprout samples were soaked in SeNPs, PGA- SeNPs, Cs-SeNPs, C-Cs, C-PGA, C-Se, and control solution at 1000 ppm for 8 h in the corresponding solutions (200 mL). This was done in quadruple, giving 28 samples. Samples were then incubated in plastic container and placed on moist at room temperature for 24 h prior to be cultivated in plastic tray containing rice husk ash: coconut coir 1:1 at 30±5 °C and 65-70 % relative humidity for 7 days. During germination, the sprouts were rinsed two times each day with 250 mL of deionized 29

water. After harvesting, all sprouts were washed carefully with deionized water to exclude contamination of the surface of the sprouts by the cultured solution. Sample were then dried in hot air oven at 45 °C for 24 h. Seedlings were randomly sampled every day to determine percentage of seed germination, shoot lengths, root lengths determination antioxidant activities and total selenium concentrations.

5.2 Sunflower sprout digestion procedure

Sunflower sporout sample 0.2 g was weighed in 100 ml digestion tube covered with plastic wrap, then carefully introduced 10 mL of HNO – HClO (1:4 3 4 v/v) and kept at room temperature overnight. The digestion tube was heated on digestion block for 1 hours at 100°C, 2 hours at 130 °C, 1 hours at 180°C and 1 hours at 210°C. After cooling, added 5 mL of HCl in digestion tube covered with plastic wrap and kept at room temperature overnight. The sample was diluted with 50 mL of Milli-Q water. The samples were filtered through a Whatman filter paper (no.42) and measured with graphite atomic absorption spectrometer.

GFAAS operating conditions

GFAAS (Varian, SpectraAA) Operating conditions for GFAAS measurements were; Wavelength = 196.0 nm, Lamp current = 10.0 mA, slit width = 1.0 nm, integration time = 2.5 sec. flow rate = 750 ml/min, carrier gas = Ar and Se standard solutions where 30, 60 and 90 ppb.

6. DPPH and ABTS free radical scavenging activity assays of sunflower sprout

Samples of sunflower sprout were then dried in hot air oven at 45 °C for 24 h. Crushed into a fine powder and stored in a plastic bag at -20°C

6.1 Extraction of sunflower sprout

Soaked of the sunflower sprout in 95% ethanol in the ratio of 1:3 for 72 hours and stir for every 4 hours. Filter to gain transparent solution. Evaporate solution with rotary evaporator at 30 °C and dry with freeze dry.

6.2 DPPH radical scavenging activity

The DPPH radical scavenging activity (Brand-Williams and Berset, 1995) of the samples was determined based on the reduction of a stable radical, 2,2- Diphenyl-1-picrylhydrazyl (DPPH) by an antioxidant of a radical species resulting in decolorization of purple into yellow color. Briefly, various ethanol dilutions of samples were mixed with 0.1 mM DPPH ethanol solution in 96 well plates and incubated at room temperature for 30 minutes. Absorbance was measured with a 30

microplate reader at the wavelengths of 517 nm and the percentage of inhibition was calculated using the following (Mensor et al., 2001) equation :

Percent inhibition = 100 – [(Absample – Abblank) x 100/ Abcontrol];

Absample = the absorbance of the sample Abblank = the absorbance of the blank Abcontrol = the absorbance of the control (solvent without sample)

Ascorbic acid was used as a positive control. IC50 value was calculated by linear regression of plots where the concentration inhibit 50% of DPPH free radicals. The experiment was performed in four replicates

6.3 ABTS radical scavenging activity

The ABTS radical scavenging activity (Re et al., 1999)of the samples was determined based on the reduction of the preformed radical mono-cation of 2,2′- azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS radical) by an antioxidant resulting in decolorization of blue-green color. Briefly, ABTS radical solution was generated by oxidation of 7 mM ABTS with 2.45 mM potassium persulfate for 18 hours. ABTS radical solution was diluted with ethanol and mixed with various ethanol dilutions of samples in 96 well plates and incubated at room temperature for 5 minutes. Absorbance was measured with a microplate reader at the wavelengths of 750 nm and the percentage of inhibition was calculated using the following equation:

Percent inhibition = 100 – [(Absample – Abblank) x 100/ Abcontrol];

Absample = the absorbance of the sample, Abblank = the absorbance of the blank Abcontrol = the absorbance of the control (solvent without sample)

(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was used as a positive control. IC50 value was calculated by linear regression of plots where the concentration inhibit 50% of DPPH free radicals. The experiment was performed in four replicates.

RESULTS AND DISCUSSION

1. Synthesis of selenium nanoparticle with sodium selenate as initial substance

Table 2 Sizes of synthesized selenium nanoparticle using different volume of sodium selenate to ascorbic acid.

volume of sodium PDI Size(nm) selenate :ascorbic acid 1:1 0.4047 225.6±5.7 1:2 0.3437 307.5±17.8 1:3 0.4603 390.5±24.9 1:4 0.2217 184.3±5.9 1:5 0.3427 279.9±22.2

The result from size measurement of the selenium nanoparticle show in Table2 found that the ascorbic acid that increased from 1 to 2 and 3 portions caused the size of selenium nanoparticle increased from 225.6 nm to 307.5 and 390.5 nm, respectively. However, the size would be smaller when the ascorbic acid increased to 4 portions and the size of selenium nanoparticle was 184.3 nm. It can be seen that when ascorbic acid concentration is higher, the final volume of solution will increase, resulting in better distribution and the particles are smaller. However, particles size probably change by various conditions such as heat, pH, reaction time, and stirring effect of the reaction. In addition, chemical components of particles are a part of such change (Xia, Y. et al., 2015). Sodium selenate to ascorbic acid in the ratio of 1:4 give the best particle distribution and also found that the average size of selenium nanoparticle is 184.3 nm.

450.0 400.0 350.0 300.0 250.0

Size (nm) Size 200.0 150.0 100.0 50.0 0.0 1:1 1:2 1:3 1:4 1:5 volume of sodium selenate : ascorbic acid

Figure 9 Sizes of selenium nanoparticle synthesized on the basis of different ratios of sodium selenate to ascorbic acid.

2. Synthesis of selenium nanoparticle with sodium selenite as initial substance

The study on effect of sodium selenite on ascorbic acid at different concentration (25mM : 100 mM, 50mM : 100 mM, 100mM : 50 mM, 200mM : 50 mM, 50mM : 25 mM) using water as solvent with sodium selenate to ascorbic acid in the ratio of 1 : 4 found that at the ratio of 200mM : 50mM, clear solution in orange red color was obtained, at the ratio of 100mM : 50mM and 25mM : 100mM, orange red color solution was obtained, at the ratio of 50mM : 25mM and 50mM : 100mM, dark orange red solution and a small amount of sediment were obtained. After the reaction was finished, the pH of solutions were determined.

Table 3 Sizes of selenium nanoparticle synthesized on the basis of different ratios of sodium selenite to ascorbic acid.

Concentrations of sodium pH PDI Size(nm) selenite : ascorbic acid

25:100 3 0.145 109.2±3.4

50:100 4 0.195 110.7±0.8

100:50 5 0.050 129.1±0.9

200:50 6 0.205 175.7±2.6

50:25 5 0.226 128.8±1.9

200 180 160 140 120 100 80 Size (nm) Size 60 40 20 0 25:100 50:100 100:50 200:50 50:25 Concentration of sodium selenite : ascorbic acid

Figure 10 Sizes of selenium nanoparticle synthesized on the basis of different ratios of sodium selenite to ascorbic acid.

The study result revealed that different concentration of sodium selenite to ascorbic acid caused different sizes of particles show in Table 3. A higher concentration of sodium selenite resulted in a bigger size of selenium nanoparticle and it would become smaller when concentration of ascorbic acid increased while the concentrations of sodium selenite to ascorbic acid are in the ratios of 25mM : 100mM, 50mM : 100mM, 100mM : 50mM, 200mM : 50mM, 50mM : 25mM for giving average sizes of selenium nanoparticles as 109.2, 110.7, 129.1, 175.7, and 128.8 nm respectively. (Gangadoo et al., 2017). It was found that high concentrations of ascorbic acid produced a clear red solution containing smaller nanoparticles, while lower concentrations of the reducing agent resulted in a cloudy, orange colored solution with larger nanoparticles size. Different effects from different concentrations of reducing agents have been observed in other papers such as (Henglein, 1999). It was found that high concentrations of a reducing agent can destabilize the nanoparticle causing the formation of irregular shapes while low concentrations of the reducing agent caused aggregates of the nanoparticles, from large particle sizes. (Kong et al., 2014) studied the synthesis and antioxidant properties of gum arabic- stabilized selenium nanoparticles. It was found that when increasing the concentration of gum arabic-stabilized can increase the size of selenium nanoparticles. Based on the pH results shown in the Table 3, it can be seen that lower concentrations of sodium selenite and higher concentrations of ascorbic acid contribute to the pH of solution is more acidic since a quite large amount of ascorbic acid left in the reaction when compared to increasing concentrations of sodium selenite and decreasing concentrations of ascorbic acid (Barnaby et al., 2013). A report showed that selenium nanoparticle has different stability in different pH and it 34

is found that at pH 6-7, it will be more stable and precipitate more slowly at other pH like pH 3 or pH 9. Besides, a study was conducted on stability of SeNP based on the ratio of sodium selenite to different concentration of ascorbic acid as 25mM : 100mM, 50mM : 100mM, 100mM : 50mM, 200mM : 50mM, 50mM : 25mM by leaving them for 7 days and 1 months and measuring the size of selenium nanoparticle by dynamic light scattering (DLS). The experimental results is shown in the Table 4 and Table 5. Table 4 Sizes of selenium nanoparticle synthesized on the basis of the ratios of sodium selenite to ascorbic acid with different concentrations when being kept for 7 days.

Concentrations of sodium PDI Size(nm) selenite : ascorbic acid 25:100 0.218 143.0±3.1 50:100 0.294 176.8±11.1 100:50 0.135 171.2±8.9

200:50 0.161 198.8±2.0 50:25 0.259 152.2±10.8

Table 5 Sizes of selenium nanoparticle synthesized on the basis of the ratios of sodium selenite to ascorbic acid with different concentrations when being kept for 22 days.

Concentrations of sodium PDI Size(nm) selenite : ascorbic acid 25:100 0.234 300.0±2.3 50:100 0.295 299.4±4.5

100:50 0.204 234.5±5.4

200:50 0.290 280.1±4.5 50:25 0.304 223.2±10.3

Table 6 Sizes of selenium nanoparticle synthesized on the basis of the ratios of sodium selenite to ascorbic acid with different concentrations when being kept for 1 month.

Concentrations of sodium PDI Size(nm) selenite : ascorbic acid

25:100 0.218 328.8±9.2 50:100 0.294 312.3±4.3 100:50 0.135 272.8±4.1 200:50 0.161 281.6±3.1 50:25 0.259 230.8±15.8

The study result of the stability by leaving it at room temperature for 1, 7, 22 and 30 days found that sizes of selenium nanoparticle were bigger on day 7 and day 30. For the concentration of sodium selenite to ascorbic acid in the ratio of 200 : 50, the size was bigger on day 30 and the concentrations of sodium selenite to ascorbic acid in the ratios of 25mM : 100mM, 50mM : 100mM, 100mM : 50mM, 200mM : 50mM, and 50mM:25mM gave the sizes of selenium nanoparticle on day 7 averagely 143.0, 176.8, 171.2, 198.8 and 152.2 nm, respectively. When it was left for 30 days, it was found that the sizes of selenium nanoparticle increased averagely 328.8, 312.3, 272.8, 281.6 and 230.8 nm respectively. Selenium nanoparticles were bigger due to the combination of particles and precipitation. The experimental result indicated that the suitable concentration of sodium selenite to ascorbic acid was 200mM : 50mM.

400 350 300 25:100 250 50:100 200

150 100:50 Size (nm) Size 100 200:50 50 50:25 0 1 7 22 30 Time (Day)

Figure 11 Sizes of selenium nanoparticle synthesized of the ratios of sodium selenite to ascorbic acid with different concentrations when being kept for 7, 22 days and 1 months. 36

A study was conducted on effect of selenium nanoparticle sizes at the 200 mM concentration of sodium selenite to 50 mM concentration of ascorbic acid. The ratios of sodium selenite to ascorbic acid volume were 1:1, 1:2, 1.3, 1.4 and 1:5 in the reaction. Stir at room temperature for 30 minutes and add 25 milliliters of DI water. It was found that after the reaction was stopped, the ratios of sodium selenite to ascorbic acid volume 1:1, 1:2, and 1.3 gave light orange color solution, the ratio of sodium selenite to ascorbic acid volume 1:4 gave orange color solution, and the ratio of sodium selenite to ascorbic acid volume 1:5 gave auburn color solution as shown in Figure 11. The results were measured with DLS as seen in the Table 6.

a b c d e

Figure 12 Solution colors of solution after selenite interacted with ascorbic acid after 30 minutes a. 1:1, b. 1:2, c. 1:3, d. 1:4, e. 1:5

Table 7 Sizes of selenium nanoparticles synthesized on the basis of the ratios of different volume of sodium selenite to ascorbic acid.

volume of sodium PDI Size(nm) selenite : ascorbic acid 1:1 0.164 160.7±2.9

1:2 0.194 175.1±11.4

1:3 0.141 178.6±0.7 1:4 0.177 207.3±2.0 1:5 0.133 270.2±4.7 37

300

250

200

150 Size (nm) Size 100

0 1:1 1:2 1:3 1:4 1:5 volume of sodium selenite : ascorbic acid

Figure 13 Sizes of selenium nanoparticles synthesized on the basis of the ratios of different volume of sodium selenite to ascorbic acid.

The ratios of sodium selenite to ascorbic acid volume were 1:1, 1:2, 1.3, 1.4 and 1:5 for giving average sizes of selenium nanoparticles as 160.7, 175.1, 178.6, 207.3 and 270.2 nm respectively. The size measurement of selenium nanoparticle found that the increasing ratios of ascorbic acid affect on sizes of selenium nanoparticles and concentrations of ascorbic acid. However, sodium selenite to ascorbic acid in the ratio of 1:4 was expected to be the ratio giving suitable reaction condition. Therefore, such condition is brought to study in terms of duration of reaction in order to study the effects of selenium nanoparticles sizes based on 15, 30, 60, 90, and 120 minutes. Study result is shown in Table 7.

a b c d e

Figure 14 Colors of solution after selenite interacted with ascorbic acid at different times a. 15 minutes, b. 30 minutes, c. 1 hour, d 90 minutes. e. 2 hours.

Table 8 Sizes of selenium nanoparticle synthesized at different reaction times.

Time (Sec) PDI Size (nm)

15 0.239 242.9±4.2 30 0.249 194.1±4.6

60 0.214 163.4±2.5 90 0.204 198.1±4.1

120 0.207 233.3±6.7

300

250

200

150

Size(nm) 100

0 15 30 60 90 120 Time(Sec)

Figure 15 Sizes of selenium nanoparticle synthesized at different reaction times.

The selenium nanoparticle were synthesized at different reaction times (15 minutes, 30 minutes, 1 hour, 90 minute and 2 hours) for giving average sizes of selenium nanoparticles as 242.9, 194.1, 163.4, 198.1 and 233.3 nm, respectively. The experimental result revealed that sizes of the particles were not different of reaction was not much different and the synthesis may occur very fast. Thus, duration on the synthesis did not have any effect on selenium nanoparticle sizes. The obtained selenium nanoparticle sizes were not much different. The study was conducted on volume of water used to stop reaction. Different volumes of water were used to see whether or not water has an effect on selenium nanoparticle sizes. 10, 25, 50, and 100 milliliters of water were used to stop reaction as shown in Table 8 and Figure 15.

Table 9 Sizes of selenium nanoparticle synthesized at Different volumes of water 10, 25, 50, 100 mL

Volume of DI PDI Size(nm) water (mL)

10 0.157 245.2±7.7

25 0.148 202.9±10.0

50 0.171 201.1±4.8

100 0.168 200.1±9.6

300

250

200

150

Size(nm) 100

0 10 25 50 100 Volume of DI(mL)

Figure 16 Sizes of selenium nanoparticle synthesized at different volumes of water 10, 25, 50, 100 mL

The selenium nanoparticle synthesized at different volumes of water 10, 25, 50, 100 mL for giving average sizes of selenium nanoparticles as 245.2, 202.9, 201.1 and 200.1 nm, respectively. It was found that water helps to make selenium particles better dispersed. Therefore result in a reduced PDI.

A study was conducted on duration of reaction to selenium nanoparticle sizes by adding water to stop reaction as per scheduled times 2, 4, 6, 8, and 10 days, as days passed by, had an effect on selenium nanoparticle sizes as shown in Table 9 and Figure 16.

Table 10 Selenium particle sizes synthesized by adding water for stopping reaction as per scheduled times 2, 4, 6, 8, and 10 days.

Time(Day) PDI Size(nm)

2 0.150 191.3±10.8 4 0.232 328.5±18.5 6 0.155 200.6±12.8

8 0.224 307.4±12.5

10 0.198 369.47±9.6

400 350 300 250 200

150 Size(nm) 100 50 0 2 4 6 8 10 Time(Day)

Figure 17 Selenium particle sizes synthesized by adding water for stopping reaction as per scheduled times 2, 4, 6, 8, and 10 days.

The study showed that duration of reaction to selenium nanoparticle sizes by adding water to stop reaction as per scheduled times 2, 4, 6, 8, and 10 days, as days passed by, had an effect on selenium nanoparticle sizes. It was found that average sizes of SeNPS were 191.3, 328.5, 200.6, 307.4, and 369.47 nm, respectively. The experimental result indicated that water could help improve selenium nanoparticle distribution and easily precipitate.

3. Synthesis of selenium nanoparticle

Synthesis of selenium nanoparticle was cited from (Lin et al., 2004) using selenous acid as initial substance and sulfur dioxide (SO2) as a reducing agent. Reaction was performed at different temperature, i.e. 40, 60, and 80 C for 30 seconds. The results are shown in Table 10. Table 11 Selenium nanoparticle sizes at 40, 60, and 80 ºC.

Temperature( ºC) PDI Size(nm) 40 0.540 193.9±6.7

60 0.519 246.6±12.9

80 0.223 120.4±5.8

300

250

200

150

100 Size(nm)

0 40 60 80 temperature(°C)

Figure 18 Selenium nanoparticle sizes at 40, 60, and 80 ºC.

Based on the experimental results, it can be seen that PDI was greater than 0.3 and had more than one peak. Peaks of 40 ºC and 60 ºC are shown in Figure 18. Thus, z-average could not be selected and sizes were chosen from peaks instead. The experimental results showed that 80 ºC was stable temperature gave rise to one peak as seen in Figure 19. It was expected that reaction was completely performed at 80 ºC and at 80 ºC also low PDI values were given at this temperature. At 40 and 60 ºC, as seen in the figure, gave several peaks with various sizes. Therefore, 40 ºC and 60 ºC were not stable. (Lin et al., 2004) conducted a study on synthesis of selenium nanoparticles using sodium dodecyl sulfate (SDS) as a stabilizer. It was found that 80 ºC was appropriate temperature for the synthesis. Figure 18 shows 100 nm selenium nanoparticle from DLS. 42

Figure 19 Shows 100 nm selenium nanoparticle measured at 80 ºC from DLS.

Figure 20 Shows 100nm selenium nanoparticle measured at a. 40 ºC and b. 60 ºC from DLS.

4. Synthesis of chitosan-coated selenium nanoparticles The experiment was performed using long chain 750 kD chitosan to study different chitosan concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 µM). The result is shown in Table 11.

Table 12 Chitosan-coated selenium nanoparticle sizes.

Concentrations of PDI Size(nm) chitosan(µM)

0.1 0.206 244.57±5.3

0.2 0.277 232.70±9.5

0.3 0.380 347.77±12.2

0.4 0.273 373.30±14.53

0.5 0.361 377.17±2.35 0.6 0.213 381.37±3.74

450 400 350 300 250

200 Size(nm) 150 100 50 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of chitosan(µM)

Figure 21 Chitosan-coated selenium nanoparticles with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 µM

The study on synthesis of chitosan-coated selenium nanoparticles with different concentrations found that an increase in concentrations of chitosan made an increase in selenium nanoparticle sizes. Concentrations at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 µM gave rise to average sizes of selenium nanoparticle as 244.57, 232.70, 347.77, 373.30, 377.17, and 381.37 nm, respectively. This is consistent with the study conducted by (Chen et al., 2015) on coating selenium with chitosan and carboxymethyl chitosan. The study found that a higher concentration of chitosan and carboxymethyl chitosan led to an increase in selenium nanoparticle sizes. (Gangadoo 44

et al., 2017) It was found that if the protecting agent concentration is too high, a larger hydrodynamic diameter of a nanoparticles is usually experienced; surface charge of nanoparticle is increased as more electrons are added onto the surface resulting in the nanoparticle having a larger diameter. Some authors have shown that higher concentrations of protecting agent produce a larger particles size range along with various shapes: triangles, rods and cylinders rather than spherical- shaped nanoparticles (100 – 300 nm in size).

A study was conducted on stability of long chain chitosan-coated SeNPs with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 µM by leaving them for 7 days and 1 month and measured the sizes of selenium nanoparticle with DLS. The experimental result is shown in Table 12 and Table 13. Table 13 Sizes of chitosan - coated selenium nanoparticles after 7 days passed.

Concentrations of PDI Size (nm) chitosan (µM)

0.1 0.297 210.93±8.7

0.2 0.323 222.07±2.3

0.3 0.284 240.73±12.8

0.4 0.279 286.03±0.7

0.5 0.286 305.40±5.6

0.6 0.271 315.23±0.8

Table 14 Sizes of chitosan - coated selenium nanoparticles after 22 days passed.

Concentrations of PDI Size (nm) chitosan (µM)

0.1 0.243 199.6±6.7

0.2 0.345 200.4±7.8 0.3 0.209 277.8±10.4

0.4 0.223 350.7±4.5

0.5 0.304 400.8±6.7

0.6 0.233 456.8±0.2

Table 15 Sizes of chitosan - coated selenium nanoparticles after 1 month passed.

Concentrations of PDI Size (nm) chitosan (µM)

0.1 0.294 186.90±8.7

0.2 0.305 163.63±2.3

0.3 0.291 236.20±12.8

0.4 0.454 424.60±0.7

0.5 0.462 436.47±5.6

0.6 0.473 572.50±0.8

700

600

500 0.1 400 0.2 300 0.3 0.4 Size(nm) 200 0.5 100 0.6 0 1 7 22 30 Time(Day)

Figure 22 Chitosan-coated selenium nanoparticles with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 µM after being kept for 7, 22 days and 30 days.

The study result on the stability after leaving them at room temperature for 1, 7, 22 and 30 days found that chitosan-coated selenium nanoparticles with different concentrations 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 µM on day 7 had average sizes as 210.9, 222.1, 240.7, 286.0, 305.4, and 315.2 nm, respectively. On day 30 had average sizes as 186.9, 163.6, 236.2, 424.6, 436.5, and 572.5 respectively. Based on the earlier results, chitosan-coated selenium nanoparticle sizes slightly decreased on day 7 and day 30 at chitosan concentrations 0.1, 0.2 and 0.3 µM. The nanoparticle sizes increased on day 30 at chitosan concentrations .4, 0.5 and 0.6 µM. It was also found that at chitosan concentration 0.1 µM the highest stability was observed. The chitosan-coated selenium nanoparticle sizes at this concentration were not much different on day 1, 7, and 30. 46

The experimental result found that when time passed by chitosan-coated selenium particle sizes decreased. Chitosan and its hydrophobic derivatives had high electrostatic repulsions and bridging effects, causing particle pushing and independent distribution as loss microstructure. When they were left, selenium nanoparticles released easily. As days passed by, chitosan-coated selenium nanoparticles slightly decreased. From previous research studies, (Zhai et al., 2017) found that using long chain chitosan to coat selenium nanoparticles the particles from 350 nm and as 30 days passed the sizes decreased to 103 nm.

4.1 Synthesis of short chain chitosan-coated selenium nanoparticles

The experiment was performed using short chain 1526 Da chitosan to study different chitosan concentrations 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 µM. The result is shown in Table 14.

Table 16 Short chain chitosan-coated selenium nanoparticle sizes.

Concentrations of PDI Size (nm) chitosan (µM)

0.05 0.189 77.47±0.4

0.1 0.274 104.87±0.7 0.2 0.374 134.33±1.4

0.3 0.144 148.70±7.9 0.4 0.238 279.13±8.6

0.5 0.238 281.90±4.3

0.6 0.376 270.70±0.7

350 300 250 200

150 Size(nm) 100 50 0 0.05 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of chitosan(µM)

Figure 23 Short chain chitosan-coated selenium nanoparticle sizes. 47

The study on synthesis of short chain chitosan-coated selenium nanoparticles with different concentrations found that an increase in short chain chitosan concentrations made an increase in the nanoparticle sizes 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 µM. Average sizes of selenium nanoparticles were 77.47, 134.33, 148.70, 279.13, 281.90, and 270.70 nm, respectively. Moreover, a study was conducted on stability of short chain chitosan-coated SeNPs with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 µM by leaving them for 7 days and 1 month and measured the sizes of selenium nanoparticle with DLS. The experimental result is shown in Table 15 and Table 16. Table 17 Sizes of short chain chitosan-coated selenium nanoparticles after 7 days passed.

Concentrations PDI Size (nm) of chitosan (µM) 0.05 0.121 101.59±5.3 0.1 0.165 120.97±17.9 0.2 0.126 204.60±0.9 0.3 0.194 175.10±11.4 0.4 0.238 283.93±4.7 0.5 0.263 283.37±4.3

0.6 0.238 284.70±1.5

Table 18 Sizes of short chain chitosan-coated selenium nanoparticles after 22 days passed.

Concentrations PDI Size (nm) of chitosan (µM)

0.05 0.234 120.7±4.5

0.1 0.105 160.5±11.3

0.2 0.165 267.9±1.3 0.3 0.230 206.7±10.3

0.4 0.245 306.9±5.5

0.5 0.277 360.8±5.6 0.6 0.208 350.6±2.5

Table 19 Sizes of short chain chitosan-coated selenium nanoparticles after 1 month passed.

Concentrations of PDI Size (nm) chitosan (µM)

0.05 0.154 160.57±2.9

0.1 0.274 191.07±6.7

0.2 0.285 307.2±6.9

0.3 0.323 327.53±1.8

0.4 0.474 321.23±4.1

0.5 0.389 364.43±4.0

0.6 0.316 417.57±.8.3

450 400 0.05 350 0.1 300 250 0.2 200 0.3 Size (nm) Size 150 0.4 100 0.5 50 0.6 0 1 7 22 30 Time (Day)

Figure 24 Short chain chitosan-coated selenium nanoparticles with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 µM after being kept for 7, 22 days and 30 days.

The study result on the stability after leaving them at room temperature for 1, 7, 22 and 30 days found that short chain chitosan - coated selenium nanoparticles with different concentrations at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 µM on day 7 had average sizes as 101.6, 121, 204.6, 175.1, 284, 283, and 284 respectively and on day 30 had average sizes as 160.6, 191.1, 307.2, 327.2, 327.5, 321.2, 364.4 and 417.6 nm, respectively. On the previous mentioned earlier result, long chain chitosan-coated selenium nanoparticle size increased when left to day 7 and day 30 and it was found that when time passed by chitosan-coated selenium particle sizes increased. Short chain chitosan may result in a small amount of hydrophobic derivatives causing less particle pushing and combination. As days passed by, chitosan-coated selenium 49

nanoparticles slightly increased. From previous research studies, (Zhai et al., 2017) found that using short chain chitosan to coat selenium nanoparticles led to an increase in the particles from 50 nm and as 30 days passed the sizes increased to 103 nm.

5. Synthesis of PGA coated selenium nanoparticles

The experiment was performed using chain 20 kDa PGA to study different concentrations of PGA (0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 mM). The result is shown in Table 17.

Table 20 PGA-coated selenium nanoparticle sizes.

Concentrations of PDI Size(nm) PGA(mM)

0.4 0.250 163.0±1.2

0.8 0.348 167.6±0.8

1.2 0.391 187.5±15.3

1.6 0.340 234.1±2.2

2.0 0.393 223.8±5.0

2.4 0.398 225.1±5.4

250

200

150

100 Size(nm)

0 0.4 0.8 1.2 1.6 2 2.4 Concentration of PGA(mM)

Figure 25 PGA-coated selenium nanoparticle sizes.

The study on synthesis of PGA coated selenium nanoparticles with different concentrations found that an increase in PGA concentrations made an increase in the nanoparticle sizes based on PGA concentrations at 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 mM. 50

Average sizes of the selenium nanoparticles were 163.0, 167.6, 187.5, 234.1, 223.8, and 225.1 nm, respectively.

Besides, a study was conducted on stability of PGA-coated SeNPs with different concentrations at 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 mM by leaving them for 7 days and 1 month and measured the sizes of selenium nanoparticle with DLS. The experimental result is shown in Table 18 and Table 19. Table 21 Sizes of PGA-coated selenium nanoparticles after 7 days passed.

Concentrations of PDI Size (nm) PGA (mM)

0.4 0.247 125.6±1.3

0.8 0.378 144.2±6.4

1.2 0.387 195.7±10.5

1.6 0.345 196.1±7.2

2.0 0.350 166±4.5

2.4 0.376 171.4±2.4

Table 22 Sizes of PGA-coated selenium nanoparticles after 22 days passed.

Concentrations of PDI Size (nm) PGA (mM)

0.4 0.201 150.7±1.6

0.8 0.400 160.7±6.2

1.2 0.334 206.8±6.7

1.6 0.235 200.5±0.2

2.0 0.302 180.2±6.2

2.4 0.313 199.7±5.3

Table 23 Sizes of PGA-coated selenium nanoparticles after 1 month passed.

Concentrations of PDI Size (nm) PGA (mM)

0.4 0.273 148.5±0.4

0.8 0.272 183.3±2.7

1.2 0.263 208.5±6.0

1.6 0.320 209.7±20.6

2.0 0.300 206.7±14.7

2.4 0.358 198.9±13.9

250

200 0.4 0.8 150 1.2 100 Size(nm) 1.6 2 50 2.4 0 1 7 22 30 Time(Day)

Figure 26 PGA-coated selenium nanoparticles with different concentrations at 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 mM after being kept for 7, 22 days and 30 days.

The study result on the stability after leaving them at room temperature for 1, 7, 22 and 30 days found that the PGA-coated selenium nanoparticles with different concentrations at 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 mM on day 7 had average sizes as 125.6, 144.2, 195.7, 196.1, 166 and 171.4 nm, respectively and on day 30 had average sizes as 148.3, 183.3, 208.5, 209.7, 206.7, and 198.9 nm respectively. It was found that the PGA-coated selenium nanoparticles had not much different increasing and decreasing sizes on day 7 and day 30. It was found that PGA concentration at 1.6 mM had the best stability. The sizes of the PGA-coated selenium nanoparticles at this concentration were not much different on day 1, 7, 22 and 30.

A study was conducted on water volume used to stop reaction of the synthesis of chitosan-coated and PGA-coated selenium nanoparticles to consider whether or not water volume had an effect on selenium nanoparticle sizes by using 52

25, 50, 100, and 200 milliliters water to stop reaction as shown in Table 20 and Table 21. Table 24 Long chain chitosan-coated selenium nanoparticle sizes that are synthesized by using different water volume

Sample PDI Size (nm)

25 0.387 242.0±0.4 50 0.212 243.7±6.3 100 0.244 147.3±6.1

200 0.145 120.0±0.6

300 0.136 118.5±1.6

Table 25 PGA-coated selenium nanoparticle sizes that are synthesized by using different water volume.

Sample PDI Size(nm) 25 0.259 194.0±0.4

50 0.242 183.0.±3.1 100 0.193 123.1±2.0

200 0.214 138.1±2.6 300 0.149 130.9±1.4

300

250

200

150 CS-SeNPs

100 PGA-SeNPs Size(nm)

0 25 50 100 200 300 Volume of DI (mL)

Figure 27 Chitosan-coated and PGA-coated selenium nanoparticle sizes using different water volume. 53

The study on water volume used to stop the synthesis reaction of chitosan- coated and PGA-coated selenium nanoparticles using 25, 50, 100, and 200 mL water to stop the reaction found that the chitosan-coated selenium nanoparticles had average sizes as 242.0, 243.7, 147.3, 120.0, and 118.5 nm. The PGA -coated selenium nanoparticles had average sizes as 194.0, 183.0, 123.1, 138.1, and 130.9 nm. The experimental result indicated that the chitosan-coated and PGA-coated selenium nanoparticle sizes decreased and had better distribution when the water volume increased.

6. The analysis of the nanoparticles Characteristics

The characterization of the synthesized selenium nanoparticles was achieved by several techniques such as UV-Vis spectrophotometer, Dynamic light scattering (DLS), Fourier transform Infrared Spectrometer (FT-IR) and Transmission electron microscopy (TEM)

6.1 UV–vis spectroscopy analysis

UV–vis spectrum of SeNPs, Cs-SeNPs, PGA-SeNPs are shown in Figure 27. In general, light wavelengths in the range of 200-400 nm are usually used for characterizing various metal nanoparticles. The absorbance band showed a sharp absorption band at 265 nm, which indicates the formation of SeNPs. It was shown that an absorbance peak of Cs-SeNPs at 265 nm and PGA-SeNPs at 265 nm. The size of nanoparticles has a significant effect on the UV–Vis absorption spectra, i.e. small size particles absorb light of lower wavelength. The UV-visible absorption maximum in the present study was found to be in agreement with previous reports. (Gangadoo et al., 2017) found that the resulting nanoparticle solution displayed a surface plasmon resonance band at 262 nm, which confirmed the presence of selenium nanoparticles. (Gunti et al., 2019) confirm of PF-SeNPs formation by plasmon resonance using UV- Visible spectroscopy. The maximum absorption was observed at 270 nm, which indicate that plant material has reduced and stabilized the PF-SeNPs formation. (Kokila et al., 2017) have prepared SeNPs from Diospyros montana leaf extract by green synthesis approach and observed UV-visible absorption maximum at 261 nm. Similarly, (Anu et al., 2016) have green-synthesized SeNPs from Allium sativum and noticed UV-visible maximum at 260 nm. Likewise, (Fesharaki et al., 2010) have observed UV-visible maximum at 218 and 248 nm (located between 200 and 300 nm) for Klebsiella pneumoniae mediated bio-synthesized SeNPs. On the other hand, (Shah et al., 2010) have synthesized polyvinyl alcohol-stabilized SeNPs by wet chemical method involving a reaction of acetone and noticed UV-visible maximum at 270 nm. The study concluded that sodium selenite was successfully converted into CS-SeNPs and PGA-SeNPs by reduction action of ascorbic acid. 54

Figure 28 UV-Vis spectrum of SeNPs, CS-SeNPs and PGA-SeNPs

6.2 FT-IR analysis

FT-IR spectra were captured in the frequency range from 500 to 4000 cm–1. FTIR allows the determination of the functional groups that exist on the surface of nanoparticles by measuring the vibrational frequencies of chemical bonds.The obtained molecular data facilitates to establish structuraland conformational changes of the coordinating self-assembled functional groups on the surface of nanoparticles. FTIR spectroscopy was performed to identify the interaction mechanism between Cs, PGA and SeNPs as displayed in Figure 28. The IR spectrum of Cs–SeNPs showed absorbance peaks at 3351 cm−1 (O-H stretch), 2943 cm−1 (C-H stretch), 1561 cm−1 (N- H bend), 1620 cm−1 (C=O stretch), 1320 cm−1 (C-N stretch), 1054 cm−1 (C-O stretch). which were similar with the absorbance peaks of CS at 3358cm−1 (O-H stretch), 2885 cm−1 (C-H stretch), 1588 cm−1 (N-H bend), 1419 cm−1 (C-N stretch), 1028 cm−1 (C-O stretch). However, the absorption peak of CS at 3373 cm−1 was blue-shifted to 3351 cm−1 in the IR spectra of Cs-SeNPs, the shift of OH group indicated a strong bonding interaction between hydroxyl groups of CS and surface atoms of SeNPs. Similarly, the absorption peak of PGA-SeNPs showed absorbance peaks at 3250 cm−1 (N-H stretch), 2929 cm−1 (C-H stretch), 1093 cm−1 (C-N stretch), 1589 cm−1 (- CONHR stretch), 1401 cm−1 (-COOH stretch). However, the absorption peak of PGA at 3288 cm−1 was shifted to 3250 cm−1 in the IR spectra of PGA-Se NPs. The combination mode of CS-SeNPs and PGA-SeNPs were similar to what was reported for GA/AHGA nanoparticles to the report of (Kong et al., 2014). 55

a. b. ,. ,.

Figure 29 (a.) The FTIR spectra of SeNPs, Cs-SeNPs, PGA-SeNPs, Cs and PGA (b) SeNPs interaction with CS 6.3 The morphology and structure of SeNPs, Cs-SeNPS and PGA-SeNPs.

SeNP, Cs-SeNPS and PGA-SeNPs of various shapes and sizes were synthesized by chemical synthesis and reducing sodium selenite with ascorbic acid. Ascorbic acid is often used as preferred reducing agent due to its biocompatibility and low toxicity in the body compared to other reducing agents. The color change from colorless to orange indicated the occurrence of reduction reaction to form SeNP. The red color of the selenium colloids implied that selenium form was either amorphous or monodinic since trigonal selenium is black show in Figure 31. Finally, monodispersed and homogeneous spherical SeNP, Cs-SeNPS and PGA-SeNPs were obtained. The colloid’s color was dependent on the concentration of SeNPs. Besides, the size distribution analysis conducted using TEM and DLS calculated the average diameters of SeNPs to be 158 and 188.4 nm, Cs-SeNPs to be 107 and 194.7 nm, PGA-SeNPs to be 102 and 191.8 nm respectively. Compared with TEM results shown in Figure 29. and Figure 30 , the diameter measured by DLS was higher than that by TEM, and the difference can be explained by the fact that TEM was only sensitive to the electron-rich particle, whereas DLS provided the hydrodynamic radius of the nanocomposites.

Figure 30 Morphology and formation of SeNPs freshly synthesized within CS solution and PGA solution. (a), (b) TEM image of SeNPs. (c), (d) TEM image of CS-SeNPs. (e), (f ) TEM image of PGA-SeNPs.

Figure 31 Morphology and formation of SeNPs freshly synthesized within CS solution and PGA solution. (a.) The size distribution of SeNPs measured DLS, (b.) The size distribution of CS-SeNPs measured DLS, (c.) The size distribution of PGA- SeNPs measured DLS.

Figure 32 Representative samples of SeNPs with chitosan and PGA

7. The effect of SeNPs uptake in sunflower sprout

Selenium fortification of foods and the use of selenium supplements are quite popular to compensate for low Se intake from diets. In the present work, the process of sprouting was investigated as an alternative to enrich selenium in sunflower sprout. Sprouting was chosen because it additionally improves the nutritional value of seeds, for example, by a higher vitamin content, a better quality of protein, and some other parameters. The selenium sensitivity of the sprouts was tested by visible germination levels measurement and seedling development. The uptake rates were determined by measuring the total Se concentration in the sprouts after 7 days of germination. Uptake rates were studied by determination of total selenium using Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) as show in Figure. 5. The results showed that the SeNPs had highest uptake 1136±0.19 g/ kg compared to CS-SeNPs and PGA-SeNPs. Cs and PGA may affect uptake of selenium in sunflower sprout, causing Cs-SeNPs and PGA-SeNPs to have less Se concentration than SeNPs 407±0.37 g/kg and 728±0.87 g/kg, respectively. From the results suggest that the encapsulation of chitosan and PGA had slowly release SeNPs, resulting in less total selenium concentration than SeNPs show in Table 22. (Thawatchai, 1994) found the plasticized coated tablets with the higher M.W. of chitosan exhibited slower drug release than those coated with the plasticized lower M.W. of chitosan.

Table 26 Total selenium concentration in sunflower sprouts after 7 days.

Treatment Total selenium concentration (g/kg)

CS-SeNPs 406.71±0.37

PGA-SeNPS 727.74±0.87

SeNPs 1135.53±0.19

Control 0

C-Cs 0

C-PGA 0

C-selenite 131.90±0.94

) 1200

kg /

g 1000 µ 800 600 400 200

Se concentration ( concentration Se 0

Figure 33 Total selenium concentration in sunflower sprouts after 7 days.

8. Determine percentage of seed germination, shoot lengths and root lengths

8.1 Germination percentage

The result on the percentage determination of seed germination found that the both CS-SeNPs and PGA-SeNPs displayed higher seed germination. As shown in Table 23. PGA-SeNPs treatment increased germination percentage at 92.49% in 7 day as compared to control - selenite 78%. PGA was found to be highly effective for enhancing seedling growth. CS-SeNPs treatment increased germination percentage at 84.59% in 7 day as compared to control-selenite.

Application of chitosan is one of the methods to decrease the negative effect of abiotic stress. Chitosan is a linear β-(1, 4)-glucosamine polymer produced by deacetylation of chitin and is an important structural component of several plant fungi cell walls (Radman et al., 2003). The positive traits of chitosan give to this polymer numerous and unique physiological and biological properties with great potential in a wide range of industries such as pharmacology, medicine, and agriculture. One of the most important bioactivity of chitosan on plants is stimulation of seed germination. In peanut, seed coated with chitosan enhance the energy of germination and germination percentage (Zhou et al., 2002). (Dzung et al., 2011) suggested that chitosan could increase growth and yield in soybean. It is reported that seed priming with two different acidic chitosan solutions improved the vigor of maize seedlings (Shao et al., 2005). In addition, other studies also supported a role of chitosan in modulating the plant response to several abiotic stresses including salt and water stress (Chularat et al., 2009) Chitosan is a natural polymer that can be applied for increasing yield and for decreasing plant disease severity in many crops. The effects of chitosan on seed germination and growth of rice (Oryza sativa L.). The result showed that chitosan at 8 g/l gave the highest germination rate of 94.6% and (Manjunatha et al., 2008) reported that seed priming with chitosan enhances seed germination and seedling vigour in pearl millet. Seed soaked with chitosan increased germination rate,length and weight of hypocotyls. Similarly (No et al., 2003) observed that treatment with 493 kDa chitosan. They found that this condition can improved growth in soybean sprout. Chitosan treatment (476 kDa) increased total weight as wel as the length and thickness of sunflower hypocotylscompared with the control

Table 27 The percentage seed germination of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS.

Germination percentage (%)

Treatment 1 2 3 4 5 6 7 Cs-SeNPs 47 53 65 77 84 84 85 PGA-SeNPS 53 66 77 90 92 92 92 SeNPs 46 51 62 71 76 80 81 Control 69 81 82 92 93 94 94 C-Cs 62 65 71 78 82 86 86 C-PGA 73 84 91 94 95 95 95 C-selenite 34 48 60 68 77 77 78

100 90 80 70 Cs-SeNPs 60 PGA-SeNPS 50 SeNPs 40 Control

30 C-Cs Seed germination (%)germination Seed 20 C-PGA 10 C-selenite 0 1 2 3 4 5 6 7 Germination time (Day)

Figure 34 The percentage seed germination of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-Cs.

8.2 Shoot lengths The results showed that in Figure 23. Soaking the seeds with the PGA and CS solution before seeding can increase the height of shoot lengths. Shoot lengths PGA-SeNPs and CS-SeNPs treatment were 8.16 and 6.86 cm on the 7 day as compared to control-selenite 5.00 cm. The result show that the CS and PGA increased the shoot lengths when compared to control-selenite and SeNPs. From the results found that selenium had affect the growth of sunflower sprout, which selenium causes plants to grow slower. It has been reported that when Se-sensitive plants are subjected to high levels of Se in the soil-root medium, they may show various symptoms such as short growth, chlorosis, withering, drying of leaves, and premature death of the entire plant. PGA is a source of carbon, nitrogen and microbial growth minerals Therefore, PGA makes plants grow well. It was reported that (Chunhachart et al., 2014) found that γ-PGA was able to alleviate lead poisoning in Cantonese cabbage which was the cause of inhibition of root length and seedling growth. (Wang et al., 2008) reported that γ-PGA substance was able to increase the amount of dry weight of cucumber roots and stems. This showed that γ-PGA was able to support plant’s growth and biological weight. (Z. Xu et al., 2013) found that γ-PGA was helped increase growth process in plants. Table 28 The shoot lengths sunflower sprout of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS.

Shoot lengths (cm) Treatment 1 2 3 4 5 6 7 Cs-SeNPs 0.98±0.1 1.88±0. 2.60±0 3.99±0 5.01±0 6.86±0. 5 80 .38 .16 .65 54 PGA-SeNPS 1.09±0.1 1.86±0. 4.44±0 5.70±0 5.98±0 8.16±0. 0 58 .42 .38 .29 61 SeNPs 0.88±0.2 1.53±0. 3.86±0 4.97±0 4.87±0 6.26±0. 7 23 .15 .23 .36 51 Control 1.41±0.4 2.10±0. 4.14±1 6.34±0 7.83±0 9.68±0. 0 79 .15 .46 .16 25 C-Cs 1.76±0.3 2.70±0. 4.13±0 6.96±0 8.67±0 9.91±0. 7 55 .89 .15 .26 25 C-PGA 2.02±0.1 3.16±0. 5.56±0 7.86±0 9.88±0 11.68± 7 47 .34 .29 .23 0.50 C-selenite 0.81±0.2 1.27±0. 2.53±0 3.12±0 4.14±0 5.00±0. 7 30 .46 .74 .73 78 63

12.00

10.00 Cs-SeNPs 8.00 PGA-SeNPS SeNPs 6.00 Control C-Cs

Shoot lengths (cm) lengths Shoot 4.00 C-PGA 2.00 C-selenite

0.00 1 2 3 4 5 6 7 Germination time (Day)

Figure 35 The shoot lengths sunflower sprout of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS.

8.3 Root lengths

Sunflower sprout have high root lengths. According to the planting time In the control set, the trunk was 4.24 cm tall on the 7 day of planting. The results showed that in Figure 34. Soaking the seeds with the PGA and CS solution before seeding can increase the height of root lengths. Root lengths PGA-SeNPs and CS-SeNPs treatment were 3.77 and 3.22 cm on the 7 day as compared to control-selenite 3.20 cm.

Table 29 The root lengths sunflower sprout of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS.

Root lengths (cm) Treatment 1 2 3 4 5 6 7 1.47±0 1.84± 2.15±0. 2.58±0. 3.06±0. 3.22±0. Cs-SeNPs .36 0.33 18 32 39 23 PGA- 1.95±0 2.64± 2.68±0. 3.11±0. 3.31±0. 3.77±0.

SeNPS .34 0.32 48 10 25 19 1.64±0 2.09± 2.41±0. 2.84±0. 3.20±0. 3.31±0. SeNPs .43 0.16 23 17 19 15 1.79±0 2.26± 2.79±0. 3.26±0. 3.84±0. 4.24±0. Control .46 0.29 49 51 21 51 2.23±0 2.76± 3.05±0. 3.27±0. 3.58±0. 3.77±0. C-Cs .36 0.42 28 27 28 23 2.15±0 2.71± 3.12±0. 3.62±0. 4.20±0. 4.61±0. C-PGA .71 0.49 35 30 25 45 1.36±0 2.34± 2.49±0. 2.92±0. 3.07±0. 3.20±0. C-selenite .39 0.35 28 17 30 26

4 Cs-SeNPs

3 PGA-SeNPS SeNPs 2 Control

C-Cs Root lengths (cm) Root 1 C-PGA C-selenite 0 1 2 3 4 5 6 7 Germination time (Day)

Figure 36 The root lengths sunflower sprout of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS.

9. Antioxidant activities

The DPPH and ABTS activity of sunflower sprouts were evaluated. Some research has suggested that SeNPs have strong antioxidant activity, which is dependent on the nanoparticle size. Moreover, smaller particles have greater activity. The scavenging mechanism of DPPH is based on the reduction of methanolic DPPH solution in the presence of a hydrogen donating antioxidant. DPPH is a stable nitrogen-centered and lipophilic-free radical that is widely used in the antioxidant activities evaluation.

The results showed that in Figure 36. The IC50 of SeNPs, CS-SeNPs and PGA-SeNPs were determined as 397.22, 660.32 and 198.68 g/mL for DPPH. The

scavenging ability of PGA-SeNPs on DPPH was stronger than Na2SeO3. And reached 82% at a concentration of 350 g/mL. Moreover, the scavenging ability of PGA- SeNPs on DPPH was higher than that of CS-SeNPs and SeNPs, It may be due to their hydrogen-donating ability and small particles. The introduction of hydroxyl groups on the surface of SeNPs may enhance the interaction between PGA-SeNPs and the radicals. The PGA fraction again seems to play a crucialrole in the antioxidant action of PGA-SeNPs. This can be related totheir different stability. The inferior stability of CS-SeNPs would lead to more aggregations of SeNPs; thus, reducing the superficialarea of SeNPs that could react with the free radicals. According to the reference, it showed that the DPPH radical scavenging ability of SeNPs decorated with gum Arabic reached about 80% at the concentration of 4.0 mg/mL. In another 66

report, (Qiu et al., 2018) have synthesized pectin decorated SeNPs of 41 nm and

quantified its IC50 value as 500 g/mL. The results represented that the DPPH scavenging activity of PGA-SeNPs is higher than other nanoparticles.

Table 30 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C- PGA and C-CS towards DPPH radicals.

Extract IC50 )µg/ml(

C-PGA 402.822 Control 157.318 CS-SeNPs 660.316

PGA-SeNPs 198.681 SeNPs 397.222

C-Se 303.266

C-CS 253.164

700 600

500

mL) /

400 (µg

50 50 300 IC 200 100 0

Figure 37 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS towards DPPH radicals.

ABTS radicals are more reactive than DPPH radicals ; unlike the reactions with DPPH radicals, which involve H-atom transfer. The reactions with ABTS radicals involve an electron-transfer process. The results were shown in Table 27. The IC50 of SeNPs, CS-SeNPs and PGA-SeNPs were determined as 418.86, 592.17 and 67

418.86 g/mL for ABTS. The scavenging ability of PGA-SeNPs on ABTS was stronger than Na2SeO3 and SeNPs. And reached 99.63% at a concentration of 700 g/mL. The free radical scavenging capacity of PGA-SeNPs might be attributed to their high surface to volume ratio and surface binding energy. And the assumption was supported by the research of Huang et al., in which they found that the smaller the SeNPs provided the better scavenging effect. It showed that the ABTS radical scavenging ability of SeNPs selenium nanoparticles using Aloe vera extract reached about 73%. It was found by (Cho et al., 2008) that soaking sunflower seeds in Chitosan solution before planting can stimulate free antioxidant, all phenolic composites, melatonin and all isoflavones in sunflower sprouts.

Table 31 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C- PGA and C-CS towards ABTS radicals.

Extract IC50(µg/ml)

C-PGA 654.24 Control 370.11 CS-SeNPs 592.20 PGA-SeNPs 256.05 SeNPs 418.86 C-Se 529.60 C-CS 783.38

900 800 700

600 mL) / 500

(µg 400

50 50 300

IC 200 100 0

Figure 38 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS towards ABTS radicals. 68

CONCLUSIONS

1. Synthesis of Selenium Nanoparticles using Sodium Selenite as a Substrate

From the study of selenium nanoparticles synthesis with ascorbic acid as the reducer, the results of sodium selenite (Na2SeO3) per ascorbic with different concentration (25mM : 100 mM, 50mM : 100 mM, 100mM : 50 mM, 200mM : 50 mM, 50mM : 25 mM) by using water as the solvent with sodium selenite per Ascorbic acid 1:4 found that the suitable concentration proportion of sodium selenite per ascorbic acid was 200mM : 50 mM with low PDI and from the study of SeNP stability found that stability of SeNPs lasts for 7 days. 2. Synthesis of Selenium Nanoparticles coated with long and short-chain Chitosan From the study on selenium nanoparticles covered with long and short-chain chitosan synthesis with ascorbic acid as the reducing agent with different concentration level of chitosan 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 µM found that the suitable concentration level of long strand chitosan stability was 0.1. µM. The study of the long chain CS-SeNPs stability found that 1 month is suitable for CS-SeNPs stability.

From the study on synthesis of selenium nanoparticles coated with long chain chitosan measured DLS and TEM found the selenium nanoparticles gave 107 nm and 194.7 nm. , respectively. 3. Synthesis of selenium nanoparticles coated with PGA.

From the study on selenium nanoparticles covered with PGA Synthesis with ascorbic acid as the reducer by studying the different concentration level of PGA 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 mM found that the suitable concentration level of PGA was 1.6 mM and from the study of the PGA-SeNP stability found that the stability of PGA-SeNPs lasted for 1 month. From the study on Synthesis of selenium nanoparticles coated with PGA measured DLS and TEM found the selenium nanoparticles 102 and 191.8 nm, respectively. 4. The effect of SeNPs uptake in sunflower sprout

SeNPs and other forms were synthesized by green synthesis method with the reduction of selenium salts and was decorated on the surface with Cs and PGA. The effect of SeNPs, Cs-SeNPs and PGA-SeNPs uptook in sunflower sprout showed that the SeNPs had highest uptake while both of Cs-SeNPs and PGA-SeNPs displayed the highly seed germination property. PGA is a source of carbon, nitrogen and microbial growth minerals, thus, PGA makes plants grow well. Moreover, the PGA-SeNP nanocomposites exhibited higher 82% at a concentration of 350 g/mL of DPPH scavenging ability and 99.63% at a concentration of 700 g/mL of ABTS scavenging 69

ability in comparison to Na2SeO3. This is a simple method for the preparation of functional selenium nanocomposites with high stability, biocompatibility, and biodegradability. These compounds have potential to be used as a food supplement or ingredient in the pharmaceutical industry.

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APPENDICES

Appendix A

Experimental Data

UV-vis

Appendix Figure A1 UV–vis spectrum of (a) SeNPs, (b). Cs-SeNPs and (c) PGA- SeNPs. 79

FT-IR

Appendix Figure A2 The FTIR spectra of (a) SeNPs, (b) Cs-SeNPs, (c) PGA- SeNPs, (d)Cs and (e) PGA

7 92.42 92.54 93.94 82.09 80.30 80.60 81.82 93.94 84.85 83.58 86.36 83.58 91.04

- 6

92.42 91.04 93.94 79.10 80.30 80.60 80.30 93.94 83.33 83.58 86.36 83.58 91.04 and

5 PGA 92.42 89.55 93.94 74.63 74.24 76.12 78.79 93.94 83.33 83.58 86.36 82.09 91.04 - C

4 92.42 89.55 89.39 70.15 72.73 70.15 69.70 92.42 75.76 76.12 78.79 77.61 89.55 selenite, -

3 77.27 77.61 77.27 61.19 60.61 64.18 63.64 83.33 62.12 67.16 63.64 65.67 74.63

percentage of seed of seed germination percentage control,

2 65.15 67.16 66.67 52.24 48.48 52.24 51.52 83.33 54.55 52.24 53.03 53.73 65.67

SeNPs, -

1 45.45 68.18 50.00 44.78 48.48 46.27 52.24 54.55 50.75 53.03 46.27 45.45 47.76 PGA

g SeNPs,

- 4.089 4.0345 4.0215 4.0103 4.0165 4.0531 4.0671 4.0456 4.0223 4.0235 4.0734 4.0621 4.0456

66 66 66 67 66 67 67 66 67 66 67 66 67 seed

SeNPs,

7 54 62 56 56 57 56 61 61 62 62 55 53 54

6 53 62 55 56 57 56 61 61 61 62 53 53 54

5 germination 52 62 55 56 57 55 61 61 60 62 50 49 51

4 seed 46 61 50 51 52 52 60 61 60 59 47 48 47

3 42 55 41 45 42 44 50 51 52 51 41 40 43 seed germination seed germination

2 percentage 35 32 35 34 55 35 35 36 44 43 45 44 36

The

1 31 30 32 30 45 30 32 31 35 36 34 35 33 A1

Table

s s rol eNPs eNPs SeNPS SeNPS SeNPS SeNPS - - - - SeNPs S S SeNPs - - - - SeNP SeNPs SeNPs SeNP Cont sameple Cs Cs Cs Cs ppendix PGA PGA PGA PGA

82 82 86.36 95.52 96.97 93.94 94.03 75.76 78.79 80.30 76.12 93.94 95.45 93.94 92.54 86.36 87.88 85.07

84.85 95.52 96.97 92.42 94.03 75.76 77.27 78.79 74.63 93.94 95.45 93.94 92.54 86.36 87.88 85.07

84.85 95.52 96.97 92.42 94.03 75.76 77.27 78.79 74.63 93.94 92.42 92.42 92.54 83.33 78.79 82.09

75.76 95.52 96.97 92.42 89.55 68.18 69.70 69.70 65.67 92.42 92.42 90.91 92.54 83.33 74.24 77.61

69.70 91.04 93.94 89.39 88.06 59.09 62.12 60.61 58.21 83.33 80.30 84.85 79.10 75.76 71.21 68.66

62.12 83.58 83.33 83.33 85.07 45.45 48.48 50.00 46.27 83.33 80.30 83.33 77.61 68.18 62.12 65.67

33.33 35.82 69.70 68.18 71.64 60.61 62.12 59.70 63.64 71.64 72.73 75.76 73.13 31.82 34.85 68.18

4.045 4.089 4.009 4.102 4.0231 4.0089 4.0324 4.0256 4.0567 4.0459 4.0987 4.0654 4.0703 4.0899 4.0925 4.0345

66 67 66 66 67 66 66 67 66 67 66 66 67 66 66 66

53 51 63 62 62 57 58 57 57 64 64 62 63 50 52 62

52 50 63 62 62 57 58 57 56 64 64 61 63 50 51 62

52 50 61 61 62 55 52 55 56 64 64 61 63 50 51 62

46 44 61 60 62 55 49 52 50 64 64 61 60 45 46 61

40 39 53 56 53 50 47 46 46 61 62 59 59 39 41 55

57 30 32 33 31 45 41 44 41 56 55 55 55 53 55 52 (Continued)

49 21 23 22 24 40 41 40 42 48 48 50 45 46 45 48 A1

Table

a a a se se se se cs cs cs cs pg pg pga pg ------C C C C C C C C C C C C Control Control Control Control

Appendix

The effect of SeNPs uptake in sunflower sprout Appendix Table A2 The effect of SeNPs uptake in sunflower sprout

same se (Conc μg/L) Se average ple Se (μg/L) average 1 2 3 (μg/L) Cs- 1.17 1.31 2.38 294.875 328.107 596.025 406.335 SeNPs 95 44 41 0 8 0 9 Cs- 1.19 1.59 2.12 296.014 396.716 528.369 407.033 SeNPs 59 48 51 9 4 0 4 Cs- 1.18 2.13 1.60 293.169 529.305 398.607 407.027 SeNPs 03 31 24 4 2 0 2 Cs- 1.74 1.66 1.87 403.972 385.942 429.491 406.468 406.7163± SeNPs 92 65 43 3 6 3 7 0.37 PGA- 3.36 2.49 2.93 823.300 624.825 733.500 727.208 SeNP 73 93 4 7 0 0 6 S PGA- 2.43 3.04 3.73 576.682 721.621 884.824 727.709 SeNP 36 38 75 5 6 8 6 S PGA- 2.13 3.05 3.73 521.425 744.160 914.478 726.688 SeNP 68 85 29 1 6 2 0 S PGA- 3.95 2.56 2.19 989.550 638.484 549.000 725.678 727.7396± SeNP 82 16 6 0 5 0 2 0.88 S SeNPs 4.32 5.05 4.27 1079.74 1263.22 1063.97 1135.64 76 29 08 05 50 61 72 SeNPs 4.82 4.53 5.17 1130.93 1067.36 1208.80 1135.70 91 63 37 68 47 84 33 SeNPs 4.28 4.70 5.18 1028.99 1079.47 1297.40 1135.28 06 65 96 04 25 00 76

Appendix Table A2 (Continuous)

SeNP 4.19 5.41 4.12 1039.50 1345.28 1021.65 1135.48 1135.5302± s 13 61 75 89 07 84 27 0.19 Contr 0 0 0 ol Contr 0 0 0 ol Contr 0 0 0 ol Contr 0 0 0 ol C-cs 0 0 0 C-cs 0 0 0 C-cs 0 0 0 C-cs 0 0 0 C- 0 0 0 pga C- 0 0 0 pga C- 0 0 0 pga C- 0 0 0 pga C-se 0.39 0.34 0.85 98.7256 86.9587 211.853 132.512 51 94 25 9 7 C-se 0.70 0.67 163.125 75.0000 159.644 132.589 0.33 47 37 0 5 8 C-se 0.97 0.48 0.23 225.000 116.309 54.4545 131.921 2 85 96 0 5 4 C-se 0.42 0.98 0.15 105.037 247.150 39.5025 130.563 131.8968±0. 12 86 88 4 0 3 93 soil 0 0 0 85

Appendix Table A2 (Continuous)

soil 0 0 0 soil 0 0 0 soil 0 0 0

DPPH Assay Appendix Table A3 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS towards DPPH radicals.

Extract IC50(µg/mL) C-PGA 402.822 Control 157.318 CS-SeNPs 660.316 PGA-SeNPs 198.681 SeNPs 397.222 C-Se 303.266 C-CS 253.164

Extract of C-PGA +99 %ETOH Appendix Table A4 DPPH assay showing enhanced antioxidant activity of C-PGA

Final inhibition Conc.(µg/ml) conc. A B C D A-B C-D (%) (µg/ml) 1100 550.000 0.391 0.040 0.168 0.053 0.352 0.115 67.28 1000 500.000 0.191 0.053 0.138 60.67 900 450.000 0.210 0.051 0.159 54.84 800 400.000 0.229 0.050 0.179 49.00 700 350.000 0.244 0.049 0.195 44.52 600 300.000 0.263 0.048 0.216 38.62 500 250.000 0.281 0.046 0.236 33.00 400 200.000 0.300 0.045 0.255 27.60 300 150.000 0.323 0.044 0.279 20.77 200 100.000 0.343 0.043 0.300 14.65 100 50.000 0.364 0.041 0.323 8.04

Appendix Figure A3 DPPH assay showing enhanced antioxidant activity of C-PGA

Extract of Control +99 %ETOH Appendix Table A5. DPPH assay showing enhanced antioxidant activity of control

Final inhibition Conc.(µg/ml) conc. A B C D A-B C-D (%) (µg/ml) 700 350 0.379 0.040 0.073 0.045 0.339 0.028 91.67 650 325 0.081 0.045 0.037 89.23 600 300 0.092 0.044 0.048 85.91 550 275 0.106 0.044 0.063 81.56 500 250 0.121 0.043 0.078 76.97 450 225 0.144 0.043 0.101 70.28 400 200 0.173 0.042 0.131 61.50 350 175 0.194 0.042 0.152 55.16 300 150 0.217 0.042 0.175 48.53 250 125 0.241 0.042 0.199 41.22 200 100 0.270 0.041 0.229 32.52 88

Appendix Figure A4. DPPH assay showing enhanced antioxidant activity of control

Extract of Cs_SeNPs + 99 %ETOH

Appendix Table A6 DPPH assay showing enhanced antioxidant activity of Cs- SeNPs

Final inhibition Conc.(µg/ml) conc. A B C D A-B C-D (%) (µg/ml)

2000 1000 0.370 0.040 0.147 0.070 0.330 0.078 76.46 1750 875 0.173 0.067 0.106 67.83 1500 750 0.197 0.064 0.133 59.75 1250 625 0.228 0.060 0.169 48.98 1000 500 0.277 0.057 0.220 33.31 750 375 0.299 0.053 0.246 25.51 500 250 0.320 0.048 0.272 17.64

Appendix Figure A5 DPPH assay showing enhanced antioxidant activity of Cs- SeNPs.

Extract of PGA-SeNPs + 99% ETOH

Appendix Table A7. DPPH assay showing enhanced antioxidant activity of PGA- SeNPs

Final inhibition Conc.(µg/ml) conc. A B C D A-B C-D (%) (µg/ml)

700 350 0.389 0.040 0.109 0.047 0.349 0.063 82.08 650 325 0.127 0.047 0.080 77.06 600 300 0.146 0.046 0.100 71.25 550 275 0.164 0.045 0.118 66.09 500 250 0.180 0.045 0.136 61.08 450 225 0.198 0.045 0.153 56.06 400 200 0.219 0.044 0.175 49.89

Appendix Figure A6. DPPH assay showing enhanced antioxidant activity of PGA- SeNPs.

Extract of SeNPs + 99% ETOH

Appendix Table A8 DPPH assay showing enhanced antioxidant activity of SeNPs

Final Conc.(µg/ml conc. inhibitio A B C D A-B C-D ) (µg/ml n (%) ) 1300 650 0.379 0.040 0.144 0.062 0.339 0.082 75.87 1200 600 0.162 0.061 0.101 70.18 1100 550 0.175 0.059 0.116 65.83 1000 500 0.190 0.058 0.133 60.81 900 450 0.206 0.056 0.151 55.57 800 400 0.223 0.054 0.170 49.96 700 350 0.238 0.052 0.186 45.17

Appendix Figure A7 DPPH assay showing enhanced antioxidant activity of SeNPs

Extract of C-Se + 99% ETOH

Appendix Table A9 DPPH assay showing enhanced antioxidant activity of C-Se

Final inhibition Conc.(µg/ml) conc. A B C D A-B C-D (%) (µg/ml)

800 400 0.370 0.040 0.167 0.049 0.330 0.119 64.04 700 350 0.182 0.047 0.135 59.06 600 300 0.219 0.046 0.173 47.54 500 250 0.235 0.045 0.190 42.46 400 200 0.259 0.044 0.216 34.57 300 150 0.285 0.043 0.242 26.76 200 100 0.311 0.043 0.269 18.50

Appendix Figure A8 DPPH assay showing enhanced antioxidant activity of C-Se.

Extract of C-CS + 99% ETOH

Appendix Table A10. DPPH assay showing enhanced antioxidant activity of C-Cs

Final inhibition Conc.(µg/ml) conc. A B C D A-B C-D (%) (µg/ml)

700 350 0.388 0.040 0.184 0.063 0.348 0.122 64.99 650 325 0.199 0.062 0.138 60.36 600 300 0.208 0.060 0.148 57.37 550 275 0.221 0.059 0.163 53.20 500 250 0.232 0.057 0.175 49.60 450 225 0.244 0.056 0.188 45.87 400 200 0.256 0.054 0.202 41.84

Appendix Figure A9 DPPH assay showing enhanced antioxidant activity of C-Cs.

ABTS Assay

Appendix Table A11 Scavenging effect of SeNPs, Cs-SeNPs, PGA-SeNPs, control, C-selenite, C-PGA and C-CS towards ABTS radicals.

Extract IC50(µg/mL)

C-PGA 654.238

Control 370.106

CS-SeNPs 592.166

PGA-SeNPs 256.049

SeNPs 418.863

C-Se 529.602

C-CS 783.384

Extract of C-PGA +99 %ETOH

Appendix Table A12 ABTS assay showing enhanced antioxidant activity of C-PGA.

Final Conc. inhibition conc . A B C D A-B C-D )%( (µg/ml) (µg/ml) 10000 1000 0.822 0.038 0.229 0.040 0.784 0.189 75.95 9000 900 0.292 0.039 0.253 67.75 8000 800 0.357 0.039 0.318 59.40 7000 700 0.407 0.038 0.369 52.95 6000 600 0.455 0.039 0.416 46.99 5000 500 0.519 0.038 0.481 38.69 4000 400 0.578 0.039 0.540 31.13

Appendix Figure A10 ABTS assay showing enhanced antioxidant activity of C- PGA.

Extract of Control + 99 %ETOH

Appendix Table A13 ABTS assay showing enhanced antioxidant activity of Control.

Final Conc.(µg/ml conc. inhibitio A B C D A-B C-D ) (µg/ml n (%) )

7000 700 0.770 0.039 0.145 0.040 0.731 0.105 85.61 6000 600 0.222 0.039 0.183 74.94 5000 500 0.293 0.038 0.255 65.13 4000 400 0.369 0.038 0.331 54.77 3000 300 0.455 0.038 0.417 43.01 2000 200 0.543 0.038 0.505 30.91 1000 100 0.632 0.038 0.594 18.77

Appendix Figure A11 ABTS assay showing enhanced antioxidant activity of Control.

Extract of CS-SeNPs + 99 %ETOH

Appendix Table A14. ABTS assay showing enhanced antioxidant activity of Cs- SeNPs.

Final Conc. inhibition conc . A B C D A-B C-D )%( (µg/ml) (µg/ml) 10000 1000 0.759 0.039 0.177 0.040 0.721 0.137 81.02 9000 900 0.235 0.039 0.195 72.90 8000 800 0.300 0.040 0.261 63.81 7000 700 0.340 0.039 0.301 58.22 6000 600 0.395 0.038 0.357 50.49 5000 500 0.445 0.039 0.406 43.65 4000 400 0.502 0.039 0.463 35.81

Appendix Figure A12 ABTS assay showing enhanced antioxidant activity of Cs- SeNPs.

Extract of PGA-SeNPs + 99 %ETOH

Appendix Table A15 ABTS assay showing enhanced antioxidant activity of PGA- SeNPs.

Final Conc.(µg/ml conc. inhibitio A B C D A-B C-D ) (µg/ml n (%) )

7000 700 0.774 0.039 0.042 0.039 0.735 0.003 99.63 6000 600 0.057 0.039 0.018 97.52 5000 500 0.138 0.038 0.100 86.46 4000 400 0.237 0.038 0.198 73.03 3000 300 0.339 0.039 0.300 59.18 2000 200 0.462 0.038 0.424 42.38 1000 100 0.601 0.038 0.563 23.39

Appendix Figure A13 ABTS assay showing enhanced antioxidant activity of PGA- SeNPs.

Extract of SeNPs + 99 %ETOH

Appendix Table A16 ABTS assay showing enhanced antioxidant activity of SeNPs

Final inhibition Conc.(µg/ml) conc. A B C D A-B C-D (%) (µg/ml)

7000 700 0.172 0.038 0.134 79.50 6000 600 0.229 0.038 0.192 70.65 5000 500 0.297 0.038 0.259 60.27 4000 400 0.373 0.038 0.335 48.70 3000 300 0.454 0.038 0.416 36.28 2000 200 0.542 0.038 0.504 22.80 1000 100 0.629 0.038 0.591 9.43

Appendix Figure A14. ABTS assay showing enhanced antioxidant activity of SeNPs.

Extract of C-Cs + 99% ETOH

Appendix Table A17 ABTS assay showing enhanced antioxidant activity of C-Cs

Fin al con inhibitio Conc.(µg/ml) A B C D A-B C-D c. n )%( (µg/ ml) 100 10000 0 0.781 0.038 0.322 0.040 0.743 0.283 61.97 9000 900 0.370 0.039 0.331 55.40 8000 800 0.397 0.039 0.359 51.73 7000 700 0.437 0.040 0.398 46.45 6000 600 0.482 0.039 0.443 40.36 5000 500 0.528 0.038 0.489 34.14 4000 400 0.579 0.038 0.541 27.16

Appendix Figure A15 ABTS assay showing enhanced antioxidant activity of C-Cs.

100

Extract of C-Se + 99% ETOH

Appendix Table A18. ABTS assay showing enhanced antioxidant activity of C-Se.

Final inhibition Conc.(µg/ml) conc. A B C D A-B C-D )%( (µg/ml) 10000 1000 0.760 0.039 0.130 0.040 0.722 0.090 87.49 9000 900 0.183 0.039 0.144 80.04 8000 800 0.237 0.039 0.198 72.59 7000 700 0.293 0.039 0.255 64.69 6000 600 0.356 0.038 0.318 55.89 5000 500 0.418 0.039 0.379 47.44 4000 400 0.481 0.040 0.441 38.84

Appendix Figure A16 ABTS assay showing enhanced antioxidant activity of C- SeNPs.

101

Appendix B

Calculation

102

Selenium concentration calculation For example, plant weighed 0.2 g made into sub-volume adjustment to 50 m. The Selenium concentration from standard graph comparing was 1.1795 μg/L. In 1000 mL solution there was 1.1795 μg. If there was 50 mL solution, the content of Selenium would be 1.1795x50/1000 = 0.0590 μg The sample plant weighed 0.201 g. The content of Selenium would be 0.0831 μg. If sample plant weighed 1000 mL, The content of Selenium would be 0.0590x1000/0.2 = 294.875 μg/kg Therefore, for the sample plant weighing 0.021 g, the Selenium concentration would equal to 294.875 μg/kg.

CURRICULU M VITAE

CURRICULUM VITAE

NAME Miss Warinda Fuangchoonuch

DATE OF BIRTH 9 September 1994

BIRTH PLACE Samitivej Hospital

ADDRESS 143/34 bang wak road bang khae District bang phai bangkok 10160 EDUCATION 2019:Master's Degree of Liberal Arts and Science, Major in chemistry, Kasetsart University, Kamphaengsean campus, Thailand 2017: Bachelor's Degree of Liberal Arts and Science, Major in chemistry, Kasetsart University, Kamphaengsean campus, Thailand WORK EXPERIENCE Pollution Control Department, Bangkok

PUBLICATION 2019: Bioaccessibility of Phenolic Compounds in Germinated Jasmine Red Rice Before and After Cooking by in vitro Simulated Digestion Models, Proceeding in the KU-KPS National Conference