Research on extraction process of total flavonid from polyscias fruticosa trunk

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LE PHAM HOANG MAY STUDY ON EXTRACTION PROCESS OF TOTAL FLAVONOIDS FROM POLYSCIAS FRUTICOSA TRUNK BACHELOR THESIS Study Mode : Full-time Major : Food Technology Faculty : Advanced Education Program Office Batch : 2018-2022 Thai Nguyen, 2022 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program: Bachelor of Food Science and Technology Student name: Le Pham Hoang May Student ID: DTN1854190020 Thesis Title: Research on extraction process of total flavonid from Polyscias Fruticosa trunk Supervisor(s): MSc Vu Thi Hanh Supervisor's Signature: This study has the purpose to consider the influence of several aspects on the total extraction of flavonoids in the Polyscias fruticosa trunk including extraction temperature, solvent consumption, concentration and extraction time, size of Polyscias fruticosa trunk and the use of magnetic stirrer In this study, the Polyscias fruticosa trunk was harvested in Thai Nguyen province The powder from the Polyscias fruticosa trunk was extracted with 70% ethanol for 20 hours at room temperature Flavonoids were detected in the trunk of Polyscias fruticosa using a 10% Pb(CH3COO)2 solution The results showed that the presence of flavonoids in the Polyscias fruticosa trunk and the influencing factors in the extraction process were determined the most suitable data to increase the flavonoid extraction efficiency Keywords: Number of pages: Polyscias fruticosa trunk, flavonoids, solvent consumption, concentration, extraction time, size, magnetic stirrer 65 Date of submission November 2nd, 2022 i ACKNOWLEDGEMENT A completed study would not be done without any assistance Therefore, I gratefully gives acknowledgeme to the support and motivation during the time of doing this research as a requirement of completing my thesis First of all, I would like to express my endless thanks and gratefulness to my supervisor Dr Vu Thi Hanh from Vietnam National University of Agriculture Her kindly support and continuous advices went through the process of completion of my thesis Her encouragement and comments had significantly enriched and improved my work Without her motivation and instructions, the project: Extraction process of total Flavonoid from Polyscias Fruticosa trunk would have been impossible to be done effectively Following that, I would like to thank MSc Nguyen Thuong Tuan from the Institute of Life – Thai Nguyen University for providing me with equipment and useful knowledge, as well as my research group colleague who assisted me throughout the course of the thesis As last, my deeply thanks come to the teachers at the Faculty of Food Technology for their assistance and knowledge throughout my time at the school Thai Nguyen, August 20th, 2022 Sincerely Lê Phạm Hoàng Mây ii Table of Contents ACKNOWLEDGEMENT ii LIST OF FIGURES v LIST OF TABLE vi LIST OF ABBREVIATIONS vii CHAPTER I: INTRODUCTION 1.1 Research rationale 1.2 Research’s objectives 1.3 Research questions and hypotheses 1.4 Limitation CHAPTER II: LITERATURE REVIEW 2.1 Overview about Polyscias Fruticosa 2.1.1 Polyscias Fruticosa Source 2.1.2 Polyscias Fruticosa Characteristics 2.1.3 Chemical component 2.1.4 Pharmacological action 2.1.5 Ecological distribution 2.1.6 Polyscias Fruticosa part for research use 2.2 Flavonoid Overview 2.2.1 Flavonoid Definition 2.2.2 Flavonoid Source 2.2.3 Structure and classification of Flavonoids 2.2.4 Properties of Flavonoid 11 2.2.5 Biological effects of Flavonoid 12 2.3 Methods for extraction of total flavonoids 14 2.3.1 Traditional methods 14 2.3.2 Modern methods 16 2.4 Researches in our country and international researches: 18 2.4.1 International researches 18 2.4.2 Researches in Vietnam 19 CHAPTER III RESEARCH CONTENT AND METHODS 20 3.1 Research subject 20 3.2 Research scope 20 3.2.1 Resarch location 20 3.2.2 Research Time 20 3.2.3 Reseach equipment and chemicals 20 3.3 Research content 22 iii 3.4 Research method 22 3.4.1 Methodology of experiment design: 22 3.4.2 Analytical method 27 3.5 Medology of data processing 29 CHAPTER IV EXPECTED RESULTS 30 4.1 The result of qualitative analysis of total flavonoid found in the Polyscias fruticosa trunk 30 Qualitative results of the presence of flavonoids in 30 4.2 Determination on factors that influence the ability to extract total flavonoid from the Polyscias fruticosa trunk 31 4.2.1 Selecting the best solvents for extracting total flavonoid from the Polyscias fruticosa trunk 31 4.2.2 The effect of concentration of extraction solvent on the total flavonoid content 32 4.2.3 The effects of extraction time on the total flavonoid content 33 4.2.4 The effects of ratio between solvent and material on the total flavonoid content 35 4.2.5 The influence of raw material size on extraction capability of total flavonoid content 36 4.2.6 The effect of magnetic stirring on the extraction capacity of total flavonoid content 37 CHAPTER V CONCLUSION & RECOMMENDATIONS 39 5.1 Conclusion 39 5.2 Recommendations 39 CHAPTER VI REFERENCE 40 References in Vietnamese 40 References in English 40 Internet references 42 CHAPTER VII APPENDIX LIST 44 7.1 Appendix statistical analysis 44 7.2 Appendix picture 52 iv LIST OF FIGURES Figure Polyscias fruticosa Figure 2.Flavonoid structures Figure Classification of flavonoid Figure Structure of flavonoid subgroups Figure Graph showing the standard curve and the standard curve equation 28 Figure Extract solution (A) 30 Figure Extraction solution + Pb(CH3COO)₂ 30 Figure Effect of extraction solvent on the extraction of total flavonoids 31 Figure Effect of extraction time on total flavonoid extraction 33 Figure 10 The effect of extraction time on the total flavonoid content 34 Figure 11 The effect of solvent : material ratio on the total flavonoid content 35 Figure 12 The influence of Material size on extraction capability of total flavonoid content 36 Figure 13 The influence of magnetic stirrer on the total flavonoid content 37 v LIST OF TABLE Table Equipments used in laboratory 20 Table Chemical used in laboratory 21 Table Laboratory equipments 21 Table The effects of solvent type on extraction capability of 23 Table The effect of concentration of extraction solvent on 24 Table The effects of extraction time on the total flavonoid content 24 Table The effects of ratio between solvent and material on 25 Table Effects of raw material size on extraction capability on 26 Table The effect of magnetic stirring on the extraction capacity of 26 vi LIST OF ABBREVIATIONS UAE Ultrasonication Assisted Extraction PEF Pulsed Electric Field EAE Enzyme-assisted Extraction MAE Microwave-assisted Extraction PLE Pressurized Liquid Extraction SFE Supercritical Fluid Extraction QE Quercetin vii CHAPTER I: INTRODUCTION 1.1 Research rationale Vietnam is a tropical country with favorable climatic conditions, resulting in a diverse and rich flora ecosystem with 12,000 species of higher plants, nearly 5,000 of which are used as medicine (Vo Van Chi, 2003) This opens up the research potential of natural compounds for pharmaceutical applications in Vietnam Polyscias fruticosa, has other names such as Nam Duong Lam, with small leaves; belongs to the ginseng family (Araliaceae), genus Polyscias, species Polyscias Fruticosa (L.) Harms (Truong Thi Dep, 2014) In Vietnam, Polyscias Fruticosa has long been used as a traditional medicine, widely known in promoting health, increasing blood circulation, reducing rheumatic pain, etc (Pham Hoang Ho, 2003) Many studies have shown that “in Polyscias Fruticosa, there are glucosides, Alkaloids, Tannins, Vitamin B1 and about 20 types of amino acids such as: Arginin, Alanin, Asparagin, glutamic acid, Leucin, Lysin, Phenylalanin, Prolin, Threonin, Tyrosin, Cysteine, Tryptophan, Methionine (Nguyen Thoi Nam et al., 1990), Oleanilic Acid (Nguyen Thi Nguyet et al., 1992), Flavonoids (Nguyen Thi Luyen et al., 2012), essential oils (Brophy JJ et al , 1990), Polyacetylene (Luto mski J, and Tran Cong Luan, 1992; Tran Cong Luan, 1996), Saponin (Chaboud A…, 1996; Vo Duy Huan, 1998)” Currently, research on active ingredients from nature is attracting the attention of researchers and users because of its effective healing Polyscias fruticosa is being focused on breeding and cultivating due to its pharmacological effects and high biological potential In our country, the bark of Polyscias Fruticosa is used to promote the removal of the placenta after childbirth, and to cure aches and pains They are also used to treat back pain, rheumatism Flavonoids found in polyscias fruticosa have been proven to be effective in the treatment of a variety of conditions, including allergic inflammation, gastric ulcer and duodenal ulcer, assisting the body in controlling metabolic processes, anti-aging, and sustaining endurance, blood vessels, and lowering cholesterol in the blood The problem of researching the active ingredients in polyscias fruticosa is still constrained today, as it is mostly used in traditional folk remedies As a key premise in the medication of active components for the purposes of medicine, pharmaceuticals, and foods, it is necessary to examine the process of extracting the significant active ingredient total flavonoids from the trunk of polyscias fruticosa Based on the above concerns, I have chosen the topic: “Study on extraction process of total flavonoid from Polyscias fruticosa trunk” 1.2 Research’s objectives  Qualitative analysis of the presence of total flavonoids in the trunk of Polyscias fruticosa  Considering potential aspects that affect the extraction process of total flavonoids in polyscias fruticosa trunk such as extraction temperature, kind of solvent, concentration and extraction time, size of Polyscias fruticosa trunk and the use of magnetic stirrer  Propose the total flavonoid extraction process in Polyscias fruticosa trunk 1.3 Research questions and hypotheses  Is it probable to obtain total flavonoid content from the trunk of Polyscias fruticosa?  What variables can greatly affect the extraction process from the trunk of Polyscias fruticosa? 1.4 Limitation Polyscias fruticosa is widely grown in Thai Nguyen, favored as a medicine This is convenient for transporting and harvesting materials for the purpose of conducting experiments with facilities and equipment in the laboratory in Thai Nguyen city Meanwhile, due to the different seasonal conditions and areas where the Polyscias fruticosa trunk was harvested, the material prepared for the (21) Celeste De Monte, Simone Carradori, Arianna Granese, Giovanni Battista Di Pierro, Costantino Leonardo, and Cosimo De Nunzio (2014) Modern extraction techniques and their impact on the pharmacological profile of Serenoa repens extracts for the treatment of lower urinary tract symptoms 14(63), pp 1471-2490 (22) Tran PT, V C P D N H C V M C L JH , (2019) Ethanol extract of Polyscias fruticosa leaves suppresses rankl - mediated osteoclastogenesis in vitro and LPS - induced bone loss in vivo (23) Panche, A N., Diwan, A D., & Chandra, S R (2016) Flavonoids: an overview Journal of nutritional science, 5, e47 (24) Tzanova, M (2020, October 23) Flavonoids—Classification and Natural Sources In Encyclopedia https://encyclopedia.pub/entry/2767 (25) Ullah, A., Munir, S., Badshah, S L., Khan, N., Ghani, L., Poulson, B G., Emwas, A H., & Jaremko, M (2020) Important Flavonoids and Their Role as a Therapeutic Agent Molecules (Basel, Switzerland), 25(22), 5243 (26) Badshah, S L., Faisal, S., Muhammad, A., Poulson, B G., Emwas, A H., & Jaremko, M (2021) Antiviral activities of flavonoids Biomedicine & pharmacotherapy - Biomedecine & pharmacotherapie, 140, 111596 (27) Hosseinzade, A., Sadeghi, O., Naghdipour Biregani, A., Soukhtehzari, S., Brandt, G S., & Esmaillzadeh, A (2019) Immunomodulatory Effects of Flavonoids: Possible Induction of T CD4+ Regulatory Cells Through Suppression of mTOR Pathway Signaling Activity Frontiers in immunology, 10, pp 51 Internet references (28) Soxhlet extraction.(n.d.) Retrieved from https://www.sciencedirect.com/topics/biochemistry-genetics-andmolecular biology/soxhlet-extraction (accessed on 18/10/ 2022) (29) The Plant List - A working list for all plant species (2013) Retrieved from http://www.theplantlist.org/ (accessed on 10/10/2022) 42 (30) Wang, T., Li, Q., & Bi, K (2017, August 15) Bioactive flavonoids in medicinal plants: Structure, activity and Biological Fate Retrieved October 20, 2022, from https://www.sciencedirect.com/science/article/pii/S1818087617301940 (31) Szalay, J (2022, February 25) What are flavonoids? Retrieved October 18, 2022, from https://www.livescience.com/52524-flavonoids.html 43 CHAPTER VII APPENDIX LIST 7.1 Appendix statistical analysis One-way ANOVA: Content versus Solvent Type Source Solvent Type Error Total S = 0.005260 Level Ethanol Water N 3 DF SS 0.0390427 0.0001107 0.0391533 MS 0.0390427 0.0000277 R-Sq = 99.72% Mean 0.29300 0.13167 StDev 0.00700 0.00252 F 1411.18 P 0.000 R-Sq(adj) = 99.65% Individual 95% CIs For Mean Based on Pooled StDev -+ -+ -+ -+ -(-*) (*-) -+ -+ -+ -+ -0.150 0.200 0.250 0.300 Pooled StDev = 0.00526 Grouping Information Using Tukey Method Solvent Type Ethanol Water N 3 Mean 0.29300 0.13167 Grouping A B Means that not share a letter are significantly different Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Solvent Type Individual confidence level = 95.00% Solvent Type = Ethanol subtracted from: Solvent Type Water Lower Center -0.17326 -0.16133 Upper -0.14941 -+ -+ -+ -+ ( *-) -+ -+ -+ -+ - -0.150 -0.100 -0.050 -0.000 One-way ANOVA: Content versus Solvent Concentration Source Solvent Concentration Error Total S = 0.01277 Level 70 80 90 N 3 DF SS 0.023818 0.000978 0.024796 R-Sq = 96.06% Mean 0.29633 0.33133 0.41867 StDev 0.00153 0.00777 0.02065 MS 0.011909 0.000163 F 73.06 P 0.000 R-Sq(adj) = 94.74% Individual 95% CIs For Mean Based on Pooled StDev + -+ -+ -+ ( -* ) ( * -) ( * -) + -+ -+ -+ 0.280 0.320 0.360 0.400 Pooled StDev = 0.01277 Grouping Information Using Tukey Method Solvent Concentration 90 80 70 N 3 Mean 0.41867 0.33133 0.29633 Grouping A B C Means that not share a letter are significantly different Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Solvent Concentration Individual confidence level = 97.80% Solvent Concentration = 70 subtracted from: Solvent Concentration 80 90 Solvent Concentration 80 90 Lower 0.00301 0.09034 Center 0.03500 0.12233 Upper 0.06699 0.15432 -+ -+ -+ -+-( * ) ( -* ) -+ -+ -+ -+ 0.070 0.000 0.070 0.140 Solvent Concentration = 80 subtracted from: Solvent Concentration 90 Solvent Concentration 90 Lower 0.05534 Center 0.08733 Upper 0.11932 -+ -+ -+ -+-( -* ) -+ -+ -+ -+ 0.070 0.000 0.070 0.140 One-way ANOVA: Content versus Extraction Time Source Extraction Time Error Total S = 0.007348 Level 16 18 20 22 24 N 3 3 DF 10 14 SS 0.0175984 0.0005400 0.0181384 R-Sq = 97.02% Mean 0.38300 0.38800 0.42367 0.37367 0.31767 StDev 0.00265 0.00265 0.00451 0.01514 0.00252 MS 0.0043996 0.0000540 F 81.47 P 0.000 R-Sq(adj) = 95.83% Individual 95% CIs For Mean Based on Pooled StDev + -+ -+ -+ (-* ) ( * ) ( * ) ( *-) ( *-) + -+ -+ -+ 0.315 0.350 0.385 0.420 Pooled StDev = 0.00735 Grouping Information Using Tukey Method Extraction Time 20 18 16 22 24 N 3 3 Mean 0.42367 0.38800 0.38300 0.37367 0.31767 Grouping A B B B C Means that not share a letter are significantly different Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Extraction Time Individual confidence level = 99.18% Extraction Time = 16 subtracted from: Extraction Time 18 20 22 24 Extraction Time 18 20 22 24 Lower -0.01473 0.02094 -0.02906 -0.08506 Center 0.00500 0.04067 -0.00933 -0.06533 Upper 0.02473 0.06039 0.01039 -0.04561 + -+ -+ -+( * ) ( * ) ( *-) ( *-) + -+ -+ -+-0.070 0.000 0.070 0.140 Extraction Time = 18 subtracted from: Extraction Time 20 22 24 Extraction Time 20 22 24 Lower 0.01594 -0.03406 -0.09006 Center 0.03567 -0.01433 -0.07033 Upper 0.05539 0.00539 -0.05061 + -+ -+ -+( * ) ( * ) ( * ) + -+ -+ -+-0.070 0.000 0.070 0.140 Extraction Time = 20 subtracted from: Extraction Time 22 24 Extraction Time 22 24 Lower -0.06973 -0.12573 Center -0.05000 -0.10600 Upper -0.03027 -0.08627 + -+ -+ -+( * ) ( * ) + -+ -+ -+-0.070 0.000 0.070 0.140 Extraction Time = 22 subtracted from: Extraction Time 24 Lower -0.07573 Extraction Time 24 Center -0.05600 Upper -0.03627 + -+ -+ -+( * ) + -+ -+ -+-0.070 0.000 0.070 0.140 One-way ANOVA: Content versus Ratio Source Ratio Error Total DF 12 17 S = 0.004295 Level 10 100 30 50 70 N 3 3 3 SS 0.0354447 0.0002213 0.0356660 MS 0.0070889 0.0000184 R-Sq = 99.38% Mean 0.43167 0.29700 0.32033 0.34800 0.32000 0.31300 StDev 0.00404 0.00500 0.00252 0.00500 0.00500 0.00361 F 384.34 P 0.000 R-Sq(adj) = 99.12% Individual 95% CIs For Mean Based on Pooled StDev -+ -+ -+ -+-(*) (*-) (*) (*) (*) (*-) -+ -+ -+ -+-0.320 0.360 0.400 0.440 Pooled StDev = 0.00429 Grouping Information Using Tukey Method Ratio 10 30 50 70 100 N 3 3 3 Mean 0.43167 0.34800 0.32033 0.32000 0.31300 0.29700 Grouping A B C C C D Means that not share a letter are significantly different Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Ratio Individual confidence level = 99.43% Ratio = 10 subtracted from: Ratio 100 30 50 70 Lower -0.14644 -0.12311 -0.09544 -0.12344 -0.13044 Center -0.13467 -0.11133 -0.08367 -0.11167 -0.11867 Upper -0.12289 -0.09956 -0.07189 -0.09989 -0.10689 + -+ -+ -+ (-*-) (-*-) (-*-) (-*-) (-*-) + -+ -+ -+ -0.120 -0.060 0.000 0.060 Ratio = 100 subtracted from: Ratio 30 50 70 Lower 0.01156 0.03922 0.01122 0.00422 Center 0.02333 0.05100 0.02300 0.01600 Upper 0.03511 0.06278 0.03478 0.02778 + -+ -+ -+ (-*-) (-*) (-*-) (-*-) + -+ -+ -+ -0.120 -0.060 0.000 0.060 Ratio = 30 subtracted from: Ratio 50 70 Lower 0.01589 -0.01211 -0.01911 Center 0.02767 -0.00033 -0.00733 Upper 0.03944 0.01144 0.00444 + -+ -+ -+ (-*-) (-*-) (-*-) + -+ -+ -+ -0.120 -0.060 0.000 0.060 Ratio = subtracted from: Ratio 50 70 Lower -0.03978 -0.04678 Center -0.02800 -0.03500 Upper -0.01622 -0.02322 + -+ -+ -+ (-*-) (-*-) + -+ -+ -+ -0.120 -0.060 0.000 0.060 Ratio = 50 subtracted from: Ratio 70 Lower -0.01878 Center -0.00700 Upper 0.00478 + -+ -+ -+ (-*-) + -+ -+ -+ -0.120 -0.060 0.000 0.060 One-way ANOVA: Content versus Size Source Size Error Total DF SS 0.014985 0.001423 0.016408 S = 0.01540 Level < M ≤ 3 < M ≤ M ≤ MS 0.007492 0.000237 R-Sq = 91.33% N 3 Mean 0.35133 0.38500 0.44967 F 31.58 P 0.001 R-Sq(adj) = 88.43% StDev 0.02021 0.01229 0.01234 Individual 95% CIs For Mean Based on Pooled StDev + -+ -+ -+( -* ) ( * -) ( * -) + -+ -+ -+0.360 0.400 0.440 0.480 Pooled StDev = 0.01540 Grouping Information Using Tukey Method Size M ≤ < M ≤ < M ≤ N 3 Mean 0.44967 0.38500 0.35133 Grouping A B B Means that not share a letter are significantly different Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Size Individual confidence level = 97.80% Size = < M ≤ subtracted from: Size < M M ≤ ≤ Size < M M ≤ ≤ Lower -0.00493 0.05974 Center 0.03367 0.09833 Upper 0.07226 0.13693 -+ -+ -+ -+ -( -* ) ( * -) -+ -+ -+ -+ -0.070 Size = < M Size M ≤ 0.000 0.070 0.140 ≤ subtracted from: Lower 0.02607 Center 0.06467 Upper 0.10326 -+ -+ -+ -+ -( * -) -+ -+ -+ -+ 0.070 0.000 0.070 0.140 One-way ANOVA: Content versus Magnetic stirrer Source Magnetic stirrer Error Total S = 0.01313 DF SS 0.023688 0.000689 0.024378 R-Sq = 97.17% Mean 0.57733 0.45167 MS 0.023688 0.000172 F 137.46 P 0.000 R-Sq(adj) = 96.47% Level magneticstirring Nomagneticstirring N 3 StDev 0.00473 0.01795 Level magneticstirring Nomagneticstirring Individual 95% CIs For Mean Based on Pooled StDev + -+ -+ -+ ( -* ) ( -* ) + -+ -+ -+ 0.450 0.500 0.550 0.600 Pooled StDev = 0.01313 Grouping Information Using Tukey Method Magnetic stirrer N Magnetic stirring No magnetic stirring Mean Grouping 0.57733 A 0.45167 B Means that not share a letter are significantly different Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Magnetic stirrer Individual confidence level = 95.00% Magnetic stirrer = magneticstirring subtracted from: Magnetic stirrer Nomagneticstirring Lower -0.15543 Magnetic stirrer Nomagneticstirring -+ -+ -+ -+ -( -* -) -+ -+ -+ -+ 0.150 -0.100 -0.050 -0.000 7.2 Appendix picture Center -0.12567 Upper -0.09591