THÔNG TIN TÀI LIỆU
THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURAL AND FORESTRY TRAN DIEU LINH STUDY ON EXTRACTION PROCESS OF TOTAL FLAVONOIDS FROM POLYSCIAS FRUTICOSA ROOT AND LEAF BACHELOR THESIS Study Mode : Full time Major : Food Technology Faculty : Advanced Education Program Office Batch : 2018 – 2022 Supervisor : Dr Vu Thi Hanh Thai Nguyen, 2022 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program: Bachelor of Food Science and Technology Student name: Tran Dieu Linh Student ID: DTN1854190021 Thesis Title: Study on extraction process of total flavonoids from Polyscias fruticosa root and leaf Supervisor(s): Dr Vu Thi Hanh Supervisor's Signature: This study aimed to investigate the effects of some factors on the extraction process of total flavonoid content from Polycias fruticosa root and leaf, including: solvent type, concentration of extraction solvent, extraction time, ratio between solvent and material Moreover, the purpose of this study is also to determine the effects of magnetic stirring on total flavonoid extraction process Finally, after investigating the factors influencing the extraction of flavonoids from the Polycias fruticosa root and leaf, the suitable solvent, concentration of solvent, extraction time, solvent/material ratio, material size, and magnetic stirring capability are detemined Before process of total flavonoid content, root and leaf were washed carefully and dried at 60oC for hours until the moisture of sample was under 10% After drying, the samples were chopped into small pieces and ground in a mill The samples were crushed to increase the permeability of the solvent into the raw components and therefore the extraction process’s effectiveness In these experiments, the samples were soaked at different solvent/material ratio, solution concentration, size, time, and tested to aid in stirring to determine the factors affecting extraction efficiency The results show that, the suitable solvent is ethanol (C2H5OH) at concentration of 90%, with a ratio of 10:1(ml/g) for root; 30:1(ml/g) for leaf, size1mm and needs the support of magnetic stirrer for 20 hours for high efficiency extraction process Keywords: Polyscias fruticosa, flavonoid content, extraction, solvent, ratio, concentration, size, room temperature, root and leaf Number of pages: 48 pages Date of submission November 2nd, 2022 i ACKNOWLEDGEMENT This experiment was carried out in the laboratory at the Institute of Life Sciences, at Thai Nguyen University of Agriculture and Forestry in the year 2022 First and foremost, I would like to thank my supervisor Dr Vu Thi Hanh from Vietnam National University of Agriculture and MSc Nguyen Thuong Tuan from Institute of Life Sciences, for their assistance in helping me to overcome the obstacles during the period under the thesis They were always willing to assist me whenever I needed help and directed me in the right direction for finishing the project Secondly, I would want to express my sincere gratitude to my family and friends for always being there for me, supporting me whenever I've encountered a challenge and assisting me in getting through the tough period of finishing this bachelor thesis Finally, due to my lack of specific expertise, I continue to have several weaknesses while studying, assessing, and presenting on the topic I am looking forward to obtaining feedback and recommendations from professors and friends to help me enhance my thesis Best regards, Thai Nguyen, November 2022 Sincerely TRAN DIEU LINH ii TABLE OF CONTENT CHAPTER INTRODUCTION 1.1 Research rationale 1.2 Research’s objective 1.3 Research question 1.4 Limitation CHAPTER LITERATURE REVIEW 2.1 Overview about Polycias Fruticosa (L.) Harms 2.1.1 Characteristics of Polycias Fruticosa (L.) Harms 2.1.2 Overview of the chemical composition of Polycias fruticosa (L) Harms 2.1.3 Overview of the pharmacological composition of Polycias fruticosa (L) Harms 2.2 Overview of flavonoids 2.2.1 Definition 2.2.2 The origin of flavonoids 2.2.3 Structure and classification of flavonoids 2.2.4 Overview of properties of flavonoids 2.2.5 The biological values of flavonoid 10 2.3 Overview of methods for the total flavonoid extraction 11 2.3.1 The traditional extraction methods 12 2.3.2 The mordern extraction methods 13 2.4 Factors influencing flavonoid extraction process 15 2.5 Research situation in the world and Vietnam 17 2.5.1 Research situation in the world 17 2.5.2 Research in Vietnam 17 CHAPTER MATERIALS, RESEARCH CONTENTS AND METHODOLOGY 18 3.1 Material and research scope 18 3.1.1 Material 18 3.1.2 Research scope 18 3.2 Workplace and time to proceed 18 3.3 Chemicals, equipment 18 3.4 Research content 19 3.5 Research methods 20 3.5.1 Experimental design method 20 3.5.2 Analytical methods 24 3.5.3 Data processing methods 26 CHAPTER RESULTS AND DISCUSSION 27 4.1 The result of qualitative analysis of the presence of total flavonoids in the Polyscias fruticosa root and leaf 27 iii 4.2 Effect of some factors on the extraction process of Polycias fruticosa root and leaf 27 4.2.1 Effect of solvents for extracting total flavonoids from the Polyscias fruticosa root and leaf 27 4.2.2 Effect of solvent concentration on extraction capability of total flavonoid content 30 4.2.3 Influence of solvent time on extraction capability of total flavonoid content 31 4.2.4 Effect of solvent/material ratio on extraction capability of total flavonoid content 33 4.2.5 Effect of material size on extraction capability of total flavonoid content 34 4.2.6 Effect of magnetic stirring on extraction capability of total flavonoid content 36 CHAPTER CONCLUSION AND RECOMMENDATION 38 5.1 Conclusion 38 5.2 Recommendation 38 REFERENCE 39 CHAPTER APPENDIX 41 6.1 Appendix ANOVA data analysis 41 6.2 Appendix picture 58 iv 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 LIST OF FIGURES Figure 2.1 Structure of flavonoids Figure 2.2 Structure of flavonoid aromatic ring Figure 2.3 Basic skeleton structure of flavonoids and their classes Figure 4.1 The results of qualitative analysis of flavonoid presence in the Polycias fruticosa root and leaf 27 Figure 4.2 The effect of extraction solvent on total flavonoid extraction 29 Figure 4.3 The effect of solvent concentration on total flavonoid extraction 31 Figure 4.4 The effect of extraction time on total flavonoid extraction capability 32 Figure 4.5 The effect of the solvent/material ratio on the total flavonoid content extraction capability 33 Figure 4.6 The effect of the material size on the total flavonoid content extraction capability 35 Figure 4.7 The effect of the magnetic stirrer on the total flavonoid content extraction capability 36 v 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 LIST OF TABLE Table 3.1 Experiment chemicals 18 Table 3.2 Laboratory instruments 19 Table 3.3 The investigation of the appropriate solvents for total flavonoid extraction 20 Table 3.4 The investigation of the influence of solvent concentration on total flavonoid extraction 21 Table 3.5 The investigation of the influence of solvent time on total flavonoid extraction 22 Table 3.6 The investigation of the influence of solvent/material ratio on total flavonoid extraction 22 Table 3.7 The investigation of the influence of sample size on total flavonoid extraction 23 Table 3.8 The investigation of the influence of magnetic stirring on total flavonoid extraction 24 vi 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 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37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 CHAPTER INTRODUCTION 1.1 Research rationale Life is more and more contemporary, people are becoming increasingly concerned about their personal health, thus they are keenly interested in the helpful substances found in nature As a result, scientists have concentrated their efforts on discovering such molecules and determining how to extract them so that they might be utilized in food and medication to benefit human health Scientists have recently focused on foods that contain antioxidants, a healthful component that can help prevent cardiovascular disease and combat aging Flavonoids are one type of chemical that is useful as an antioxidant Flavonoids also have antibacterial, anti-inflammatory, analgesic, sedative, and other biological effects Flavonoids are found in many plants, fruits and vegetables Vietnam is located in the tropical monsoon hot and humid belt of Asia with ¾ of the continent's area of hills and mountains stretching from North to South These natural conditions have really favored our country with a rich and diverse forest ecological system Our country has up to 12,000 species of higher plants that not only regulate the climate but also have great potential for medicinal resources From distant past to present day, medicinal plants have always played an important role in maintaining human health, so the research on medicinal plants has been conducted very early Herbal medicine is known to be suitable for the body's physiology, effectively treating many complex diseases with little or no side effects Therefore, the search for natural active ingredients with high biological activity is a trend that is of great interest to scientists Polyscias fruticosa (L.) Harms (Araliaceae) is an herbal plant having a myriad of medicinal purposes In Vietnam, this plant has been used for centuries in traditional medicine, which is seen as being analgesic, febrifuge, and diuretic Polyscias fruticosa is widely cultivated in Vietnam, China, and other tropical countries Polycias fruticosa has been utilized as folk medicine in Vietnam to cure ischemia and inflammation and to accelerate blood flow in the brain (Tran Phuong Thao, 2019) Moreover, Polyscias fruticosa has long been widely known 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medicine to treat a number of diseases such as allergic inflammation, gastric ulcer, and duodenal ulcer, helping the body to regulate metabolic processes, anti-aging, strengthen blood vessels and reduce blood cholesterol Currently, research on the active ingredients in Polyscias fruticosa is still limited, usually only used in traditional folk remedies It is necessary to study the method of extracting the important active ingredient total flavonoids from Polyscias fruticosa root and leaf as a basic premise in the preparation of active ingredients for application in medicine, pharmaceuticals, and health foods Considering the abovementioned problems, the topic of bachelor’s thesis is chosen as: "Study on extraction process of total flavonoids from Polyscias fruticosa root and leaf" 1.2 Research’s objective 1.2.1 Overall objectives The overall objective of this research is to find out the factors affecting the extraction process of Polycias Fruticosa root and leaf with the aim towards improving the pure flavonoid extraction capacity of Polycias Fruticosa root and leaf 1.2.2 Detail objectives The main objectives of this thesis are briefly summarized in the followings: - To determine total flavonoid content in Polycias Fruticosa - Qualitative analysis of total flavonoid content found in Polyscias fruticosa root and leaf 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 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confidence level = 97.80% Solvent Concentration = 70 subtracted from: Solvent Concentration Lower Center Upper 80 -0.0129 0.2780 0.5689 90 0.4034 0.6943 0.9852 Solvent Concentration + -+ -+ -+ - 80 ( -* ) 90 ( -* -) + -+ -+ -+ -0.50 0.00 0.50 1.00 Solvent Concentration = 80 subtracted from: Solvent Concentration Lower Center 0.1254 0.4163 Upper + -+ -+ -+ 90 0.7072 ( * -) 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 + -+ -+ -+ 0.50 0.00 0.50 1.00 One-way ANOVA: Content versus Extraction Time Source DF SS MS F P 0.2337 0.0584 4.90 0.019 Error 10 0.1191 0.0119 Total 14 0.3528 Time S = 0.1091 R-Sq = 66.23% R-Sq(adj) = 52.72% Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev 16 0.7447 0.0606 18 0.9540 0.0547 20 1.1180 0.1242 22 1.0350 0.0408 24 0.9970 0.1892 + -+ -+ -+ ( * ) ( * ) ( * ) ( * ) ( * ) + -+ -+ -+ 0.60 0.80 1.00 1.20 Pooled StDev = 0.1091 Grouping Information Using Tukey Method Time N Mean 20 1.1180 Grouping A 22 1.0350 A B 24 0.9970 A B 18 0.9540 A B 16 0.7447 B Means that not share a letter are significantly different Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Time Individual confidence level = 99.18% Time = 16 subtracted from: Time Lower Center Upper 18 -0.0837 0.2093 0.5024 20 0.0803 0.3733 0.6664 -+ -+ -+ -+ -( -* -) ( * -) 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 22 -0.0027 0.2903 0.5834 24 -0.0407 0.2523 0.5454 ( -* ) ( * -) -+ -+ -+ -+ 0.30 0.00 0.30 0.60 Time = 18 subtracted from: Time Lower Center Upper 20 -0.1290 0.1640 0.4570 22 -0.2120 0.0810 0.3740 24 -0.2500 0.0430 0.3360 -+ -+ -+ -+ -( * -) ( -* ) ( * -) -+ -+ -+ -+ 0.30 0.00 0.30 0.60 Time = 20 subtracted from: Time Lower Center Upper 22 -0.3760 -0.0830 0.2100 24 -0.4140 -0.1210 0.1720 -+ -+ -+ -+ -( -* -) ( -* -) -+ -+ -+ -+ 0.30 0.00 0.30 0.60 Time = 22 subtracted from: Time 24 Lower Center Upper -0.3310 -0.0380 0.2550 -+ -+ -+ -+ -( -* -) -+ -+ -+ -+ 0.30 0.00 0.30 0.60 One-way ANOVA: Content versus Ratio Source DF SS MS F P Ratio 1.65922 0.33184 46.97 0.000 Error 12 0.08478 0.00706 Total 17 1.74400 S = 0.08405 R-Sq = 95.14% R-Sq(adj) = 93.11% Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev 0.8953 0.0647 10 1.4100 0.1037 30 1.7313 0.1357 50 1.2090 0.0594 -+ -+ -+ -+ -( -* ) ( -* -) ( -* ) ( * -) 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 70 0.8703 0.0566 100 1.0407 0.0482 ( -* -) ( -* ) -+ -+ -+ -+ -0.90 1.20 1.50 1.80 Pooled StDev = 0.0841 Grouping Information Using Tukey Method Ratio N Mean Grouping 30 1.7313 10 1.4100 B 50 1.2090 B C 100 1.0407 C D 0.8953 D 70 0.8703 D A 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 = Ratio subtracted from: Lower Center Upper 10 0.2842 0.5147 0.7452 30 0.6055 0.8360 1.0665 50 0.0832 0.3137 0.5442 70 -0.2555 -0.0250 0.2055 100 -0.0852 0.1453 0.3758 + -+ -+ -+( -* ) ( -* -) ( -* -) ( -* ) ( * -) + -+ -+ -+-0.60 Ratio = Ratio 0.00 0.60 1.20 10 subtracted from: Lower Center Upper 30 0.0908 0.3213 0.5518 50 -0.4315 -0.2010 0.0295 70 -0.7702 -0.5397 -0.3092 100 -0.5998 -0.3693 -0.1388 + -+ -+ -+( * -) ( -* ) ( -* -) ( -* -) + -+ -+ -+-0.60 Ratio = 0.00 0.60 30 subtracted from: 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 1.20 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 Ratio Lower Center Upper 50 -0.7528 -0.5223 -0.2918 70 -1.0915 -0.8610 -0.6305 100 -0.9212 -0.6907 -0.4602 + -+ -+ -+( -* -) ( -* ) ( * -) + -+ -+ -+-0.60 Ratio = Ratio 0.00 0.60 1.20 50 subtracted from: Lower Center Upper 70 -0.5692 -0.3387 -0.1082 100 -0.3988 -0.1683 0.0622 + -+ -+ -+( * -) ( -* -) + -+ -+ -+-0.60 Ratio = 70 subtracted from: Ratio Lower Center Upper -0.0602 0.1703 0.4008 100 0.00 0.60 1.20 + -+ -+ -+( -* -) + -+ -+ -+-0.60 0.00 0.60 1.20 One-way ANOVA: Content versus Size Source DF SS MS F P Size 0.427096 0.213548 257.15 0.000 Error 0.004983 0.000830 Total 0.432079 S = 0.02882 R-Sq = 98.85% R-Sq(adj) = 98.46% Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev < M ≤ 3 1.0890 0.0413 < M 1.0013 0.0275 1.5010 0.0050 M ≤ ≤ + -+ -+ -+ ( *-) ( *-) ( * ) + -+ -+ -+ 1.05 1.20 1.35 1.50 Pooled StDev = 0.0288 Grouping Information Using Tukey Method Size N Mean Grouping 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 M ≤ 1.50100 < M ≤ 3 1.08900 < M 1.00133 ≤ A B C 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 ≤ Lower Center Upper -0.15987 -0.08767 -0.01546 0.33979 0.41200 0.48421 M ≤ Size < M -+ -+ -+ -+ ≤ (-*-) M ≤ ( *-) -+ -+ -+ -+ -0.30 Size = < M Size M ≤ 0.00 0.30 0.60 ≤ subtracted from: Lower Center Upper 0.42746 0.49967 0.57187 -+ -+ -+ -+ ( *-) -+ -+ -+ -+ -0.30 0.00 0.30 0.60 One-way ANOVA: Content versus Magnetic stirrer Source DF SS MS F P Magnetic stirrer 0.15424 0.15424 85.73 0.001 Error 0.00720 0.00180 Total 0.16144 S = 0.04242 R-Sq = 95.54% R-Sq(adj) = 94.43% Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev -+ -+ -+ -+ - magneticstirring 1.7877 0.0453 Nomagneticstirring 1.4670 0.0393 ( -* -) ( * -) 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 -+ -+ -+ -+ 1.44 1.56 1.68 Pooled StDev = 0.0424 Grouping Information Using Tukey Method Magnetic stirrer N Mean magneticstirring 1.78767 Nomagneticstirring 1.46700 Grouping A 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 Lower Center Upper Nomagneticstirring -0.41682 -0.32067 -0.22451 Magnetic stirrer + -+ -+ -+- Nomagneticstirring ( * -) + -+ -+ -+-0.30 -0.15 0.00 0.15 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 1.80 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 6.2 Appendix picture 0 20 40 60 80 100 Figure 1: The quercetine standard curve ROOT LEAF PRECIPITA PRECIPITAT Figure 2: The results of qualitative analysis of flavonoids presence in the Polycias fruticosa root and leaf 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 Leaf in Root in C2H5OH C2H5OH Leaf in H2O Root in H2O Figure 3: To select the best solvents for extracting total flavonoid from the Polyscias fruticosa root and leaf 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 70/1 80/1 90/1 Leaf sample 70/1 80/1 90/1 Root sample Figure 4: To determine of solvent concentration on extraction capability of total flavonoid Content 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 16h 18h 20h 22h 24h Leaf sample 16h 18h 20h 22h 24h Root sample Figure 5: To investigate on the influence of solvent time on extraction capability of total flavonoid content 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 5/1 10/1 30/1 50/1 70/1 100/1 Leaf sample 5/1 10/1 30/1 50/1 70/1 100/1 Root sample Figure 6: To investigate on the influence of solvent/material ratio on extraction capability of total flavonoid content 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 1𝑚𝑚 size 3𝑚𝑚 1mm size 5𝑚𝑚 Leaf sample 1𝑚𝑚 1mm size 3𝑚𝑚 3𝑚𝑚 size 5𝑚𝑚 Root sample Figure 7: To investigate on the influence of material size on extraction capability of total flavonoid content 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 No Magnetic Stirrer Magnetic Stirrer Leaf sample No Magnetic Stirrer Magnetic Stirrer Root sample Figure 8: To investigate on the influence of magnetic stirring on extraction capability of total flavonoid content 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99
Ngày đăng: 08/09/2023, 00:17
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