Optimizing the extraction of gynnostemma pentaphyllum to initially produce capsules for storage

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY HUNG LE SY OPTIMIZING THE EXTRACTION OF GYNNOSTEMMA PENTAPHYLLUM TO INITIALLY PRODUCE CAPSULES FOR STORAGE BACHELOR THESIS Study mode: Full-time Major: Food Technology Faculty: Advanced Education Program Office Batch: 2017 - 2021 Thai Nguyen, 2022 Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Food Technology Student name Hung Le Sy Student ID DTN 1753070015 Optimizing the extraction of Gynnostemma pentaphyllum Thesis Title to initially produce capsules for storage MSc Trinh Thi Chung Supervisor (s) MSc Luu Hong Son Signature (s) signed Abstract: This study focused on the optimal conditions for extracting in-vitro Gynostemma pentaphyllum, subsequently storing the extract in the form of pharmaceutical flour The method of extraction was ultrasonically-assisted liquid-solid extraction using 70% ethanol as the solvent Using batch experiments and the Response Surface Methodology, the optimal conditions included minutes of 37 kHz ultrasound processing, 101 minutes of extraction time, 69oC, and a material – solvent ratio of : 18 g/ml The optimal extraction process produced a maximum saponin extract of 15.01 g/100g The G pentaphyllum extract was also evaluated in terms of DPPH Radical Scavenging Activity and the cell proliferation inhibitory activity Results show that the concentration for scavenging 50% of DPPH free radical (IC50) was 7.21 mg/ml The G pentaphyllum extract also exhibited anti-liver cancer and anti-blood cancer activities To store the extract in the form of flour, the mixing ratio was 45 ml of extract in 75 g of glucose The appropriate drying temperature was 70oC in 75 minutes, which produced pharmaceutical flour with 1.45% of saponin extract and 8.61% of moisture for encapsulation The data were processed using SPSS at a confidence level of 95% The results have proven the potential of in-vitro G pentaphyllum in Thai Nguyen and created a foundation for further development stages, particularly encapsulation Keywords: Gynostemma pentaphyllum, herbal extraction, saponins, bioactivity, utilization Number of pages: 75 Date of Submission: 27th May 2022 i ACKNOWLEDGEMENT The completion of this study could never happen without the enormous contribution of many individuals and parties For the two distinguished lecturers/instructors who have directly supervised my study, Ms Trinh Thi Chung and Mr Luu Hong Son, I owe them the utmost gratitude that a student can offer They have provided much knowledge regarding the techniques and the directions of research as well as thesis writing Not only that, they have been more than supervisors as they have taught me humility, enthusiasm, diligence, and life experience It has been one of my greatest honors having worked with Ms Chung and Mr Son I am also grateful to the personnel of Faculty of Biotechnology and Food Technology, especially Dr Luong Hung Tien and MSc Vi Dai Lam Dr Tien, Mr Lam, and the staff were not my supervisors Yet, they have enthusiastically given me some of the most precious advice during implementation, leading to the results of this work They are indeed among the best lecturers of this university, if not the best The effort of the Advanced Education Program’s officers in particular and the Rector Board of Thai Nguyen University of Agriculture and Forestry in general is also a key factor for finalizing this thesis Thank you very much Last but not least, to my family and my friend, I thank you for being with me Things would have been much different in a bad way without you May you all receive the best out of everything you want Thai Nguyen, 2022 Hung Le Sy ii Table of contents List of Tables List of Figures PART 1: INTRODUCTION 1.1 Research rationale 1.2 Research objectives 1.3 Research questions and hypotheses 1.4 Limitations 1.4.1 Raw material 1.4.2 Research conditions 1.4.3 Literature PART LITERATURE REVIEW 2.1 Gynostemma pentaphyllum 2.1.1 Morphology, classification, and distribution 2.1.2 Artificial cultivation 2.1.3 Compositional bioactive compounds in Gynostemma pentaphyllum 10 2.1.4 Health effects 13 2.2 Extraction methods 15 2.2.1 Solvents 15 2.2.2 Laboratory-scale extraction methods 17 2.3 Encapsulation 20 2.4 Research status and usage of Gynostemma pentaphyllum 20 2.4.1 International status 20 2.4.2 Vietnamese markets 21 PART METHODOLOGY 23 3.1 Sample collection and preparation 23 3.1.1 Sample collection 23 3.1.2 Chemicals 23 3.1.3 Instruments 23 3.1.4 Research location and scope 24 3.2 Experiment design and methods 24 3.2.1 Research contents 24 3.2.2 The input quality of Gynostemma pentaphyllum 24 3.2.3 The effects of single factors on extracting G pentaphyllum 26 iii 3.2.4 Optimization experiment 28 3.2.5 Bioactivity of the G pentaphyllum extract 30 3.2.6 Pharmaceutical flour and encapsulation 32 3.4 Statistical Analysis 33 PART 4: RESULT AND DISCUSSION 34 4.1 Determination of input quality 34 4.2 The effects of single-factors on G pentaphyllum extraction 34 4.2.1 The effects of ultrasonic processing time on extraction 34 4.2.2 The effects of different solvents 36 `4.2.3 The effects of ethanol concentration 38 4.2.4 The effects of extraction time 40 4.2.5 The effects of extraction temperatures 42 4.2.6 The effects of solvent – material ratios on extraction 44 4.3 Response surface optimization 46 4.4 Bioactivity of the G pentaphyllum extract 49 4.4.1 Anti-oxidation capability 49 4.4.2 Anti-cancer capability 50 4.5 Pharmaceutical flour quality and encapsulation 51 4.5.1 Mixing ratio of G pentaphyllum extract and glucose 51 4.5.2 The effect of drying temperatures 52 4.5.3 Encapsulation and quality determination 53 PART 5: CONCLUSION 55 REFERENCES 56 APPENDICES 65 Data analysis 65 1.1 Ultrasonically processing time 65 1.2 Type of solvent 66 1.3 Solvent concentration 67 1.4 Extraction time 68 1.5 Extraction temperature 70 1.6 Material – Solvent ratio 72 1.7 Effects of drying temperature 73 1.8 DPPH assay analysis 75 Photos during implementation 76 iv List of Tables Table 3.1 Coding of factors for optimization 29 Table 3.2 Experiment designs for optimization experiments of Box - Behnken 30 Table 3.3 The demand of total moisture content for herbal pharmacy 33 Table 4.1 Initial indexes of the raw material 34 Table 4.2 Total saponin extract from different ultrasonic processing time 35 Table 4.3 Total saponin extract using water, ethanol, and methanol 366 Table 4.4 Total saponin extract using different ethanol concentrations 38 Table 4.5 Total saponin extract using different extraction time in 120 minutes 40 Table 4.6 Total saponin extract using different temperatures (60 – 80oC) 42 Table 4.7 Total saponin extract obtained different solvent amount (1/10 – 1/25) 44 Table 4.8 The responses of 17 experiments in the Box-Behnken design 46 Table 4.9 ANOVA analysis of response surface optimization 47 Table 4.10 Absorbance obtained from anti-oxidation capability experiment 49 Table 4.11 Cell proliferation inhibitory effect of G pentaphyllum extract 500 Table 4.12 Mixing ratios and descriptions 501 Table 4.13 Total saponin extract of G pentaphyllum extract flour after drying 52 Table 4.14 Indexes of a Gynostemma pentaphyllum capsule Error! Bookmark not defined List of Figures Figure 2.1 The classification of Gynostemma pentaphyllum (Credit: Ho Trang) Figure 2.2 Rb1, the saponin present in G pentaphyllum and Panax Ginseng 10 Figure 2.3 Polysaccharides identified from G pentaphyllum 12 Figure 4.1 The effects of ultrasonic time on extracting G pentaphyllum 36 Figure 4.2 The effects of solvents on extracting G pentaphyllum 37 Figure 4.3 The effects of solvent contrentations on extracting G pentaphyllum 39 Figure 4.4 The effects of extraction time on extracting G pentaphyllum 41 Figure 4.5 The effects of extraction temperatures on extracting G pentaphyllum 43 Figure 4.6 The effects of material-solvent ratios on extracting G pentaphyllum 45 Figure 4.7 Specific solution and desirability of optimization 48 Figure 4.8 Linear fitting and indexes of the intercept, slope, and R square 48 Figure 4.9 Production process of pharmaceutical flour from G pentaphyllum 544 PART I INTRODUCTION 1.1 Research rationale Gynostemma pentaphyllum (G pentaphyllum, Jiaogulan, Giao Co Lam) is an herb usable as a type of pharmaceutical food in many regions worldwide Some documents showing evidence of the Chinese using this plant from the 14th-15th centuries claimed that G pentaphyllum was capable of significant effects as an antitumor medicine, a pain killer, and an antibiotic (Zhu, 1991; Blumert & Liu, 1999) Through time, this herb is even reputed with the abilities of health enhancement and life prolongation (Linfu et al., 2016) Furthermore, people who used G pentaphyllum were also cured from their other sufferings General information regarding Gynostemma pentaphyllum has been verified Particularly, Morphology and classification, growth conditions and cultivation, chemical composition, health effects, and applications were studied in Vietnam, China, and some Western countries such as Croatia, Italy, and The United States (Huyen et al., 2012; Lin-Na & Yong-Xiu, 2014; Gelen et al., 2017; Mastinu et al., 2021) Studies on this subject have achieved certain positive results, unravelling scientific fundaments and mechanisms behind the miraculous effects of the plant (Tanner et al., 1999; Razmovski-Naumovski et al., 2005) However, despite having been acknowledge worldwide, the employment of G pentaphyllum remains limited in both spatial scales and usage forms Specifically, the plant is only popular in Japan, Korea, China, and Vietnam It is used daily but only in forms of infusions In many markets worldwide, G pentaphyllum can only be found as tea bags and dried plants In a scientfic view, infusion is an extraction method Nevertheless, although it is operationally simple and provides relative adequacy, it is far less specialized compared to other extraction methods The aforementioned information incites requirements for more academic studies on Gynostemma pentaphyllum and utilization matters Furthermore, it is uncertain if the potentials of the subject has been fully reached Therefore, it is safe to assert that a research on the extraction of G pentaphyllum to produce a specialized product, optimizing the benefits from this plant, assures necessity and topicality Simultaneously, it offers solutions for some issues concerning raw material waste and spoilage In the laboratory scale of Thai Nguyen University of Agriculture and Forestry, there are two bases for conducting such a research The first one is that the facility can support the quantification of saponins This is a group of compounds that are inherent in Gynostemma pentaphyllum and Panax Ginseng, making up primary bioactivities in those plants Tanner et al (1999) As a result, the determination of saponins in G pentaphyllum is a firm foundation for clarifying the product’s quality and inferring health effects The second basis for research is the availability of raw materials as the faculty of Biotechnology – TUAF has successfully cultivated Gynostemma pentaphyllum with comparable traits and qualities to wild 5-leaf G pentaphyllum found in Ha Giang Province (Bui et al., 2015) Therefore, a study on the extraction of the plant in TUAF, creating a specialized product, is not only necessary and topical but also highly feasible “Optimizing the extraction of Gynnostemma pentaphyllum to initially produce capsules for storage” is a study set for utilizing such advantages Furthermore, it is going to unravel the capability of G pentaphyllum in bioactivity The success of this study will also pave a way for future researches involving the issue 1.2 Research objectives This study aims to the following objectives: • Determine the quality of input G pentaphyllum (leaves and stems) • Determine the factors affecting G pentaphyllum extraction • Optimize the extraction process • Determine the bioactivity and anti-cancer effects of G pentaphyllum extract • Retrieve G pentaphyllum extract in the form of capsules for storage 1.3 Research questions and hypotheses The background introduced prior to this section has formulated the primary question for this study, corresponded by hypotheses as follows: • Question (Primary): Is there a way to utilize (extract) Gynostemma pentaphyllum better than what appears in the market (infusion) nowadays? This question leads to hypotheses that are particularly about extraction methods, solvents, and operational factors including time, temperatures, and ratios between the raw material and the solvent Based on this hypothesis, question is inferred as follows: • Question 2: What are the optimal conditions for the extraction of Gynostemma pentaphyllum? From question 1, the form of utilization is also set as a question for this study Particularly: • Question 3: What kind of a realistic product is suitable for storing and utilizing Gynostemma pentaphyllum extract? Additionally, the secondary questions set above also incite other questions and hypotheses regarding methodology, implementation, and equipment Those are the foundations for the direction of this study in order to attain sufficient scientific data and significances 1.4 Limitations 1.4.1 Raw material Gynostemma pentaphyllum was selected for this study among many other plants with pharmaceutical potentials That is because this subject has been in the market for a relatively long time Moreover, it has also been researched in a certain number of scientific articles Nevertheless, the form of utilization, which is based on infusing Homogeneous Subsets Saponin CT Subset for alpha = 0.05 N Duncana 192733 3 10 241600 244033 211567 228567 Sig 1.000 1.000 1.000 487 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 1.2 Type of solvent Test of Homogeneity of Variances Saponin Levene Statistic df1 606 df2 Sig 576 ANOVA Saponin Sum of Squares df Mean Square Between Groups 015 008 Within Groups 000 000 Total 015 F 738.009 Sig .000 Post Hoc Tests 66 Multiple Comparisons Dependent Variable:Saponin (I) Formula (J) Formula J) LSD Std Error Sig Lower Bound Upper Bound -.0889667* 0026262 000 -.095393 -.082541 -.0857000* 0026262 000 -.092126 -.079274 0889667* 0026262 000 082541 095393 0032667 0026262 260 -.003159 009693 0857000* 0026262 000 079274 092126 -.0032667 0026262 260 -.009693 003159 dimension3 dimension2 95% Confidence Interval Mean Difference (I- dimension3 dimension3 * The mean difference is significant at the 0.05 level Homogeneous Subsets Saponin Formula Subset for alpha = 0.05 N Duncana 240033 3 325733 329000 dimension1 Sig 1.000 260 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 1.3 Solvent concentration Test of Homogeneity of Variances Saponin Levene Statistic df1 125 df2 Sig 943 ANOVA Saponin Sum of Squares df Mean Square Between Groups 003 001 Within Groups 000 000 Total 003 11 F 118.253 Sig .000 Post Hoc Tests 67 Multiple Comparisons Dependent Variable:Saponin (I) Formula (J) Formula 95% Confidence Interval Mean Difference (IJ) LSD 50 dimension3 60 dimension3 dimension3 80 dimension3 Sig Lower Bound Upper Bound 60 0024950 000 -.030487 -.018980 70 -.0391333* 0024950 000 -.044887 -.033380 80 -.0419667* 0024950 000 -.047720 -.036213 50 0247333* 0024950 000 018980 030487 70 -.0144000* 0024950 000 -.020153 -.008647 80 -.0172333* 0024950 000 -.022987 -.011480 50 0391333* 0024950 000 033380 044887 60 0144000* 0024950 000 008647 020153 80 -.0028333 0024950 289 -.008587 002920 50 0419667* 0024950 000 036213 047720 60 0172333* 0024950 000 011480 022987 70 0028333 0024950 289 -.002920 008587 dimension2 70 Std Error -.0247333* * The mean difference is significant at the 0.05 level Homogeneous Subsets Saponin Formula Subset for alpha = 0.05 N Duncana dimension1 50 297300 60 70 336433 80 339267 322033 Sig 1.000 1.000 289 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 1.4 Extraction time Test of Homogeneity of Variances Saponin Levene Statistic 890 df1 df2 Sig 12 517 68 ANOVA Saponin Sum of Squares df Mean Square Between Groups 031 006 Within Groups 000 12 000 Total 031 17 F 319.947 Sig .000 Post Hoc Tests Homogeneous Subsets 69 Saponin Formula Subset for alpha = 0.05 N Duncana dimension1 45 299433 60 75 90 405200 105 410633 120 410967 340333 378100 Sig 1.000 1.000 1.000 153 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 1.5 Extraction temperature Test of Homogeneity of Variances Saponin Levene Statistic df1 345 df2 Sig 10 842 ANOVA Saponin Sum of Squares df Mean Square Between Groups 004 001 Within Groups 000 10 000 Total 005 14 F 45.324 Sig .000 Post Hoc Tests 70 Multiple Comparisons Dependent Variable:Saponin (I) Formula (J) Formula J) L 60 S D 95% Confidence Interval Mean Difference (IStd Error Sig Lower Bound Upper Bound 65 -.0098333* 0040174 034 -.018785 -.000882 70 -.0233000* 0040174 000 -.032251 -.014349 75 -.0040000 0040174 343 -.012951 004951 80 0286667* 0040174 000 019715 037618 60 0098333* 0040174 034 000882 018785 70 -.0134667* 0040174 007 -.022418 -.004515 75 0058333 0040174 177 -.003118 014785 80 0385000* 0040174 000 029549 047451 60 0233000* 0040174 000 014349 032251 65 0134667* 0040174 007 004515 022418 75 0193000* 0040174 001 010349 028251 80 0519667* 0040174 000 043015 060918 60 0040000 0040174 343 -.004951 012951 65 -.0058333 0040174 177 -.014785 003118 70 -.0193000* 0040174 001 -.028251 -.010349 80 0326667* 0040174 000 023715 041618 60 -.0286667* 0040174 000 -.037618 -.019715 65 -.0385000* 0040174 000 -.047451 -.029549 70 -.0519667* 0040174 000 -.060918 -.043015 75 -.0326667* 0040174 000 -.041618 -.023715 dimension3 65 dimension3 70 dimension2 dimension3 75 dimension3 80 dimension3 * The mean difference is significant at the 0.05 level 71 Homogeneous Subsets Saponin Formula Subset for alpha = 0.05 N Duncana 80 60 405433 75 409433 65 70 376767 409433 dimension1 415267 428733 Sig 1.000 343 177 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 1.6 Material – Solvent ratio Test of Homogeneity of Variances Saponin Levene Statistic 1.914 df1 df2 Sig 228 ANOVA Saponin Sum of Squares df Mean Square Between Groups 018 009 Within Groups 001 000 Total 019 F 81.534 Sig .000 Post Hoc Tests 72 Multiple Comparisons Dependent Variable:Saponin (I) Formula (J) Formula 95% Confidence Interval Mean Difference (I-J) L 10 S 15 dimension2 Sig Lower Bound Upper Bound 15 0086218 000 -.113230 -.071037 20 -.0982667* 0086218 000 -.119363 -.077170 10 0921333* 0086218 000 071037 113230 20 -.0061333 0086218 504 -.027230 014963 10 0982667* 0086218 000 077170 119363 15 0061333 0086218 504 -.014963 027230 dimension3 D Std Error -.0921333* dimension3 20 dimension3 * The mean difference is significant at the 0.05 level Homogeneous Subsets Saponin Formula Subset for alpha = 0.05 N Duncana 10 336433 15 428567 20 434700 dimension1 Sig 1.000 504 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 1.7 Effects of drying temperature Test of Homogeneity of Variances Levene Statistic df1 df2 Sig Weight 1.676 10 231 Saponin 2.089 10 157 73 ANOVA Sum of Squares Weight Mean Square F Between Groups 962 240 Within Groups 334 10 033 1.296 14 Between Groups 000 000 Within Groups 000 10 000 Total 000 14 Total Saponin df Sig 7.198 005 23.650 000 Homogeneous Subsets Weight Duncana Formula Subset for alpha = 0.05 N 90 6.332700 80 6.365300 6.365300 70 6.478000 6.478000 60 50 dimension1 6.709100 6.709100 7.011600 Sig .375 052 070 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 Saponin Duncana Formula Subset for alpha = 0.05 N 90 086900 80 70 094500 60 096200 50 091100 dimension1 Sig .096200 098300 1.000 1.000 222 139 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 74 1.8 DPPH assay analysis Test of Homogeneity of Variances OD Levene Statistic df1 597 df2 Sig 10 673 ANOVA OD Sum of Squares df Mean Square Between Groups 012 003 Within Groups 000 10 000 Total 012 14 F Sig 1232.095 000 Homogeneous Subsets OD Duncana Concentration Subset for alpha = 0.05 N 2.5 2.0 1.5 1.0 3 26500 28200 29800 dimension1 Sig .32100 34567 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000 75 Photos during implementation Weighing the samples before and after experimenting 76 Chemicals and instruments for this study 77 Experiment design in triplicate and implementation 78 Removal of solvent and achievement of saponin extract glue 79 DPPH assay, encapsulation, and results 80