Investigation of antibacterial, pholyphenol content and antioxidant activities of chinese wedelia, camphor laurel and vietnamese yellow chrysanthemum

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS INVESTIGATION OF ANTIBACTERIAL, POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITIES OF CHINESE WEDELIA, CAMPHOR LAUREL AND VIETNAMESE YELLOW CHRYSANTHEMUM Hanoi- 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TITLE: INVESTIGATION OF ANTIBACTERIAL, PHOLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITIES OF CHINESE WEDELIA, CAMPHOR LAUREL AND VIETNAMESE YELLOW CHRYSANTHEMUM Student : Hoang Thi Hong Student code : 620581 Class : K62CNSHE Instructor : Assoc Prof Nguyen Thanh Hai Dr Nguyen Thi Thanh Ha Hanoi- 2022 COMMITMENT I hereby guarantee the truthfulness and objectivity of the report’s data, images, and results All data, images, and results have never been published in any scientific research I hereby declare that all help for the completion of this thesis has been thanked, all citations and references are indicated in the list of references of the thesis Hanoi, May 2022 Sincerely, Hoang Thi Hong i ACKNOWLEDGMENTS First, I would like to thank the Board of Directors of The Vietnam National University of Agriculture, the teachers in the Department of Biotechnology, and the teachers in the Department of Plant Biotechnology for their help and for creating good conditions best for me during the process of doing my graduate thesis I would like to express my deepest gratitude to my supervisor, Assoc Prof Nguyen Thanh Hai, Ph.D Nguyen Thanh Ha for their support, inspiring guidance, and valuable advice throughout this study, and most of all, their patience during my writing up I am delighted to be supervised by Ph.D Nguyen Thanh Ha thank you for your inspiring guidance, knowledge, and critical comments on the thesis I also wish to express my gratitude and appreciation to the following people for their contribution to the successful completion of this study: To the lab members of Dept of Toxicology-Pharmacology DiagnosticsInternal Medicine, past and present Thank you for everyone's enthusiastic help, encouragement, and advice To my parents for their do, love, and support, and also to my siblings for their encouragement Hanoi, May 2022 Sincerely, Hoang Thi Hong ii CONTENTS COMMITMENT i ACKNOWLEDGMENTS ii CONTENTS iii LIST OF ABBREVIATIONS vi LIST OF TABLES vii LIST OF FIGURES ix ABSTRACT xi PART I: INTRODUCTION 1.1.The urgency of the subject 1.2 Purposes and Requirements PART II: OVERVIEW 2.1 Role of plant natural products in antibacterial 2.2.Research situation of antibacterial effect, pollyphenol content, and antioxidant activity of medicinal plants in the world and Vietnam 2.2.1 In the word 2.2.2 In Vietnam 2.3 Medicine 2.3.1 Chinese wedelia (Sphagneticola calendulacea) 2.3.2 Camphor laurel (Cinnamomum camphora), 2.3.3 Vietnamese yellow chrysanthemum (Chrysanthemum Indicum) 2.4 Type of bacteria 2.4.1 Group of Gram-positive bacteria 2.4.2 Group of Gram-negative bacteria 11 2.5 Overview of polyphenol 13 2.6 Overview of antioxidant activities 14 PART III: RESEARCH MATERIALS, CONTENTS AND METHODS 17 3.1 Materials 17 3.1.1 Materials 17 3.1.2 Time and place of the study 18 iii 3.2 Research content 18 3.3 Research methods 19 3.3.1 Method of extracting medicinal herbs 19 3.3.2 Determination of antibacterial of herbal extracts and essential oils 21 3.3.3 Method for determination of polyphenol content 26 3.3.4 Determination of antioxidant activity 27 3.3.5 Data processing methods 28 PART IV: RESULTS AND DISCUSSION 29 4.1 Results of surveying antibiotics of medicinal herbs 29 4.1.1 The results of determining the diameter of inhibition zones of extracts of Vietnamese yellow chrysanthemum 29 4.1.2 The results of determining the diameter of inhibition zones of extracts of Chinese wedelia 34 4.1.3 The results of determining the diameter of inhibition zones of extracts of Camphor laurel 38 4.2 Results of surveying antibiotics of essential oil 43 4.2.1 The results of the diameter of inhibition zones of Vietnamese yellow chrysanthemum essential oils 43 4.2.2 The results of the diameter of inhibition zones of Camphor laurel, Chinese wedelia essential oils 44 4.3 Results of determination of polyphenol content of medicine herb 46 4.3.1 The results of building a standard Figure between chlorogenic acid content and the increase in optical density measured when reacting with Folin - Ciocalteu reagent 46 4.3.2 Results of total polyphenol content converted to chlorogenic acid (mg) of herbal extracts Camphor laurel, Chinese wedelia, Vietnamese yellow chrysanthemum (content 100mg/ml) when extracted with different solvents 48 4.3.3 Results of total polyphenol content converted to Chlorogenic acid (mg) of essential oils 50 4.4 Results of determining the antioxidant of medicine 51 iv 4.4.1 Determination of antioxidant capacity of standard VTME (Alphatocopherol) 51 4.4.2 Results of determining the antioxidant capacity of medicinal herbs 52 4.4.3 Results of determining the antioxidant capacity of essential oil 54 PART V: CONCLUSIONS AND RECOMMENDATIONS 56 REFERENCES 57 v LIST OF ABBREVIATIONS ATCC American Type Culture Collection AMR Antimicrobial resistance B subtilis Bacillus subtilis DMSO Dimethyl Sunfoxide DPPH 1,1-diphenyl-2-picrylhydrazyl DW Distilled water E coli Eschrichia coli Ethyl Ethyl acetate G philus Geobacillus stearothemophilus P seudo Pseudomonas aeruginosa S aureus Staphylococcus aureus Sal Salmonella enterica VTME Vitamin E vi LIST OF TABLES Table 4.1 Diameter of inhibition zones (mm) of Vietnamese yellow chrysanthemum extract with hot water, ethanol, methanol on E coli, P.seudo and : Salmonella 29 Table 4.2 Diameter of inhibition zones (mm) of Vietnamese yellow chrysanthemumextract with Ethyl acetate, Acetone, Hexane on E coli, P.seudo and Salmonella 30 Table 4.3 Diameter of inhibition zones (mm) of Vietnamese yellow chrysanthemum extract with hot water, ethanol, methanol on B.subtilis, S aureus and G phillus .31 Table 4.4 Diameter of inhibition zones (mm) of Vietnamese yellow chrysanthemum extract with Ethyl acetate, Acetone, Hexane on B.subtilis, S aureus and G phillus 32 Table 4.5 Diameter of inhibition zones (mm) of Chinese wedelia extract with hot water, ethanol, methanol on E coli, P.seudo and Salmonella 34 Table 4.6 Diameter of inhibition zones (mm) of Chinese wedelia extract with Ethyl acetate, Acetone, Hexane on E coli, P.seudo and : Salmonella 35 Table 4.7 Diameter of inhibition zones (mm) of Chinese wedelia extract with hot water, ethanol, methanol on B.subtilis, S aureus and G phillus 36 Table 4.8 Diameter of inhibition zones (mm) of Chinese wedelia extract with Ethyl acetate, Acetone, Hexane on B.subtilis, S aureus and G phillus 37 Table 4.9 Diameter of inhibition zones (mm) of Camphor laurel extract with hot Water, Ethanol, and Methanol on E coli, P.seudo, and Salmonella 38 Table 4.10 Diameter of inhibition zones (mm) of Camphor laurel extract with Ethyl acetate, Acetone, and Hexane on E coli, P.seudo and Salmonella .39 Table 4.11 Diameter of inhibition zones (mm) of Camphor laurel extract with hot water, ethanol, methanol on B.subtilis, S aureus and G phillus 40 vii Table 4.12 Diameter of inhibition zones (mm) of Camphor laurel extract with Hot water, Ethanol, Methanol on B.subtilis, S aureus and G phillus .41 Table 4.13: The results of the diameter of inhibition zones of Vietnamese yellow chrysanthemum essential oils .44 Table 4.14 Variation of OD values according to Chlorogenic acid standard concentration (mg/ml) .47 Table 4.15: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/100 mg of medicinal herbs) when extracted with different solvents 48 Table 4.16: Polyphenol content of essential oils converted to chlorogenic acid (mg/100 mg of medicinal herbs) 50 Table 4.17 The antioxidant activity of the VTME standard was determined by the method using DPPH at different concentrations AA% 51 Table 4.18 Antioxidant capacity of extracts 53 Table 4.19 Antioxidant capacity of essential oils extracted 54 viii Figure 4.3 Image results of experiments with essential oils by the steam method Figure 4.4 Image results of experiments with essential oils by agar diffusion method Thus, in the experiment to evaluate the antibacterial ability of essential oils, our three herbal plants did not give antibacterial results with the bacteria we tested This could be explained as: First, The concentrations of active substances in plant essential oils may depend on the plant genotype and is greatly contributed by several factors such as geoFigureical origin and environmental and agronomic conditions (Rota et al 2004; Yesil et al 2007) The method of extraction also affects the activity of the essential oil 45 The second is about the mechanism of action: when essential oils are considered antibacterial, it is important to consider that their effectiveness will depend not only on the concentration and chemical nature, but also on the susceptibility and concentration of the pathogen, and even of the microbial strain characteristics This capacity will vary depending on the composition and nature of the microbial cell surface (Melgarejo & Postilla, 2011; Rao et al., 2019) In the case of essential oils, due to their hydrophobic nature, they could penetrate through bacterial cell outer membranes and cytoplasmic membranes into the interior of cell and thus disintegrate its structures It renders them more permeable, causing the leakage of cellular components or inactivating the enzymes responsible for the cell wall synthesis and provoking molecular changes in their structure In general, essential oils may provoke a physical, chemical, or biochemical change in the microorganisms, and different components may operate by different mechanisms and may target different kinds of microbes, such as Gram-positive and Gram-negative bacteria, yeasts, or molds, because they differ in the composition of their cell membranes (Laura et al.,2020) Therefore, it is difficult to predict how susceptible pathogens are and why the susceptibility to various from strain to strain after applying these compounds 4.3 Results of determination of polyphenol content of medicine herb 4.3.1 The results of building a standard Figure between chlorogenic acid content and the increase in optical density measured when reacting with Folin - Ciocalteu reagent In the experiment to determine the polyphenol content, we use Chlorogenic acid as a standard to convert the polyphenol content of medicinal herbs We set up a Figure of the correlation between the concentration of chlorogenic acid and the increase in optical density value, assessed by measuring the OD value of the 46 solutions created after the reaction with the Folin-Ciocalteu reagent The obtained results are collected in table 4.14 and shown in Figure 4.5 Figure 4.5 The colored cuvettes represent the color change of Folin Ciocalteu reagent produced by Chlorogenic acid (standard substance) at different concentrations Table 4.14 Variation of OD values according to Chlorogenic acid standard concentration (mg/ml) OPTICAL DENSITY Blank Blank 0.058 0.056 0.1 0.203 0.184 0.145 0.128 0.137 0.0120208 0.0085 0.2 0.326 0.310 0.268 0.254 0.261 0.0098995 0.007 0.3 0.412 0.438 0.354 0.382 0.368 0.019799 0.4 0.570 0.553 0.512 0.497 0.505 0.0106066 0.0075 0.5 0.655 0.683 0.597 0.627 0.612 0.0212132 0.015 0.6 0.843 0.844 0.785 0.788 0.787 0.0021213 0.0015 0.7 0.990 0.995 0.932 0.939 0.936 0.0049497 0.0035 0.8 1.110 1.113 1.052 1.057 1.055 0.0035355 0.0025 0.9 1.264 1.267 1.206 1.211 1.209 0.0035355 0.0025 1.0 1.443 1.393 1.385 1.337 1.361 0.0339411 0.024 Blank n=1 n=2 Mean SD SE Chlorogenic acid 47 0.014 Optical density value 1,6 y = 0,1367x - 0,029 R² = 0,9974 1,4 1,2 0,8 0,6 0,4 0,2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Chlorogenic acid(mg/ml) Figure 4.6 Correlation between chlorogenic acid standard content (mg/ml) and increase in optical density value (OD value) The results showed that there is a positive correlation between acid content chlorogenic and increased optical density with the coefficient of determination R² = 0.9939 and p-value < 0.001 This correlation function is used to convert the polyphenol content in the experimental samples 4.3.2 Results of total polyphenol content converted to chlorogenic acid (mg) of herbal extracts Camphor laurel, Chinese wedelia, Vietnamese yellow chrysanthemum (content 100mg/ml) when extracted with different solvents Table 4.15: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/100 mg of medicinal herbs) when extracted with different solvents Medicine Chinese wedelia Camphor laurel Polyphenols (mg chlorogenic acid/100mg medicinal herbs) Ethyl Hot water Ethanol Methanol Acetone Hexane Acetate 12.107±0.243 8.138±0.091 19.971±0.402 2.048±0.110 4.279±0.475 2.158±0.512 6.529±0.165 13.277±0.841 21.964±0.055 4.792±0.329 9.181±0.293 1.518±0.018 Vietnamese 6.053±0.128 yellow chrysanthemum 5.852±1.097 12.747±0.530 2.871±0.201 3.749±0.311 1.481±0.201 48 Experimental results show that all the investigated medicinal herbs contain polyphenols with different concentrations, depending on the type and extraction solvent To compare the polyphenol content of the three medicinal herbs, we compared the contents of the extracts extracted by the same solvent The results are shown in figure 4.7 Chinese wedelia Camphor laurel Vietnamese yellow chrysanthemum 25 20 15 10 Hot water Ethanol Methanol Ethyl Acetone Hexan SOLVENTS Figure 4.7 Total polyphenol content converted to Chlorogenic acid (mg) of three medicinal herbs (content 100mg/ml) when extracted with different solvents From table 4.15 and figure 4.7 we found that: in all solvents except Hexane and Hot Water, Camphor laurel extract had the highest polyphenol content, Chinese wedelia In Hexane and Hot Water solvents, the content was higher than the other two medicinal herbs In general, Methanol extracts from all three medicinal herbs gave the highest polyphenol concentration, the highest in Camphor laurel with a concentration of 21,964mg (chlorogenic acid/100g of medicinal herbs) Extract from Hexan gave the lowest results 49 4.3.3 Results of total polyphenol content converted to Chlorogenic acid (mg) of essential oils We measure the polyphenol concentration of essential oils, the results are shown in tables 4.16 and figure 4.8 Table 4.16: Polyphenol content of essential oils converted to chlorogenic acid (mg/100 mg of medicinal herbs) Essential oils Chinese wedelia 1/10 Camphor laurel 1/10 Vietnamese yellow chrysanthemum 1/10 Total polyphenol content 63.277 92.538 56.693 Total polyphenol content 100,000 90,000 80,000 70,000 60,000 50,000 40,000 30,000 20,000 10,000 0,000 Chinese wedelia 1/10 Camphor laurel 1/10 Vietnamese yellow chrysanthemum 1/10 Figure 4.8 Total polyphenol content converted to chlorogenic acid (mg) of essential oils (content 100mg/ml) From the data table we see that: The total phenolic contents of Camphor laurel 1/10 essential oil is the largest (92.538/100 mg of medicinal herbs) next is the essential oil of > Chinese wedelia 1/10> Vietnamese yellow chrysanthemum 1/10 50 4.4 Results of determining the antioxidant of medicine 4.4.1 Determination of antioxidant capacity of standard VTME (Alphatocopherol) VTME (Alpha-tocopherol) is used as a standard to convert the antioxidant capacity of medicinal herbs First, we first established a standard Figure between the concentration of VTME and the antioxidant capacity of DPPH The obtained results are shown in Table 4.17 and Figure 4.9 Figure 4.9 The cuvettes show the color change of the DPPH solution produced by the antioxidant activity of VTME (standard substance) at different concentrations Table 4.17 The antioxidant activity of the VTME standard was determined by the method using DPPH at different concentrations AA% OD control value Concentration of VTME Control Control 1.113 1.110 0.05 0.10 0.15 0.20 0.25 0.30 0.40 0.45 0.50 OD sample value 0.982 0.802 0.748 0.610 0.505 0.375 0.154 0.134 0.132 51 0.990 0.889 0.750 0.606 0.526 0.409 0.174 0.132 0.128 OD blank value 0.022 0.025 0.024 0.025 0.025 0.025 0.025 0.025 0.023 0.028 0.024 0.024 0.024 0.025 0.030 0.022 0.024 0.025 AA% 13.657 26.235 34.861 47.574 55.930 67.251 87.376 90.252 90.476 120 y = 11,581x + 1,2322 R² = 0,986 Antioxidant activity 100 80 60 40 20 Vitamin E concentration(mg/ml) Figure 4.10 Correlation between the content of VTME standard (standard substance) and antioxidant activity (scavenging activity) The results show that a positive correlation between VTME content and the increase in optical density value is generated when reacting with DPPH reagent, where the coefficient of determination R² is 0.986 and p value < 0.001 This correlation will be used to equivalently convert the increase in optical density value produced by the extracts reacting with the DPPH reagent 4.4.2 Results of determining the antioxidant capacity of medicinal herbs To investigate the antioxidant capacity of extracts of medicinal herbs, we compared the degree of oxidation generated by the extracts The results are shown in Table 4.18 and Figure 4.11 52 Table 4.18 Antioxidant capacity of extracts Medicine Chinese wedelia Hot water Ethanol Antioxidant capacity of extracts Ethyl Methanol Acetone Acetate Hexane 29.337±0.083 23.761±0.525 8.866±0.456 1.132±0.108 6.93±1.182 0.171±0.00 Camphor laurel 12.648±2.290 25.636±1.002 45.094±0.400 2.857±0.272 18.496±2.097 Not detect Vietnamese yellow 11.765±1.162 11.286±1.016 34.42±1.180 3.669±2.578 5.707±5.707 Not detect chrysanthemum 50 45 40 35 30 25 20 15 10 DW Ethanol Chinese wedelia Methanol Camphor laurel Ethyl Aceton Hexan Vietnamese yellow chrysanthemum Figure 4.11 Total antioxidant activity of the extract converted to VTME content (mg/100 mg of medicinal herbs) From the results of tables and figure, we found that all extracts from medicinal herbs have antioxidant activities, at different concentrations, depending on concentration and extraction solvent Methanol extract gave the highest results in the three medicinal plants, especially the methanol extract of Camphor laurel (45.094±0.400) This finding is proven that camphor with high 53 antioxidant properties is suitable for medicine composition This was also mentioned in the study of Salmi et al (2019) It was also found that Camphor laurel exhibited the highest antioxidant activity of the three studied medicinal plants The extracts of Camphor laurel, Chinese wedelia, Vietnamese yellow chrysanthemum on solvents DW, methanol and ethanol showed high polyphenol content and antioxidant activity In the cases where the concentration was increased to 100 mg/ml to 200 mg/ml but still no antioxidant activity was determined, while the blank tube was dark in color, it was the same as in previous studies (Fany Nelt), 2019; Zeynep Akar 2017); we write in the result table not detect (below quantification limit or unobserved) According to these researchers, besides the possibility that the antioxidant activity of the test sample was too low, there was also a case where DPPH could not determine the antioxidant activity because the color of the medicinal herbs affected the color of the collection obtained after DPPH has been freed, causing the sample tube to have a higher OD value than the blank tube, but due to the color of the drug, not the color of the reaction 4.4.3 Results of determining the antioxidant capacity of essential oil We measured the antioxidant activity of essential oils, the results are shown in the following table and figure Table 4.19 Antioxidant capacity of essential oils extracted Essential oils Antioxidant activity content according to VTME (mg/ ml of pure essential oil) Chinese wedelia 108,130±0,035 Camphor laurel 60,392±1,258 Vietnamese yellow chrysanthemum 52,166±2,727 54 To compare the antioxidant activity of the three essential oils, we compared the antioxidant activities of the essential oils The results are shown in the figure 140 120 100 80 60 40 20 Chinese wedelia Camphor laurel Vietnamese yellow chrysanthemum Essential oils Figure 4.12 Total antioxidant activity of extracted essential oils converted to VTME content (mg/1 ml of pure essential oil) Based on the figure and data table, we draw the conclusion that: Chinese wedelia has the highest antioxidant activity, followed by Camphor laurel and Vietnamese yellow chrysanthemum According to this study, Chinese wedelia essential oils have high antioxidant activity A Manjamalai, VM Berlin Grace with in vivo studies also showed Chinese wedelia essential oil has very good antioxidant properties in the process of cancer development From these results, essential oils can be recommended to treat diseases related to free radicals and prevent the development of cancer From there, we realized the potential of Chinese wedelia essential oil in the treatment of disease, and the preservation of food from toxic effects of oxidants 55 PART V: CONCLUSIONS AND RECOMMENDATIONS Conclusions Through the results of our study, we have preliminary demonstrated the antibacterial effect of Chinese wedelia, Camphor Laurel and Vietnamese yellow chrysanthemum on bacteria: Escherichia coli ATCC 25922; Escherichia coli ATCC 35218; Escherichia coli ATCC 85922; Staphylococcus aureus ATCC 25923; Staphylococcus aureus ATCC 25023; Pseudomonas aeruginosa ATCC 9027; Bacillus subtilis ATCC 7953; Geobacillus stearothemophilus ATCC 7953; Salmonella ATCC 11311 Ethanol solvent extracts showed the most impressive antibacterial ability when capable of forming an inhibition zone in both gram-negative and grampositive bacteria Especially the Ethanol extract of chrysanthemum when the inhibition zone diameter ranges from 12.22 to 26.88mm The essential oil extracted by these three herbal plants according to our study had no antibacterial effect against the nine tested bacteria Besides, our survey also showed that Chinese wedelia, Camphor Laurel and Vietnamese yellow chrysanthemum contain polyphenol components and antioxidant activity in both extract and essential oil In medicine, these are ingredients that are said to be anti-aging, health-promoting and diseasepreventing Recommendations We believe that more research is still needed to determine the main active ingredients in each medicinal herb as well as to determine the efficiency of the medicinal herbs after extraction 56 REFERENCES Vietnamese document Nguyen Thi Thanh Ha, Le Phuong Hong Thuy, Nguyen Van Thanh, Nguyen Thi Thu Hang, Đao Van Cuong, Nguyen Van Cuong, Nguyen Huy Thai Nguyen Thanh Hai (2021) Khảo sát hàm lượng polyphenol, hoạt tính chống oxy hóa kháng khuẩn vi khuẩn Escherichia coli ATCC 25922 số lồi thảo dược Tạp chí Khoa học Nơng nghiệp Việt Nam 19(12): 1628-1639 Phung Tuan Giang (2017) Đa dạng sinh học tiềm to lớn thuốc Việt Nam, Bài viết tin tức Y học cổ truyền ngày 14/01/2017, Truy cập ngày 22/05/2022 từ http://benhvienquan4.vn/news/y-hoc-co- 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