Investigation of antibacterial effect, polyphenol content and antioxidant activity of lemongrass, cinnamon and anise

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS INVESTIGATION OF ANTIBACTERIAL EFFECT, POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF LEMONGRASS, CINNAMON AND ANISE HA NOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TILLE: INVESTIGATION OF ANTIBACTERIAL EFFECT, POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF LEMONGRASS, CINNAMON AND ANISE Student Name : Pham Thi Huong Class : K62CNSHE Student Code : 620495 Supervisor : Associate Prof Nguyen Thanh Hai Ph.D Nguyen Thi Thanh Ha HA NOI – 2022 COMMITMENT I hereby declare that the data, images, and results in this report are honest, and objective and have never been published in any scientific work I hereby declare that all help in the preparation of this thesis has been thanked and that all citations and references are acknowledged in the list of references of the thesis Hanoi, May 2022 Sincerely, Pham Thi Huong i ACKNOWLEDGEMENTS Firstly, I would like to thank the Vietnam National University of Agriculture, the Department of Biotechnology, and the teachers in the Department of Plant Biotechnology for their help and for creating the best conditions throughout my graduation thesis Secondly, I want to appreciate my supervisor, Prof Dr Nguyen Thanh Hai, and Dr Nguyen Thi Thanh Ha, for providing me with great knowledge about my graduation thesis, an opportunity to work as a team and combat difficulties together, and especially to learn as well as to take responsibilities for ourselves Then, I would like to express my gratitude to every member in the laboratory of the Department of Internal Medicine - Diagnosis - Pharmacology Toxicology, Faculty of Veterinary Medicine for the help and companionship Last but not least, my sincere gratefulness is dedicated to my loving ones for their perpetual and unparalleled love and their emotional support throughout hardships in my life My project would not be complete if not for them, and I dedicate this milestone to them Hanoi, May 2022 Sincerely, Pham Thi Huong ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES vi LIST OF FIGURES viii LIST OF GRAPH AND CHART……………………………………… ………….x LIST OF ABBREVIATIONS x ABSTRACT xi PART I INTRODUCTION 1.1 Preface 1.2 Objective and requirements PART II LITERATURE OVERVIEW 2.1 Potential for development of herbal medicine 2.2 Overview of medicinal herbs 2.2.1 Lemongrass 2.2.2 Cinnamon 2.2.3 Anise 2.3 Overview of bacteria 2.3.1 The gram-positive bacteria 2.3.2 The Gram-negative bacteria 10 2.4 Overview of polyphenol 13 2.4.1 What is polyphenols ? 13 2.4.2 Types of polyphenols 13 2.4.3 Health benefits of polyphenols 13 2.5 Overview of antioxidant activity 13 2.5.1 Antioxidant activity 13 2.5.2 Method for determination of antioxidant activity 14 PART III MATERIAL, CONTENTS, AND METHODS 16 3.1 Material 16 iii 3.1.1 Medicinal 16 3.1.2 Bacteria 16 3.1.3 Time and place of study 16 3.2 Research contents 17 3.3 Research Methods 18 3.3.1 Method of extracting medicinal herbs 18 3.3.2 Method of determining the antibacterial activity of medicinal herbs 21 3.3.3 Method of determining polyphenol content 25 3.3.4 Method of determining antioxidant activity 27 3.3.5 Data processing methods 28 PART IV RESULTS AND DISCUSSION 29 4.1 The results of the investigation of antibacterial activity of medicinal herbs 29 4.1.1 The results of determining the diameter of the inhibition zone of extracts of lemongrass 29 4.1.2 The results of determining the diameter of the inhibition zone of extracts of cinnamon 32 4.1.3 The results of determining the diameter of the inhibition zone of extracts of anise 33 4.2 Results of the investigation on the antibacterial activity of essential oils 37 4.2.1 Results of antibacterial activity of lemongrass essential oil 37 4.2.2 Results of antibacterial activity of cinnamon essential oil 38 4.2.3 Results of antibacterial activity of anise essential oil 40 4.3 The results of determining polyphenol contents 42 4.3.1 The result of building a standard graph between chlorogenic acid content and the increase in optical density measured when reacting with Folin Ciocalteu reagent 42 4.3.2 The results of polyphenol content of herbal extracts of cinnamon, anise, lemongrass (content 100mg/ml) 44 4.3.3 The results of polyphenol content of essential oils of cinnamon, anise, lemongrass 45 iv 4.4 Results of determining the antioxidant activity of medicine extracts and essential oils 47 4.4.1 The results of the determination of the antioxidant capacity of the standard VTME (Alpha tocopherol) 47 4.4.2 The results of the determination of antioxidant activity of extracts of cinnamon, anise, lemongrass 49 PART V CONCLUSION AND SUGGESTION 53 REFERENCES 54 v LIST OF TABLES Table 4.1 Results of inhibition zone diameter (mm) of lemongrass extracts 30 Table 4.2 Results of inhibition zone diameter (mm) of lemongrass extracts 31 Table 4.3 Results of inhibition zone diameter (mm) of cinnamon extracts 32 Table 4.4 Results of inhibition zone diameter (mm) of cinnamon extracts 33 Table 4.5 Results of inhibition zone diameter (mm) of anise extracts 35 Table 4.6 Results of inhibition zone diameter (mm) of anise extracts 36 Table 4.7 Antibacterial activity of lemongrass essential oil based on steam method 37 Table 4.8 Antibacterial activity of lemongrass essential oil based on steam method 38 Table 4.9 Antibacterial activity of cinnamon essential oil based on steam method 38 Table 4.10 Antibacterial activity of cinnamon essential oil based on diffusion method 39 Table 4.11 Antibacterial activity of anise essential oil based on steam method.40 Table 4.12 Antibacterial activity of anise essential oil based on diffusion method 40 Table 4.13 The results of antibacterial activity of trans-cinnamalehyde with different concentrations 41 Table 4.14 Variation of OD values according to chlorogenic acid standard concentration (mg/ml) 43 Table 4.15: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/100 mg of medicinal herbs) when extracted with different solvents 44 Table 4.16: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/ml pure essential oil) 45 vi Table 4.17 Antioxidant activity of standard VITAMIN E determined by DPPH method at different concentrations AA% 48 Graph 4.4 Correlation between content of VTME standard (standard substance) and antioxidant activity (scavenging activity) 48 Table 4.18 Antioxidant activity of extracts 50 Table 4.19 Antioxidant capacity of essential oils extracted 52 vii LIST OF FIGURES Figure 2.1 Lemongrass and lemongrass powder Figure 2.2 Cinnamon and cinnamon powder Figure 2.3 Anise and anise powder Figure 2.4 DDPH (1,1-diphenyl-2-picrylhydrazyl) 15 Figure 3.1 Steps to perform the method of extracting medicinal herbs 19 Figure 3.2 Essential oil distillation kit 20 Figure 3.3 Extraction flask 21 Figure 3.4 Essential oils of cinnamon, anise, lemongrass 21 Figure 3.5 Describe the method of mixing bacteria into agar and punching a small well of antibacterial agent on agar plates 22 Figure 3.6 The ability to inhibit bacteria of medicinal herbs within 24 hours of culture 23 Figure 3.7 Antibacteria of essential oil based on steam method 24 Figure 3.8 Antibacteria of essential oils within 24 hours of culture based on diffusion method 25 Figure 3.9 Diagram of the experiment to determine the total polyphenol content, using Folin-Ciocalteu's Phenol Reagent reagent 26 Figure 3.10 Mechanism of the color change reaction in the determination of total polyphenol content, using Folin-Ciocalteu's phenol reagent (Sadeer et al., 2020) 26 Figure 3.11 2, 2-diphenyl-1-picrylhydrazyl (DPPH) reaction mechanism Mechanism of the color change reaction in the determination of antioxidant activity using the DPPH reagent 28 Figure 4.1 The diameter of the inhibition zone produced on S aureus ATCC 25923 by trans-cinnamaldehyde (left image) and cinnamon (right image) at 1/10 concentration after 24 h of culture on Muller Hinton Agar agar 42 viii Figure 4.1 The diameter of the inhibition zone produced on S aureus ATCC 25923 by trans-cinnamaldehyde (left image) and cinnamon (right image) at 1/10 concentration after 24 h of culture on Muller Hinton Agar agar The Table 4.12 confirm that trans-cinnamaldehyde had inhibitory effects on all bacteria However, cinnamon essential oil was also inhibited on all investigated bacteria, and the inhibition zone was also larger Figure 4.1 This demonstrates that the antibacterial effect of cinnamon is not only induced by trans-cinnamaldehyde, but also other unidentified components in cinnamon The above results confirm the need for more in-depth studies to identify the main active ingredients in cinnamon when used in the direction of inhibiting the growth of bacteria, because the components are considered to play a role The most important factor in producing the pharmacological effects of these medicinal plants are only partially active against bacteria 4.3 The results of determining polyphenol contents 4.3.1 The result of building a standard graph between chlorogenic acid content and the increase in optical density measured when reacting with Folin Ciocalteu reagent In the polyphenol content determination experiment, we used chlorogenic acid as a standard to convert the polyphenol content of medicinal herbs To 42 so, we first set up a graph of the correlation between the concentration of chlorogenic acid and the increase in optical density value, assessed by measuring the OD value of the solutions created after the reaction with Folin Ciocalteu reagent The obtained results are collected in the table and shown on the graph Table 4.12 Variation of OD values according to chlorogenic acid standard concentration (mg/ml) Acid chloro 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Blank Blank Optical density 0,058 0,056 n=1 n=2 0,203 0,326 0,412 0,570 0,655 0,843 0,990 1,110 1,264 1,443 0,184 0,310 0,438 0,553 0,683 0,844 0,995 1,113 1,267 1,393 0,145 0,268 0,354 0,512 0,597 0,785 0,932 1,052 1,206 1,385 0,128 0,254 0,382 0,497 0,627 0,788 0,939 1,057 1,211 1,337 Medium SD SE 0,137 0,261 0,368 0,505 0,612 0,787 0,936 1,055 1,209 1,361 0,012 0,010 0,020 0,011 0,021 0,002 0,005 0,004 0,004 0,034 0,009 0,007 0,014 0,007 0,015 0,002 0,004 0,002 0,002 0,024 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 4.2 Correlation between content of chlorogenic acid standard substance (mg/ml) and the degree of increase in optical density value (OD value) 43 The results showed that there was a positive correlation between the content of chlorogenic acid and the increase in optical density with the coefficient of determination R² = 0.9974 and p value < 0.001 This correlation function is used to convert the polyphenol content in the experimental samples 4.3.2 The results of polyphenol content of herbal extracts of cinnamon, anise, lemongrass (content 100mg/ml) Table 4.13: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/100 mg of medicinal herbs) when extracted with different solvents Polyphenol (mg chlorogenic acid/100mg medicine) Medicine Hot water Ethanol Methanol Ethyl Acetone Hexane acetate Cinnamon 23,446±2,743 30,852±0.165 42,996±6,090 2,542±0,055 19,824±0.146 1,719±0,439 Anise 5,486±0,146 8,559±0,073 14,210±0,091 4,371±0,128 6,419±0,347 3,456±0,165 Lemongrass 3,932±0,201 2,652±0,055 5,852±0,000 1,298±0,018 2,981±0,128 2,762±0,347 Experimental results show that all the investigated medicinal herbs contain polyphenols with different concentrations, depending on the species 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 the Figure 44 Figure 4.3 Total polyphenol content converted to chlorogenic acid (mg) of cinnamon, anise, lemongrass (content 100mg/ml) when extracted with different solvents From Table 4.13 and Figure 4.3, we confirm that with the solvents methanol, ethanol, DW, acetone, cinnamon extract gave the highest polyphenol content, followed by anise and lemongrass In the solvent extracts of ethyl acetate and hexane, the anise extract showed higher polyphenol content than the other extracts The polyphenol content was highest in the case of the methanol cinnamon extract 42,996 mg (chlorogenic acid/100g medicinal herbs) In the case of all three medicinal herbs, methanol, ethanol, DW and acetone solvents all gave higher polyphenol content than the other two solvents The polyphenol content of the hexane extract in the three medicinal herbs was the lowest at 1,719 mg, respectively; 3,456 mg and 1,298 mg (chlorogenic acid/100g medicinal herbs) 4.3.3 The results of polyphenol content of essential oils of cinnamon, anise, lemongrass (1ml content) Table 4.14: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/ml pure essential oil) 45 Essential oil Polyphenol content Cinnamon 1/10 26,225±1,792 Anise 1/10 43,599±1,244 Lemongrass 1/10 62,692±4,170 Experimental results show that all the investigated medicinal herbs contain polyphenols with different concentrations, depending on the species and extraction solvent To compare the polyphenol content of anise, cinnamon, and lemongrass, we compared the content The results are shown in the chart Figure 4.4 Total polyphenol content converted to chlorogenic acid (mg) of essential oils (converted to ml of pure essential oil) From table 4.14 and Figure 4.4, we found that lemongrass essential oil at 1/10 concentration gave the highest polyphenol content, followed by anise and cinnamon The highest polyphenol content in lemongrass at the concentration of 1/10 was 62,692 mg (chlogenic acid/100g of medicinal herbs) and the lowest in cinnamon at the concentration of 1/10 was 26.225 mg (chlogenic acid/100g of medicinal herbs) 46 4.4 Results of determining the antioxidant activity of herbal extracts and essential oils 4.4.1 The results of the determination of the antioxidant capacity of the standard VTME (Alpha tocopherol) VTME (Alpha tocophenol) is used as a standard to convert the antioxidant capacity of medicinal herbs To so, the control first established a standard graph between the concentration of VTME and the antioxidant capacity of DPPH The obtained results are shown in tables and figures Figure 4.5 The color cups represent the color change of DPPH solution induced by the antioxidant activity of VTME ) at different concentrations (from right to right: Blank, VTM E 0.05mg/ml; 0.1 mg/ml; 0.15mg/ml; 0.2mg/ml; 0.25mg/ml; 0.3mg /ml; 0.4mg/ml; 0.45mg/ml; 0.5mg/ml) 47 Table 4.15 Antioxidant activity of standard VITAMIN E determined by DPPH method at different concentrations AA% OD OD control value VTME No.1 No.2 Concentration 1.113 1.110 sample OD blank Antioxidant value value activity content 0.05 0.986 0.025 13.657 0.10 0.846 0.025 26.235 0.15 0.749 0.024 34.861 0.20 0.608 0.025 47.574 0.25 0.516 0.025 55.93 0.30 0.392 0.028 67.251 0.40 0.164 0.024 87.376 0.45 0.133 0.025 90.252 0.50 0.13 0.024 90.476 120 100 y = 11,581x + 1,2322 R² = 0,986 80 60 40 20 Figure 4.6 Correlation between content of VTME standard (standard substance) and antioxidant activity (scavenging activity) 48 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 11,581 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 The results of the determination of antioxidant activity of extracts of cinnamon, anise, lemongrass Figure 4.7 Color change of cinnamon at a concentration of 20mg/ml of medicinal herbs upon color reaction with DPPH The darker the color of medicinal herbs when reacting with DPPH, the lower the antioxidant index, as shown in Figure 4.3 The medicinal colors in the 2nd and 3rd cuvettes from the left are hexane cinnamon and ethyl cinnamon, respectively, with antioxidant activity converted to 1,167mg and 1,112mg in 20mg/ml of medicinal herbs The yellower the color of the medicinal herbs tested for antioxidant activity, the greater the antioxidant activity, as shown in Figure 4.4, the color reaction with DPPH of cinnamon medicinal herbs after reducing the concentration to 10mg/ml of medicinal herbs 49 Figure 4.8 Color change of cinnamon medicinal herbs at a concentration of 10mg/ml of medicinal herbs upon color reaction with DPPH After adjusting the concentration of extracts to ensure the measurement of antioxidant activity of medicinal herbs, the results of investigation of antioxidant activity of medicinal herbs are summarized in the Table 4.18 Table 4.16 Antioxidant activity of extracts Antioxidant activity converted to VTME (mg) Medicine DW Ethanol Methanol Ethyl Acetone Hexane Cinnamon 64,202±1,128 77,339±0,399 76,796±0,800 4,327±1,085 59,897±1,805 1,104±0,070 Anise 13,192±0,588 20,687±1,600 27,019±0,494 0,875±0,172 4,159±0,102 Lemongrass 5,406±0,613 1,378±0,131 0,729±0,300 0,893±0,643 6,342±0,050 4,441±1,024 To compare the antioxidant activities of the three medicinal herbs, we compared the antioxidant activities of extracts extracted by the same solvent The results are shown in the figure 4.5 50 Figure 4.9 Total antioxidant activity of lemongrass, cinnamon and anise extracts was converted to VTME content (mg/100 mg of medicinal herbs) From table 4.18 and chart 4.3, we confirm that with solvents methanol, ethanol, DW, acetone, cinnamon extract gave the highest total antioxidant activity, followed by anise and lemongrass In the solvent extracts of ethyl acetate and hexane, the lemongrass extract showed less total antioxidant activity than the other extracts, even none for the solvent hexane The highest polyphenol content in the case of methanolic cinnamon extract was 76,796 mg (chlorogenic acid/100g medicinal herb) In the case of all three medicinal herbs, methanol, ethanol, DW and acetone solvents all showed higher total antioxidant activity than the other two solvents The extracts of lemongrass, cinnamon, and anise showed good antibacterial ability on solvents DW, methanol and ethanol, and also showed high polyphenol content and antioxidant activity This result is consistent with previous studies on the relationship between antibacterial activity and polyphenol content and antioxidant activity Specifically, Nguyen Thi Thanh Ha et al., (2021) asserted that medicinal herbs capable of inhibiting the growth of bacteria are also medicinal herbs with high polyphenol content and strong antioxidant activity and Thanh Van Nguyen and Hai Thanh Nguyen (2019) 51 have shown a positive correlation between polyphenol content, antioxidant activity of medicinal herbs and their antibacterial ability 4.4.3 Results of determining the antioxidant capacity of essential oils Table 4.17 Antioxidant capacity of essential oils extracted Essential oil Antioxidant activity converted to VTME (mg) Cinnamon 10,163±0,246 Anise 32,543±2,768 Lemongrass 41,143±1,548 To compare the antioxidant activity of three essential oils of lemongrass, anise, and cinnamon, we compared the antioxidant activities of the essential oils The results are shown in the figure 45 40 35 30 25 20 15 10 Quế Cinnamon Hồi Anise Essential oil Sả Lemongrass Figure 4.10 Total antioxidant activity of medicines essential oils converted to VTME content (mg/ml of pure essential oil) From Table 4.19 and Figure 4.4 , we confirm that lemongrass essential oil at a concentration of 1/10 gave the highest antioxidant activity, followed by anise and cinnamon The highest antioxidant activity in lemongrass at a concentration of 1/10 was 41,143 mg (chlorogenic acid / 100g of medicinal herbs) and the lowest in cinnamon at a concentration of 1/10 was 10,163 mg (chlorogenic acid / 100g of medicinal herbs) 52 PART V CONCLUSIONS AND SUGGESTIONS Our study demonstrated the antibacterial effects of cinnamon, anise, and lemongrass, which in part provided a pharmacological basis to explain their traditional uses for the treatment of diseases associated with bacterial infections The antibacterial effects of these plant extracts were strongly correlated with their polyphenol content and antioxidant activity, suggesting that these compounds play an important role in the effects of plants for bacteria Among all the plant materials tested, cinnamon showed the highest antibacterial activity, and they also inhibited strains of E coli, S aureus, B.subtilis and G.Philus that were already resistant with antibiotics, suggesting that they may be candidates to replace antibiotics used in bacterial infections In addition, the antibacterial and biological activities of cinnamon, anise and lemongrass in the volatile part (diffusion method) were stronger than in the non-volatile 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