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. 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. 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. iii LIST OF TABLES.
vi LIST OF FIGURES. viii LIST OF GRAPH AND CHART……………………………………….x LIST OF ABBREVIATIONS. Objective and requirements. Potential for development of herbal medicine.
Overview of medicinal herbs. Overview of bacteria. The gram-positive bacteria. The Gram-negative bacteria.
Overview of polyphenol. What is polyphenols ?. Types of polyphenols. Health benefits of polyphenols.
Overview of antioxidant activity. Method for determination of antioxidant activity. MATERIAL, CONTENTS, AND METHODS. Time and place of study.
Method of extracting medicinal herbs. Method of determining the antibacterial activity of medicinal herbs. Method of determining polyphenol content. Method of determining antioxidant activity.
Data processing methods. RESULTS AND DISCUSSION. The results of the investigation of antibacterial activity of medicinal herbs. The results of determining the diameter of the inhibition zone of 6 extracts of lemongrass.
The results of determining the diameter of the inhibition zone of 6 extracts of cinnamon. The results of determining the diameter of the inhibition zone of 6 extracts of anise. Results of the investigation on the antibacterial activity of essential oils. Results of antibacterial activity of lemongrass essential oil.
Results of antibacterial activity of cinnamon essential oil. Results of antibacterial activity of anise essential oil. The results of determining polyphenol contents. The result of building a standard graph between chlorogenic acid content and the increase in optical density measured when reacting with Folin Ciocalteu reagent.
The results of polyphenol content of herbal extracts of cinnamon, anise, lemongrass (content 100mg/ml). The results of polyphenol content of essential oils of cinnamon, anise, lemongrass. Results of determining the antioxidant activity of medicine extracts and essential oils. The results of the determination of the antioxidant capacity of the standard VTME (Alpha tocopherol).
The results of the determination of antioxidant activity of extracts of cinnamon, anise, lemongrass. CONCLUSION AND SUGGESTION. 54 v LIST OF TABLES Table 4. Results of inhibition zone diameter (mm) of lemongrass extracts.
Results of inhibition zone diameter (mm) of lemongrass extracts. Results of inhibition zone diameter (mm) of cinnamon extracts. Results of inhibition zone diameter (mm) of cinnamon extracts. Results of inhibition zone diameter (mm) of anise extracts.
Results of inhibition zone diameter (mm) of anise extracts. Antibacterial activity of lemongrass essential oil based on steam method. Antibacterial activity of lemongrass essential oil based on steam method. Antibacterial activity of cinnamon essential oil based on steam method.
Antibacterial activity of cinnamon essential oil based on diffusion method. Antibacterial activity of anise essential oil based on steam method. Antibacterial activity of anise essential oil based on diffusion method. The results of antibacterial activity of trans-cinnamalehyde with different concentrations.
Variation of OD values according to chlorogenic acid standard concentration (mg/ml).15: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/100 mg of medicinal herbs) when extracted with different solvents.16: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/ml pure essential oil). Antioxidant activity of standard VITAMIN E determined by DPPH method at different concentrations AA%. Correlation between content of VTME standard (standard substance) and antioxidant activity (scavenging activity). Antioxidant activity of extracts.
Antioxidant capacity of essential oils extracted. 52 vii LIST OF FIGURES Figure 2. Lemongrass and lemongrass powder. Cinnamon and cinnamon powder.
Anise and anise powder. Steps to perform the method of extracting medicinal herbs. Essential oil distillation kit. Essential oils of cinnamon, anise, lemongrass.
Describe the method of mixing bacteria into agar and punching a small well of antibacterial agent on agar plates. The ability to inhibit bacteria of medicinal herbs within 24 hours of culture. Antibacteria of essential oil based on steam method. Antibacteria of essential oils within 24 hours of culture based on diffusion method.
Diagram of the experiment to determine the total polyphenol content, using Folin-Ciocalteu's Phenol Reagent reagent. Mechanism of the color change reaction in the determination of total polyphenol content, using Folin-Ciocalteu's phenol reagent (Sadeer et al. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) reaction mechanism. Mechanism of the color change reaction in the determination of antioxidant activity using the DPPH reagent.
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. Correlation between content of chlorogenic acid standard substance (mg/ml) and the degree of increase in optical density value (OD value). Total polyphenol content converted to chlorogenic acid (mg) of cinnamon, anise, lemongrass (content 100mg/ml) when extracted with different solvents.
Total polyphenol content converted to chlorogenic acid (mg) of 3 essential oils (converted to 1 ml of pure essential oil). 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. Correlation between content of VTME standard (standard substance) and antioxidant activity (scavenging activity). Color change of cinnamon at a concentration of 20mg/ml of medicinal herbs upon color reaction with DPPH.
Color change of cinnamon medicinal herbs at a concentration of 10mg/ml of medicinal herbs upon color reaction with DPPH. Total antioxidant activity of lemongrass, cinnamon and anise extracts was converted to VTME content (mg/100 mg of medicinal herbs). Total antioxidant activity of medicines essential oils converted to VTME content (mg/1 ml of pure essential oil). 52 ix LIST OF ABBREVIATIONS Abbreviation Explanation ATCC American Type Culture Collection AMR Antimicrobial resistance B.subtilis Bacillus subtilis DMSO Dimethyl Sunfoxide DPPH 1,1-Diphenyl-2-Picrylhydrazyl DW Distilled Sunfoxide E.coli Eschrichia Coli G.philus Geobacilus stearothermophilus P.seudo Pseudomonas aeruginosa S.aureus Staphylococcus aureus Sal Salmonella enterica VTME Vitamin E x ABSTRACT Our study evaluated the inhibitory effects of lemongrass, cinnamon, anise on bacterial growth, so as to explain their uses in bacterial infections.
In addition, the correlation between antibacterial effects of plants and the polyphenol contents or antioxidant activities was also verified to investigate if these properties were responsible for the plant antibacterial effects. We tested extracts with 9 bacterial strains (Bacillus subtilis ATCC 6633, Geobaccillus stearothermophilus ATCC 7953, Staphylococcus aureus ATCC 25023, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 85922, Escherichia coli ATCC 35218, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella ATCC 13311). After polyphenol contents and antioxidant activities were measured, we examined their correlations with antibacterial effects. The results showed that all extracts had an inhibitory effect on the bacteria tested and to varying degrees.
Among all the samples, the cinnamon extract had the strongest effect and also inhibited 9 bacterial strains. The antibacterial and biological activities of cinnamon, anise, and lemongrass in the volatile part (diffusion method) were stronger than in the non-volatile part (steam method). In addition, direct and significant correlations between the antibacterial effect and the value of polyphenol content were established and thus demonstrated that the antibacterial effects of cinnamon, anise, lemongrass, at least in part, is due to the presence of polyphenol compounds. Our study demonstrated the antibacterial effects of cinnamon, anise, and lemongrass and thus partly provided a pharmacological basis to explain their traditional uses in diseases associated with bacterial infections.
In addition, since cinnamon extracts showed the highest effect, we suggest that they may be the best candidates for bacterial treatment. However, further studies are still required to evaluate their potential. Preface Since very ancient times, humans have used plants to treat infectious diseases; scientific investigations of plant material have clearly proved the therapeutic efficacy of plants over time. Today, many countries use plants to treat different maladies including infectious diseases of the respiratory, gastrointestinal, urinary and biliary systems (Rios and Recio, 2005).
Although significant progress has been made in microbiological research and in the control of many diseases caused by infectious organisms such as bacteria, recurrent epidemics due to drug-resistant bacteria as well as the appearance of new bacterial pathogenic strains demand the discovery of new antibiotics. The investigation of medicinal plants using frontier technologies is now being reconsidered to be a feasible approach for discovering novel bioactive agents to solve widespread public health problems (Cos et al., 2006, Harvey et al. The search for natural active ingredients with high biological activity to make medicine is a trend that is of great interest to scientists. Many medicinal materials have been used to extract medicinal ingredients such as: berberine extract from Coscinium fenestrated, morphine from poppy (Papaver somniferum), beta-carotene and lycopene from gac fruit (Mormodica cochichinensis), Strychnos nux-vomica, papain (Carica papaya), diosdenin (Dioscorea deltoidea), curcumin from turmeric (Curcuma longa), peppermint menthol (Metha arvesis)… There are many types of important active ingredients such as quinine, morphine, strychnin, etc., which must be extracted from medicinal herbs but cannot be taken by chemical synthesis (Phung Tuan Giang, 2017).
Therefore, the analysis and investigation of antibacterial activity, polyphenol content and antioxidant activity of medicinal herbs is essential in order to properly evaluate the capabilities and activities of medicinal herbs. Medicinal herbs such as lemongrass, cinnamon, and anise have long been used in folk medicine as medicines to cure diseases, increase resistance, improve 1 health, and are used to treat infections very well. Therefore, these medicinal herbs have great potential in the research and development of pharmaceuticals, and are an ideal source of pharmaceutical substitutes for antibiotics and synthetic chemicals due to their superiority in cost, effectiveness and quality. For that reason, we carried out a study to extract and evaluate the antibacterial ability, polyphenol content and antioxidant activity of the medicinal plants lemongrass, cinnamon, and anise.
Objective - Proven antibacterial effect of medicinal herbs lemongrass, cinnamon, and anise on bacteria Bacillus subtilis ATCC 6633, Geobaccillus stearothermophilus ATCC 7953, Staphylococcus aureus ATCC 25023, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 85922, Escherichia coli ATCC 35218, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella ATCC 13311. - Investigation of polyphenol content and antioxidant activity of extracts and essential oils. - Choose the right solvent to use in the extraction process to get the best results.