Evaluate the effect of honey and choerospondias axillaris mixture on necrotic bacteria (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - THESIS EVALUATE THE EFFECT OF HONEY AND CHOEROSPONDIAS AXILLARIS MIXTURE ON NECROTIC BACTERIA Hanoi, 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - THESIS TITLE EVALUATE THE EFFECT OF HONEY AND CHOEROSPONDIAS AXILLARIS MIXTURE ON NECROTIC BACTERIA Student : Bui Duc Hiep Faculty : Biotechnology Supervisors : Nguyen Thi Thuy Hanh, PhD Hanoi, February 2021 COMMITMENT I hereby declare that: This is my study, which was conducted under the guidance of supervisors; All of information provided in this study is true and accurate All published data and information have been duly cited Hanoi, February 2021 Student Bui Duc Hiep i ACKNOWLEDGEMENTS First, I am grateful to express my gratitude to the Thai Nguyen University of Medicne and Pharmacy for granting me support to pursue my thesis as well as the Department of Biotechnology, Vietnam National University of Agriculture, who provided me opportunities to pursue a higher education, anh to prepare myself to better I also would like to thank my supervisor, Dr Nguyen Thi Thuy Hanh who has given me her encouragement, and help during my writing and revising process I owe a debt of gratitude to many people whose help has been important to my success in completing this dissertation Firstly, I am deeply grateful to my major Bs Nguyen Thi Hien and Mas Nguyen Van Dung for their critical comments and useful suggestion Special for their help in collectiong data of my experiments It would be difficult to mention everyone who has contributed to the research reported in this dissertation I appreciate you all It is also give my thankfulness to my family giving me various used advises during the process of learning and studying Thank you very much! Hanoi,February 2021 Student Bui Duc Hiep ii TABLE OF CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vii ABSTRACT viii I INTRODUCTION 1.1 Problem: 1.2 Evaluate the effect of Honey and Choerospondias axillaris mixture on necrotic bacteria 1.3 Purpose and Requirement 1.3.1 Purpose 1.3.2 Requirement .2 II OVERVIEW 2.1 Introduction of Choerospondias axillaris 2.1.1 General introduction of Choerospondias axillaris 2.1.1.1 Distribution 2.1.1.2 Chemical composition 2.1.2 Botanical characteristic of Choerospondias axillaris 2.2 Honey 2.3 Necrosis of human skin 10 2.3.1 General information 10 2.2.2 The main types of bacteria cause necrosis of human skin 11 2.2.2.1 Streptococcus 11 2.2.2.2 Pseudomonas aeruginosa 13 2.3 The application of Choerospondias axillaris on folk drug 15 III: MATERIALS AND METHODS 17 3.1 Materials 17 3.2 Time and place 17 iii 3.3 Methods 17 3.3.1 Extraction of material from Choerospondias axillaris 17 3.3.2.Implementation of experiment In Vitro 18 3.3.3 Implementation of experiment on white mice 19 3.4 Experiments to assess the ability to kill bacteria Streptococcus and Pseudomonas aeruginosa cause skin necrosis of honey products and Choerospondias axillaris on white mice 20 IV RESULTS AND DISCUSSION 22 4.1 Extraction results for Choerospondias axillaris 22 4.2 Test in vitro 22 4.2.1 Research results on properties of Streptococcus of Honey and Choerospondias axillaris on a sterile ring 22 4.2.2 Research results on antibacterial properties of Pseudomonas aeruginosa of Honey product + Choerospondias axillaris extract on sterile ring 24 4.2.3 Research results of the experiment determine the dose of bacteria that can cause necrosis on an individual mouse .27 4.2.3.1 Streptococcus .27 4.3.2 Pseudomonas aeruginosa 29 4.4 Research results of the experiment evaluating the ability to kill Streptococcus and Pseudomonas aeruginosa cause skin necrosis of honey products - Choerospondias axillaris in white mice 33 V CONCLUSION AND PROPOSAL .35 5.1 Conclusion 35 5.2 Proposal 35 REFERENCE 36 iv LIST OF TABLES Table 3.1 Mix with honey according to different ratios to get formulas for studies 18 Table 3.2 Evaluate the general situation of white mice before causing streptococus 20 Table 3.3 Evaluate the general situation of mice before causing illness by the Pseudomonas aeruginosa 20 Table 3.4 Assess the general situation of mice before causing disease 21 Table 4.1 Results of bacterial inhibition loop on experimental agar with Streptococcus 23 Table 4.2 Variance and standard deviation of the formulas Resistant to Streptococcus 23 Table 4.3 Anti-streptococcal effect of Preparations of Honey Choerospondias axillaris 24 Table 4.4 Results of bacterial inhibition loop on experimental agar plates with Pseudomonas aeruginosa 25 Table 4.5 Variance and standard deviation of the formula resistant to Pseudomonas aeruginosa 25 Table 3.1 Mix with honey according to different ratios to get formulas for studies 18 Table 3.2 Evaluate the general situation of white mice before causing streptococus 20 Table 3.3 Evaluate the general situation of mice before causing illness by the Pseudomonas aeruginosa 20 Table 3.4 Assess the general situation of mice before causing disease 21 Table 4.1 Results of bacterial inhibition loop on experimental agar with Streptococcus 23 Table 4.2 Variance and standard deviation of the formulas Resistant to Streptococcus 23 Table 4.3 Anti-streptococcal effect of Preparations of Honey - Choerospondias axillaris 24 Table 4.4 Results of bacterial inhibition loop on experimental agar plates with Pseudomonas aeruginosa 25 v Table 4.5 Variance and standard deviation of the formula resistant to Pseudomonas aeruginosa25 vi LIST OF FIGURES Figure 2.1 Leaves and Fruits Figure 2.2 Young Leaf Figure 2.3 Honey Figure 2.4 Necrosis of the hand 11 Figure 2.5 Necrosis of the feet 11 Figure 2.6 Butt necrosis 11 Figure 2.7 Leg necrosis 11 Figure 2.8 Streptococcus 12 Figure 2.9 Pseudomonas aeruginosa 14 Figure 3.1 Medicinal herbs have been dried 17 Figure 3.2 Measure the diameter of the punching tool and the agar was perforated 19 Figure 4.2 Measure the bacterial inhibitory ring for the bacterial connective system 22 Figure 4.3 Direct antibacterial resistance of regime 25 vii ABSTRACT Nowadays, the use of natural products to treat necrosis is of great interest to many scientists and pharmaceutical companies The use of products derived from nature shows many advantages over the use of compounds and drugs that are synthesized by chemical methods namely environmentally friendly, safe for users, low irritation rate, etc However, besides that, it also has some limitations such as the need for an abundant source of raw materials, not much in-depth research works on those preparations Most natural preparations used to treat necrosis are on the basis of folk remedies Based on these advantages, thesis “Evaluate the effect of Honey and Choerospondias axillaris mixture on necrotic bacteria” was carried out to study and evaluate the effect in the treatment of necrosis disease for two main factors, streptococcus and Pseudomonas aeruginosa The research results of the project show that the product from honey and choerospondias axillaris can treat necrosis in in vitro experiments and experiments performed on living individuals (white mice) The medicinal use of the preparation is outstanding and is likely to be widely used in the future viii Table 4.1 Results of bacterial inhibition loop on experimental agar with Streptococcus Formula value Formula Formula 24 25 25 24 25 26 23 24 1 Formula Formula 25 23 11 3 (mm) frequency 1 1 (* In the table above, the row of values is the diameter of the bacterial inhibition rings (in mm) After obtaining raw data (measurements of bacterial inhibition ring), data processing is carried out: Table 4.2 Variance and standard deviation of the formulas Resistant to Streptococcus Formula Formula Formula Formula Formula Value n (sum of frequencies) Ā (average value of the 3 3 24,67 25,00 24,00 23,00 11,00 0 0 data sheet) Variance: S x 2= n (n x + n x 2) 0,06 - (Ā)2 Standard deviation: x ) 0,24 0,82 0,82 From there, it can be initially commented that, for streptococci, the antibacterial ability of formula is most stable (the variance and standard deviation are both 0) but the mean diameter of the inhibitory ring bacteria has the lowest value (23mm) The average variation is formula (standard deviation is 0.24mm), the average diameter of the bacterial inhibitory ring is also average (24.67mm) The strongest fluctuations were in formula and (standard deviation were both 23 0.82mm), however, the mean diameter of the bacteria inhibitory ring in formula was superior (25mm).) Table 4.3 Anti-streptococcal effect of Preparations of Honey - Choerospondias axillaris Diameter of aseptic ring (mm) Honey – Choerospondias axillaris extractum products under Papers research formulas impregnated with antibiotic Formula Formula Formula Formula Formula 24,67±0,24 25,00±0,82 24,00±0,82 23,00±0,00 mm mm mm mm 24,00 mm 11,00±0,00 mm From the result table 4.3, we can see that both the formula from Choerospondias axillaris inoculant has the ability to inhibit Streptococcus - one of the causes of necrosis of the skin However, the degree of inhibition of formulations with different ingredients differs Specifically, the lowest efficiency was formula with a bacterial inhibition diameter of 11mm and no fluctuation; The average efficiency is formula (100% Choerospondias axillaris) and formula (70% honey - 30% Choerospondias axillaris) with the bacteria inhibition ring diameter ranging from 24-25mm and achieve efficiency Formula is highest (30% honey - 70% Choerospondias axillaris) with a diameter ranging from about 25-26mm, superior inhibitory ability of the antibiotic Ceftriaxone 4.2.2 Research results on antibacterial properties of Pseudomonas aeruginosa of Honey product + Choerospondias axillaris extract on sterile ring After incubating in the oven at a temperature of 370C, for a period of 24 hours, similar to cocci, we will get the most accurate results on the agar plate 24 Occurrence of bacteria inhibitory rings for the Pseudomonas aeruginosa Measuring the diameter of the bacteria inhibition ring, we obtained the results with each formula as shown in figure 4.3: Figure 4.3 Direct antibacterial resistance of regime Table 4.4 Results of bacterial inhibition loop on experimental agar plates with Pseudomonas aeruginosa Formula Formula Formula Formula Formula Value 15 16 17 17 14 15 10,8 Frequency 1 3 Perform data processing steps for the experiment of resistance to Pseudomonas aeruginosa of honey product - Choerospondias axillaris Table 4.5 Variance and standard deviation of the formula resistant to Pseudomonas aeruginosa Value n (sum of frequencies) Ā (average value of the data sheet) Variance: S x 2= n (n x + n x 2) (Ā)2 Standard deviation x ) Formula Formula Formula Formula Formular 3 3 15,00 16,67 14,00 15,00 10,80 0,11 0 0,33 0 0 25 From the table above, we can initially comment that, for the green pus bacillus, the antibacterial ability of formula 1, 2, is the most stable (variance, standard deviation are all 0), but The mean diameter of the bacterial inhibitory ring was low (14mm) and medium (15mm) The strongest oscillation is formula (standard deviation is 0.33mm), however, the average diameter of the bacteria inhibitory ring in formula is superior (16.67mm)) Table 4.6 Antibacterial effects of Pseudomonas aeruginosa of honey product Choerospondias axillaris Diameter of aseptic ring (mm) Honey – Choerospondias axillaris extractum products under research formulas Papers impregnated with antibiotic Formula Formula Formula Formula Formula 13,00 mm 15,00±0,00 16,67±0,33 14,00±0,00 mm mm mm 15,00±0,00 10,80±0,00 mm mm The result in table 4.6 shows that, all four formulas from Honey and Choerospondias axillaris extract are capable of inhibiting Pseudomonas aeruginosa one of the causes of skin necrosis However, inhibition levels of formulations with different ingredients were not equal Specifically, the lowest efficiency is formula with the inhibitory ring diameter of 14.00mm and no fluctuation; The average efficiency was formula (100% Choerospondias axillaris and formula (50% Choerospondias axillaris + 50% honey) with the inhibitory ring diameter of 15mm and no fluctuation Achieving the highest efficiency is formula (70% Choerospondias axillaris + 30% honey) with a diameter ranging from about 16-17mm, which is superior to the inhibitory ability of Ceftriaxone antibiotics 26 4.2.3 Research results of the experiment determine the dose of bacteria that can cause necrosis on an individual mouse 4.2.3.1 Streptococcus Wound assessment: The progression of wounds is shown in the figures 4.4 a1-3: Representative individual batch 1: No signs of necrosis Representative individual batch 2: No signs of necrosis Representative individual batch 3: No signs of necrosis Representative individual batch 4: No signs of necrosis Representative individual batch 5: No signs of necrosis Figure 4.4 a1 The wound after 24 hours Representative Representative Representative Representative Representative individuals of individuals of individuals of individuals of individuals of lot 1: small lot 2: small lot 3: small lot 4: big pus lot 5: big pus pus appear pus appear pus appear appear appear Figure 4.4 a2 The wound after 48 hours 27 Representative Representative Representative Representative Representative individuals of individuals of individuals of individuals of individuals of lot 1: big pus lot 2: big pus lot 3: big pus lot 4: Broken lot 5: broken appear pus, clear pus, necrotic necrotic appears, but appear some of them appear appear die Figure 4.4 a3 The wound after 72 hours Body weight rating: The change in average weight of all bodies after 72 hours is shown in table 4.7 Table 4.7 Average weight change of individuals after 72 hours (Unit: gram) Lots Lot Lot Lot Lot Lot 24 hours 26,13 28,71 27,22 26,71 29,98 48 hours 26,11 28,65 27,12 25,86 27,78 72 hours 26,11 28,43 26,94 24,72 Time Base temperature rating: The change in average temperature of all people after 72 hours can be seen in table 4.8: 28 Table 4.8 The average temperature change of all bodies after 72 hours Lots Lot Lot Lot Lot Lot 24 hours 35,6 35,5 35,8 36,2 37,7 47 hours 35,3 35,7 36,1 35,9 38,1 72 hours 35,4 35,2 36,9 37,8 Time From the assessment of the wound, temperature and body weight of the individual mice used for the above experiment, the team made the following conclusions: In all lots, no individual died With a dosage of 0.1ml; 0.15ml and 0.2ml (concentration of 106 bacteria / ml): Temperature and body weight did not change significantly However, signs of necrosis appear late (after 72 hours to appear large pustules).With a dose of 0.25ml (concentration of 106 bacteria / ml): Temperature and body weight did not change significantly However, signs of necrosis appeared earlier, after 72 hours a marked necrotic lesion appeared With a dose of 0.3ml (concentration of 106 bacteria / ml): Temperature and body weight also did not change significantly However, signs of necrosis appeared earlier (after 72 hours of appearance of large necrosis) but at the same time a number of mice died So the dose of Streptococcus appropriate to cause necrosis on an individual mouse in the shortest time (72 hours) without causing death is 0.25ml / individual 4.3.2 Pseudomonas aeruginosa Wound assessment: The progression of wounds is shown in the figures 4.5.a1-3: 29 Representative Representative Representative Representative Representative individual individuals of individuals of individuals of individuals of batch 1: No lot 2: small lot 3: big pus lot 4: died signs of pus appear appear lot 5: died necrosis Figure 4.5 a1 The wound after 24 hours Representative Representative individuals Representative individuals individuals of lot 1: small of lot 2: Broken pus, clear pus appea necrotic appear of lot 3: died Figure 4.5 a2 The wound after 48 hours 30 Representative individuals of lot 1: small Representative individuals of lot 2: big pus appear pus appear Figure 4.5 a3 The wound after 72 hours Body weight rating: The change in average weight of all bodies after 72 hours is shown in the table 4.9: Table 4.9 Average weight change of the bodies after 72 hours (Unit : Gram) Lots Lot Lot Lot 24 Hours 26,18 29,72 27,44 48 Hours 26,16 27,43 72 Hours 26,13 25,58 Lot Lot Time Assessment of body temperature: The change in average temperature of all bodies after 72 hours is shown in the table 4.10: Table 4.10 The average temperature change of all bodies after 72 hours (Unit: 0C) Lots Lot Lot Lot Time 24 Hours 35,8 35,4 40,6 48 Hours 35,5 35,6 72 Hours 35,4 35,2 31 Lot Lot From the evaluation of the wound, temperature and body weight of the individual mice used in the above experiment, the team discussed the following: Individuals in Lot and live, individuals in Lot and die after 24 hours, individuals in Lot die after 48 hours With a dosage of 0.1ml; (concentration 106 bacteria / ml): Temperature and body weight did not change significantly However, signs of necrosis appear late (after 48 hours to appear small pustules).With a dose of 0.15ml (concentration of 106 bacteria / ml): Temperature and body weight did not change significantly However, signs of necrosis appeared early, after 48 hours appeared a small necrotic lesion, after 72 hours appeared a large necrosis.With a dose of 0.2ml (concentration of 106 bacteria / ml): The body temperature increases, with symptoms of fever Body weight did not change significantly However, the individual died after 48 hours, so the study could not be conducted With doses of 0.25ml and 0.3ml (concentration of 106 bacteria / ml): Because individuals died immediately after 24 hours, it was impossible to continue monitoring So the appropriate dose of Pseudomonas aeruginosa to cause necrosis on an individual rat in the shortest time (72 hours) without causing death is 0.15ml / individual 32 4.4 Research results of the experiment evaluating the ability to kill Streptococcus and Pseudomonas aeruginosa cause skin necrosis of honey products - Choerospondias axillaris in white mice Wound assessment: The progression of wounds is shown in the figure 4.6: Time Represent Lot A1 Represent Lot B1 Represent Lot A2 Represent Lot B2 days Repre Represent sent Lot B3 Lot A3 12 18 rats rats died died 28 The days rats mouse is died dead over Figure 4.6 Evaluate a change of the wound Results of wound assessment in table 4.12 showed: For lots A1, A2 and B1, B2 After days, the wound shows signs of drying back to young skin Not growing feathers After days, the wound has healed completely The hair has begun to grow again, no individuals died and the progression of wounds on individuals was similar Batches A3 and B3 after days, the wound is wet, widespread, pus, no hair growth and bad smell There were 12 deaths in Lot A3 and 18 deaths in Lot B3 After days the wound is more widespread, the pus cracks rupture, with a very bad smell, began to be invaded by ants, flies and maggots A total of 28 mice died in Lot A3 and all mice in Lot B died Assessment of body weight: The change in average weight of the cardfish is 33 shown in the following table: Table 4.11 Variation in average weight of individuals (Unit: Gram) Lots Lot A Lot B Lot A Lot B Lot A Lot B 3 days 28,12 30,32 27,44 29,34 24,58 21,74 days 28,67 29,83 28,56 27,73 19,62 Time Results of assessing body weight in table 4.11 showed: For lots A1, A2 and B1, B2: After days and after days, the body weight does not change significantly Batches A3 and B3 after days and after days, the body weight is significantly reduced Evaluation of body temperature: The change in average temperature of the cardamom is shown in the following table: Table 4.12 Variation in average body temperature of individuals (Unit: 0C) Lots Time days days Lot A Lot B Lot A Lot B Lot A Lot B 38,8 35,7 39,2 37,8 39,7 38,5 38,4 35,9 39,8 40,3 40,7 Results of body temperature assessment in Table 4.12 show: For lots A1, A2 and B1, B2 after days, the rat's body temperature increased, and fever appeared After days, the body temperature returns to normal, there is no fever For batches A3 and B3 after days and after days, the body temperature in the living individuals is very high From the results of research and evaluation above, I can confirm that: Honey products - Choerospondias axillaris (with the most effective rate is 70% high Choerospondias axillaris - 30% Honey) actually has ability to treat necrosis in experimental rats The product not only kills bacteria that cause necrosis but also helps the wound quickly heal 34 V CONCLUSION AND PROPOSAL 5.1 Conclusion From the research results obtained above, I draw the following three conclusions: High-grade honey products are capable of killing Streptococcus, Pseudomonas aeruginosa Successful treatment of necrosis in experimental mice Formula with a high rate of 70% Choerospondias axillaris - 30% Honey has the highest effectiveness in treating necrosis For mice with an average body weight ranging from 25-30g, the best dose of Streptococcus is 0.25ml / individual; The best dosage of bacillus green pus is 0.15ml / individual 5.2 Proposal Continue to draw the most accurate and reasonable dosage from the above experiments Continue to improve the product Conducted testing on voluntary patients Research on how to transfer research results to the product for application in clinical practice and outside medical facilities 35 LIST OF IMAGE Image : Measure the diameter of the perforations on the plate Image 2: Put the plates of agar in the oven 36 Image 3: mouse injection instructions REFERENCE [1] Central Dermatology Hospital 2013, Results of treatment of a case of necrosis of the skin - necrotizing fasciitis (necrotizing fasciitis) in the lower leg with surgical cleaning combined with a thin skin graft [2] Department of Microbiology - Thai Nguyen University of Medicine and Pharmacy 2015, Lecture on medical microbiology (materials used for training students of the University of Medicine and Pharmacy) [3] Vietnamese Pharmacopeia Vietnamese medicinal plants and animals (2003), Volume II, Science and Technology Publishing House in Hanoi [4] Nguyen The Hanh (2015), Doctoral thesis of Medicine: Clinical research, bacteriology and evaluation of antiseptic effectiveness in the treatment of Baume IV tooth marrow diseases with Calcium Hydroxide and Camphorated Parachlorophenol [5] Faculty of Medicine, Bac Ninh University of Sports, General outline of bones, joints, and muscles [6] Minh Nghiem (2014), Skin diseases, prevention and treatment of General Medicine Bookcase, Culture and Information Publishing House [7] Dang Thi Thuy Trang, Tran Minh Truong (2009), "Survey of necrotizing fasciitis in the neck and face", Journal of Medicine, Ho Chi Minh City [8] Ho Chi Minh City University of Agriculture and Forestry - Institute for Biotechnology and Environment (2012), Ribe Honey, http://ribe.hcmuaf.edu.vn/ribe-20012-1/vn/mat -ong-ribe-img-src imagesnewgif-border - 0.html [9] Pham Duc Tuan et al (2002), Introduction to some forest tree species grown in limestone mountains Department of Forestry Development, Ministry of Agriculture and Rural Development - Hanoi [10] Vo Quang Yen, Book of homegrown plants - medicinal plants and herbs (volume 4, author Vo Quang Yen) [11] http://www.nacurgo.com/; [12] http://www.duoclieu.org/2012/01/tac-dung-va-cong-dung-cua-tanin-vaduoc.html 37