THAI NGUYEN UNIVERSITY THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY LUONG YEN NHI RESEARCH ON MAKING CALLUS FROM BLACK GINGER ROOT FOR CULTURING THE SUSPENSION PRODUCING SECONDARY COMPOUND 5.7 DIMETHOXYL FLAVONE IN LABORATORY SCALE. BACHELOR THESIS Full name : Luong Yen Nhi Study Mode : Full time Major : Food Technology Faculty : Advanced Education Program Office Batch : K48 Type : Research (Lab work) Supervisor : Dr. Luong Hung Tien Thai Nguyen,2023. 1 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program: Bachelor of Food Science and Technology Student name: Luong Yen Nhi Student ID: DTN1654190004 Thesis Title: Research on making callus from black ginger root for culturing the suspension producing secondary compound 5.7 dimethoxyl flavone in laboratory scale.Luong Hung Tien - Thai Nguyen University of Agriculture and Forestry, Vietnam.
Supervisor's Signature: This study aimed to build a process for industrial production of the bioactive ingredient 5.7-dimethoxy flavone using suspension cell culture from black ginger (Kaempferia parviflora). The research results are the premise for providing more scientific knowledge and important experience for producing biologically active substances in black ginger to provide raw materials for the pharmaceutical industry using suspension culture technology on the industrial scale. Black ginger root that is 10 months old or older, has no signs of infection and must have a growth peak. It was treated with Antracol 70WP 2.5% solution, after incubation of Antracol 70WP, move to further incubation in clean sand, and waited for 20-30 days for the top to grow and turn into sprouts.
The result gives information that the best method of sample sterilization is povidone iodide 10% for 2 minutes and HgCl 0.1% for 9 minutes, giving a survival rate of 96. The best medium for callus formation was MS supplemented with 1 mg/liter of 2.4-D, 1 mg/liter of NAA, and 1 mg/liter of Kinetin, achieving a rate of 70.00% of samples and MS supplemented with 1 mg/liter of 2.4D after 30 days of culture when the culture reaches 20 grams fresh. Keywords: Black Ginger (Kaempferia parviflora), 5.7- dimethoxyflavone, callus, flavonoids, methoxyflavons Number of pages: 52 pages Date of submission 10/07/2023 2 ACKNOWLEDGEMENT This experiment was carried out at the Vietnam Indigenous Medical Institute-No 166, group 1, Tích Lương ward, Thai Nguyen city. With the deepest and most sincere affection, allowing me to express my gratitude to all the individuals and organizations who have facilitated support and assistance throughout the learning and research process.
First of all, I would like to thank my supervisor, Dr.Luong Hung Tien for his interest in helping and guiding me over the period under study. He always assisted me whenever I needed it and directed me in the right direction to finish the project. Secondly, I would like to thank Mr. Hoang Van Nam and the Vietnam Indigenous Medical Institute for providing me with a lot of information and professional advice related to my thesis.
Besides, they also created good conditions, ready to provide me with a full range of equipment and machinery to ensure research. Thirdly, I would want to convey my heartfelt appreciation to my family and friends, those who have always been there for me and supported me. Due to the limited theoretical level and practical experience, the report inevitably has shortcomings, I look forward to receiving your comments so that I can learn from experience to improve the article report to the best results. Once again, I would like to say thank you so much and wish you good health and success in your noble career.
Best regards, Thai Nguyen, January, 2𝑛𝑑 ,2023 Sincerely Luong Yen Nhi 3 TABLE OF CONTENT DOCUMENTATION PAGE WITH ABSTRACT. 3 LIST OF FIGURES. 4 LIST OF TABLE. 8 LIST OF ABBREVIATIONS.
Error! Bookmark not defined. Aim(s) of the study. Purpose and Objective. Error! Bookmark not defined.
Meaning of the research. 11 CHAPTER II: LITERATURE REVIEW .Overview of black ginger plant. Some domestic and foreign studies on black ginger. Anti-inflammatory effect.
Anti-allergic effect. Effects on the genitourinary system. Effects on the heart. Inhibition of metabolic enzymes in the liver.
There are a few products that contain the active ingredient of black ginger on the market. Bioactive of 5,7-dimethoxyflavone (DMF),. The concept of stem cells and plant stem cells. Method of culturing tuberous black ginger by cell suspension method.
Plant cell suspension culture concept. History of suspension culture research. Characterization of plant cells in suspension culture. 23 CHAPTER III: METHODOLOGY.
Materials and chemicals. Image of the source of the research object. Prepare research subjects:. Location and time.
Culture environment and conditions. Research content and methods. Research process for black ginger callus culture and biomass production from suspension. 32 CHAPTER IV:RESULTS.
Results Studying the effect of sample sterilization time on the survival of callus specimens. Research results on the effects of 2,4D, NAA, and Kinetin on the formation of callus. Research results on the effects of 2,4D, NAA, Kinetin on the growth of single cell biomass containing compound 5. 36 CHAPTER V: DISCUSSION, CONCLUSION AND RECOMMENDATION.
Error! Bookmark not defined. CHAPTER VII : APPENDIX. 1 Appendix 1: Sample sterilization results after 45 days of culture. 1 Appendix 2: Study on the effect of growth regulators on the formation of Calus.
2 Appendix 3: Effect of growth regulators on growth of single cell biomass 40 days. 4 Appendix 4: Results of growth curve study. 5 6 LIST OF FIGURES Figure 1: Botanical characteristics of the genus Kaempferia L. 12 Figure 2: Black Ginger (Kaempferia parviflora).
13 Figure 3: Some compounds found in Black Ginger. 14 Figure 4: General chemical formula of flavones in Black Ginger. 16 Figure 6: Chemical structure of 5. 20 Figure 7: Experimental explants after sterilization.
34 Figure 8: a) Callus formed from the studied samples; b) rapid multiplication of scar tissue. 35 Figure 9: c) callus; d) suspension culture; e) single cell biomass. 37 Figure 10: Single cell suspension growth graph. 38 Figure 11: Qualitative image by TLC-TLC image captured by Qualitative TLC.
38 Figure 12: Analytical chromatogram of 5.7-Dimethoxyflavone extracted from the suspension culture. 39 7 LIST OF TABLE Table 1: Materials and chemicals used in the study. 24 Table 2: Some basic tools, means and equipment used in the research. 25 Table 3: Sample sterilization results after 45 days of culture.
33 Table 4: Effect of 2,4D, NAA, Kinetin CCPs on callus formation. 34 Table 5: The results of the effect of plant growth regulator on the growth of single cell biomass for 40 days. 36 8 LIST OF ABBREVIATIONS KP Kaempferia parviflora Wall.7-dimethoxyflavone MS Murashige và Skoog, 1962 2.4D Dichlorophenoxyacetic acid NAA Aldrich, Naphthaleneacetic acid TLC Thin layer chromatography HPLC High Performance Liquid Chromatography 9 CHAPTER I INTRODUCTION 1. Research rationale Black ginger, with the scientific name Kaempferia parviflora (family Zingiberaceae), also known as "Thailand Ginseng," is used as a folk medicine with the effects of stimulating digestion, improving blood flow, and promoting male sexual health.
The bioactive compounds in the black ginger root have strong antibacterial effects to help treat allergies, peptic ulcers, and fungal infections. Besides, black ginger can enhance vitality and stress resistance, reduce triglycerides, fight atrial fibrillation, and prevent diabetes. In black ginger, methoxyflavone is the main group of substances found. Reports also indicate that 5.7-dimethoxyflavone (DMF) is one of the three main compounds, containing many biological and pharmacological effects.
With such uses, the exploitation of natural medicinal herbs is becoming an important issue. In most of the world, black ginger has not been grown on a large scale. There has not been much research on propagation. In addition, black ginger grows slowly, does not seem difficult to branch and grow, and takes a long time to harvest.
The price of this medicine is also high. Thus, the production of plant secondary compounds by traditional farming methods and chemical synthesis will have many limitations, so it will be difficult to meet the increasing demand for medicinal herbs in the future. Aim(s) of the study 1. Overall objectives Building a process for industrial production of the bioactive ingredient 5.7-dimethoxy flavone using suspension cell culture from black ginger (Kaempferia parviflora) to provide raw materials for the production of KD-01 Saman products used to treat erectile dysfunction.Detail objectives - Determine the best method of sample sterilization for maximum survival.
10 - Determine the growth regulator ratio for maximum callus formation efficiency. - Determination of the growth regulator ratio in suspension culture in order to produce secondary compounds5.7-dimethoxy flavone with high efficiency. Meaning of the research - Provides new scientific knowledge on the study of cell suspension culture for black ginger plant. - Applying the results to the production of food products, health foods, or medicinal products - Reduce the financial strain on agricultural land funds.
11 CHAPTER II: LITERATURE REVIEW 1.Overview about Black Ginger 2.Name, origin Figure 1: Botanical characteristics of the genus Kaempferia L. Ginger family According to the Takhtajan plant taxonomy published in 2009, Kaempferia parviflora belongs to the phylum Magnoliophyta, class Liliopsida, subclass Liliidae, order Zingiberales, family Zingiberaceae, and genus Kaempferia L. The genus Kaempferia L is distinguished by its small herbaceous plant, with the root tip frequently bulging into a bulbous form.The false stem is very short or absent, and the leaf blade is sometimes mottled or pink on the underside. inflorescence apex, growing between leaf sheaths or in soil from rhizomes, with flowers appearing before or after leaves.
Lateral stamens have white or pink lips, sometimes with spots and other colors near the base of the lip. They usually live in moist humus soil, along the fields, in rock holes, and under the forest canopy. There are about 50 species in the world, distributed in tropical Asia; there are eight species in Vietnam. Kaempferia parviflora was originally found in northern and northeastern Thailand.
12 This plant's rhizome has been used as food and medicine in Thailand for over 1000 years.They are not only considered a health-promoting herb in general and for male sexual health in particular, but they are also used by folk as a medicine to treat ailments. The dried Kaempferia parviflora root is usually ground into a powder and used to make tea bags. The fresh root is used to make alcohol. In addition, black ginger has been made into various preparations such as tinctures, tinctures with honey, capsules, etc.
Today, Kaempferia parviflora is found distributed in China, India, and some Southeast Asian countries such as Thailand, Laos, and Malaysia. In a study on the botanical taxonomy of the ginger family in the region of Central Vietnam, this plant was discovered in Krong Bong district, Dak Lak province. And until now, there have been some provinces that have grown this black ginger tree, such as Thanh Hoa, Son La, Lao Cai, Lai Chau. Characteristic of Black Ginger (Kaempferia parviflora) Figure 2: Black Ginger (Kaempferia parviflora) 13 Kaempferia parviflora is an herbaceous, up to 90cm tall, dark purple rhizome with several branched succulent roots.
The leaves are thin, ovate or oblong, 7-11cm wide, smooth on the upper surface, hairy on the lower surface, with long petioles and webbed, burgundy leaf margins. Their inflorescences are surrounded by many leaves. Young flowers are white. The only difference between black ginger and normal ginger is that the inner part of black ginger is dark purple.
Black ginger is also often confused with black turmeric (Curcuma caesia) because they look similar; however, the inside of sliced black turmeric has a bluish color. Chemical composition Figure 3: Some compounds found in Black Ginger Some studies on the chemical composition of Kaempferia parviflora The main compounds found in black ginger rhizomes are flavonoids, glycosides, essential oils, etc. The main ones are methoxyflavons.