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
INTRODUCTION
LITERATURE REVIEW
Some domestic and foreign studies on black ginger
1.4 2.2 Some domestic and foreign studies on black ginger
Black ginger extract has been shown to have anti-inflammatory effects with the ability to inhibit NO and PGE2 activities More specifically, the inhibition of PGE2 production in RAW 264.7 cells gave an IC50 value of 9.2 g/mL Regarding the inhibition of NO release, an inflammatory mediator (IC50 = 3.6 g/ml), the researchers conducted an anti-inflammatory model test with chloroform and n-hexane extract fractions at a dose of 150 mg/kg The percentage inhibition of edema at 3 and 5 hours after injection of carrageenan was 25.4 and 25.3%, respectively; 18.3% higher than the control group using Indomethacin at 10 mg/kg after 3 hours.Meanwhile, with ethanol, ethyl acetate, and water extract fractions, the percentage of inhibition was lower, respectively, at 12.9, 5.6, and 6.2 (Sae-wong, 2009)
The antifungal activity of black ginger rhizome has been tested against human pathogens like bacteria, yeast, ringworm, etc in a model using a diffuser agar plate
The ethanol extract exhibited strong antifungal activity against ringworm, with an antifungal circle size of 10.7–19.8 mm at a concentration of 2 mg/ml of black ginger extract Studies showed that ethanol extracts of rhizomes exhibited potent antifungal activity against Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum with MICs of 62.5 g/ml, 125 g/ml, and 250 g/ml With the three fungal species mentioned above, the 3,5,7-trimethoxyflavone has significant antifungal activity, with a MIC value of 250 g/ml (Chavi Yenjai a, 2003)
Shoko Kobayashi's research group investigated the antiallergic effects of methoxyflavone compounds in black ginger rhizomes These flavone compounds were tested for their ability to inhibit -Hexosaminidase release and activity in RBL-2H3 cells.With an IC50 of 10.9 g/mL, it has a strong antiallergic effect against hexosaminidase antigen release (Supinya Tewtrakul, 2008)
2.2.4 Effects on the genitourinary system
In a study on the ability of Kaempferia parviflora to inhibit Phosphodiesterase 5 (PDE5—a substance that regulates cGMP levels), Prapapan Temkitthawon et al showed that the ethanol extract from black ginger rhizomes was able to inhibit PDE5 and PDE6, and the group of compounds with this effect are the polymerthoxyflavons
5.7-dimethoxyflavone has an extremely high ability to inhibit PDE5 (IC50 = 10.64 M) among this group of flavone compounds (Temkitthawon, 2011)
Tep-Arechan et al studied the effects of 5.7-dimethoxyflavone (DMF) isolated from the black ginger rhizome Kaempferia parviflora The study was performed on the aortas of rats The results showed dilation of the aorta in mice with DMF concentrations ranging from 1àM to 100àM This effect was significantly reduced when the endothelium was removed (Somruedee Yorsin, 2016)
Recently, there have been many studies on the anticancer effects of this plant A study by Saranyapin Potikanond et al showed that a 95% ethanol extract of black ginger rhizomes reduced the viability of HeLa cells (Banjerdpongchai, 2009)
2.2.7 Inhibition of metabolic enzymes in the liver
5.7-dimethoxyflavone (5.7-DMF), one of the main components of black ginger rhizome, has a direct inhibitory effect on the cytochrome P450 (CYP) 3A metabolizing enzyme (Prapapan Temkitthawon a, 2011)
Many other studies have also shown the use of this "panacea."
2.3 There are a few products that contain the active ingredient of black ginger on the market
Kaempferia parviflora Herbal Powder Extract 50 G
ORIENTAL HERITAGE HERBALISTS Improved Sexual & Physical Capacity, Improved Blood Circulation, Relieve Anti-metabolic
Supports healthy circulation and physical vigor Promotes mental and emotional well-being Standardized to 4% 5,7-dimethoxyflavone
The Best Premium Kaempferia Parviflora Natural Herbal Powder 500mg 100 Vegetarian Capsules
Promotes vitality benefit increase the body’s ability to cope with daily environment Benefits for health promotes vitality and stamina and skin radiant
Kaempferia Parviflora 500 mg 100 Caps -Black Ginger-Krachai Dum Original from Thailand (3 Bottle 300 Caps.)
Kongka Herb Kaempferia Parviflora Capsules (Kra Chai Dum), 60 g
Kra Chai Dum tones, improves endurance, provides energy, improves potency, relieves muscle spasms, relaxes the body after exercise, relieves tension from muscles, improves performance.
KD-01 Saman- Vietnam Indigenous Medicine Co., Ltd
Figure 6: Chemical structure of 5.7-dimethoxyflavone
5,7-dimethoxyflavone (DMF), a major flavone found in Kaempferia parviflora It is a white solid with the molecular formula C17H14O4, molecular weight 282.295, and melting point 157-158 o C
5.7-dimethoxyflavone (DMF) possesses a wide range of pharmacological and biological activities:
The function of anti-obesity and melanogenesis, anti-inflammatory and anti-cancer;
DMF inhibits cytochrome P450 (CYP) 3A in the liver; DMF is a powerful anti-breast cancer protein inhibitor; inhibiting the accumulation of fat cells should be used to combat obesity; improving sexual satisfaction and treats erectile dysfunction by dilating the cavernous artery, the spongy body in the penis, helping to produce longer and stronger erections
2.5 The concept of stem cells and plant stem cells
21 The essence of the cell suspension culture process is to culture plant stem cells until the cells are mature and capable of producing secondary biological substances as in the natural environment
Stem cells are cells that can develop into many different types of cells in plants and animals Plant stem cells can divide endlessly to fill and make up for the lack of cells caused by death and organ damage in living plant and animal tissues
Stem cells have the unique ability to self-renew and regenerate functional tissues
2.6 Method of culturing tuberous black ginger by cell suspension method
2.6.1 Plant cell suspension culture concept
Each plant cell is itself an independent unit that contains all the genetic information specific to the species from which it is born So each cell can rebuild a whole new being through omnipotence Higher plants are an important source of chemical and pharmaceutical compounds The plant cell suspension culture method has the potential to contribute to providing bioactive substances as raw materials for production
Culture a single-cell suspension or a multi-cell cluster in suspension in a liquid nutrient medium The suspension was made by culturing a piece of ceased callus in a liquid medium and was shaken and rotated for the duration of the culture The cells separated from the callus and dispersed in a liquid medium, after a while the suspension would be a mixture of single cells, cell clusters, culture remnants, and dead cells
2.6.2 History of suspension culture research
- In 1949, Caplin and Stewart cultured plant cells on a liquid medium with stirring;
Muis (1954) and Nickell (1956) found that plant cells can grow as organisms; In 1959, Melchen and Beckman successfully isolated and cultured single cells and cell suspensions
- The history of previous studies shows that the process of creating callus and nourishing the suspension has the same model design, but the research results are very different
Depending on the conditions and purposes, there are different methods of cell suspension culture Today, the main method of culture is the dynamic humoral culture
In a dynamic culture with a gas supply system (air blower), the gases are filtered through the system to ensure sterility
- Continuous submerged culture: cells are exposed to the nutrient medium because they are completely immersed in the nutrient solution Ventilation was performed by a shaker running at 100 - 150 rpm Inlet gas must be sterile Ventilation also prevents and reduces the adhesion of cells to each other
According to Thomas and Davey (1975), in cell suspension culture with a capacity of 25 ml, the most suitable speed of the shaker is 100 – 120 rpm The volume of the liquid medium should also be suitable for the size of the culture vessel with approximately 20% of the flask volume For small-scale cultures and short periods, a magnetic stirrer can be used at 250 rpm and an incubation period of 10-15 days Then, the cultures must be transferred to a new medium to ensure the growth and development of the cells
- Circulatory submerged culture: Cells are immersed in the humoral medium, alternating with periods of being exposed to the medium The process is carried out using the “rocking” movement of the culture vessels When in motion, the cell mass at one end sinks, and the cell mass at the other end emerges to contact the air Steward et al (1952) also designed special culture vessels according to the method of circulating submerged culture
2.6.3.2 Research on industrial-scale cell suspension culture using Bioreactor:
The culture system is ventilated or gas-exchanged Plant cell culture media can be easily changed from time to time Plant cells are sensitive to a lack of oxygen and light, so the bioreactor system regularly ensures enough oxygen, light, and agitation of the nutrient medium
Bioreactors used in plant tissue culture are modified from bioreactors used in microbial cell cultures
2.6.4 Characterization of plant cells in suspension culture
There are a few products that contain the active ingredient of black ginger on
Kaempferia parviflora Herbal Powder Extract 50 G
ORIENTAL HERITAGE HERBALISTS Improved Sexual & Physical Capacity, Improved Blood Circulation, Relieve Anti-metabolic
Supports healthy circulation and physical vigor Promotes mental and emotional well-being Standardized to 4% 5,7-dimethoxyflavone
The Best Premium Kaempferia Parviflora Natural Herbal Powder 500mg 100 Vegetarian Capsules
Promotes vitality benefit increase the body’s ability to cope with daily environment Benefits for health promotes vitality and stamina and skin radiant
Kaempferia Parviflora 500 mg 100 Caps -Black Ginger-Krachai Dum Original from Thailand (3 Bottle 300 Caps.)
Kongka Herb Kaempferia Parviflora Capsules (Kra Chai Dum), 60 g
Kra Chai Dum tones, improves endurance, provides energy, improves potency, relieves muscle spasms, relaxes the body after exercise, relieves tension from muscles, improves performance.
KD-01 Saman- Vietnam Indigenous Medicine Co., Ltd
Figure 6: Chemical structure of 5.7-dimethoxyflavone
5,7-dimethoxyflavone (DMF), a major flavone found in Kaempferia parviflora It is a white solid with the molecular formula C17H14O4, molecular weight 282.295, and melting point 157-158 o C
5.7-dimethoxyflavone (DMF) possesses a wide range of pharmacological and biological activities:
The function of anti-obesity and melanogenesis, anti-inflammatory and anti-cancer;
DMF inhibits cytochrome P450 (CYP) 3A in the liver; DMF is a powerful anti-breast cancer protein inhibitor; inhibiting the accumulation of fat cells should be used to combat obesity; improving sexual satisfaction and treats erectile dysfunction by dilating the cavernous artery, the spongy body in the penis, helping to produce longer and stronger erections.
The concept of stem cells and plant stem cells
21 The essence of the cell suspension culture process is to culture plant stem cells until the cells are mature and capable of producing secondary biological substances as in the natural environment
Stem cells are cells that can develop into many different types of cells in plants and animals Plant stem cells can divide endlessly to fill and make up for the lack of cells caused by death and organ damage in living plant and animal tissues
Stem cells have the unique ability to self-renew and regenerate functional tissues.
Method of culturing tuberous black ginger by cell suspension method
2.6.1 Plant cell suspension culture concept
Each plant cell is itself an independent unit that contains all the genetic information specific to the species from which it is born So each cell can rebuild a whole new being through omnipotence Higher plants are an important source of chemical and pharmaceutical compounds The plant cell suspension culture method has the potential to contribute to providing bioactive substances as raw materials for production
Culture a single-cell suspension or a multi-cell cluster in suspension in a liquid nutrient medium The suspension was made by culturing a piece of ceased callus in a liquid medium and was shaken and rotated for the duration of the culture The cells separated from the callus and dispersed in a liquid medium, after a while the suspension would be a mixture of single cells, cell clusters, culture remnants, and dead cells
2.6.2 History of suspension culture research
- In 1949, Caplin and Stewart cultured plant cells on a liquid medium with stirring;
Muis (1954) and Nickell (1956) found that plant cells can grow as organisms; In 1959, Melchen and Beckman successfully isolated and cultured single cells and cell suspensions
- The history of previous studies shows that the process of creating callus and nourishing the suspension has the same model design, but the research results are very different
Depending on the conditions and purposes, there are different methods of cell suspension culture Today, the main method of culture is the dynamic humoral culture
In a dynamic culture with a gas supply system (air blower), the gases are filtered through the system to ensure sterility
- Continuous submerged culture: cells are exposed to the nutrient medium because they are completely immersed in the nutrient solution Ventilation was performed by a shaker running at 100 - 150 rpm Inlet gas must be sterile Ventilation also prevents and reduces the adhesion of cells to each other
According to Thomas and Davey (1975), in cell suspension culture with a capacity of 25 ml, the most suitable speed of the shaker is 100 – 120 rpm The volume of the liquid medium should also be suitable for the size of the culture vessel with approximately 20% of the flask volume For small-scale cultures and short periods, a magnetic stirrer can be used at 250 rpm and an incubation period of 10-15 days Then, the cultures must be transferred to a new medium to ensure the growth and development of the cells
- Circulatory submerged culture: Cells are immersed in the humoral medium, alternating with periods of being exposed to the medium The process is carried out using the “rocking” movement of the culture vessels When in motion, the cell mass at one end sinks, and the cell mass at the other end emerges to contact the air Steward et al (1952) also designed special culture vessels according to the method of circulating submerged culture
2.6.3.2 Research on industrial-scale cell suspension culture using Bioreactor:
The culture system is ventilated or gas-exchanged Plant cell culture media can be easily changed from time to time Plant cells are sensitive to a lack of oxygen and light, so the bioreactor system regularly ensures enough oxygen, light, and agitation of the nutrient medium
Bioreactors used in plant tissue culture are modified from bioreactors used in microbial cell cultures
2.6.4 Characterization of plant cells in suspension culture
- Cell suspension culture requires a fairly large amount of callus, approximately 2-3 g/100ml of medium solution (Helgeson, 1979) The degree of separation of cells depends on the characteristics of the porous mass, agitation, oxygen, light, and nutrient concentrations of the medium Because of many factors, according to King and Street (1977), there is no universal standard procedure for all cultures of cell suspension species; outcome variations also seem to be influenced by many random factors
- The maximum cell density after about 18-25 days of inoculation, with strong growth suspension may be shorter, in the range of 6-9 days According to King (1980), cells that undergo culture, growth, and metabolism in suspension are called cell lines
Characteristics of the cell line such as high cell separation capacity, uniform cell morphology, clear nucleus and dense cytoplasm, and many starch granules
Application to research works: Based on the above knowledge, I take it as a scientific basis and an understanding foundation for future research 5,7-dimethoxyflavone (DMF), a major flavone found in Kaempferia parviflora It is a white solid with the molecular formula C17H14O4, a molecular weight of 282.295, and a melting point of 157–158 oC
METHODOLOGY
Materials and chemicals
Table 1: Materials and chemicals used in the study
No Name Origin Expiry date Notes
Equipment
Table 2: Some basic tools, means and equipment used in the research
No Name Origin Expiry date Notes
In addition, there are glass cups, measuring tubes, rotary inoculation, surgical scalpels, lights, violet lamps, sterile sample handling chambers, and sterile warm ventilation chambers
Research object
The growth peak of the sprouts of black ginger root is still fresh, just harvested 3 days ago; is being cultivated at the medicinal garden of the Institute of Indigenous Medicine of Vietnam
3.3.2 Image of the source of the research object
Sample of black ginger root from the Institute of
Indigenous Medicine of Vietnam bought in Lao Cai (the sample has been tested for medicinal herbs)
Selection of varieties: choose black ginger root that is 10 months old or older, has no signs of infection, and must have a growth peak
Treatment: mix Antracol 70WP 2.5% solution, and spray selected black ginger root for 120 minutes to kill pathogens;
Sand incubation: After incubation of Antracol 70WP, move to further incubation in clean sand, and wait for 20-30 days for the top to grow and turn into sprouts;
Harvest sprouted tubers, clean with running water to remove dirt, and let dry naturally;
Use a sterile surgical knife to separate and collect the sprouts for research use.
Research scope
This study was carried out in a laboratory setting; is a branch of the institute-level project of the Institute of Indigenous Medicine of Vietnam: “Study on suspension culture of black ginger (stem cells) growth stem cells; extracting secondary compound 5.7 – Dimethoxy Flavone as raw materials for the production process of
27 products KD 01 – Saman” announced registration number: 3896/2021/DKSP – Food Safety Authority issued on May 4, 2021
Subject code: E21-001-YHBDVN of the Institute of Indigenous Medicine in Vietnam;
Project leader Hoang Van Nam, principal investigator of the Institute of Indigenous Medicine in Vietnam.
Location and time
- Location: The study was carried out at the Institute of Indigenous Medicine of Vietnam; Address: No 166, Group 1, Tich Luong Street, City Thai Nguyen, Thai Nguyen
- Research period: 12 months (From July,2022 to July.2023) - Laboratory manual: Hoang Van Nam.
Culture environment and conditions
The background medium was MS medium (Murashige and Skoog, 1962), with the addition of 7 grams/liter agar, and 30 grams/liter sucrose Growth regulators: 2,4- Dichlorophenoxyacetic acid (2.4D) containing 99% of Sigma-Aldrich's active ingredients, Naphthaleneacetic acid (NAA) containing 99% of Merck's active ingredients, Kinetin containing 99% of active ingredients from Merck Active ingredients of Sigma-Aldrich Depending on the experiments, the addition or absence of CBTs at different concentrations The pH of the medium is 5.7 – 5.8 Autoclaved 121 o C, 1 atm, time 20 minutes
Culture conditions: Lighting time 16 hours/day; light intensity 2000 – 3000 lux; temperature 25±2 o C, shaking number 110 rpm
Research content and methods
- Content 1: Evaluation of sample sterilization time to the survival of research specimens
- Content 2: Evaluation of the effects of 3 growth regulators (2, 4D, NAA, and Kinetin) on callus formation
- Content 3: Evaluation of the effects of substances (2,4D, NAA, Kinetin) on the biomass growth of the studied specimens and the sample's ability to produce the secondary compound 5.7 Dimethoxy flavone
3.7.2 Research Methods 3.7.2.1 Evaluation of sample sterilization techniques affecting the survival rate of samples
Step 1: The selected tubers are soaked in dilute soapy water for 15 minutes;
Step 2: Samples are washed under running water for 15 minutes;
Step 3: The sample is washed with 70% Ethanol before being placed in the inoculation box for sterilization;
Step 4: In the inoculation box, the samples are divided equally into 2 groups A and
B, and continue to be disinfected by soaking in Povidon Iod 10% with time: group A soaking time is 1 minute; group B soaking time is 2 minutes; grouped by marking A, B, all washed immediately with sterile distilled water twice;
Step 5: Each group A, B is divided into 3 smaller sample clusters including, A = A1,
B = B1, B2, B3; Record and label each sample to avoid confusion
+ In which, small clusters of samples belonging to A (A1, A2, A3) are disinfected and soaked in 0.05% HgCl2 solution; however, the soaking time is different, respectively A1 = 7 minutes; A2 = 9 minutes and A3 = 11 minutes
+ Small clusters of samples belonging to B (B1, B2, B3) are disinfected and soaked in 0.1% HgCl2 solution (double the concentration of group A); soaking time is also different, respectively B1 = 7 minutes; B2 = 9 minutes and B3 = 11 minutes
+ After the above-mentioned samples have been soaked for enough time as designed, they should be washed 5 times with sterile distilled water, then left to dry naturally, and stored in sterile jars
Step 6: In a sterile inoculation chamber, use a scalpel to separate the growth tips and inoculate the plates containing MS agar according to each study
Step 7: Culture, the incubation time after inoculating on an agar plate is 45 days, monitoring samples daily, discarding contaminated samples, and recording statistics
- The experiment was arranged in a completely randomized design, each treatment was repeated 3 times, and each time 10 samples were cultured in 1 Petri dish
- Nutrition of culture and culture medium includes:
29 i) for callus: MS + 7 gr Agar + 30 g/l sugar + CSF, pH = 5.7-5.8 ii) for cell suspension: H2O MS + 30 g/l sugar+ CSF in pH = 5.7-5.8
3.7.2.2 Study and evaluate the effect of 3 growth regulators 2, 4D, NAA, and Kinetin on the formation of Callus (callus cells)
The growth peaks were transplanted into a soft agar nutrient medium supplemented with a combination of 3 growth regulators and monitored and treated as follows:
Substance 2,4D with 3 concentrations of 0.5mg/L, respectively; 1mg/L and 2mg/L;
NAA also has 3 concentrations of 0.5mg/L; 1mg/L and 2mg/L;
Kinetin also has 3 concentrations of 0.5mg/L; 1mg/L and 2mg/L;
Monitoring: paraffin-coated Petri dishes, cultured upside down in a standard room; lighting time 16 hours/day; light intensity 2000 – 3000 lux; temperature 25±2 o C;
Monitor callus formation up to 115 days, record statistics
The callus samples were then separated and transferred to the best growth medium to increase the mass of the callus as material for further study
Note: The experiment was arranged in a completely randomized design, each treatment was repeated 3 times, 10 cultures each time in 1 Petri dish
3.7.2.3 Method to evaluate the effect of 3 substances 2,4D, NAA, and Kinetin on the growth of single-cell biomass-producing compound 5.7 Dimethoxy flavone in the suspension medium
1) Experiment method: standardize each recipe with 4 500ml conical flasks, sterile, pour 150ml of the same nutrient solution into each, add 3 flasks, each bottle 01g of cultured black ginger callus, collect planned in previous experiments Add to 3 flasks, each using a different type of growth regulator, with 3 concentrations from low to high; only flask (4) does not add any chemicals, the purpose is as a control:
+ Environment (1): use substance 2,4D with 3 concentrations of 0.5mg/L, respectively; 1mg/L and 2mg/L;
+ Environment (2): Set up 3 experimental groups combining 2,4D and NAA, each group has 3 experimental formulas as follows:
Group 1: The concentration of 2.4D is 0.5mg/l combined with the concentrations of NAA: 0.5mg/L, respectively; 1mg/L and 2mg/L
30 Group 1: The concentration of 2,4D is 1mg/l combined with the concentrations of NAA: 0.5mg/L, respectively; 1mg/L and 2mg/L
Group 1: Concentration of 2,4D is 2mg/l combined with concentrations of NAA:
0.5mg/L, respectively; 1mg/L and 2mg/L
+ Environment (3): Set up 3 experimental groups combining 2,4D and Kinetin, each group has 3 experimental formulas as follows:
Group 1: The concentration of 2.4D is 0.5mg/l in combination with the concentrations of Kinetin: 0.5mg/L, respectively; 1mg/L and 2mg/L
Group 1: Concentration of 2,4D is 1mg/l combined with Kinetin concentrations:
0.5mg/L, respectively; 1mg/L and 2mg/L
Group 1: Concentration of 2,4D is 2mg/l combined with Kinetin concentrations:
0.5mg/L, respectively; 1mg/L and 2mg/L
+ Environment (4): without adding any chemicals, for control
The flasks were fed to the rotating shaker, and the switch was switched on to initiate continuous shaking in the standard room
2) Monitoring, nurturing, and harvesting: daily monitoring of room temperature, ventilation, nutrition, light nutritional supplements; carefully record the day 0 - 10 - 20 - 30 - 40, when seeing the strongest growth of biomass, the most in each conical flask, filter out the biomass to collect Weigh fresh, dried to constant weight, dry weight, and calculate the ratio fresh/dry
3) Data collection: based on recorded data, monitor tables, process raw data and make growth graphs, compare preliminary samples, and compare samples of flasks with control flasks
Note on culture conditions: Lighting time 16 hours/day; light intensity 2000 – 3000 lux; temperature 25±2 o C, number of swings 110 rpm; Each experiment was repeated 3 times
4) Extraction of the polymerthoxyFlavone group compound:
- Weigh 01 gram of dry biomass, extract Soxhlet with 95% Ethanol alcohol, recover the alcohol, and dry it with a rotary evaporator at ≤ 45 o C; The total extract obtained was dissolved in pure Methanol (according to German standards) using HPLC analysis at a concentration of 100pp;
31 - Filter through 0.45 àm nylon membrane, collect the expected substrate, predicted to be 5.7 - DimethoxyFlavone;
- Attachment: qualitative determination of 5.7 - DimethoxyFlavone by the expected substrate control with standard 5.7 - DimethoxyFlavone (99.9% purity of Sigma- Aldrich) through thin layer chromatography (TLC) and High-performance pressurized chromatography (HPLC)
The collected raw data was entered into the computer, using Excel software to process the data Statistical analysis of the data using SPSS software version 20.0 and the Duncan test to compare the difference between treatments
3.8 Research process for black ginger callus culture and biomass production from suspension
Results Studying the effect of sample sterilization time on the survival of callus specimens
Table 3: Sample sterilization results after 45 days of culture
Sterilization time by Povidone iodide
From the results of the study in Table 3, it can be seen that the tuber shoot samples sterilized at different concentrations and times gave different survival rates of the samples
It seems that in this experiment, with the same concentration of 0.05% HgCl2 with an increasing soaking time of 7-9-11 minutes, the survival rate of proportional samples also increased by 43.33% to 70% and 73 respectively ,33%
It seems that in this experiment, with the same concentration of 0.1% HCl, increasing the soaking time, the survival rate of samples is inversely proportional: 9 minutes is 86.67%, 11 minutes is only 66.67%; that disinfectants, disinfectants are toxic to cells, so high concentrations, long infiltration time can cause cell death;
34 The above results seem to be correct, as King and Street have commented, they vary without being able to determine the law of development
Figure 7: Experimental explants after sterilization
Research results on the effects of 2,4D, NAA, and Kinetin on the formation of
Table 4: Effect of 2,4D, NAA, Kinetin CCPs on callus formation
Time to appearance of callus (days)
Note: +++: Strong growth; ++: Average growth; +: Weak growth; -: no growth a b
Figure 8: a) Callus formed from the studied samples; b) rapid multiplication of scar tissue
- From the results of Table IV, the concentration of the designed CIs follows the increasing rule, but the formation of scar tissue does not increase gradually, without any rule as King and Street commented
- The combination of 2,4D with NAA according to increasing concentration, and increasing time, but the result of scar tissue development seems to be random, not regular
- The combination of all three substances 2, 4D, NAA, and Kinetin at high/low concentrations also gave very different results; The sharp growth of a certain sample or the decline of a certain sample of callus both seem very random
- Compared with the control treatment without using biostimulants, it is clear that the treatments using biostimulants create scar tissue with differences, without controversy
Research results on the effects of 2,4D, NAA, Kinetin on the growth of single
Table 5: The results of the effect of plant growth regulator on the growth of single cell biomass for 40 days
Kinetin (mg/L) Fresh weight (g) Dried weight
Note: Means in the same column with different letters are significantly different P 0.05
Figure 9: c) callus; d) suspension culture; e) single cell biomass
From the results of Table V, it seems that using 2,4D alone as a growth regulator, the growth of biomass has the best results compared to all other combinations With different concentrations of biodiesel and the combination of biosynthetic agents in different models, the results of single-cell biomass are also different
Figure 9 it is shown that the CCP affects the growth of the suspension biomass, which also shows that it is possible to grow the suspension to create biomass on the laboratory scale
Media supplemented with 2.4D 1mg/l was used for the cell growth curve study
Accordingly, (Figure 10), in the 40-day follow-up study, it was clear that the biomass increased in proportion to the time: the acclimatization phase in the first 10 days; middle 20-day growth phase; about the last 10 days it seems that growth has waned and growth won't last forever
Qualitative determination of the obtained biomass containing the secondary compound 5.7 – Dimethoxyflavone from cell suspension culture; according to qualitative results by thin layer chromatography (TLC): Mobile phase includes Toluene: Chloroform: Acetone: Formic acid (5: 4: 1: 0.2); Under UV lamp at 365 nm, a light streak appeared consistent with the original sample from black ginger root and also with the standard sample (Figure 11)
Qualitative on a high-performance liquid chromatograph Hitachi Chromater CM5000
Set up the analytical method as follows: Mobile phase, Acetonitrile B channel, D channel water supplemented with 0.5% Formic acid, flow 0.5ml/min, wavelength
38 350nm; Method of running analysis: 0 - 15 minutes 5%B, 15-35 minutes 50%B, 35 - 50 minutes 80%B, 50 - 65 minutes 100%B, 65 - 70 minutes 100%B (Figure 12)
Figure 10: Single cell suspension growth graph
GD: Black ginger root C: Standard substance 5.7- Dimethoxy Flavone
Figure 11: Qualitative image by TLC-TLC image captured by Qualitative TLC
NUMBER OF DAYS OF CULTURE
Figure 12: Analytical chromatogram of 5.7-Dimethoxyflavone extracted from the suspension culture
From the qualitative results of Figure 11 and Figure 12, it can be seen that the black ginger growth peak cultured by the suspension method produces the secondary compound 5.7-Dimethoxyflavone as in the authentically cultivated black ginger root and similar to the German standard
DAD-CH1 350 nm 5,7chuan200ppm041121
DISCUSSION, CONCLUSION AND RECOMMENDATION
Discussion
This is a new study in Vietnam The research process is serious and honest, and students can carry out the entire research process by themselves
At the laboratory of the Institute of Indigenous Medicine of Vietnam, the tools, means, equipment, chemicals, etc are complete and have an expiration date, so the research results are reliable
The design of the research is suitable for the research content; the studies were conducted carefully, meticulously, and accurately recorded, so the above results have a scientific basis
The research results are the premise for providing more scientific knowledge and important experience for the production of biologically active substances in black ginger to provide raw materials for the pharmaceutical industry using suspension culture technology in the industry industrial scale.
Conclusion
1) 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 96.67%
2) 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
3) MS supplemented with 1 mg/liter of 2.4D after 30 days of culture when the culture reaches 20 grams fresh is the best medium for culturing a single-cell suspension that produces the secondary compound 5.7-dimethoxyflavone
Recommendations
- Proposing additional bioreactor research to improve the process on an industrial scale
- Conduct research to optimize the bioreactor culture conditions in order to produce the target biological compound as efficiently as possible
1 Weerateerangkul P, Palee S, Chinda K, Chattipakorn S C, et al (2012),
"Effects of Kaempferia parviflora Wall Ex Baker and sildenafil citrate on cGMP level, cardiac function, and intracellular Ca2+ regulation in rat hearts", J Cardiovasc Pharmacol, 60 (3), pp 299-309
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APPENDIX
Sample sterilization results after 45 days of culture
Number of live samples at 1 st trial (10 samples inoculated)
Number of live samples at 2 nd trial (10 samples inoculated)
Number of live samples at 3 rd trial (10 samples inoculated)
Average number of viable samples after
Study on the effect of growth regulators on the formation of Calus
Create Callus for the 1st time
Create Callus for the 2nd time
Create Callus for the 3rd time
3 Note: +++: Strong growth; ++: Average growth; +: Weak growth; -: no growth