Summary of Biological doctoral thesis: Research on increasing the accumulation of bioactive alkaloid compound from Eurycoma longifolia Jack breeded on 20- lít bioreactor

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Based on the above scientific bases and arguments, I conduct a research project to search for bioactive alkaloids while also researching new activities from alkaloids, thereby increasing cumulative growth culture. Content of alkaloids by adding elicitors.

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Nội dung Text: Summary of Biological doctoral thesis: Research on increasing the accumulation of bioactive alkaloid compound from Eurycoma longifolia Jack breeded on 20- lít bioreactor

MINISTRY OF EDUCATION AND VIET NAM ACADEMY OF TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SIENCE AND TECNOLOGY ----------------------------- Name Ph.D: Tran Thu Trang PROJECT NAME RESEARCH ON INCREASING THE ACCUMULATION OF BIOACTIVE ALKALOID COMPOUNDS FROM EURYCOMA LONGIFOLIA JACK BREEDED ON 20-LITER BIOREACTOR Major: Biochemistry Code: 9.42.01.16 SUMMARY OF BIOLOGICAL DOCTORAL THESIS Ha Noi-2020 1
The work was completed in: Graduate university of sience and techology- Viet Nam academy of science and technology Science instructor 1: TS. Chu Nhatt Huy Science instructor 2: PGS. TS. Pham Bich Ngoc Reviewer 1: … Reviewer 2: … Reviewer 3: …. The thesis will be defended in front of the Academy Thesis Evaluation Council, meeting at Graduate university of sience and techology- Viet Nam academy of science and technology at ... hour .. ’, date ... month ... year 201 .... The thesis can be found at: - Library of Academy of Science and Technology -Vietnam National Library 2
PREAMBLE The urgency of the subject Eurycoma longifolia Jack is a common medicinal plant in our country, mainly distributed in the Central provinces, Kon tum, Dong Nai, Phu Quoc and Bai Tu Long National Park. E. longifolia is used to improve health, increase testosterone in men, has strong anti-inflammatory activity, good anti-cancer and some other effects such as malaria treatment, anti- osteoporosis, diabetes ... research work on E. longifolia shows that the plant contains many alkaloid active substances that are valuable in anti-malarial, anti-inflammatory, carcinogenic and invigorating cancer... Currently, E.longifolia is being over-exploited in the wild due to the increasing demand for herbal use. Moreover, E.longifolia is difficult to grow and replicate, with the time to harvest quality roots takes at least 5-6 years, leading to difficulties in raw materials for pharmaceutical production. To date, root culture is considered a potential alternative to stable collection of large amounts of clean biomass in a short period of 30-40 days as a source of raw materials for pharmaceutical production. Scar tissue culture technology or cell suspension culture requires the addition of growth regulators. However, the residual value of the biotechnology community directly affects the health of users. This problem can be overcome in hairy root biomass culture because hairy roots can grow and develop continuously without the addition of growth regulators. Moreover, hairy roots have many other advantages, such as being able to produce a large number of secondary compounds and have a more stable inheritance than suspension culture and scar tissue. In vitro culture, inducing resistance or the addition of elicitors greatly increases the ability to accumulate secondary compounds during the rapid multiplication of root biomass. Cultivation of hairy roots of E.longifolia, although a nutrient-rich environment helps roots grow quickly, the alkaloid content analyzed is very low. Moreover, the process of 1
accumulating some substances does not take place due to the lack of a number of stimulating factors, leading to lower endogenous alkaloid content than the wild-collected root. Since stressors due to changes in light, temperature, or impacts from plant pathogens have been discovered to stimulate the accumulation of secondary compounds through the signal pathways of elicitors such as jasmonic acid (JA), salicylic acid (SA), yeast extract (YE)... in turn activate the expression of defense genes, and biosynthesis of secondary compounds. The elicitor has been widely applied in culture to increase the saponin active substance in the cultivation of many valuable medicinal plants. Additional studies of elicitors for increasing the ability to accumulate secondary compounds in in vitro culture are not available. The tissue culture technique was born to revolutionize plant breeding. But with the traditional method of culturing on agar, it is difficult to meet the demand of plant seeds supplied in the market, the price is high; Due to the fact that the transfer and separation of samples inside the incubator are almost done by hand, it is labor-intensive to become infected. Therefore, a new culture system that can be automated can help reduce labor, time and the number of plants. In liquid culture, people are divided into three types: static liquid culture, shaking liquid culture and Bioreactor culture, all of which are used to cultivate cell suspension, organogenesis. Currently, there is a tendency to use Bioreactor to multiply hairy roots biomass to obtain secondary active ingredients. Based on the above scientific bases and arguments, I conduct a research project to search for bioactive alkaloids while also researching new activities from alkaloids, thereby increasing cumulative growth culture. Content of alkaloids by adding elicitors. After that, the hairy roots were studied for large biomass for the orientation of medicinal herbs by optimizing the 20 liter bioreactor system: “Research on increasinh the accumulation of 2
bioactive alkaloid compound from Eurycoma longifolia Jack breeded on 20- lít bioreactor” Objectives of the study Survey of biological activity of alkaloid isolated from hairy roots of E.longifolia to find new activities. Determining optimal culture conditions for E.longifolia hairy roots to grow and develop. Determining the concentration of three elicitors (Salicylic acid, Jasmonic acid and Yeat extract) on the production and accumulation of alkaloids isolated from hairy roots of E.longifolia in vitro. Building a 20 liter bioreactor for multiplication of hairy roots biomass. Research content Content 1: Research on chemical composition and biological activity of alkaloid extract in hairy root biomass of Eurycoma longifolia. Content 2: Researching some culture conditions affecting hairy root growth and accumulation of alkaloids (1) 7-methoxy- (9H-β-carbolin-1-il) - (E) -1-propenoic acid) (7-MCPA), (2) 9-methoxycanthin-one, (3) 9- hydroxycanthin-6-one in hairy root of Eurycoma longifolia. Content 3: Building a culture technique to collect E.longifolia hairy root biomass on a 20-liter bioreactor. Evaluation of the content of alkaloids (1) 7-MCPA, (2) 9-methoxycanthin-one, (3) 9-hydroxycanthin-6-one in biomass obtained cultured in 20-liters bioreactor compared to roots wild. CHAPTER 1. OVERVIEW 1.1. Introduction of Eurycoma longifolia Eurycoma longifolia thuộc họ Simaroubaceae, also called mật nhân, bách bệnh, mật nhơn, bá bịnh. In terms of plant morphological characteristics, E.longifolia is a small tree, small stems, 2-8 m high with few branches, young when rarely have branches, bark and roots are very bitter. 3
The leaves are long, consisting of more than 10 pairs of leaflets, opposite, oval, petioles very short, oblong base, pointed tip, glossy upper surface, gray lower surface. The whole plant (except the ripe fruit) has a bitter taste. 1.2. Application of Eurycoma longifolia in Traditional Medicine E. longifolia is used extensively in traditional medicine to treat back pain, indigestion, and postpartum tonic, used to treat fever, jaundice, cachexia and ascites . E.longifolia is one of the popular folk remedies for its aphrodisiac and antimalarial effects. Cooked leaf juice is used to treat itchy skin, while the fruit is used to treat dysentery, the skin is used as a worming remedy. Roots are used to treat sexual dysfunction, aging ... 1.3. Hairy roots biomass culture 1.3.1. Introduction of hairy roots biomass culture Hairy roots is the name used to refer to the small hairy roots that are strongly produced at the site infected by Agrobacterium rhizogenes. The tissue culture technique was born to revolutionize plant breeding. But with the traditional method of culturing on agar, it is difficult to meet the demand of plant seeds supplied in the market, the price is high. In 1981, Takayama and Misawa proposed a liquid culture system with an active external aeration system called the Bioreactor. The bioreactor system is commonly used, mainly to cultivate cell suspensions and produce secondary active ingredients on a variety of research subjects, and there is a tendency to use bioreactor to cultivate hairy roots for collection. secondary active substance. (Hệ thống bioreactor là thường được dùng nhiều, chủ yếu để nuôi cấy huyền phù tế bào và sản xuất hoạt chất thứ cấp trên nhiều đối tượng nghiên cứu khác nhau và hiện nay đang có xu hướng dùng bioreactor để nuôi cấy rễ tơ nhằm thu nhận hoạt chất thứ cấp.) 1.3.2. Factors affecting the ability to synthesize HCTC in hairy root culture Selection of lines, morphology of hairy roots after gene transfer, interaction between plants and microorganisms, development stages of hairy 4
roots during culture, other factors ... 1.3.3 Effect of elicitor on accumulation of HCTC during hairy root culture Elicitor are compounds that stimulate all forms of plant protection. The broad definition of elicitors includes substances of a pathogen (exogenous) or released by plants themselves in response to a pathogen (endogenous). The elicitor can cause a series of defense reactions that lead to the gene being expressed and stimulated accumulation of HCTC in conditions that do not affect plants or cell culture. 1.3.4 The situation of domestic and foreign research on hairy root biomass culture receiving financial credits In the world, there have been some authors studying hairy roots of several different species, but there have been no studies on hairy roots of E.longifolia. Domestic and foreign studies on E. Longifolia have just stopped at analyzing the chemical composition and pharmacological effects, the research on hairy roots of some species is still very limited. The research of hairy root biomass collection of medicinal plants and the collection of HCTC is not only limited at the laboratory scale but has been industrialized by bioreactor culture. CHAPTER II: MATERIALS AND METHODS 2.1. Research materials We use dried root biomass of E.longifolia (90 g) and hairy root of E.longifolia cultured on agar medium provided by Plant Cell Technology Department, Institute of Biotechnology. Wild roots of E.longifolia are collected in Bai Tu Long National Park, Dong Son - Ky Thuong Nature Reserve, Hoanh Bo, Quang Ninh. 2.2. Research Methods 2.2.1. Method of isolating and determining the chemical structure of alkaloids from hairy root biomass. Isolation of substances from alkaloid extract 5
Alkaloid (651mg) SKC SiO2, Grad CH2Cl2 /MeOH (25:1) F1 F2 F3 F4 F5-F11 F12 SKC SiO2, Grad Rửacrystallization kết tinh bằng SKC SiO2, Grad MeOH/H20 (3:1) CHwash 2Cl2 CH2Cl2 CH2Cl2 /MeOH (20:1) Chất 3 Chất 1 F2.1 F2.2-F2.4 15 mg 29,6 mg Rửacrystallization kết tinh bằng New substance 9-hydroxycanthin-6-one Chất mới wash n-Hecxan n-hexane 7-methoxy-(9H-β-carbolin-1- yl)-(E)-3-propenoic acid Chất 2 96 mg 9-methoxycanthin-6-one Methods of determining chemical structure The chemical structure of compounds is determined by a combination of modern spectroscopic methods such as mass spectrometry (ESI-, HR-ESI-MS), one-way magnetic resonance spectra (1H-, 13C-NMR). , DEPT) and bidirectional (HSQC, HMBC, 1H-1H-COZY). 2.2.2. In vitro biological activity research methods 2.1.2.1 Cytotoxic assay (cytotoxic assay) This test was performed using the method of A Monks (1991). This test determines the total cellular protein content based on the optical density (OD) measured when the cellular protein content is stained with Sulforhodamine B (SRB). 2.1.2.2 Testing the ability to inhibit the production of IL-6 and TNF-α in RAW264.7 macrophages, mice and THP-1 stimulated by LPS Cell lines were cultured in 48 well plates, containing 5x105 cells / ml. Add 1 ml of the cell to 1 µl of the sample so that the final concentration of the test compound is (1, 3, 10, 30 mM). Incubate the mixture at 37°C, 5% CO2 for 30 minutes before being stimulated with 1 µg/mL LPS (Sigma, Tokyo, Japan). The supernatant is collected after 24 hours. The cytokine IL- 6 and TNF-α concentrations of mouse macrophages, RAW264.7, THP-1 6
were determined by ELISA (Quantikine ELISA of R&D) according to the manufacturer's instructions. The data is expressed as an average of at least 3 replicates. The value of IC50 will be determined by the computer software ImageJ 1.50i 2.2.3 Experimental and quantitative methods of alkaloids through HPLC- DAD method. Develop analytical method of e.longifolia sample: designation DK-Bb Eclipse Zorbax Chromatographic Column XDB-C18 (4.6 x 250 mm, 5 µm) Detector DAD, detection wavelength 272 nm. 25oC column temperature Flow rate of 0,5 ml/min Sample concentration of 10 mg/ml Injection volume of 10 µl Mobile phase ACN-H2O, solvent gradient program: Time (minutes) 0 10 35 45 60 ACN (% volume) 10 20 50 90 90 Quantify clean substances (1), (2), (3) by comparing the peak area of the sample with the peak area value on the calibration line. => Quantification of clean substances (1), (2) , (3) by comparing the peak area of the sample with the peak area value on the calibration graph 2.2.4. Evaluate factors affecting the growth and accumulation of alkaloids of hairy root of E.longifolia in 500 ml flask. Several factors such as environmental status, light, initial sample length, weight of transplanted roots were assessed to affect the growth and development of E.longifoli hairy roots. Several factors such as the basic medium, jasmonic acid elicitor (JA), salicylic acid (SA), yeast extract (YE) were assessed to affect the growth and accumulation of compounds (1), (2), (3) in hairy root culture. 7
2.2.5. Establishing the process of cultivating and collecting E.longifolia hairy root biomass on 20-liter Bioreactor system Ống thoát khí Ống dẫn khí Ống tiếp môi trường Màng lọc khí ống nối Đầu sục khí Figure 2.5. Simulate parts of a self-created spherical 20-liter effervescent bioreactor system Based on the available microbial fermentation bioreactor system, a number of bioreactor samples have been designed to be used for biomass culture experiments with capacities of 5 - 20 liters. Based on reference [66], I selected the effervescent spherical bioreactor model to cultivate E.longifolia hairy roots. CHAPTER III: RESULTS AND DISCUSSION 3.1. Determine the chemical structure of substances isolated from alkaloid extract This is the first time the chemical composition of the alkaloid extract of the hairy roots of E.longifolia has been studied. Results of isolation and structure determination of compounds from alkaloid extract showed that plants contain β-Carboline alkaloids (7-methoxy- (9H-β-carbolin-1-il) - (E) -1 -propenoic acid), Canthin-6-one alkaloids (9-methoxycanthin-6-one and 9-hydroxycanthin-6-one) 8
Substance (1): 7- Substance (2): 9- Substance (3): 9- methoxy-(9H-β- methoxycanthin-6-one methoxycanthin-6-one carbolin-1-il)-(E)-1- propenoic acid 3.2. Biological activity of 7-MCPA, 9-methoxycanthin-6-one, 9- hydroxycanthin-6-one 3.2.1. Carcinogenicity of the test substances (1), (2), (3) Table 3.1. Results of cytotoxic activity of substances isolated from alkaloids of hairy roots Test Cell % inhibition at IC50 No. reagent line concentration (µg/ml) 100 20 4 0,8 1 (1) MCF-7 98,8 90,6 27,7 2,9 6,3 KB 95,7 73,9 20,8 9,1 10,3 2 LU-1 97 76,5 25,2 9,3 8,6 (2) MCF-7 98,2 72,8 29,8 8,5 8,4 HepG2 96,9 66,7 32,3 9,2 9,3 KB 95,4 47,2 14,2 0,11 23,5 3 LU-1 92,8 61,3 32,3 11,4 11,2 (3) MCF-7 75,5 36,6 19,4 9,8 39,7 HepG2 84,2 39,9 15,1 9,2 34,2 KB: Epithelial cancer; LU-1 lung cancer; MCF-7 breast cancer; HepG2 liver cancer The test results show that: 9
Thus, substance (2) exhibits better cytotoxic activity than substance (3) on the KB, LU-1, MCF-7, HepG2 lines. Comparing compound structure (2) and (3) shows that these two substances differ only in methoxy and hydroxy group attached at carbon position 7. It can be said that methoxy functional group can play an important role in activity. Calculate inhibition of KB, MCF7, LU-1, HepG2 cell lines of the substance (2). Substance (1) has good cytotoxic activity on MCF-7 strain with IC50 reaching 6.3 (µg / ml). 3.2.2. Testing the ability to inhibit the production of IL-6 and TNF-α in mouse and human macrophages stimulated by LPS of substances (1), (2), (3) Table 3.2. The results determine the inhibitory concentration of 50% production IL-6 and TNF-α of the cell Concentration of inhibiting production of 50% IL-6 and TNF-α of IC50 cells (µM) IL-6 TNF-α N Test o reagent RAW ĐTB THP RAW ĐTB THP 264.7 in mice -1 264.7 in mice -1 1 (1) 4,5 12,8 9,9 6,6 12,4 16,0 2 (2) 4,2 3,7 17,2 10,2 3,5 10,0 3 (3) 1,4 4,1 53,7 10,1 0,95 45 The results in Table 3.2 show that b-carboline alkaloid 7-MCPA (1), 9-methoxycanthin-6-one (2) isolated from hairy root culture of E.longifolia inhibited IL-6 and TNF-α production in macrophages of RAW264.7, mice and humans THP-1 stimulates LPS. 7-MCPA has been elucidated by the research group Dang 2016 to explain the molecular mechanism of anti- inflammatory activity through ROS-dependent p38 MAPK and anti- inflammatory effect combined with activation of Nrf2 / HO-1 pathway. It is necessary to continue studying whether the 9-methoxycanthin-6-one active 10
substance interacts with the NF-κB pathway in LPS-stimulated macrophages. Substance (3) exhibited IL-6 and TNF-α inhibition in RAW264.7 and mice macrophages, poorly inhibited on THP-1 macrophages. 3.3. Constructing a quantitative calibration curve for alkaloids in HPLC-DAD methods Table 3.3. Results of building up the calibration curve of three alkaloid (1), (2), (3) (1) 7-MCPA (2) 9- (3) 9- Standard methoxycanthin-6- hydroxycanthin- one Standard 6one Standard No Concentr Concen Concen Pic area Pic area Pic area ation tration tration (mAU.s) (mAU.s) (mAU.s) (µg/ml) (µg/ml) (µg/ml) 1 0 0 0 0 0 0 2 1 44,62 10 371,63 10 427,03 3 5 310,79 25 1004,75 25 1049,06 4 10 504,07 50 1848,17 75 3692,95 5 25 1434,35 125 4285,08 125 6549,70 6 50 2821,10 250 8528,45 250 12948,40 (A) (B) (C) Figure 3.10. Calibration curve showing correlation between peak area and concentration of standard substance A) Standard (1); B) Standard (2); C) Standard (3) 11
Survey results show a close linear correlation between analyte concentration and peak area in the range of the investigated compounds. 3.4. Evaluation of factors affecting the growth and accumulation of substances (1), (2), (3) of hairy root culture in a 500 ml flask 3.4.1. Influence the environmental state on the growth and development of E.longifolia hairy roots Table 3.4. Development of E.longifolia hairy roots on liquid and agar media Mean Describe Dry No. mean State of root roots after CT weight branches /1 medium length 30 days of (g) root (cm) culture Gelrile 0,49 ± Small roots, CT1 7,1 ± 1,2 7,7 ± 1,5 medium 0,04 yellow Liquid 0,63 ± Large roots, CT2 5,3 ± 1,3 7,5 ± 2,1 mediu 0,03 yellow Môi trường thạch Gelrile medium Môi trường lỏng Liquid mediu Figure 3.11. Growth of hairy roots in liquid and gelrile media In summary, the liquid medium is most suitable for growth and development of rooting culture for E.longifolia, for root biomass culture purposes. 12
3.4.2. Effect of light on the growth and development of E.longifolia hairy roots Light Dark Figure 3.12. The development of hairy roots in dark and light conditions As such, light can inhibit the growth of hairy roots of E.longlifolia and dark growing conditions are most suitable for the growth and development of hairy roots. Table 3.5. The development of hairy roots in dark and light conditions Light Dry Mean root No. mean Describe roots CT conditions Weight length branches after 30 days of (g) (cm) /1 root culture CT1 Light 0,38 ± 4,3 ± 0,9 4,3 ± 1,5 Small roots, 0,07 dark yellow CT2 Dark 0,66 ± 8,1 ± 0,8 6,0 ± 1 Big roots, bright 0,04 yellow P1-2 < P1-2 < 0,001 P1-2 < Value P 0,001 0,001 3.4.3. Study on the effect of the length of transplanted roots on the growth and development of E.longlifolia hairy roots In this experiment, cut root samples with the size from 1-1.2 cm gave the highest survival rate (97%) and the highest weight was obtained (0.74 ± 0.05 g), CT3 was statistically significant. Statistics compared with formulas 1 and 2 13
with p
Table 3.7. Development of hairy roots on 100 ml of medum Original Dry The rate of Mean root No. mean fresh weight increase length (cm) branches /1 CT weight (g) chief (times) root (g) CT1 0,1 3,0 ± 0,13 30 8,0 ± 0,45 6,4 ± 0,8 CT2 0,2 5,8 ± 0,1 29 8,1 ± 0,63 6,0 ± 0,4 CT3 0,3 7,9 ± 0,18 26,3 8,1 ± 0,31 6,1 ± 0,56 CT4 0,4 7,8 ± 0,14 19,5 7,8 ±0,4 6,1 ± 0,4 0,1 0,2 0,3 0,4 (g) Figure 3.15. The development of hairy roots on 100 ml of media with different initial amounts 3.4.5. Effect of mineral environment on growth and accumulation of compounds (1) 7-MCPA, (2) 9-methoxycanthin-6-one, (3) 9- hydroxycanthin-6-one of E.longifolia hairy roots. Table 3.8. Effect of the mineral environment on the growth and development of hairy roots CT 0 day 5 day 10 day 15 day 20 day 25 day 30 day DW-SH 0,03 ± 0,06 ± 0,07 ± 0,25 ± 0,50 ± 0,78 ± 0,79 ± (g) 0,006 0,02 0,02 0,02 0,03 0,04 0,03 DW-WP 0,03 ± 0,06 ± 0,08 ± 0,26 ± 0,23 ± 0,58 ± 0,59 ± (g) 0,006 0,01 0,02 0,01 0,01 0,02 0,05 DW-MS 0,03 ± 0,06 ± 0,05 ± 0,22 ± 0,38 ± 0,68 ± 0,67 ± (g) 0,006 0,01 0,01 0,01 0,02 0,03 0,04 15
DW: Dry weight Table 3.9. Effect of mineral environment on 7-MCPA accumulation CT 0 day 5 day 10 day 15 day 20 day 25 day 30 day 0,0057 ± 7-SH 0,0006 - - - - - - 7- 0,0057 ± 0,0013 ± 0,007 ± 0,01 ± 0,02 ± 0,009 ± 0,0069 WP 0,0006 0,0011 0,0015 0,0021 0,0051 0,0007 ±0,0009 0,0057 ± 7-MS 0,0006 - - - - - - Sign: 7-SH: % of 7-MCPA compound in dry weight was obtained when cultured on SH medium 7-WP: % 7-MCPA compound in dry weight obtained when cultured on WP medium 7-MS: % 7-MCPA compound is in dry weight obtained when cultured on MS medium Figure 3.16. Effect of mineral environment on the growth and development of hairy roots after 30 days of culture. The KLK gained after 30 days of culture on SH medium (0.79 ± 0.03 g) reached the highest value statistically significant compared to culturing on WP medium (0.59 ± 0.05 g) and MS (0.67 ± 0.04 g) with P
conducted to determine the concentration of three substances (1), (2), (3) at each stage of growth of hairy roots. The results showed the presence of substances (2), (3) in all treatments with different content changes in each stage, substance (1) was little or no in the treatments different. Table 3.10. Effect of mineral environment on 9-methoxycanthin-6-one accumulation. CT 0 day 5 day 10 day 15 day 20 day 25 day 30 day 0,04 ± 0,011 ± 0,022 ± 0,023 ± 0,133 ± 0,106 ± 0,102 ± 9Me-SH 0,014 0,012 0,015 0,015 0,032 0,037 0,024 9Me- 0,04 ± 0,057 ± 0,088 ± 0,089 ± 0,143 ± 0,429 ± 0,241 ± WP 0,014 0,023 0,012 0,022 0,033 0,059 0,068 0,04 ± 0,055 ± 0,042 ± 0,106 ± 0,126 ± 0,145 ± 0,057 ± 9Me-MS 0,014 0,017 0,017 0,054 0,055 0,012 0,034 Sign: 9Me-SH:% 9-methoxycanthin-6-one is present in root cultures on SH medium 9Me-WP:% 9-methoxycanthin-6-one is present in root cultures on WP medium 9Me-MS:% 9-methoxycanthin-6-one is present in root cultures on MS medium Table 3.11. Effect of mineral environment on 9-hdroxycanthin-6-one accumulation CT 0 day 5 day 10 day 15 day 20 day 25 day 30 day 9Hy- 0,075 ± 0,018 ± 0,027 ± 0,026 ± 0,05 ± 0,05 ± 0,066 ± SH 0,005 0,002 0,003 0,003 0,003 0,005 0,005 9Hy- 0,075 ± 0,044 ± 0,031 ± 0,15 ± 0,137 ± 0,147 ± 0,3 ± WP 0,005 0,005 0,005 0,018 0,07 0,011 0,036 9Hy- 0,075 ± 0,025 ± 0,039 ± 0,087 ± 0,089 ± 0,091 ± 0,037 ± MS 0,005 0,005 0,004 0,006 0,08 0,007 0,04 Sign: 17
9 Hy-SH:% 9-hydroxycanthin-6-one is in root cultures on SH medium 9 Hy-WP:% 9-hydroxycanthin-6-one is in root culture on WP medium 9 Hy-MS:% 9-hydroxycanthin-6-one is in root culture on MS medium Hairy roots cultured for 30 days on WP medium produced the highest content (1) of the 20th day (0.02 ± 0.0051% DW), the highest (2) substance for 25 days (0.429 ± 0.059) % DW), the highest (3) substance for 30 days (0.3 ± 0.036% DW) In summary, hairy roots were cultured on SH liquid medium for 30 days most suitable for growth and development of hairy roots of E.longifolia. Hairy roots cultured on WP liquid media for 20, 25, 30 days are best suited for accumulation of the corresponding substances (1) 7-MCPA, (2) 9- methoxycanthin-6-one, (3) 9-hydroxycanthin -6-one during the cultivation of hairy roots of E.longifolia 3.4.6. The effect of jasmonic acid (JA) on the growth and accumulation of substances (1) 7-MCPA, (2) 9-methoxycanthin-6-one, (3) 9- hydroxycanthin-6-one of hairy roots E. longifolia Table 3.12. The effect of JA on the growth and accumulation of alkaloids of hairy roots JA Dry weight % Dry weight (mg/l) (g) Substance (1) Substance (2) Substance (3) 0 0,67 ± 0,04 0,018 ± 0,007 0,337 ± 0,100 0,26 ± 0,040 1 0,61 ± 0,06 - 0,388 ± 0,051 0,111 ± 0,064 4 0,6 ± 0,07 - 0,513 ± 0,114 0,147 ± 0,021 8 0,54 ± 0, 05 - 0,874 ± 0,125 0,465 ± 0,043 16 0,41 ± 0,06 - 0,119 ± 0,056 0,052 ± 0,009 The signaling elicitor (JA) derived from endogenous hormones has an effect that greatly increases alkaloid accumulation, but inhibits root growth. Therefore, a supplementary culture medium of 0.8 mg / l JA is most suitable for accumulation of (2) 9-methoxycanthin-6-one and (3) 9- 18