Research on increasing the accumulation of bioactive alkaloid compounds from eurycoma longifolia jack breeded on 20 - Liter bioreactor

- (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. 4 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 5 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 6 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 Alkaloid (651mg) F1 F2 F3 F4 F5-F11 F12 F2.1 F2.2-F2.4 SKC SiO2, Grad MeOH/H20 (3:1) Rửa kết tinh bằng n-hexane Chất 2 96 mg 9-methoxycanthin-6-one Rửa kết tinh bằng CH2Cl2 Chất 3 15 mg SKC SiO2, Grad CH2Cl2 /MeOH (20:1) Chất 1 29,6 mg Chất mới 7-methoxy-(9H-β-carbolin-1- yl)-(E)-3-propenoic acid 9-hydroxycanthin-6-one SKC SiO2, Grad CH2Cl2 /MeOH (25:1) crystallization wash CH2Cl2 crys allization wash n-Hecxan New substance 7 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. 8 2.2.5. Establishing the process of cultivating and collecting E.longifolia hairy root biomass on 20-liter Bioreactor system 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) Màng lọc khíỐng tiếp môi trường Ống thoát khí Ống dẫn khí ống nối Đầu sục khí 9 Substance (1): 7- methoxy-(9H-β- carbolin-1-il)-(E)-1- propenoic acid Substance (2): 9- methoxycanthin-6-one Substance (3): 9- methoxycanthin-6-one 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 No. Test reagent Cell line % inhibition at concentration IC50 (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: 10 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 N o Test reagent Concentration of inhibiting production of 50% IL-6 and TNF-α of IC50 cells (µM) IL-6 TNF-α RAW 264.7 ĐTB in mice THP -1 RAW 264.7 ĐTB in mice THP -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 11 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) No (1) 7-MCPA Standard (2) 9- methoxycanthin-6- one Standard (3) 9- hydroxycanthin- 6one Standard Concentr ation (µg/ml) Pic area (mAU.s) Concen tration (µg/ml) Pic area (mAU.s) Concen tration (µg/ml) Pic area (mAU.s) 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 Figure 3.10. Calibration curve showing correlation between peak area and concentration of standard substance A) Standard (1); B) Standard (2); C) Standard (3) (A) (B) (C) 12 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 CT State of medium Dry weight (g) No. mean branches /1 root Mean root length (cm) Describe roots after 30 days of culture CT1 Gelrile medium 0,49 ± 0,04 7,1 ± 1,2 7,7 ± 1,5 Small roots, yellow CT2 Liquid mediu 0,63 ± 0,03 5,3 ± 1,3 7,5 ± 2,1 Large roots, yellow Gelrile medium 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. Môi trường thạch Môi trường lỏng 13 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 CT Light conditions Dry Weight (g) Mean root length (cm) No. mean branches /1 root Describe roots after 30 days of culture CT1 Light 0,38 ± 0,07 4,3 ± 0,9 4,3 ± 1,5 Small roots, dark yellow CT2 Dark 0,66 ± 0,04 8,1 ± 0,8 6,0 ± 1 Big roots, bright yellow Value P P1-2 < 0,001 P1-2 < 0,001 P1-2 < 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 14 with p <0.001. As such, I select the root to be cut with size of 1-1.2 cm which is most suitable for biomass collection and saving the original sample source. Table 3.6. Effect of length of transplanted roots on growth and development of hairy roots CT Root length (cm) Dry weight (g) Mean root length (cm) No. mean branches /1 root 10-day survival rate (%) CT1 0,2-0,4 0,35 ± 0,05 8,1 ± 0,5 6,1 ±1,4 32 CT2 0,6-0,8 0,57 ± 0,03 8,2 ± 0,6 6,5 ± 1,0 61 CT3 1,0-1,2 0,74 ± 0,05 8,2 ± 0,5 6,3 ± 1,3 97 0,2-0,4 cm 0,6-0,8 cm 1,0-1,2 cm Figure 3.14. Effect of length of transplanted roots on growth and development of hairy roots 3.4.4. Effect of weight of transplanted roots (khối lượng rễ ban đầu) on growth and development of E.longifolia hairy roots The rooting volume transferred 0.3 g is best suited to 100 ml of growth medium for the growth and development of hairy roots for bioreactor culture. 15 Table 3.7. Development of hairy roots on 100 ml of medum CT Original fresh weight (g) Dry weight (g) The rate of increase chief (times) Mean root length (cm) No. mean branches /1 root 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 (g) 0,03 ± 0,006 0,06 ± 0,02 0,07 ± 0,02 0,25 ± 0,02 0,50 ± 0,03 0,78 ± 0,04 0,79 ± 0,03 DW-WP (g) 0,03 ± 0,006 0,06 ± 0,01 0,08 ± 0,02 0,26 ± 0,01 0,23 ± 0,01 0,58 ± 0,02 0,59 ± 0,05 DW-MS (g) 0,03 ± 0,006 0,06 ± 0,01 0,05 ± 0,01 0,22 ± 0,01 0,38 ± 0,02 0,68 ± 0,03 0,67 ± 0,04 16 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 7-SH 0,0057 ± 0,0006 - - - - - - 7- WP 0,0057 ± 0,0006 0,0013 ± 0,0011 0,007 ± 0,0015 0,01 ± 0,0021 0,02 ± 0,0051 0,009 ± 0,0007 0,0069 ±0,0009 7-MS 0,0057 ± 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 <0.05. The root morphology in all three media is elongated, heavily branched and yellowish- white (màu vàng trắng). However, in the SH medium the roots last longer and are larger in the MS and WP environments. Thus the SH medium is the most suitable environment for the growth and development of hairy roots. Accumulation of alkaloids, after assessing root growth and biomass growth of hairy roots at different growth stages, HPLC analyzes were 17 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 9Me-SH 0,04 ± 0,014 0,011 ± 0,012 0,022 ± 0,015 0,023 ± 0,015 0,133 ± 0,032 0,106 ± 0,037 0,102 ± 0,024 9Me- WP 0,04 ± 0,014 0,057 ± 0,023 0,088 ± 0,012 0,089 ± 0,022 0,143 ± 0,033 0,429 ± 0,059 0,241 ± 0,068 9Me-MS 0,04 ± 0,014 0,055 ± 0,017 0,042 ± 0,017 0,106 ± 0,054 0,126 ± 0,055 0,145 ± 0,012 0,057 ± 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- SH 0,075 ± 0,005 0,018 ± 0,002 0,027 ± 0,003 0,026 ± 0,003 0,05 ± 0,003 0,05 ± 0,005 0,066 ± 0,005 9Hy- WP 0,075 ± 0,005 0,044 ± 0,005 0,031 ± 0,005 0,15 ± 0,018 0,137 ± 0,07 0,147 ± 0,011 0,3 ± 0,036 9Hy- MS 0,075 ± 0,005 0,025 ± 0,005 0,039 ± 0,004 0,087 ± 0,006 0,089 ± 0,08 0,091 ± 0,007 0,037 ± 0,04 Sign: 18 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 (mg/l) Dry weight (g) % Dry weight 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- 19 hydroxycanthin-6-one, not suitable for accumulated substances (1). In the next study, it is necessary to find out more elicitors capable of accumulating (1) more during culture Figure 3.18. The effect of JA on the growth and accumulation of alkaloids of hairy roots of E.longifolia 3.4.7. Effect of salicylic acid (SA) 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 3.13. Effect of SA on growth and accumulation of alkaloids of hairy roots. SA (mg /l) Dry weight (g) 30 day % Dry weight Substance 1 (7-MCPA) 20 day Substance 2 (9- methoxycanthin -6-one) 25 day Substance 3 (9- hydroxycanthin- 6-one ) 30 day 0 0,67 ± 0,04 0,018 ± 0,007 0,337 ± 0,100 0,26 ± 0,04 5 0,66 ± 0,1 - 0,912 ± 0,034 0,464 ± 0,034 10 0,65 ± 0,09 - 0,245 ± 0,076 0,208 ± 0,087 20 0,54 ± 0,08 - 0,083 ± 0,006 0,084 ± 0,009 40 0,43 ± 0,10 - 0,083 ± 0,005 0,182 ± 0,058 Signal transduction elicitors (SA) derived from endogenous hormones have a greater effect on alkaloid accumulation, but inhibit root growth, therefore, an additional 5 mg/l culture medium SA is best suited for the accumulation of (2) 9-methoxycanthin-6-one and (3) 9-hydroxycanthin- 6-one but inhibiting the accumulation of (1) 7-MCPA. 0 1 4 8 16 (mg/l) 20 Figure 3.20. The effect of SA on the growth and accumulation of alkaloids of hairy roots E.longifolia 3.4.8. Effect of yeast extract (YE) on the growth and accumulation of (1) 7-MCPA, (2) 9-methoxycanthin-6-one, (3) 9-hydroxycanthin-6-one of hairy roots E.longiflolia. Table 3.14. The effect of YE on the growth and accumulation of alkaloids of hairy roots YE (mg /l) Dry weight (g) % Dry weight Substance 1 (7-MCPA) 20 day Substance 2 (9- methoxycanthi n-6-one) 25 day Substance 3 (9- hydroxycanthin -6-one ) 30day 0 0,67 ± 0,04 0,018 ± 0,007 0,337 ± 0,100 0,26 ± 0,04 10 0,77 ± 0,13 0,0112 ± 0,0065 0,381± 0,153 0,274 ± 0,095 20 0,82 ± 0,10 0,0239 ± 0,0056 0,694 ± 0,202 0,428 ± 0,019 40 0,72 ± 0, 2 0,0254 ± 0,0047 1,139 ± 0,341 0,657 ± 0,098 80 0,69 ± 0,12 0,0251 ± 0,0043 0,689 ± 0,220 0,578 ± 0,045 Figure 3.21: The effect of YE on the growth and accumulation of alkaloids of hairy roots 0 5 10 20 40 (mg/l) 0 10 20 40 (80 mg/l) 21 Yeast extract elicitors have a great effect on alkaloid accumulation and root growth, so a 40 mg/l YE supplement culture medium is best suited for alkaloid accumulation (1), (2), (3). The supplemented medium of 20 mg/l YE was most suitable for the growth of E.longifloia hairy roots 3.5. Establishing the process of cultivating and collecting E.longifloia hairy root biomass on 20-liter bioreactor system Figure 3.26. Global root culture procedure on 20-liter Bioreactor system Results after 30 days of hairy root culture on a self-created bioreactor system of 20 liters In this experiment, E.longifoila hairy roots of 30 g fresh weight were transplanted into a 20 liter s