Production of triterpenoids in liquid-cultivated hairy roots of Galphimia glauca

The production of three triterpenoids from Galphimia glauca hairy root cultures, the sedative principle galphimine E (2), the recently described glaucacetalin A (3), and maslinic acid (6), was quantified by HPLC in the biomass and the culture medium. Batch cultures of the hairy root line VYT, obtained through infecting cotyledons with Agrobacterium rhizogenes ATCC 15 834, were grown for 41 days in shake flasks containing B5 medium without phytohormones. A maximum biomass of 11 g/L DW was obtained on day 33, while the doubling time was 6 days. Throughout the growth cycle fresh and dry weights as well as triterpene production were registered. Glaucacetalin A (3), excreted into the culture media, reached a maximum amount of 2.14 mg/L after 21 days while galphimine E (2) and maslinic acid (6) were recovered from the root biomasses reaching maximum concentrations of 0.11 and 0.43 mg/g, respectively, on day 39.     

A study of the production of essential oils in chamomile hairy root cultures.

The active substances in chamomile (Matricaria recutita L.) belong to chemically different structural types. The largest group of medically important compounds forming the essential oils are primarily chamazulene, (-)-alpha-bisabolol, bisabololoxides, bisabolonoxide A, trans-beta-farnesene, alpha-farnesene, spathulenol and the cis/trans-en-in-dicycloethers. Flavonoids, coumarins, mucilages, mono- and oligosaccharides also have pharmacological effects. We studied the production of essential oils in genetically transformed cultures. Sterile juvenile chamomile plants were infected with A4-Y strains of Agrobacterium rhizogenes. They are known plant pathogens and are capable of inducing so-called hairy roots. The transfer DNA segment of the Ri-virulence plasmid of A. rhizogenes becomes integrated in the genome of the plant cells. The isolated hairy roots grow rapidly on hormone-free media. In order to obtain bacteria-free media, we cultured the transformed roots on Murashige-Skoog (MS) medium supplemented with carbenicillin (800 mg/l). To study the production of essential oils, the clones were propagated on liquid and solid MS and Gamborg (B5) media, respectively. According to gas chromatography, the composition of the essential oil of hairy root cultures on different media was found to be similar, but differing in proportion. The main component of the essential oil which was identified by gas chromatography and mass spectrometry was trans-beta-farnesene, as in the intact roots.     

Technology of compact MAb and its application for medicinal plant breeding named as missile type molecular breeding

Single chain fragment-variable (scFv) enhanced solasodine glycoside accumulation in Solanum khasianum hairy root cultures transformed by the ScFv solamargine (As)-scFv gene. The scFv protein was expressed at a high level in inclusion bodies of E. coli. After being renatured, the scFv protein was purified in a one-step manner by metal chelate affinity chromatography. The yield of refolded and purified scFv was 12.5 mg per 100 ml of cell culture. The characteristics of the As-scFv expressed in E. coli and transgenic hairy roots were similar to those of the parent monoclonal antibody (MAb). The expression of scFv protein provides a low cost and a high yield of functional scFv antibody against solamargine. The full linear range of the ELISA assay using scFv was extended from 1.5-10 μg/ml. The expressed anti-solamargine scFv protein could be useful for determination of total solasodine glycoside content in plant samples by ELISA. Solasodine glycoside levels in the transgenic hairy root were 2.3-fold higher than that in the wild-type hairy root based on the soluble protein level and binding activities. The As-scFv expressed in S. khasianum hairy roots enhanced solasodine glycosides accumulation and provide a novel medicinal plant breeding methodology that can produce a high yield of secondary metabolites.     

Decreased scopolamine yield in field-grown Duboisia plants regenerated from hairy roots

Hairy root cultures were obtained from hybrid clones of Duboisia myoporoides x D. leichhardtii following transformation by Agrobacterium rhizogenes strain A4. Shoots spontaneously regenerating from the hairy root cultures were rooted and transferred to soil. The plants displayed typical morphological alterations known as hairy root syndrome to varying degrees. PCR analysis confirmed that all transformed plants contained the rolA, rolB and rolC genes, irrespective of the degree of morphological alterations. A field test of the transformed regenerated plants revealed that those plants displaying the strongest hairy root syndrome symptoms had the highest content of the tropane alkaloid scopolamine. However, the overall scopolamine and hyoscyamine yield of all transformed plants was clearly reduced compared to untransformed control plants. These results demonstrate that the A. rhizogenes-transformed plants tested in this study do not provide a viable alternative to agricultural farming of hybrid clones of D. myoporoides x D. leichhardtii obtained by conventional breeding.     

Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids

Hairy roots, transformed with Agrobacterium rhizogenes, have been found to be suitable for the production of secondary metabolites because of their stable and high productivity in hormone-free culture conditions. A number of plant species including many medicinal plants have been successfully transformed with Agrobacterium rhizogenes. Transformed root cultures have also been found to be a potential source of high-value pharmaceuticals. In this article the most important alkaloids produced by hairy roots are summarised. Several different methods have been used to increase the alkaloid accumulation in hairy root cultures. The selection of high productive root lines based on somaclonal variation offers an interesting option to enhance the productivity. Elicitors and modification of culture conditions have been shown to increase the growth and the alkaloid production in some cases. Genetic engineering is a modern tool to regulate the secondary metabolism also in hairy roots. However, our knowledge on biosynthesis of many alkaloids is still poor. Only a limited number of enzymes and their respective genes which regulate the biosynthetic pathways are fully characterised.     

唐古特山莨菪毛状根对去氢表雄酮的生物转化

目的通过生物转化对去氢表雄酮进行结构修饰，以期获得更有意义的转化产物。方法利用唐古特山莨菪毛状根液体悬浮培养体系对去氢表雄酮进行生物转化，通过柱色谱及制备薄层色谱进行分离纯化，利用核磁共振、ESI-MS等光谱鉴定结构。结果分离得到了5个转化产物，即：androst-4-ene-3,17-dione ( I)； 6α-hydroxyandrost-4-ene-3, 17-dione (II)；6α,17β-dihydroxyandrost-4-ene-3-one(III)； androst-4-ene-3,6,17-trione (IV)；17β-hydroxyandrost-4-ene-3-one (V)。结论所得到的5个化合物均为首次通过植物组织培养生物转化的方法从唐古特山莨菪毛状根液体培养体系中分离得到。     

Genetic transformation of Glaphimia glauca by Agrobacterium rhizogenes and the production of norfriedelanes

Transformed root cultures of Galphimia glauca (Malpighiaceae) were established by infecting cotyledons and hypocotyls with Agrobacterium rhizogenes ATCC 15 834. Cotyledon-derived cell lines were grown in liquid B5 nutrient medium without phytohormones and have shown the typical hairy roots phenotype over two years of continuous subculturing. PCR analysis was used to confirm the integration of rol A and rol C genes into the plant genome. The transformed cultures synthesized three major norfriedelanes, the new glaucacetalins A-C (1-3), which were secreted into the nutrient medium. The structural elucidation of these in vitro produced metabolites was performed by the application of high resolution NMR techniques that proved them to be triterpenoids related to the known galphimines, the sedative principles of this plant species. These results suggest the possibility of further biotechnological exploration of sedative friedelane biosynthesis by in vitro plant organ cultures.     

Medicinal plant biotechnology: the Apiaceae family as the example of rapid development

This article reviews the results of research representative of different trends in biotechnological studies of the Apiaceae species. These species are a well-known source of many important herbal products. A number of investigations focus on the biosynthesis of secondary metabolites in plant in vitro cultures. There are also numerous reports concerning the methods of plant micropropagation, especially using somatic embryogenesis. The papers dealing with the usage of the enzymatic potential of plant cells cultured in vitro, in biotransformation processes are less copious. Several studies are related to genetic engineering, exploring mainly the possibility of plant transformation with Agrobacterium rhizogenes to obtain hairy root cultures. The present review demonstrates the significance of biotechnological methods in the studies of medicinal plants taking species of the Apiaceae family as an example.     

Artemisinin, Related Sesquiterpenes, and Essential Oil in Artemisia annua During a Vegetation Period in Vietnam

The active principle of ARTEMISIA ANNUA L., artemisinin, is currently being developed to a registered antimalarial drug. For production purposes, plants with a high artemisinin content are required. We followed the development of the artemisinin content and of the biosynthetically related sesquiterpenes artemisinic acid, arteannuin B, and artemisitene in A. ANNUA plants, during a vegetation period in Vietnam, where this species is indigenous. In addition, the essential oil content and composition were studied. Samples of leaves, buds, flowers, or post-bloom flowers and fruits were taken at different stages: vegetative (5, 6, and 8 months old), at mass formation of buds (9 months), at full bloom (10 months), and post-bloom (10S months). The highest artemisinin content (0.86% dry wt) was present in the leaves of 5 months-old plants. At this stage also the highest leaf yield was found. Subsequently, the artemisinin content gradually dropped. At the age of 5 months the highest artemisinic acid and arteannuin B contents, 0.16 and 0.08% dry wt, respectively, were found as well. Artemisitene was present at all stages of development, ranging from 0.002 to 0.09% dry wt. With 1.9% v/w, the essential oil content was maximal just before flowering and was composed of 55% monoterpenes and 45% sesquiterpenes. At all other stages (0.4 - 1.0% v/w oil) this ratio was ca. 30%/70%. The main components of the oil were camphor and germacrene-D.     

抗疟新药青蒿素及其类似物的合成

青蒿素(1)是中药青蒿中提出的一个抗疟活性成分,可从R(+)-香草醛(16)通过关键中间体17进行全合成,利用光氧化反应把氢过氧基引入17的 C_6位构成1的过氧基团。同时合成了植物青蒿所含的已被分离鉴定的九个倍半萜化合物中的青蒿甲素(7)、青蒿乙素(3)青蒿丙素(脱氧青蒿素(?))、青蒿戊素(6)以及青蒿己素(5)。     

Influence of Agrobacterium rhizogenes Strains on Biomass and Alkaloid Productivity in Hairy Root Lines of Hyoscyamus muticus and H. albus *

Using leaf explants of IN VITRO grown HYOSCYAMUS ALBUS and H. MUTICUS plantlets, hairy roots were induced following inoculation with AGROBACTERIUM RHIZOGENES strains A (4) and LBA-9402. The transformed roots, appearing after 14 - 17 days incubation on hormone-free MS medium containing 1 g/L cephalexin, were excised and maintained in the same medium. Ten randomly selected hairy root lines from each bacterial treatment of the two plant systems were compared for growth and alkaloid production in half-strength, hormone-free MS medium on 25 (th) day of culture. A. RHIZOGENES strain - A (4) induced hairy root lines of both H. ALBUS and H. MUTICUS were comparatively faster growing than those induced by strain LBA-9402. In contrast to earlier reports, some of the hairy root lines of H. ALBUS induced by A. RHIZOGENES strain A (4) were as fast growing as the hairy root lines of H. MUTICUS. The atropine yields of A (4) induced lines of H. ALBUS were significantly higher (3.5 fold) than the LBA-9402 induced lines. No such relationship between the bacterial strain and alkaloid productivity could, however, be obtained in case of hairy root lines of H. MUTICUS.     

Expression of Vitreoscilla hemoglobin enhances growth of Hyoscyamus muticus hairy root cultures

The Vitreoscilla hemoglobin gene (vhb) was introduced into Hyoscyamus muticus with the aim of investigating its effect on growth and alkaloid production of Agrobacterium rhizogenes-induced hairy root cultures. We were able to generate several VHb-expressing hairy root lines with different integration patterns. Substantial somaclonal variation was observed in growth and hyoscyamine production amongst both VHb-expressing lines and controls. Despite this variation, the growth properties of single lines remained stable over time. Expression of VHb was found to improve growth of H. muticus hairy roots in shake-flask cultures. The dry weights of the root cultures expressing Vitreoscilla hemoglobin were on average 18 % higher than those of the controls. VHb expression also increased the volumetric hyoscyamine production, mainly due to the improved growth properties. However, this difference was not statistically significant due to the wide somaclonal variation and fluctuations over time in both VHb and control hairy root lines.     

Stimulation of tanshinone production in Salvia miltiorrhiza hairy roots by β -cyclodextrin-coated silver nanoparticles

Diterpenoid tanshinones, major bioactive constituents of Salvia miltiorrhiza roots are drug candidates against cardiocerebral diseases. To develop sustainable and effective elicitation strategies for the enhanced production of tanshinones, the spherical shaped β-cyclodextrin-coated silver nanoparticles (AgNP-β-CDs, Ø35.3 ± 8.3 nm) were synthesized and applied as elicitor to hairy root cultures of S. miltiorrhiza. The content of total tanshinones including tanshinone I, IIA and cryptotanshinone in the cultures was stimulated by AgNP- β-CD at 30 mg/L. The elicitation effects of AgNP-β-CD on tanshinone were found to be related to the released dissolved Ag⁺ and nanoparticle bumping to the root surface in the cultures. AgNP-β-CD treatment induced reactive oxygen species (ROS) including H₂O₂ and O₂^? production in the hairy roots, leading to enhance activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), and up-regulated expressions of key biosynthetic genes for tanshinones in hairy roots. The nano-elicitor increased total production of tanshinones in 18-day-old hairy root cultures to 10.8 mg/L, a 1.8-fold over that of the control. These results indicate that AgNP- β-CD could be a novel effective elicitor in hairy root cultures for the production of pharmaceutical secondary metabolites.     

Analysis of 6-methoxy podophyllotoxin and podophyllotoxin in hairy root cultures of Linum album Kotschy ex Boiss

Linum album is a herbaceous medicinal plant that contains some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, hairy root cultures of L. album were established by transformation with Agrobacterium rhizogenes strains LBA9402, A4, AR15834 and Agrobacterium tumefaciens strain C58C1 (pRiA4). The presence of PTOX and MPTOX in the hairy roots was verified by ESI/MS in positive ion mode. MPTOX was confirmed and its enantiomer determined by nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy, respectively. PTOX and MPTOX production was determined by HPLC, in different lines of hairy roots. The results showed that all obtained hairy root lines produced higher yield of lignans than mother plant roots. In addition, the lignan content in the roots derived from A. rhizogenes strain LBA9402 was higher than in those obtained from A. tumefaciens strain C58C1.     

黄花蒿发根的液体培养及青蒿素合成的动态特征研究

目的:探讨黄花蒿发根生长及青蒿素生物合成的动态特征,建立高效、稳定的黄花蒿发根液体培养体系。方法:测定不同培养基以及MS培养基中不同营养元素对黄花蒿发根生物量和青蒿素含量的影响。结果:筛选出优化的黄花蒿发根液体培养基,获得拟合的黄花蒿发根生长和青蒿素合成的Logistic方程。结论:在优化的MS培养基中黄花蒿发根生长迅速且能稳定合成青蒿素,为工业化大规模生产青蒿素提供了可能。     

丹参毛状根中多种丹酚酸类化合物的鉴定与生物合成(英文)

应用发根农杆菌9402侵染丹参叶片获得了一株可以稳定产生多种丹酚酸类化合物的丹参毛状根。除迷迭香酸和丹酚酸B外还含有丹酚酸K、丹酚酸L、乙基丹酚酸B、甲基丹酚酸B以及一个分子量为538的未知化合物(化合物538)。LC-MS对这些化合物进行了鉴定。对茉莉酸甲酯(MeJA)和酵母诱导子对几种主要化合物积累的影响进行了分析:MeJA促进丹酚酸B、迷迭香酸、丹酚酸K和化合物538的积累,分别从干重4.21%、2.48%、0.29%和0.01%提高到7.11%、3.38%、0.68%和0.04%;酵母诱导子提高迷迭香酸含量,从干重2.83%上升到5.71%,抑制了丹酚酸B、丹酚酸K和化合物538的生物合成。实验结果表明所获得的丹参毛状根可以作为生产丹酚酸B、迷迭香和丹酚酸K以及化合物538的替代资源。对这些酚酸类化合物对诱导子的生物累积效应变化趋势进行分析推测:丹参毛状根中丹酚酸K和化合物538可能是从迷迭香酸合成丹酚酸B的中间产物。     

Transformation of ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834. Southern blot analysis of the established roots revealed that only the TL-DNA was integrated into the plant genome without incorporation of the TR-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degrees C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.     

In vitro cultures of Bacopa monnieri and an analysis of selected groups of biologically active metabolites in their biomass

Context: Bacopa monnieri L. Pennell (Scrophulariaceae) is one of the most important plants in the system of Indian medicine (Ayurveda).

Objective: This paper studies the optimal growth of B. monnieri for effective accumulation of metabolites. Biomass growth of this plant could be accomplished in liquid cultures on Murashige & Skoog medium.

Materials and methods: Powdered shoots of in vitro cultures of B. monnieri were extracted by methanol for indole compounds, phenolic compounds and bacosides for RP-HPLC analysis. Fatty acid analysis was performed via gas chromatography. Anti-inflammatory effect of B. monnieri extracts was evaluated in the A549 cells. COX-2 and cPGES expression was analyzed using Western blots.

Results: l-Tryptophan and serotonin were found in biomass from in vitro cultures of B. monnieri on MS medium and in biomass from the MS mediums enriched with the different additions such as of 0.1?g/L magnesium sulphate, 0.1?g/L zinc hydroaspartate, 0.1?g/L l-tryptophan, 0.25?g/L serine, 0.5?g/L serine and 0.5?mg/L anthranilic acid. The content of l-tryptophan and serotonin compounds was significant in biomass from medium with the addition of 0.1?g/L zinc hydroaspartate (0.72?mg/g dry weight and 1.19, respectively). Phenolic compounds identified in biomass from the same variants of MS medium were chlorogenic acid (ranging from 0.20 to 0.70?mg/g dry weight), neochlorogenic acid (ranging from 0.11 to 0.40?mg/g dry weight) and caffeic acid (ranging from 0.01 to 0.04?mg/g dry weight). The main group of fatty acids in biomass was saturated fatty acids (53.4%). The predominant fatty acid was palmitic acid. A significant decrease of COX-2 and cPGES expression was observed in the A549 cells activated with LPS and treated with B. monnieri extracts.

Discussion and conclusions: As far as we know, this is the first analysis of indole compounds and phenolic acids in this plant. The multi-therapeutic effect of B. monnieri is expressed by the activity of bacosides. Information about the presence of indole and phenolic compounds, and fatty acids in this plant is limited, but the content of these compounds might participate in the physiological activity of B. monnieri.     

Auxins affected ginsenoside production and growth of hairy roots in Panax hybrid

Hairy roots of interspecific hybrid ginseng (Panax ginseng x P. quinquefolium), induced by Agrobacterium rhizogenes ATCC 15834, grew well in B5 liquid media supplemented with 2.5 microM auxins (3-indole butyric acid (IBA), 1-naphtaleneacetic acid (NAA) and 3-indoleacetic acid (IAA)). The hairy roots cultured in B5 liquid medium supplemented with 2.5 microM IBA showed best growth (6.39 g fresh weight per a flask, at week 8). The highest content of the total ginsenosides was 1.63% as dry weight at week 8 when cultured with 2.5 microM NAA. The different auxins affected the numbers and lateral branching roots. Especially, 2.5 microM IBA promoted the lateral root formation (43.7+/-4.0 roots, at week 8), and 2.5 microM NAA promoted the lateral root growth (45.3+/-5.6 mm, at week 8). The growth and ginsenosides production of 8-week old hairy roots cultured in B5 liquid media supplemented with IBA and NAA combinations were also investigated. Hairy roots produced higher amounts of ginsenosides in B5 liquid media supplemented with 0.5-1.0 microM IBA and NAA combinations than that cultured in B5 liquid media supplemented with only IBA and NAA. The highest yield of ginsenoside was obtained when cultured with 0.5 microM IBA and 1.0 microM IBA combination (6.38 mg per a flask, at week 8).     

Effect of medium composition and light on root and rhinacanthin formation in Rhinacanthus nasutus cultures

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) has long been used in Thai traditional medicine for treatment of tinea versicolor, ringworm, pruritic rash, and abscess. The active constituents are known as a group of naphthoquinone esters, rhinacanthins. This work focused on establishment of R. nasutus root cultures and determination of rhinacanthin production. Induction of R. nasutus root formation was accomplished on solid Gamborg’s B5 (B5) medium, supplied with 0.1?mg/L indole-3-butyric acid (IBA) and 20?g/L sucrose. The effects of explants (whole leaf explants and four-side excised leaf explants), light and medium composition on root and rhinacanthin formation were investigated. The root formation from the whole leaf explants was 10 times higher than that from the four-side excised leaf explants. In addition, light possessed an inhibitory effect on the root and rhinacanthin formation of R. nasutus. Medium manipulation found that Murashige and Skoog (MS) medium supplied with 3?mg/L IBA and 30?g/L sucrose was the most suitable for induction of the root formation. Unfortunately, the obtained root cultures produced only rhinacanthin-C in very low amount, 0.026?mg/g dry weight (DW), when they were transferred into the same MS liquid medium. With semisolid medium (4?g/L agar) of the same MS composition, however, the root cultures appeared to produce higher content of rhinacanthin-C, -D and -N (3.45, 0.07 and 0.07?mg/g DW, respectively). Our finding suggests that culturing in semisolid medium is capable of improving of rhinacanthin production in R. nasutus root cultures.