Flavonoid Production in Transformed Scutellaria baicalensis Roots and Ways of Its Regulation

A root culture of skullcap (Scutellaria baicalensisGeorgi) transformed with pRi T-DNA was initiated by the inoculation of sterile seedlings with Agrobacterium rhizogenes(wild-type strain A-4). The flavonoid concentration in cultured roots comprised 5% of the root dry weight and was maintained essentially constant during a subculture. For four weeks of culturing, the weight of the roots increased by 20–30 times; when the roots were cultured for a longer time and with periodic enrichment of the nutrient medium, their weight increased 50-fold. Skullcap roots were shown to synthesize flavones characteristic of intact roots (wogonin, baicalein, and baicalin). The addition of 0.01–1 mM L-phenylalanine (a precursor of flavonoids) to the nutrient medium affected neither root growth, nor their flavonoid concentration. Root elicitation with 100 M methyl jasmonate for 72 h increased the flavonoid content per flask and per root dry weight by 1.8 and 2.3 times, respectively.     

The Relationship Between Endogenous β‐Glucuronidase Activity and Biologically Active Flavones‐Aglycone Contents in Hairy Roots of Baikal Skullcap

Here, we examine the relationship between contents of principal flavones in hairy roots of Scutellaria baicalensis with the activity of the β‐glucuronidase (sGUS) enzyme during a culturing cycle. Using RP‐HPLC, we show that the highest contents of aglycones, baicalin and wogonin is observed at the growth days 8, 14, and 71 and reach 45, 41, and 62% (based on the total weight of hairy roots of the Baikal skullcap), correspondingly. Their accumulation is accompanied by increase of the sGUS activity, which we determined fluorometrically. Moreover, the enzyme activity is characterized by significant and reasonable correlation only with the wogonin contents. Our results confirm a significant role of sGUS at the final steps of the metabolism in root‐specific flavones of Baikal skullcap and suggest how one can optimize the conditions of culturing the hairy roots for biotechnological production of individual flavonoids. For example, at the culturing day 71 wogonin constituted over 80% of all flavones extracted from cells.     

Morphological and biochemical characteristics of genetically transformed roots of Scutellaria andrachnoides

Genetically transformed roots (hairy roots) and callus tissue of skullcap (Scutellaria andrachnoides Vved.) were for the first time introduced in the in vitro culture. S. andrachnoides is the endemic plant of the Kyrgyzstan. These cultures were characterized by active and stable growth in the hormone-free liquid Gamborg nutrient medium. The growth rate of undifferentiated callus tissue was higher than that of hairy roots, which were the source of this callus. The composition of secondary metabolites in hairy roots, callus tissue, and also the roots of seedlings and adult S. andrachnoides plants was analyzed. It was found that S. andrachnoides hairy roots and callus culture retained the ability for the synthesis of flavones typical for the roots of intact plants. Substantial quantitative differences in secondary metabolites were observed between the roots of juvenile and adult plants. In the seedling roots, which like hairy roots have no secondary thickening, wogonoside, a wogonin glucuronide, predominated among flavones. In the roots of adult plants growing due to the secondary thickening, balcalin, a baicalein glucuronide, was a dominating flavon. It is proposed to use the large-scale in vitro cultivation of roots and especially the rapidly growing callus tissue of S. andrachnoides with a profitable content of only one group of flavones for the development of the biotechnological method for producing wogonin and creating on its basis a new drug — a valuable anticancer agent of plant origin with selective cytotoxic activity.     

Artificial seeds as a way to produce ecologically clean herbal remedies and to preserve endangered plant species

Influence of entrapment of the segments of isolated pRi T-DNA transformed roots of skullcap (Scutellaria baicalensis Georgi) in alginate gel capsules on the morphological, anatomical, physiological, and biochemical parameters of root cultures obtained from so-called artificial seeds (ASs) has been studied. The obtained ASs remain viable even after long-term storage at 4°C. Using the encapsulation technique, we have renewed a skullcap root culture and made it healthier. During anatomical study of the renewed root culture, the formation of chloroplasts has been observed in the cells of roots cultivated under lighting. Total chlorophyll content, chlorophyll a: chlorophyll b ratio, and the flavone content in the green roots have been determined.     

Glycyrrhizin production by in vitro cultured <Emphasis Type="Italic">Glycyrrhiza glabra</Emphasis> elicited by methyl Jasmonate and salicylic acid

Licorice (Glycyrrhiza glabra L. var. glabra, Fabaceae) is considered as a model plant synthesizing triterpenoid secondary compounds. It is known that glycyrrhizin is accumulated in thickened intact licorice roots. The effects of methyl jasmonate (MeJa) and salicylic acid (SA) on plant growth and production of glycyrrhizin in the roots of in vitro cultured 65-day-old plants were studied. Increasing amounts of glycyrrhizin in the roots treated with MeJa inhibited root growth, while SA increased the amount of glycyrrhizin without negative effects on growth. Treatment of plantlets with 0.1–2 mM MeJa and 0.1 and 1 mM SA enhanced the production of glycyrrhizin by 3.8 and 4.1 times, respectively, as compared to the controls. Results support the hypothesis that production of glycyrrhizin is related to a defense response system of the licorice.     

Artificial seed preparation as the efficient method for storage and production of healthy cultured roots of medicinal plants

The technique for the refinement of pRi T-DNA-transformed root cultivation by the root fragment encapsulation in the gel coat, i.e., so-called “artificial seed” (AS) production, was studied. AS were produced from genetically transformed roots of Baikal skullcap (Scutellaria baicalensis Georgi) and common rue (Ruta graveolens L.). The effects of duration of AS storage at 4°C on their subsequent growth activity and a capability for resumption of actively growing root cultures were analyzed. Encapsulation of contaminated Baikal skullcap root culture with the addition of antibiotic and storage during 2–5 weeks at low above-zero temperature resulted in a complete elimination of infection, i.e., obtaining the healthy root culture. Growth activity and total flavone concentration were markedly increased in this culture, so that total productivity of this renewed root culture increased substantially. Using AS produced from the root fragments of common rue, it was shown that, after long-term storage at low above-zero temperature, they are capable of not only root growth resumption but also active shoot formation, which is of interest for plant micropropagation. Long-term retaining growth activity of AS produced from root cultures of valuable medicinal plants permits their usage as a reserve and also, in the case of necessity, for long-distance transport as compact axenic root inocula. The storage of viable root fragments within AS also helps to optimize intervals between numerous subculturings of root cultures required for the maintenance of IPPRAS collection in the active state.     

Molecular characterization of flavonoid biosynthetic genes and accumulation of baicalin,baicalein, and wogonin in plant and hairy root of Scutellaria lateriflora

Scutellaria lateriflora is well known for its medical applications because of the presence of flavanoids and alkaloids. The present study aimed to explore the molecular aspects and regulations of flavanoids. Five partial cDNAs encoding genes that are involved in the flavonoid biosynthetic pathway: phenylalanine ammonia lyase (SlPAL), cinnamate 4-hydroxylase (SlC4H), 4-coumaroyl CoA ligase (Sl4CL), chalcone synthase (SlCHS), and chalcone isomerase (SlCHI) were isolated from S. lateriflora. Organ expression analysis showed that these genes were expressed in all organs analyzed with the highest levels correlating with the richest accumulation of wogonin in the roots. Baicalin and baicalein differentially accumulated in S. lateriflora plants, with the highest concentration of baicalin and baicalein detected in the leaves and stems, respectively. Exogenous methyl jasmonate (MeJA) significantly enhanced the expression of SlCHS and SlCHI, and accumulation of baicalin (22.54 mg/g), baicalein (1.24 mg/g), and wogonin (5.39 mg/g) in S. lateriflora hairy roots. In addition, maximum production of baicalin, baicalein, and wogonin in hairy roots treated with MeJA was approximately 7.44-, 2.38-, and 2.12-fold, respectively. Light condition increased the expression level of SlCHS, the first committed step in flavonoid biosynthesis in hairy roots of S. lateriflora after 3 and 4 weeks of development compared to the dark condition. Dark-grown hairy roots contained a higher content of baicalin and baicalein than light-grown hairy roots, while light-grown hairy roots accumulated more wogonin than dark-grown hairy roots. These results may helpful for the metabolic engineering of flavonoids biosynthesis in S. lateriflora.     

Two types of O-methyltransferase are involved in biosynthesis of anticancer methoxylated 4′-deoxyflavones in Scutellaria baicalensis Georgi

The medicinal plant Scutellaria baicalensis Georgi is rich in specialized 4′-deoxyflavones, which are reported to have many health-promoting properties. We assayed Scutellaria flavones with different methoxyl groups on human cancer cell lines and found that polymethoxylated 4′-deoxyflavones, like skullcapflavone I and tenaxin I have stronger ability to induce apoptosis compared to unmethylated baicalein, showing that methoxylation enhances bioactivity as well as the physical properties of specialized flavones, while having no side-effects on healthy cells. We investigated the formation of methoxylated flavones and found that two O-methyltransferase (OMT) families are active in the roots of S. baicalensis. The Type II OMTs, SbPFOMT2 and SbPFOMT5, decorate one of two adjacent hydroxyl groups on flavones and are responsible for methylation on the C6, 8 and 3′-hydroxyl positions, to form oroxylin A, tenaxin II and chrysoeriol respectively. The Type I OMTs, SbFOMT3, SbFOMT5 and SbFOMT6 account mainly for C7-methoxylation of flavones, but SbFOMT5 can also methylate baicalein on its C5 and C6-hydroxyl positions. The dimethoxylated flavone, skullcapflavone I (found naturally in roots of S. baicalensis) can be produced in yeast by co-expressing SbPFOMT5 plus SbFOMT6 when the appropriately hydroxylated 4′-deoxyflavone substrates are supplied in the medium. Co-expression of SbPFOMT5 plus SbFOMT5 in yeast produced tenaxin I, also found in Scutellaria roots. This work showed that both type I and type II OMT enzymes are involved in biosynthesis of methoxylated flavones in S. baicalensis.     

Purification and characterization of UDP-glucuronate: baicalein 7-O-glucuronosyltransferase from Scutellaria baicalensis Georgi. cell suspension cultures

UDP-glucuronate: baicalein 7-O-glucuronosyltransferase (UBGAT) catalyzes the transfer of glucuronic acid from UDP-glucuronic acid to the 7-OH of baicalein. UBGAT was purified from cultured cells of Scutellaria baicalensis Georgi (Lamiaceae). It was purified 95-fold using various chromatography and chromatofocusing procedures to apparent homogeneity. The Mr was estimated to be 110 kDa by gel filtration chromatography with a 52 kDa subunit by SDS-PAGE. The isoelectric point was pH 4.8. UBGAT was specific to UDP-glucuronic acid as a sugar donor and flavones with substitution ortho- to the 7-OH group such its baicalein (6-OH), scutellarein (6-OH) and wogonin (8-OMe).     

Composition of essential oil in genetically transformed roots of <Emphasis Type="Italic">Ruta graveolens</Emphasis>

The composition of essential oil obtained by hydrodistillation from genetically transformed roots of common rue (Ruta graveolens L.) was analyzed. Using gas chromatography and complex gas chromatography-mass spectrometry, it was established that the major component of rue essential oil was a root-specific sesquiterpene geijerene comprising 67% of total amount of volatile compounds. In essential oil of cultured rue roots, furocoumarins characteristic of intact plant roots were found, viz. osthole, halepensin, and rutacultin. The content of essential oil in genetically transformed rue roots was 0.23% of root dry weight, which is comparable with that in the roots of intact plants. The long-term maintenance in the in vitro cultured rue roots of a capability for the synthesis of essential oil major components characteristic of intact plants allows their usage for studying the physiological activity of these volatile compounds and their putative role in the plant root interaction in biocenoses.     

Formation of phenolic compounds in the roots of <Emphasis Type="Italic">Hedysarum theinum</Emphasis> cultured in vitro

A steadily growing culture of genetically transformed roots of a valuable medicinal Altaic plant Hedysarum theinum Krasnob. was established using a Ri-plasmid (pRi) T-DNA of A4 wild strain of Agrobacterium rhizogenes. The composition of secondary substances accumulated in the in vitro roots and the roots of H. theinum intact seedlings was investigated. Isoflavonoids were found to represent by their main low-molecular metabolites. Preparative HPLC made it possible to isolate four substances from the methanolic extract of H. theinum cultured roots. Using ¹H- and ¹³C-NMR-spectrometry, these substances were identified as formononetin, ononin (formononetin glycoside), malonyl ononin, and texasin glucoside. The qualitative composition of secondary metabolites of the genetically transformed roots and the roots of H. theinum seedlings was essentially the same, except that malonyl ononin was not found in the latter. The technique of producing artificial seeds on the basis of H. theinum roots cultured in vitro was tested, and the possibility of their use as a rhizogenic inoculum was substantiated. The culture of H. theinum roots is considered as a potential source of ecologically pure raw material for medicinal preparations, and the artificial seeds with root inoculum are a promising vehicle for propagation and conservation of this valuable plant.     

Flavonoid production in Scutellaria baicalensis callus cultures

St-20 and St-7 lines were isolated from the stem callus of Scutellaria baicalensis Georgi on indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid media, respectively. The flavonoid content of St-20 line was superior to that of St-7 line. The growth and flavonoid (baicalin, baicalein, wogonin and wogonin-7-0-glucuronide) content in St-20 line were best on Linsmaier-Skoog's basal medium containing 10^-7 M–10^-5 M kinetin. St-20 line showed the same flavonoid content and pattern as the root of parent plant after the culture period of 70 days.     

Comparative analysis of bioactive phytochemicals from Scutellaria baicalensis, Scutellaria lateriflora, Scutellaria racemosa, Scutellaria tomentosa and Scutellaria wrightii by LC-DAD-MS

Scutellaria is a geographically widespread and diverse genus of the Lamiaceae family of herbaceous plants commonly known as skullcaps. Scutellaria is used widely as an ethnobotanical herb for the treatment of various ailments ranging from cancers, cirrhosis, jaundice, hepatitis, anxiety and nervous disorders. We used (1) reverse-phase liquid chromatography coupled to a diode array detector (LC-DAD), and (2) multiple reaction monitoring (MRM) using mass spectrometry (LC-MS/MS) to quantify the levels of acteoside, scutellarin, scetellarein, baicalin, baicalein, wogonin, wogonoside, apigenin, chrysin, and oroxylin A in aqueous methanolic extracts of roots, shoots and leaves of S. baicalensis, S. lateriflora, S. racemosa, S. tomentosa and S. wrightii. Our results indicate that both methods (LC-DAD and LC-MS/MS) were robust for the detection of the 10 analytes from Scutellaria extracts although greater sensitivities were achieved using LC-MS/MS in MRM mode. MRM enabled the detection of low levels of analytes which were otherwise undetected using LC-DAD. The baicalin content of S. wrightii roots were 5-fold higher than the commonly used S. baicalensis. Additionally, we also showed that leaves of both S. wrightii and S. tomentosa are good sources of scutellarin compared to S. baicalensis. Our data clearly demonstrated that previously uncharacterized species, S. wrightii and S. tomentosa are both good sources of flavonoids, particularly scutellarin, baicalin, wogonin and baicalein.     

Interaction of methyl jasmonate, wounding and fungal elicitation during sesquiterpene induction in Hyoscyamus muticus in root cultures

The ability of methyl jasmonate (MeJa) to induce sesquiterpene production in root cultures of Hyoscyamus muticus has been studied. Although MeJa alone could not induce sesquiterpene in unwounded culture, MeJa added in the presence of wounding displayed a dose-dependent response, saturating at 50 μM. The ability to respond to MeJa declined with an increase in time between MeJa contact and wounding; however, responsiveness could be recovered by re-wounding of tissue prior to MeJa contact, suggesting that additional signaling related to wounding is required for sesquiterpene pathway induction. The saturation level of sesquiterpene induction with fungal elicitor was four times higher than the saturation level achieved by MeJa, with clear differences in sesquiterpene composition. Fungal elicitation results in a higher level of lubimin and a lower level of solavetivone production; whereas, methyl jasmonate induces predominantly solavetivone and little or no lubimin production. This suggests that fungal elicitation induces enzymes further down the sesquiterpene pathway which are not affected by MeJa. The induction of roots in contact with subsaturated levels of elicitor can be enhanced to saturation production levels by the addition of small amounts of MeJa (5–10 μmoles/l). In these studies, MeJa was consistently found to favor the earlier metabolite (solavetivone), while fungal elicitation promoted conversion to subsequent metabolites in the pathway (lubimin). The interactive role of MeJa in signal transduction for secondary metabolic production is discussed. Received: 8 June 1997 / Revision received: 13 August 1997 / Accepted: 13 September 1997     

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis

 A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53 094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in  E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4′-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. Received: 8 September 1999 / Accepted: 4 October 1999     

Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi

Free radical scavenging and antioxidant activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in the radix of Scutellaria baicalensis Georgi, were examined in different systems. ESR results showed that baicalein and baicalin scavenged hydroxyl radical, DPPH radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals. Ten micromol/l of baicalein and baicalin effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe(2+)-ascorbic acid, AAPH or NADPH, while wogonin and wogonoside showed significant effects only on NADPH-induced lipid peroxidation. In a study on cultured human neuroblastoma SH-SY5Y cells system, it was found that 10 micromol/l of baicalein and baicalin significantly protected cells against H(2)O(2)-induced injury. Baicalein was the most effective antioxidant among the four tested compounds in every system due to its o-tri-hydroxyl structure in the A ring. Compared with a well-known flavonoid, quercetin, the antioxidant activity of baicalein was lower in DPPH or AAPH system, but a little higher in those systems which might associate with iron ion. These results suggest that flavonoids in the radix of Scutellaria baicalensis with o-di-hydroxyl group in A the ring, such as baicalein and baicalin, could be good free radical scavengers and might be used to cure head injury associated with free radical assault.     

Occurrence of jasmonates during cystocarp development in the red alga Grateloupia imbricata

In this study, we highlight the effects of methyl jasmonate (MeJa) on cystocarp development in the red macroscopic alga Grateloupia imbricata. In G. imbricata, jasmonate release is related to the reproductive state, as fertile thalli (i.e., those that have cystocarps) released significant amounts of this volatile compound (1.27 ± 0.20 mM · mg fw^?1 · h^?1) compared with infertile thalli (0.95 ± 0.12 mM · mg fw^?1 · h^?1). Treating G. imbricata thalli with MeJa revealed a significant increase in cystocarp number (1.5 ± 0.27 cystocarps · mm^?2), which was ~7.5‐fold greater than in untreated thalli (0.2 ± 0.07 cystocarps · mm^?2). Maturation was completed within 48 h with MeJa treatment, a shortening of the typical >3‐week maturation period, and included the opening of cystocarps and the presence of dehiscent cavities. Release rates of jasmonates after exogenous MeJa treatment were also modified based on the cystocarp maturation level. All of these effects were reduced in the presence of phenidone, which blocks MeJa production, indicating that the MeJa action is genuine. The effects of MeJa during cystocarp maturation were not replicated by derivatives of reactive oxygen species from the same jasmonic acid biosynthetic pathway, as the activities of scavenger enzymes and lipid peroxidation were unchanged between infertile and fertile thalli. Therefore, a reactive oxygen species–based mechanism is not involved during cystocarp development. We conclude that MeJa has an independent function as a growth regulator during G. imbricata reproduction.     

High stilbenes accumulation in root cultures of <Emphasis Type="Italic">Cayratia trifolia</Emphasis> (L.) Domin grown in shake flasks

The Root cultures of Cayratia trifolia (Vitaceae) a tropical lianas, were maintained in liquid Murashige and Skoog’s medium containing 0.5 mg l⁻¹ NAA, 0.1 mg l⁻¹ kinetin with 3% sucrose. These root cultures when grown with 6% sucrose accumulated stilbenes (piceid, resveratrol, viniferin, ampelopsin) in high amounts, which on elicitation by 500 mg l⁻¹ yeast extract, 50 μM salicylic acid (SA), 50 μM methyl jasmonate (MeJa), 500 μM ethrel added at 25th day, increased up to ninefolds (7.1 mg l⁻¹). Addition of alar or phenylalanine along with the elicitors further enhanced the stilbenes content. In the present study, stilbenes accumulation up to 12 folds (9.2 mg l⁻¹) was obtained with SA and alar. The SA was the most effective in increasing the stilbenes contents while less than control values were recorded in the cells treated with MeJa. The roots could be grown up to 2 l flasks. The present work demonstrates that presence of precursor and sucrose during elicitation at an appropriate time combined with growth retardation significantly increased the production of stilbenes in C. trifolia cell cultures.     

Light-induced inhibitors from intact and cultured caps of Zea roots

Growth inhibitors were assayed from extracts of intact (attached) and of excised (cultured) root caps of Zea mays L., cv. Merit, the roots of which show a positive geotropic response only after exposure to light. If caps are intact at the time of illumination, at least two inhibitory substances are produced, an acid inhibitor and a neutral inhibitor, whereas if caps are detached from roots, placed in culture and then illuminated only the neutral inhibitor is formed. Cycloheximide retards inhibitor production in both intact and cultured caps. When [¹⁴C]mevalonic acid is included in the culture medium and the caps are illuminated, 15–25% of the recoverable ¹⁴C cochromatographs with the neutral inhibitor, whereas in caps cultured in the dark, this radiolabelling pattern is not observed. Cyloheximide in the light reduces the incorporation of ¹⁴C into compounds cochromatographing at the R_f of the neutral inhibitor. It is suggested that the neutral inhibitor may be important in the light-induced bending of roots.Abbreviations ABA abscisic acid - CH cycloheximide