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Flavonoid Production in Transformed Root Cultures of Scutellaria baicalensis
Abstract
Agrobacterium rhizogenes LBA 9402 transformed root cultures of Scutellaria baicalensis Georgi (Lamiaceae) were established and examined in respect to their capability to produce flavonoids characteristic of roots of the intact plant. The flavonoids were quantified by RP-HPLC after previous acid hydrolysis of glycosides to the corresponding aglycones. An effect of a nutrient medium composition on growth and flavonoid content in the cultures was studied. Optimum for flavonoid production was half-strength Gamborg B5 medium containing 5-7% sucrose. Roots grown in the medium yielded up to 7% dry weight of baicalein, 1.9 % dry weight of wogonin and 1.3 % dry weight of oroxylin A.
... The agrobacterial strains used in this study were grown on YEB medium for 48 h 28 ± 0.5 °C [36]; a suspension of agrobacteria with an optical density of 0.8-1 determin One of the risk factors for chronic diseases such as atherosclerosis is oxidative stress. This study focused on protecting the nematodes from oxidative stress. ...
... The agrobacterial strains used in this study were grown on YEB medium for 48 h at 28 ± 0.5 • C [36]; a suspension of agrobacteria with an optical density of 0.8-1 determined at a wavelength of 600 nm was used to infect seedlings [37]. The composition of the YEB medium is presented in the article of G. Vervliet [38]. ...
... Major salts B5, 20×, cm 3 Primary roots were separated and transplanted to agar medium with 250 mg/dm 3 of claforan. The hairy roots were then transferred to antibiotic-free liquid medium and transplanted every three weeks by inoculating the crude root mass into conical flasks containing liquid nutrient medium [24,36,39]. Plant material was cultivated in the dark at 23 ± 0.5 • C on an orbital shaker (shaker incubator ES-20/80, BioSan, Riga, Latvia) at 90 rpm. ...
This study aimed to investigate the anti-atherosclerotic properties of quercetin isolated from the extract of Hedysarum neglectum Ledeb hairy roots. During the study, the hormonal composition of the nutrient medium for cultivation of H. neglectum hairy root biomass was selected: Gamborg’s medium enriched with the cytokine 6-benzylaminopurine (1.5 mg/1 dm3). It was found that the extraction of hairy root biomass with a 50% water–ethanol solution (40:1 1 h at 60 ± 2 °C) yielded an extract that contained the highest amount of quercetin (an average of 2.1 times higher than in extracts obtained at other parameters). It was determined that 100 µM quercetin solution showed the greatest bioactivity on Caenorhabditis elegans: on day 61, the percentage of surviving nematodes was 2.06 times higher compared to other samples and 6 times higher compared to control, resulting in a 12.5-fold increase in SOD-3 expression compared to control (without biologically active substance (BAS) addition). Meanwhile, the 10 µM quercetin solution exhibited the best ability to inhibit the accumulation of lipid fractions; the accumulation was 1.06 times less compared to the control. The results of this study show that quercetin, which was isolated from the biomass of H. neglectum hairy roots, can be used as a component of anti-atherosclerotic dietary supplements.
... However, the yields of biologically active flavones from the cultures were unsatisfactory (up to 1.2% DW of baicalein 7-O-glucuronide). The HRs that emerged as a result of S. baicalensis transformation with R. rhizogenes LBA 9402, in optimum culture conditions produced 6.9% DW of total baicalein glycosides and 1.5% DW of total wogonin glycosides, measured by HPLC after the acidic hydrolysis of the native compounds [96]. Kuzovkina et al. [97,98], by a transformation of S. baicalensis with R. rhizogenes A4 obtained HRs that produced 5% DW of flavonoids (spectrophotometrically measured). ...
... Heterologous expression of the transcription factor Lc from Zea mays in the HRs of Baikal skullcap led to the enhanced accumulation of baicalin, baicalein and wogonin in the roots, but higher yields were achieved when the Arabidopsis PAP1 transcription factor was overexpressed in the same experimental system (up to 102 mg/g DW of baicalin) [104]. Some improvement in yields of flavones from the cultures was achieved as well by an optimization of the nutrient medium composition [96,105,106]. Aglycones:glucuronides ratios in the cultures may be at least partially controlled by the activities of the endogenous β-glucuronidase of Baikal skullcap (baicalinase, sGUS) and baicalein 7-O-glucuronosyltransferase (UBGAT) [107,108]. Hydroxylation of chrysin is the step in the biosynthesis of baicalein and wogonin that requires the respective CYP450 enzymes: flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H). ...
Due to their chemical properties and biological activity, antioxidants of plant origin have gained interest as valuable components of the human diet, potential food preservatives and additives, ingredients of cosmetics and factors implicated in tolerance mechanisms against environmental stress. Plant polyphenols are the most prominent and extensively studied, albeit not only group of, secondary plant (specialized) metabolites manifesting antioxidative activity. Because of their potential economic importance, the productive and renewable sources of the compounds are desirable. Over thirty years of research on hairy root cultures, as both producers of secondary plant metabolites and experimental systems to investigate plant biosynthetic pathways, brought about several spectacular achievements. The present review focuses on the Rhizobium rhizogenes-transformed roots that either may be efficient sources of plant-derived antioxidants or were used to elucidate some regulatory mechanisms responsible for the enhanced accumulation of antioxidants in plant tissues.
... Interest in the conservation and sustainable use the plant led to an introduction of the hairy root culture (HRC) of S. baicalensis (Kuzovkina et al., 2001;Nishikawa and Ishimaru, 1997;Stojakowska and Malarz, 2000;Zhou et al., 1997). Manipulations with genes, media, and nutrition of the HRC have allowed researchers to identify a remarkable content of main flavones (Kim et al., , 2014Park et al., 2011Park et al., , 2012Park et al., , 2016. ...
... An environment of HRC sharply differs from the soil naturally supporting the growth of plant roots. Biosynthesis of the flavones in HRC is largely controlled genetically, but it also can be regulated by nutritional and environmental factors Stojakowska and Malarz, 2000). Therefore, the question arises whether HRC has the same rich repertoire of methylated flavones as the wild root of S. baicalensis. ...
Perennial plants in northern Dauria (Zabaikalsky region) grow in low temperatures in winter and in a dry hot summer. The prairies of northern Dauria are rich in a variety of medicinal herbs, including S. baicalensis, which has roots that are in demand for traditional Chinese medicine. In addition to two monomethylated flavones (wogonin and oroxylin A), determining the pharmacological significance of the root, there is also a minority of their polymethylated congeners. Little is known about their role in the plant or their connection with the conditions of growth and cultivation of their hairy root culture (HRC). Therefore, the purpose of this study was to determine whether and to what extent the biosynthesis of the latter is retained in the hairy root culture established from wild plants of Dauria. The composition of the main methylated flavones of HRC was established using LC–MS and a previously unknown pentamethylated flavone was found in the roots. This study showed a more significant accumulation of polymethylated flavones in the root of the wild plant than in HRC.
... Considering the industrial production of S. baicalensis-derived flavonoids for medical use, production yield is still low and the product cost is high. To increase the production of flavonoids by cultivation of S. vaicalensis cells, several trials in vitro using callus cultures and Agrobacteriuminduced hairy root cultures have been reported (Hirotani et al., 1998;Stojakowska and Malarz, 2000;Yamamoto et al., 1986;Yamamoto, 1991). However, the production of flavonoids by liquid culture of S. baicalensis cells has not been studied. ...
... In other studies of flavonoid production by root-derived cells of S. baicalensis, Hirotani et al. (1998) reported that the callus tissue produced 2 mg baicalein/g dry cell weight and 6 mg baicalin/g dry cell weight in 3week culture. Stojakowska and Malarz (2000) achieved 70 mg baicalein/g dry cell weight and 19 mg wogonin/g dry cell weight of production in 3week culture using Agrobacterium rhizogenestransformed hairy root culture cells. By culturing in the present liquid condition, the culture period to obtain the maximum production of baicalein and wogonin was shortened. ...
Production of baicalein, baicalin and wogonin by liquid culture of Scutellaria baicalensis cells derived from the plant root was studied. The maximum production obtained were 119 mg/L of baicalein at two week, 1372 mg/L of baicalin at eight week, and 14 mg/L of wogonin at two week. In addition, the production of baicalin was drastically increased to 1000 mg/L level at 3-week culture, and the extremely high production rate (339 mg/Lweek) was obtained. In the comparison of total antioxidative activities among baicalein, baicalin and wogonin, evaluated by thiocyanate method, it was suggested that the location of hydroxyl groups both at 5- and 6-position contributed to enhancement of radical scavenging activity, and/or methoxylation at 8-position diminished the activity. The possibility of utilizing these flavonoids for natural antioxidants and medicine is also discussed.
... A comparison of basic morphological and biochemical data obtained for various skullcap cultures by various authors [7,[14][15][16][17][18] shows that the growth rate of all these cultures was approximately similar in spite of the fact that they were produced using different wild strains of the agrobacterium (15834, 9402, and A4) and also modified A1s strain with a Ri plasmid bearing the GUS gene [16]. Thus, a final dry weight of skullcap roots attained 250-450 mg/flask by the 4-5th week of culturing. ...
... In the studies performed with the skullcap root culture initiated by an LBA 9402 strain of soil agrobacterium [18], it was shown that an increase in the sucrose concentration in the nutrient medium up to 5-7% resulted in the doubling of root weight and a fourfold increase of the content of aglycons, baicalein and wogonin, in roots. However, these authors first performed the hydrolysis of flavones and then estimated the concentration of aglycons. ...
The composition and content of flavones were estimated in pRi T-DNA-transformed skullcap (Scutellaria baicalensis Georgi) roots obtained by the inoculation of axenically grown seedlings with a wild A4 strain of the soil bacterium Agrobacterium rhizogenes. It was elucidated by analytical and preparative HPLC of phenolic compounds in the extracts from the pRi T-DNA-transformed roots and also by ultraviolet spectroscopy and 1H and 13C NMR that cultured skullcap roots contained similar basic flavones as intact roots of this plant species, i.e., baicalein and wogonin and corresponding glucuronides, baicalin and wogonoside. The content of these flavones in cultured roots was threefold lower than in the roots of intact five-year-old plants. When skullcap roots were cultured on B5 or Murashige and Skoog medium, the ratios between major flavones changed but their total content remained unchanged. The treatment of three-week-old cultured roots with methyl ether of jasmonic acid (MeJa) doubled the total concentration of major flavones in roots; the content of aglycons, baicalein and wogonin, increased to a greater degree, e.g., by 2.3 and 3.3 times, respectively. The induction of flavone production by elicitors indicates that flavones behave as phytoanticipins because major flavones of skullcap manifest a distinct antimicrobial activity. The results of the short-term treatment of skullcap roots with MeJa show that stress biotic factors can considerably increase the content of physiologically active flavones.
... Agrobacterium rhizogenes is a soil bacterium that has morphogenic effects on higher plants. A. rhizogenes induces adventitious root growth at the wound site by incorporating a series of hormone related rol genes [16][17][18][19]. Like many plant pathogens, A. rhizogenes has been known to enhance phenolic levels [20,21]. ...
... However, the low GPX activity observed in inoculated MPH-4 clonal lines was likely caused by a deficit in phenolics, which may have been used for auxin production. Reduced peroxidase activity could also be due to phenolics being synthesized into metabolites that fend off A. rhizogenes, such as flavanoids and phytoalexins [18]. Further, inoculation may also be inhibiting root growth which in turn would inhibit dye sequestration and increased peroxidase activity. ...
Polymeric dyes are environmental pollutants widely used in the textile industry. Polymeric dyes have a structure analogous to common polycyclic aromatic hydrocarbons (PAHs). Plants that possess an inherent tolerance to these dyes are currently being investigated for use in phytoremediation. Tolerant plants create a healthy rhizosphere for microbial degradation and contribute to uptake of pollutants from soil or water. Tolerant plants have been found to contain high levels of endogenous phenolic compounds, which contribute to plant stress response mechanisms. Peroxidases are a ubiquitous group of enzymes known to participate in the oxidative coupling of phenolic compounds during the lignification process. Four elite clonal lines of Mentha pulegium were screened for high phenolic content on different concentrations of polymeric dye labeled R-478, a violet colored anthraquinone dye. One of the four clonal lines, MPH-4, showed a significant correlation between phenolic content and peroxidase activity in response to the polydye R-478. MPH-4 was chosen for further screening with polydye R-478 in conjunction with Agrobacterium rhizogenes. The use of A.rhizogenes was based on the rationale that the bacterium would induce root development and enhance dye uptake. Although the expected results were not observed, the changes seen in the innoculated plants may be due to the diversion of key metabolites from the primary metabolism towards cytokinins.
... Although the exact mechanism of the genes transferred from the Ri plasmid is still not known, they are believed to be associated with the auxin biosynthetic pathway. In addition to adventitious root growth at the wound site, A. rhizogenes has also been shown to lead to the production of secondary metabolites in many plant varieties, such as several phenolic compounds and even peroxidases, that can be monitored by several methods including HPLC [2,3]. ...
... This procedure is easy to perform by inoculation and the bacterium is then removed by antibiotic treatment [20]. Genes from the Ri plasmid have been shown to increase rooting and host phenolic content and some peroxidases in plants that have been transformed [2]. A practical application for this would be the development of ornamental and food grade plants for nutraceuticals. ...
Mentha pulegium elite clonal line MPH-4 is known to contain high levels of endogenous phenolic compounds. These compounds are secondary metabolites of the phenylpropanoid pathway that aid in several metabolic processes, including stress response and hormone production. Agrobacterium rhizogenes is a soil bacterium utilized for its ability to transfer its Rol genes via the Ri plasmid on t-DNA. A. rhizogenes is also known to increase secondary metabolites in several plant varieties. In this study, MPH-4 and A. rhizogenes were used to demonstrate how secondary metabolite production can be effected. Total plant phenolics and GPX activity were enhanced by inoculation of tissue culture explants with A. rhizogenes. Enhancement of phenolic production by A. rhizogenes was more apparent when explants were also treated with polymeric dye R-478. The response of MPH-4 to A. rhizogenes and polydye R-478 are useful to determine the use of this clonal line in phytoremediation.
... Scutellaria baicalensis, the favorite natural drug of traditional Oriental medicines, contains in the roots probably the highest known amount of flavones (up to 20% dw) of which the strong lipophilic antioxidants: baicalin, baicalein, wogonoside and wogonin are the most abundant. In cell suspension and the hairy root cultures of S. baicalensis the accumulation of these compounds was reported (Morimoto et al., 1998;Nishikawa et al., 1999;Stojakowska and Malarz, 2000). The in vivo antioxidant function of baicalein in metabolizing hydrogen peroxide has been proposed as a result of experiments using cell cultures (Morimoto et al., 1998). ...
... Baikal skullcap (S. baicalensis) is one of the richest natural sources of flavones reaching up to 20% in the root. Transformed roots produced baicalein and wogonin glucuronides, but also a phenylethanoid acteoside, a compound unknown from plants growing in natural conditions (Nishikawa et al., 1999;Stojakowska and Malarz, 2000). In S. sclarea hairy roots some abietane diterpenoids are also reported, although not in large enough amounts for them to be considered as a source of antioxidants (Kuźma et al., 2006). ...
Plant in vitro cultures are able to produce and accumulate many medicinally valuable secondary metabolites. Antioxidants are an important group of medicinal preventive compounds as well as being food additives inhibiting detrimental changes of easily oxidizable nutrients. Many different in vitro approaches have been used for increased biosynthesis and the accumulation of antioxidant compounds in plant cells. In the present review some of the most active antioxidants derived from plant tissue cultures are described; they have been divided into the main chemical groups of polyphenols and isoprenoids, and several examples also from other chemical classes are presented. The strategies used for improving the antioxidants in vitro production efficiency are also highlighted, including media optimization, biotransformation, elicitation, Agrobacterium transformation and scale-up.
... The first works in obtaining hairy roots of species of the genus Scutellaria date back to the end of the twentieth and beginning of the twenty-first century [31,32]. The hairy roots of S. baicalensis were the first to be studied. ...
We measured and studied the growth parameters and the qualitative and quantitative
composition of the flavones of hairy roots of the Scutellaria genus: S. lateriflora, S. przewalskii and S.pycnoclada. Hairy roots were obtained using wild-type Agrobacterium rhizogenes A4 by co-cultivation of explants (cotyledons) in a suspension of Agrobacterium. The presence of the rol-genes was confirmed by PCR analysis. The hairy roots of the most studied plant from the Scutellaria genus, S. baicalensis, were obtained earlier and used as a reference sample. HPLC-MS showed the predominance of four main flavones (baicalin, baicalein, wogonin and wogonoside) in the methanol extracts of the studied hairy roots. In addition to the four main flavones, the other substances which are typical to the aerial part of plants were found in all the extracts: apigenin, apigetrin, scutellarin and chrysin-7-O-�-Dglucuronide. According to the total content of flavones, the hairy roots of the studied skullcaps form the following series: S. przewalskii (33 mg/g dry weight) > S. baicalensis (17.04 mg/g dry weight) > S. pycnoclada (12.9 mg/g dry weight) > S. lateriflora (4.57 mg/g dry weight). Therefore, the most promising producer of anti-coronavirus flavones is S. przewalskii.
... Numerous studies have been conducted on the genetic transformation of the Baikal skullcap in order to obtain hairy root cultures, due to the fact that it is a species accumulating biologically active metabolites in the roots [45][46][47]. ...
Scutellaria baicalensis (Baikal skullcap) has been used for centuries in traditional Chinese medicine (TCM). The root of the Baikal skullcap (Scutellariae baicalensis radix) is also a recognized raw material in European therapeutic practice. It is mentioned in European Pharmacopoeia since 2010 and also in the WHO monograph. This raw material is a rich source of specyfic flavonoids, especially from the group of flavones, such as baicalein, baicalin, wogonin, and wogonoside. These compounds show a number of valuable biological activities. The aim of the presented research was to propose in vitro cultures of Scutellaria baicalensis as an alternative to the raw material obtained from cultivated plants for providing apart from flavonoids also phenylpropanoid glycosides and phenolic acids. Standard biotechnological treatments, such as a selection of the composition of the basal medium, selection of the concentration and mutual proportions of plant growth regulators, and different types of culture cultivation (solid, agitated and bioreactor’s cultures), were used. In addition, elicitation and feeding with biosynthetic precursors were tested. The optimization of culturing conditions led to increased accumulation of species-specific flavonoids. Additionally in vitro cultures proved to be a particularly rich source of the phenylpropanoid glycoside - verbascoside.
... В связи с исключительной ценностью корней шлемника байкальского, относительно малыми природными запасами, медленными темпами роста растений в естественных зарослях (5-10 лет) и сложностью получения экологически чистого сырья возникла необходимость получения культуры в условиях in vitro. С 80-х годов прошлого века проводятся исследования культуры ткани корня S. baicalensis и изучается ее химический состав[140][141][142][143][144][145]. Качественный состав флавонов культивируемых корней S. baicalensis, в основном, представлен теми же, которые накапливаются в корнях целого, дифференцированного растения: байкалином(19), вогонозидом(41), байкалеином (17) и вогонином(40). ...
In the scientific world, interest in flavonoids is very high, due to their high biological activity and low toxicity. The book is a guide for the study of natural flavonoids of plants of the genus Scutellaria L. The monograph provides information on the classification, methods of detection, isolation and identification of flavonoids, including qualitative reactions, thin-layer and high-performance liquid chromatography, spectral and chemical methods. The structures of the most representative groups of compounds and the main data concerning their biosynthesis are presented. The results of a scientometric analysis of data on the degree of knowledge and chemodiversity of flavonoids of species of the genus Scutellaria L. of the world flora, as well as information on the composition of flavonoids of 63 species of Scutellaria, distribution in plants, structure and sources of obtaining 301 flavonoids belonging to the groups of flavones, flavanones, flavanonols, are presented. flavonols, chalcones, isoflavones, flavolignans and biflavonoids. It is addressed to specialists in the field of bioorganic chemistry, phytochemistry, pharmacology, as well as university professors, graduate students, undergraduates and students of relevant specialties.
... Transformed root culture of S. baicalensis induced by A. rhizogenes (ATCC 15834) produced both non-glucuronidated-type flavonoids (baicalein, wogonin) and glucuronidated type flavonoids (baicalin). A. rhizogenes strain LBA 9402 induced hairy root cultures in the explants of S. bacailensis. Flavonoid accumulation was optimized to produce up to 7% baicalein, 1.9% wogonin, and 1.3% oroxylin on dry weight basis in this system (Stojakovska and Malarz 2000). ...
Abstract. Plants of the genus Scutellaria (Family Lamiaceae) constitute one of the common
components of Eastern as well as traditional American medicine. Skullcap is a North American
perennial plant belonging to the genus Scutellaria. The genus is widespread in Northern Hemisphere
represented by close to 400 species. Many species are rare, threatened, or endangered. Habitat
destruction, urbanization, and poor seed set are the few reasons behind diminishing population of
many skullcaps. Many skullcaps have showy, beautiful blooms with great potential as ornamental
plants. Skullcap are used in alternative medicine as anti-inflammatory, antispasmodic, emmenagogue,
nervine, sedative and strong tonic. We have developed a germplasm collection at Fort Valley State
University and the populations are maintained in the greenhouse and through micropropagation. We
have made significant headway in the areas of micropropagation, transformation for desired gene
transfer and hairy root induction, extraction and HPLC analysis of targeted flavonoids, and clinical
role of select flavonoids using glioma cell lines.
... The line 5 of the induced hairy roots demonstrated a vigorous growth rate and active elongation associated with tendency to produce profuse branches (Fig. 2a, b, c and d). Studies conducted on other species of Scutellaria revealed that stem explants are appropriate for hairy root induction (Marsh et al., 2014;Stojakowska and Malarz, 2000;Wang et al., 2008). Additionally, the effects of two co-cultivation medium (full and halfstrength MS) were evaluated. ...
For the purpose of the current study, hairy root induction in S. bornmuelleri, which is an important medicinal plant, was examined using a particular protocol. Accordingly, some factors such as four strain types of Agrobac-terium rhizogenes (A4, A3, MSU440 and ATCC15834), three different explants, namely stem, petiole and leaf, two co-cultivation media, i.e. full and half-MS were studied. Besides, two inoculation methods including injection and immersion as well as three inoculation times (5, 7 and 10 min) were closely taken into account. Utilizing injection method by MSU440 strain, hairy root induction took place in stem explants, and a remarkable increase in transformation frequency (100%) was observed in half-strength MS medium. Methyl jasmonate (MeJA, 100 μM), methyl-b-cyclodextrin (b-CD, 0.7, 7 and 14 mM) and Chitosan (Chi, 50, 100 and 200 mg/l) were used either individually or in a combined way to elicitation. Based on the HPLC results, production of chrysin, wogonin and baicalein increased 9.15, 10.56 and 13.25 times after elicitation of hairy roots by MeJA + Chi. In addition, transcripts of FNSП2 and MYB7, two important genes involved in the flavonoid biosynthesis pathway, were studies. By applying Chi and MeJA + Chi elicitor, the expression of both genes increased noticeably. It can be concluded that the mentioned hairy root culture system of S. bornmuelleri can be an alternative to flavonoids production. Moreover, there is a direct and positive relationship between the expression of FNSП2 and MYB7 genes as well as the level of three flavonoids.
... Numerous studies have been conducted on the genetic transformation of the Baikal skullcap in order to obtain hairy root cultures, due to the fact that it is a species accumulating biologically active metabolites in the roots [45][46][47]. ...
... Another hairy root culture of S. baicalensis was obtained using A. rhizogenes strain LBA 9402 [100]. For all received clones, B5 medium was found to be the most suitable (especially at 5-7% sucrose) for culture growth and flavone production. ...
Background:
This review is intended to draw the attention of pharmaceutical and biotechnological communities to the untapped potential of the Scutellaria genus. Skullcaps, as they are more widely known, are found in one of the oldest materia medica in the world, that of ancient Chinese pharmacology, and their numerous wide range of medicinal bioactivities have been studied both in vivo and in vitro. For thousands of years, chemical compounds from the Scutellaria species have been safely used as antitumor, antibacterial, antiviral, anti-inflammatory, antioxidant or hepatoprotective factors.
Objective:
As these effects are well known, reflected in the presence of Scutellaria plants in national pharmacopoeias, it is clear that the plant has yet enormous unexploited potential. The European pharmacological market has turned to the resources of Scutellaria only in the last two decades, and although the construction and clinical processing of a new drug is a long process, the general impression is that very few medical products in pharmacies have been inspired by the phytochemistry of skullcaps.
Conclusion:
This paper presents the current state of knowledge on the wealth of Scutellaria chemical compounds with treatment applications, its tissue culture and biotechnological achievements, especially in the context of the production of secondary metabolites.
... Due to the exceptional value of Baikal skullcap roots, its relatively limited natural reserves, slow growing speed of the plants in natural brush (5-10 years), and difficulties associated with cultivation of ecologically pure raw material, a need for in vitro culture arose. Starting from the 1980s, studies on the S. baicalensis root culture were performed investigating its chemical composition [109][110][111][112][113][114][115][116][117]. Qualitative composition of flavones in cultured roots of S. baicalensis is mainly represented by the same flavones as are accumulated in roots of the whole differentiated plant: baicalin (19), wogonoside (41), baicalein (17), and wogonin (40). ...
The review presents the results of scientometric analysis of data on the level of study and chemical diversity of flavonoids of the Scutellaria L. genus species of the world’s flora. Flavonoid composition in 63 species of skullcap is reported, together with the data on distribution in plants, structure, and sources of 301 flavonoids belonging to the groups of flavones, flavanones, flavanonols, flavonols, chalcones, isoflavones, flavolignans, and bioflavonoids. The greatest number of flavonoids was shown to be isolated from plants of S. indica, S. baicalensis, S. barbata, S. amoena, S. prostrata, S. galericulata, S. discolor, S. ramosissima, and S. supina. Scientometric studies indicate the constantly growing interest in the study of species of the Scutellaria L. genus by scientists of various branches of science, including phytochemists, biologists, and pharmacologists. Information provided in the review can be used to address issues of chemosystematics of plants of the Scutellaria L. genus.
... Supplementation of acetosyringone during co-cultivation of plant tissue and A. rhizogenes enhanced the transformation efficiency further [64]. Hairy root cultures of S. baicalensis have a similar metabolite pattern to natural roots and the major flavones can be enhanced by treatment of cultures with methyl jasmonate [65][66][67]. Over-expression of PAL or CHI in hairy roots of Scutellaria leads to enhanced levels of root-specific flavones [63,68] (Table 1). ...
Scutellaria baicalensis Georgi, or Chinese skullcap, has been widely used as a medicinal plant in China for thousands of years, where the preparation from its roots is called Huang-Qin. It has been applied in the treatment of diarrhea, dysentery, hypertension, hemorrhaging, insomnia, inflammation and respiratory infections. Flavones such as baicalin, wogonoside and their aglycones baicalein wogonin are the major bioactive compounds extracted from the root of S. baicalensis. These flavones have been reported to have various pharmacological functions, including anti-cancer, hepatoprotection, antibacterial and antiviral, antioxidant, anticonvulsant and neuroprotective effects. In this review, we focus on clinical applications and the pharmacological properties of the medicinal plant and the flavones extracted from it. We also describe biotechnological and metabolic methods that have been used to elucidate the biosynthetic pathways of the bioactive compounds in Scutellaria. © 2016 Science China Press and Springer-Verlag Berlin Heidelberg
... These differences could be due to the age of the cultures. Previous studies with hairy root cultures of related species such as S. baicalensis (Stojakowska and Malarz, 2000;Kuzovkina et al., 2005) and wild-type root cultures of S. barbata (Wilczań ska-Barska et al., 2011) have not reported the presence of scutellarein (4) or scutellarin (2). Nonetheless, the study herein is the first to report the production of scutellarein (4) in hairy root cultures of S. lateriflora. ...
Scutellaria lateriflora(American skullcap) has been used in traditional medicine to treat several medicalconditions including nervous disorders and cancer. Previous studies have associated these medicinalproperties to flavones present in roots and leaves of this species. In order to develop a production systemand study the biosynthesis of these bioactive compounds, hairy root cultures ofS. lateriflorawere estab-lished and line 4 was selected for further studies based on its growth performance in a modified Murash-ige and Skoog’s medium supplemented with 0.5 mg/l indole-3-butyric acid. Scanning electronmicroscopy of the hairy roots showed a high profusion of hairs along the root. Several phenolic com-pounds, including verbascoside, and the flavones wogonin, baicalein, scutellarein and their respectiveglucuronides were identified by high performance liquid chromatography–tandem mass spectrometryin the root tissue, but not in the culture medium. Among these compounds, verbascoside accumulatedat the highest levels. Interestingly, cultures incubated under continuous light and treated with 15 mMmethyl-b-cyclodextrin for 24 h produced significantly higher levels of the aglycones, baicalein and wogo-nin, but not scutellarein, compared to cultures incubated under continuous darkness. This work demon-strates that hairy root cultures ofS. lateriflorahave the biosynthetic capacity to produce knownScutellariaflavones and suggest that light may have a selected regulatory effect on the synthesis or accumulation ofthese phenolic compounds.
(PDF) Metabolic Engineering and Biotransformation of Flavonoids in Hairy Root Cultures of Scutellaria lateriflora. Available from: https://www.researchgate.net/publication/298358890_Metabolic_Engineering_and_Biotransformation_of_Flavonoids_in_Hairy_Root_Cultures_of_Scutellaria_lateriflora [accessed Sep 01 2021].
... Transformed root culture of S. baicalensis induced by A. rhizogenes (ATCC 15834) produced both non-glucuronidated-type flavonoids (baicalein, wogonin) and glucuronidated type flavonoids (baicalin). A. rhizogenes strain LBA 9402 induced hairy root cultures in the explants of S. bacailensis. Flavonoid accumulation was optimized to produce up to 7% baicalein, 1.9% wogonin, and 1.3% oroxylin on dry weight basis in this system (Stojakovska and Malarz 2000). ...
Plants of the genus Scutellaria (family Lamiaceae) are distributed globally and they are integral part of Eastern as well as American traditional medicine. The genus Scutellaria, commonly referred to as skullcap, is considered as a North American perennial plant. At present this genus is represented by 350–360 species. Many species are rare, threatened, or endangered. Habitat destruction, urbanization, and poor seed set are a few reasons behind the diminishing population of many skullcaps. Many skullcap species have showy, beautiful blooms with great potential as ornamental plants. Skullcaps are used in alternative medicine as antiinflammatory, antispasmodic, emmenagogue, nervine, sedative, and strong tonic. More than 295 compounds have been isolated from Scutellaria species and the majority of the compounds are flavonoids and diterpenes. We present a review of research carried out by various groups, covering aspects of phytochemical screening, biomedical studies, conservation, plant biotechnology, and development of Scutellaria as a premium crop. We have developed a germplasm collection at Fort Valley State University that includes 19 species. These species are maintained in the greenhouse and through micropropagation in the lab. Biotechnology presents significant advances for the use of Scutellaria. We will review progress in micropropagation, transformation for desired gene transfer, and hairy root induction, extraction, and HPLC analysis of targeted flavonoids, and preclinical and clinical studies on select extracts and isolated flavonoids using various cancer models.
... 5,27,28 Considering wogonin's pharmacological relevance and the fact that it occurs together with other flavonoids in the different parts of Scutellaria species, [29][30][31][32][33] several trials have been carried out for its isolation and production, even at large scale. Among them, increasing the growth rate and flavonoid content of Scutellaria species with CO 2 enrichment in a controlled environment, 34 cultivation of Scutellaria cells, 16,[35][36][37] synthesis methods, 38 biotechnology production, 4,39,40 classical column chromatography methods, analytical assay, 31,33,41 and preparative high-performance liquid chromatography, 22 high-speed countercurrent chromatography 42 and a lowpressure preparative chromatography (LPPC) after endogenous baicalinase catalyzed hydrolysis. 43 However, these methods have disadvantages of a long extraction time, low yield of wogonin, high solvent consumption, multiple steps for the isolation, high cost of the procedures and very laborious ones. ...
Pure Wogonin (5,7-dihydroxy-8-methoxyflavone) as well as part of different extracts from Scutellaria (Lamiaceae) genus plants, has demonstrated to possess anti-inflammatory, anti-cancer, anxiolytic, neuroprotective, anti-atherosclerotic, anti-arthritic, anti-tumor, anti-allergic, anticoagulant, antioxidant and antiviral properties. Because of the growing interest in this substance, many methods for its isolation and high purity have been developed and reported in the literature. However, such methods are laborious,
inefficient and expensive. In the present investigation, two new fast, selective and ship methods for isolating pure wogonin with high yields from Scutellaria havanensis were developed. The methods involve fresh aerial parts extraction with ethyl ether by sonication (5 min) and electrical shaker (2h), filtration and solvent evaporation without using any chromatographic techniques. Quantities of ca 0.8 to 1.2 g of wogonin (>95 % purity) were obtained from 50 g of plant material, yielding 8 to 12 % of recoveries (dry weight). The structure of wogonin was confirmed by PMR and 13C-NMR, UV, FT-IR and GC-MS. This is the first report on the
wogonin isolation and characterization from this endemic Cuban plant by simple extraction and direct crystallization from the extract.
... These differences could be due to the age of the cultures. Previous studies with hairy root cultures of related species such as S. baicalensis (Stojakowska and Malarz, 2000;Kuzovkina et al., 2005) and wild-type root cultures of S. barbata (Wilczań ska-Barska et al., 2011) have not reported the presence of scutellarein (4) or scutellarin (2). Nonetheless, the study herein is the first to report the production of scutellarein (4) in hairy root cultures of S. lateriflora. ...
... Infection with A. rhizogenes modulates the host cell metabolic state by, for example, altering the expression of plant defense genes and secondary metabolite synthesis (Stojakowski and Malarz, 2000;Strycharz and Shetty, 2002). Many plant defense pathways interfere with reactive oxygen species (ROS) and the cellular prooxidant/antioxidant equilibrium (Foyer and Noctor, 2009;Kuźniak et al., 2009). ...
We compared the biochemical profiles of Physalis ixocarpa hairy roots transformed with Agrobacterium rhizogenes ATCC and A4 strains with non-transformed root cultures. The studied clones of A4- and ATCC-induced hairy roots differed significantly: the latter showed greater growth potential and greater ability to produce secondary metabolites (tropane alkaloids) and to biotransform hydroquinone to arbutin. We compared glucose content, alanine and aspartate aminotransferase activity, and L-phenylalanine ammonia-lyase activity. We analyzed markers of prooxidant/antioxidant homeostasis: catalase, ascorbate peroxidase, oxidase, glutathione peroxidase and transferase activity, and the levels of ascorbate, glutathione, tocopherol and lipid peroxidation. We found that transformation induced strain-specific regulation, including regulation based on redox signals, determining the rate of allocation of carbon and nitrogen resources to secondary metabolism pathways. Our results provide evidence that A. rhizogenes strain-specific modification of primary metabolites contributed to regulation of secondary metabolism and could determine the ability of P. ixocarpa hairy root clones to produce tropane alkaloids and to convert exogenously applied hydroquinone to pharmaceutically valuable arbutin. Of the studied parameters, glucose content, L-phenylalanine ammonia-lyase activity and alanine aminotransferases activity may be indicators of the secondary metabolite-producing potential of different P. ixocarpa hairy root clones.
... These differences could be due to the age of the cultures. Previous studies with hairy root cultures of related species such as S. baicalensis (Stojakowska and Malarz, 2000;Kuzovkina et al., 2005) and wild-type root cultures of S. barbata (Wilczań ska-Barska et al., 2011) have not reported the presence of scutellarein (4) or scutellarin (2). Nonetheless, the study herein is the first to report the production of scutellarein (4) in hairy root cultures of S. lateriflora. ...
... Many of these properties are explained by the presence of specific terpenes and sesquiterpene lactones (Abdin et al. 2003; Cheriti and Belboukhari 2007; Drewes et al. 2006; Mathekga et al. 2000; Scio et al. 2003; Tereschuk et al. 1997). Significant biological activity has also been noted in the family Lamiaceae due to the presence of large concentrations of essential oils with terpenoids and some flavonoids that have anti-leishmaniasis, antiviral, antibacterial, antiinflammatory , and antioxidant actions (Kamatou et al. 2006; Matasyoh et al. 2007; Moharram et al. 2006; Stojakowska and Malarz 2000; Tan et al. 2002a, b). In analyzing the continents separately, five of the nine citations of the most important families in Africa were for the family Fabaceae. ...
Local Markets and Medicinal Plant Commerce: A Review with Emphasis on Brazil. Local markets unite, concentrate, maintain, and diffuse empirical knowledge about plant and animal resources and therefore help guarantee the resilience and maintenance of folk knowledge concerning useful species. A critical review is presented here that focuses on the importance of these local markets, emphasizing the diversity of medicinal products offered, the different approaches to this subject, and the evolution of the research approaches taken in studying the medicinal plant products sold. To this end, the scientific literature was examined for research on these markets so as to provide a panoramic view of the different approaches taken, the diversity of plants being sold, the methodological procedures employed in collecting research information, and the specific nature of the studies. From our point of view, it is necessary to intensify the ethnobotanical and ethnopharmacological investigation of these markets, especially with comparable methods and techniques.
... There have been numerous reports of the production of phytochemicals from cell cultures of S. baicalensis [17][18][19][20][21][22][23]. However, the large scale production of differentiated tissues in sterile bioreactor systems has not been previously evaluated. ...
The study was conducted to establish a model system for in vitro biomass production, biochemical characterization and evaluation of chemo-preventive properties against mutagenicity in transgenic rats of a selected germplasm line (HQ 111) of Chinese medicinal plant species Scutellaria baicalensis Georgi (Huang-qin). Biomass production was assessed in different in vitro growing systems as a means of large-scale, biotic or abiotic contaminant-free production. Result revealed that the plantlets grown in a temporary immersion bioreactor showed the highest accumulation of biomass, while plantlets grown on solid medium in a CO2 enriched environment had the highest concentrations of bioactive metabolites. Supplementation of feed with S. baicalensis decreased the mutagenicity of aflatoxin-B1 in the liver of lacI transgenic rats providing evidence of efficacy of the optimized germplasm as an anticancer agent. The system described in the current study represents a novel approach to medicinal plant research highlighting the optimized production conditions for biomass production and phytochemical content in plant tissues of a selected superior plant germplasm and the evaluation of whole plant preparations in an animal model system.
... For both plant types tested, four of five and six of seven flavone analytes, in shoot and root tissue respectively, had higher concentrations in greenhouse versus in vitro-grown plant types. This was consistent with a study by Stojakowska and Malarz [26] and Kuzovkina et al. [27], which found flavonoid concentrations to be much lower in in vitro-grown tissue to that of greenhouse plants of S. baicalensis. ...
A method of analysis of eight flavones using high-performance liquid chromatography (HPLC)-diode array detection (DAD)-mass spectrometry (MS) in root and aerial tissues of the medicinal plant Scutellaria baicalensis was developed. The identity of the analytes was confirmed using retention time, UV-vis and mass spectral comparisons to commercial standards. Both UV-vis and mass spectral patterns were characterized for glycosylated flavones. Two additional flavone glycosides were tentatively identified as chrysin-7-glucuronide and wogonoside, but not quantified. Greenhouse and in vitro-grown tissues were analyzed with flavone concentrations ranges of 0.14-150 and 0.030-1.7 microg/mg for greenhouse root and shoot tissue, respectively, and 0.0068-6.4 and 0.082-1.5 microg/mg for in vitro-grown roots and shoots, respectively.
The metabolically most active compounds are mainly secondary types of metabolites, which have therapeutic as well as pharmaceutical importance and are basically acquired from the plant through HR-based root culture. This thin, like-hairy root emerged from the injured site after the infection of the soil bacteria Agrobacterium rhizogenes. HR-based root culture is considered a valuable bio-production technique for the improvement of diverse kinds of plant-derived natural compounds. The plant-derived compounds found in the hairy roots are equivalent to those of their mother plant. However, the major limitations of the commercial manufacturing of pharmaceutical compounds via HR-based root culture do not occur under suitable protocols. Apart from this, it is an economically feasible option for sustainable enrichment of vital metabolically active compounds through this recent technique because their roots are normally high producers of pharmaceutical compounds because of their huge growing roots, well-grown in any type of media, and stability in terms of genetic value. The efficacy and commercial utilisation of hairy root culture could be enhanced through implementing novel biochemical methods such as elicitations for improved yield and bioreactor-founded uplifting; however, media composition, nanoparticles, and metabolic engineering have also been beneficial for large-scale production of secondary metabolites. This book chapter outlines the developments of hairy root culture, its application, challenges, and future prospects, as well as recent micropropagation methodologies for the enhancement of pharmaceuticals through HR-based root culture.
Исследование посвящено сравнительной характеристике флавоноидов и фенилэтаноидов корня дикого растения шлемника байкальского ( Scutellaria baicalensis ) и культуры корневых волосков. Культура корневых волосков, полученная из семян дикого растения, демонстрирует практически идентичный с корнем набор метаболитов, при этом показывая различия в обильности метилированных и гликозилированных производных обоих классов соединений. Отмечено существенное накопление сахарозы корнями интактного растения. Метилированные флавоны, ответственные за защиту растения от биотических и абиотических стресс-факторов, более обильны в культуре корневых волосков. В обоих образцах впервые идентифицирован 7-сульфат 6-ОМе вогонин. Визуализация масс-спектрометрических данных метаболома посредством селективного ионного мониторинга показала себя как удобный инструмент обнаружения изменения метаболизма, вызванного факторами внешней среды или повреждением растения.
Plant cell and organ cultures are potential sources of valuable secondary metabolites that can be used as food additives, nutraceuticals, cosmeceuticals, and pharmaceuticals. Phytochemical biosynthesis in various in vitro plant cultures, in contrast to that in planta, is independent of environmental conditions and free from quality fluctuations.
Pharmaceutical application of plant biotechnology is of interest to almost all departments of the Faculty of Pharmacy and Institute of Pharmacology in Poland with a botanical profile (Pharmaceutical Botany, Pharmacognosy, and Pharmacology).
This study discusses the advances in plant biotechnology for the production of known metabolites and/or biosynthesis of novel compounds in plant cell and organ in vitro cultures in several scientific centers in Poland.
The review presents the results of scientometric analysis of data on the level of study of flavo- noids of species of the genus Scutellaria L. of the world's flora, compiled information on hemia- trophia flavonoids. Presents data on the composition of flavonoids 63 species of skullcap, about distribution in plants, structure and sources 301 of flavonoids belonging to the groups of flavo- nes, flavanones, flavanonols, flavonols, chalcones, isoflavones, flavolignans and bioflavonoids. The greatest number of flavonoids isolated from plants of S. indica S. baicalensis, S. barbata, S. amoena, S. prastrata, S. galericulata, S.
discolor, S. ramosissima and S. supina. Scientometric studies indicate the constantly growing interest in the study of species of the genus Scutellaria L. by scientists of various branches of science – phytochemical, biologists, pharmacologists, and others. Listed in the review information can be used to address issues of chemosystematic plants of the genus Scutellaria L.
Keywords: Scutellaria L., Lamiaceae, flavonoids, scientometric analysis, hemiatrophia.
A phytochemical screening revealed the presence of phytoconstituents like flavonoid, anthraquinone and phenol in the callus and suspension culture of Oroxylum indicum (L.) Vent. (Bignoniaceae). The quantitative estimation using TLC technique reflected the presence of two flavonoids viz. baicalein and chrysin in three fractions (chloroform, ethyl acetate and n-butanol) of callus extract and in intra and extracellular suspension cultures. Protocol was developed for quantification of phytoconstituents by HPLC with methanol: water (70:30) mobile phase and c-18 column in all the three fractions of callus extract. Both baicalein and chrysin flavonoids were there in all the three callus fractions. Maximum amount of baicalein was present in nbutanol fraction of callus while maximum amount of chrysin was present in chloroform fraction. Cell suspension cultures of O. indicum were established in shake flask system to optimize medium formulation for cell growth and flavonoid content. Murashige and Skoog’s basal medium (MS) supplemented with 1 mg/L BAP, 0.5 mg /LNAA , 0.1 mg /L 2,4-D and 1 mg /L AgNO3 was found optimum for growth parameters viz. cell counting, PCV, fresh weight and dry weight. Presence of baicalein and chrysin in intracellular and extracellular fractions of this suspension culture is a successful event of present work.
The study was conducted to establish a model system for in vitro biomass production, biochemical characterization and evaluation of chemo-preventive properties against mutagenicity in transgenic rats of a selected germplasm line (HQ 111) of Chinese medicinal plant species Scutellaria baicalensis Georgi (Huang-qin). Biomass production was assessed in different in vitro growing systems as a means of large-scale, biotic or abiotic contaminant-free production. Result revealed that the plantlets grown in a temporary immersion bioreactor showed the highest accumulation of biomass, while plantlets grown on solid medium in a CO2 enriched environment had the highest concentrations of bioactive metabolites. Supplementation of feed with S. baicalensis decreased the mutagenicity of aflatoxin-B1 in the liver of lacI transgenic rats providing evidence of efficacy of the optimized germplasm as an anticancer agent. The system described in the current study represents a novel approach to medicinal plant research highlighting the optimized production conditions for biomass production and phytochemical content in plant tissues of a selected superior plant germplasm and the evaluation of whole plant preparations in an animal model system.
Plants produces diversity of secondary metabolites which not only plays an important role in adaption accordering to the environment but also represent an important source of active pharmaceuticals. Flavonoids are a group of polyphenols having a wide array of biological activities including anti-inflammatory, anti-carcinogenic, antioxidant, anti mutagenic etc. Biotechnology offers a great opportunity to exploit these secondary metabolites via different methods. Plant cell and tissue culture is one of the emerging fields of biotechnology to investigate and enhance the production of secondary metabolites. Undifferentiated cell cultures like callus cultures and suspension cultures have been studied widely for flavonoid production. Along with this hairy root culture and transformation techniques have also been studied for the production of different flavonoids as they are more genetically stable. The focus of the present review is the application of the tissue culture technology, type of medium and explants used for the production of flavonoids in different plant species.
In a search for biologically active phenolics, a hydroalcoholic extract from the hairy roots of Lactuca virosa was fractionated by chromatographical methods. The procedure led to the isolation of a substantial amount of 3,5-dicaffeoylquinic
acid (3,5-DCQA)—a potent free radical scavenger. An analytical RP-HPLC separation of the hydroalcoholic extract from the hairy
roots allowed identification of further hydroxycinnamates: caftaric acid (CTA), chlorogenic acid (5-CQA) and cichoric acid
(DCTA), as well as small amounts of unbound phenolic acids. A time course of growth and caffeic acid derivatives accumulation
in the hairy root culture was also investigated. The highest contents of the compounds in the examined roots were detected
at the logarithmic phase of growth. The average content of 3,5-DCQA in the roots (ca. 2.5% DW) was at least one order of magnitude
higher than that found in roots of Lactuca species and callus culture of L. virosa.
Plants of the genus Scutellaria (Family Lamiaceae) constitute one of the common components of Eastern as well as traditional American medicine. Skullcap is a North American perennial plant belonging to the genus Scutellaria. The genus is widespread in Northern Hemisphere represented by close to 400 species. Many species are rare, threatened, or endangered. Habitat destruction, urbanization, and poor seed set are the few reasons behind diminishing population of many skullcaps. Many skullcaps have showy, beautiful blooms with great potential as ornamental plants. Skullcap are used in alternative medicine as anti-inflammatory, antispasmodic, emmenagogue, nervine, sedative and strong tonic. We have developed a germplasm collection at Fort Valley State University and the populations are maintained in the greenhouse and through micropropagation. We have made significant headway in the areas of micropropagation, transformation for desired gene transfer and hairy root induction, extraction and HPLC analysis of targeted flavonoids, and clinical role of select flavonoids using glioma cell lines.
We investigated changes in physiological characteristics of micropropagated juvenile avocado (Persea americana Mill.) cultured in three different sucrose concentrations (5, 30 and 50 g/l) for 4 weeks and subsequently acclimatized to ex vitro conditions. No significant differences in foliar concentrations of total chlorophylls, Nmass, flavonoids and total soluble proteins were observed during in vitro growth or after acclimatization. However, ex vitro transfer exerted multiple effects on foliar ratios of chlorophyll a to b, total chlorophylls to carotenoids and stem N and C/N ratio. Both varying sucrose concentrations during in vitro growth and ex vitro transfer, affected the concentration of rubisco subunits independent of other photosynthetic components, viz. total chlorophylls, foliar N and total soluble proteins. These results suggest that preconditioning juvenile avocado microplants at different concentrations of sucrose modify several physiological parameters during ex vitro acclimatization, like concentrations of carotenoids and rubisco, which can improve vigour of plants in autotrophic conditions.
A simple and sensitive liquid chromatographic method has been established for determination of the main flavonoid components of Scutellaria baicalensis Georgi (Lamiaceae) roots and hairy-root cultures. Wogon, the dried root of the plant, is used in traditional Chinese medicine for treatment of bronchitis, hepatitis, tumors, and inflammatory diseases. Lyophilized hairy roots were extracted with methanol. The crude extracts were purified by SPE on Supelco LC-8 cartridges. HPLC separations were performed on a Eurospher 100-C8 reversed-phase column. The mobile phase was a gradient prepared from mixtures of acetonitrile and 0.1% trifluoroacetic acid. Peaks were identified by addition of standards and/or by diode-array detection. Baicalein 7-O-glucuronide (baicalin), wogonin 7-O-glucuronide (wogonoside), baicalein, wogonin, and acteoside were determined by the external standard method at 280 nm. We found that the aglycon (baicalein and wogonin) content of the transformed roots was consistently higher than that of the intact root from Siberia.
A transformed root culture of Cichorium intybus L. (Asteraceae) was found to produce sesquiterpene lactones of guaiane and germacrane type. Lactucopicrin, 8-desoxylactucin and three sesquiterpene lactone glycosides: crepidiaside B, sonchuside A and ixerisoside D were isolated from the roots. The yield of 8-desoxylactucin reached 0.03 g l(-1) at the early stationary phase of the culture.
Transformed root lines of Nicotiana species, containing NPTII and Gus genes, were used to study the parameters affecting the use of the Polymerase Chain Reaction as a routine analytical tool for quickly analysing plant transformants for the presence of a foreign gene. The basic reaction mix as described by Cetus Corporation (Saiki 1989) was close to optimal for successful PCR amplification of internal sequences of both NPTII and Gus from genomic plant DNA. The temperature of primer annealing in the PCR protocols was found to be the most important variable, as low temperatures caused amplification of artefact bands and smearing after analysis on ethidium bromide agarose gels. Various formulae for calculating the Tm for binding of primers of various lengths (20–30 bases) are described in relation to predicting suitable annealing temperatures in the PCR.
For tobacco species the PCR reaction worked efficiently with up to 2 μg of genomic DNA. However, with DNA from Mentha species (mint), an inhibitor of the PCR process was co-extracted with the DNA which prevented amplification of target sequences, if more than 10 ng of genomic DNA was present in the reaction.
SUMMARY A mutant of A. tumefaciens strain ~6~3, carrying the R factor RP4, was able to transfer its TI plasmid to various avirulent Agrobacterium strains and to a strain of Rhizobium. Strains carrying the TI(~6s3) plasmid were selected by their ability to utilize octopine. The isolates were able to induce tumours and exclude phage API . The tumours induced on Kalanchoe daigremontiana were rough and contained octopine. It was concluded that octopine utilization, phage exclusion, induction of rough tumours and synthesis of octopine in the tumours are determined by the TI(~6s3) plasmid. Tumorigenicity was determined by this plasmid both in Agro- bacteria and in Rhizobium, suggesting that all the genes necessary for tumour induction are plasmid-borne.
We have determined the nucleotide sequence of the Ri TL-DNA region from an Agrobacterium rhizogenes agropine-type plasmid using subcloned regions from the essentially identical Ri TL-DNAs from strains A4 and HRI. This sequenced region of 21,126 base pairs (bp) contains the complete TL-DNA region of the Ri plasmid as determined by analysis of TL-DNA borders in the genome of infected, clonal, Convolvulus arvensis plants. The left and right borders of the TL-DNA are flanked by 25-bp sequences which match the 25-bp terminal sequences found near the borders of T-DNA regions of Agrobacterium tumefaciens Ti plasmids. Other DNA sequences similar to these 25-bp terminal sequences are found within the TL region, and some of these sequences appear to be associated with Ri TL-DNA structures found in transformed tobacco plants. The TL-DNA region contains 18 open reading frames, many of which have 5' and 3' regulatory elements similar to those found in eukaryotic genes. In many cases, CCAAT and TATA elements were found upstream from putative transcriptional initiation codons, and poly(A) addition (AATAAA) elements were observed in presumed 3'-noncoding regions. Comparison of Ri TL-DNA coding and noncoding sequence regions with T-DNA sequence regions from octopine type Ti plasmid pTi15955 reveals no extensive sequence homologies.
The antioxidant effect of a trihydroxyflavone extract from Scutellaria baicalensis on oxidation induced by ultraviolet light, was studied with phosphatidylcholine liposome membrane. Also, as standards, the antioxidative activity of baicalin, wogonin, baicalein and butylated hydroxytoluene (BHT) was investigated. Comparison of the protective effects of the compounds studied against photoinduced lipid peroxidation in lecithin liposome membranes showed that: (1) the inhibitory effect of those compounds (at 1.2 mol% antioxidant content in liposomes) on TBA reactive materials from lipid peroxidation decreased in the order of baicalin > BHT approximately equal to Scutellaria baicalensis. These were found much greater than wogonin and baicalein; (2) the depressed effect of those compounds (at 1.1 mol% compounds content in liposomes) on the production of conjugated dienes (proportional to oxidation index) could be classified as follows: Scutellaria baicalensis approximately equal to baicalin > BHT, these three were found more active much greater than baicalein and wogonin. Results obtained by ESR measurement confirm that Scutellaria baicalensis extract and the BHT compound significantly depressed the effect of liposome oxidation. It was found that the new trihydroxyflavones of Scutellaria baicalensis, ensured a very satisfactory concentration-dependent protection of the liposome membrane against UV-induced oxidation. These findings suggest that some of the beneficial effects of the extract of the Scutellaria baicalensis can be mediated in certain diseases (for example in skin diseases) by their ability to scavenge free radicals and by their protective effect on lipid peroxidation caused by sunlight irradiation.
The genus Scutellaria belongs to the family Labiatae and subfam. Scutellarideae. Scutellaria is widely distributed all over the world except for South Africa, and there are about 300 species. The calyx of this genus is remarkably specialized to become two-lip-shaped, and characterized by the upper lip having a flat or dish-shaped upper surface on which a small swelling forms; the dish-shaped part peels off at fruit maturation to allow a seed to fall.
Micropropagation of plants is a multibillion dollar industry being practiced in hundreds of small and large nurseries and biotech labs throughout the world. Presently, it is the only component of plant biotechnology which has been commercially exploited on such a large scale. There are about 1000 plant species that can now be micropropagated (Murashige 1989). Micropropagation has been employed for about half a century, especially for ornamental plants (see Ammirato et al. 1989). Millions of plants are produced, sold locally, or exported to different countries (Chu and Kurtz 1989). Presently, interest has been extended to agricultural/plantation crops (e. g., potato, strawberry, oil palm, banana, etc.), medicinal and aromatic plants (Bajaj et al. 1988), and trees (see Bajaj 1986a, 1989a, 1991), (especially those which are difficult to propagate. Micropropagation of fruit and forest trees offers not only means for the mass multiplication of existing stocks of germplasm for biomass energy production, but also for the conservation of important, elite, and rare plants that are threatened with extinction.
A high yielding cell line of Scutellaria baicalensis G. has successfully been developed to produce flavonoids. Major components of the flavonoids were identified as baicalin and wogonin-7-O-glucuronic acid by a series of instrumental analyses using UV, IR, MASS, and NMR. After 12 days in suspension culture, the cell growth reached 14 g
DW/l, and baicalin and wogonin-7-O-glucuronic acid were obtained in concentrations of 2.9 g/l and 1.07 g/l, respectively. The culture temperature was found to be an important parameter for improving production yield of the flavonoids. The yield of baicalin was observed to increase to 4.2 g/l by shifting the temperature from 30 °C to 25 °C after 72 h of suspension culture.
Hairy root cultures of Scutellaria baicalensis transformed by Agrobacterium rhizogenes A13 harboring β-glucuronidase (GUS) genes were established and the flavonoid production in the tissues was determined. In six clones of the hairy roots, the contents of the glucuronide-type flavonoids were fairy low compared with those observed in the adventitious (non-transformed) roots of this plant. On the contrary, the content levels of the glucose-type flavonoids in the hairy roots were almost similar to those of the adventitious roots. The modified accumulation of the glucuronide-type flavonoids in the hairy roots was supposed to be caused by the expression of GUS genes integrated by Agrobacterium. This Agrobacterium-mediated transformation seemed to be useful for the biosynthetic study on successful modification of the flavonoid metabolism in this species.
We successfully established hairy root cultures of Scutellaria baicalensis by direct inoculation on sterile seedlings with Agrobacterium rhizogenes pRil5834 harbouring pBI121. Transformation was proved by direct detection of the inserted T-DNA by the polymerase chain reaction. To determine the optimal medium for growth of hairy roots, the effects of five basal media were investigated; growth was best in B5 liquid medium. A new flavone glucoside, 5,7,2′,6′-tetrahydroxyflavone 2′-O-β-d-glucopyranoside 15 known flavonoids and five known phenylethanoids were isolated from the hairy root cultures of S. baicalensis for the first time. Their structures were elucidated by various spectroscopic methods.
An intermediate vector, pAMNeo10, was constructed containing the replication origin and carbenicillin-resistance gene of pBR322, an homology region to allow insertion into the TL-DNA of pRiA4 in Agrobacterium A4T, and a chimaeric kanamycin-resistance gene (nop. neoΔ) for identification of TL-DNA::pAMNeo10 transformed roots. Roots produced by inoculating stem explants of Lycopersicon esculentum, L. hirsutum × L. esculentum (KNVF Rootstock) and L. peruvianum with an exconjugant stain, A4T (pRiA4::pAMNeo10), were resistant to kanamycin at levels that completely inhibit the growth of transformed roots produced with wild-type A4T. When transformed by the exconjugant strain, roots of the three tomato hosts were resistant to different levels of kanamycin, and, in the case of L. peruvianum, regenerated plants were tolerant to much higher levels (10×) of kanamycin than the transformed roots from which they were derived. Kanamycin-resistant transformed roots expressed aminoglycoside phosphotransferase activity, and Southern blotting confirmed the presence of the intermediate vector sequence in transformed roots and in shoots of regenerated plants. TR-DNA was shown to be present in most transformed roots and regenerated shoots by testing for agropine and mannopine. The application of Ri plasmid vectors to the study of foreign gene expression in plants is discussed.
To search for possible anti-tumor-promoters, fourteen flavones obtained from the root of Scutellaria baicalensis were examined for their inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation by a short-term in vitro assay. Among these flavones, 5,7,2'-trihydroxy- and 5,7,2',3'-tetrahydroxyflavone showed remarkable inhibitory effects on the EBV-EA activation, and the effect of the latter on Raji cell cycle was also examined by flow cytometer. These two flavones exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.
The nutrient requirements of suspension cultures from soybean root have been investigated, and a simple medium consisting of mineral salts, sucrose, vitamins and 2,4-dichlorophenoxyacetic acid (2,4-d) has been designed.The cells required thiamine, 2,4-d and ammonium in addition to the usual mineral salts and sucrose.Optimum concentrations of nitrate and ammonium were 25 and 2 mM respectively. The highest yield of cells was achieved at an initial pH of 4.5–5.5. During the growth cycle the pH gradually increased to 6.0–6.2.
We investigated effects of isoscutellarein-8-methylether (5,7,4'-trihydroxy-8-methoxyflavone, F36) from the roots of Scutellaria baicalensis on the single-cycle replication of mouse-adapted influenza viruses A/Guizhou/54/89 (H3N2 subtype) and B/Ibaraki/2/85 in Madin-Darby canine kidney (MDCK) cells. The agent suppressed replication of these viruses from 6 to 12 h after incubation in a dose-dependent manner by 50% at 20 microM and 90% at 40 microM, respectively. F36 (50 microM) reduced the release of B/Ibaraki virus in the medium by 90-93% when it was added to the MDCK cells at 0 to 4 h after incubation. The cell-associated virus determined by sialidase activity was also reduced by the treatment at 0 to 4 h. F36 (120 microM) inhibited the low pH-dependent membrane fusion of both the viruses with the liposome containing mixed gangliosides from bovine brain. However, the agent little affected the hemagglutination and RNA-dependent RNA polymerase activities of these viruses in vitro. These results suggest that F36 inhibits the replication of A/Guizhou and B/Ibaraki viruses at least partly by inhibiting the fusion of viral envelopes with the endosome/lysosome membrane which occurs at the early stage of the virus infection cycle. F36 (0.5 mg/kg) showed no antiviral activity against A/Guizhou and B/Ibaraki viruses in mice when administered intranasally 5 min prior to virus inoculation, whereas it significantly inhibited their proliferation in the mouse lung when administered intranasally 7 times (total 3.5 mg/kg) from 18 h before to 54 h after virus infection.
Studies on the effects of flavonoids isolated from the roots of Scutellaria baicalensis on the fibrinolytic system induced by trypsin in cultured human umbilical vein endothelial cells (HUVECs) showed that baicalein (1) strongly inhibited the reduction of t-PA production and the elevation of PAI-1 production induced by trypsin. The IC50 for PAI-1 production was 3.7 microM. In addition, wogonin (3), oroxylin A (5), skullcapflavone II (6), and 2',5,5',7-tetrahydroxy-6',8-dimethoxyflavone (7) inhibited the elevation of PAI-1 induced by trypsin, though less strongly; their IC50 were 105, 61, 110, and 88 microM, respectively. These findings suggest that baicalein prevents the thrombotic tendency induced by trypsin.
A benzodiazepine binding assay directed separation led to the identification of 3 flavones baicalein (1), oroxylin A (2), and skullcapflavone II (3) from the water extract of Scutellaria baicalensis root. Compounds 1, 2, and 3 interacted with the benzodiazepine binding site of GABAA receptors with a Ki value of 13.1, 14.6 and 0.36 micromol/L, respectively.
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