Comparative analysis of flavonoids and polar metabolites from hairy roots of Scutellaria baicalensis and Scutellaria lateriflora

Baicalin, baicalein, and wogonin were accumulated in hairy roots derived from Scutellaria lateriflora and Scutellaria baicalensis. The levels of baicalein and baicalin were 6.8 and 5.0 times higher, respectively, in S. baicalensis than in S. lateriflora. A total of 47 metabolites were detected and identified in Scutellaria species by GC-TOF MS. The metabolites from the two species were subjected to principal component analysis (PCA) to evaluate differences. PCA fully distinguished between the two species. The results showed that individual phenolic acids and phenylalanine, precursors for the phenylpropanoid biosynthetic pathway, were higher in S. baicalensis than in S. lateriflora. This GC-TOF MS-based metabolic profiling approach was a viable alternative method to differentiate metabolic profiles between species.     

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.     

Deep Sequencing of the Scutellaria baicalensis Georgi Transcriptome Reveals Flavonoid Biosynthetic Profiling and Organ-Specific Gene Expression

Scutellaria baicalensis Georgi has long been used in traditional medicine to treat various such widely varying diseases and has been listed in the Chinese Pharmacopeia, the Japanese Pharmacopeia, the Korean Pharmacopoeia and the European Pharmacopoeia. Flavonoids, especially wogonin, wogonoside, baicalin, and baicalein, are its main functional ingredients with various pharmacological activities. Although pharmaological studies for these flavonoid components have been well conducted, the molecular mechanism of their biosynthesis remains unclear in S. baicalensis. In this study, Illumina/Solexa deep sequencing generated more than 91 million paired-end reads and 49,507 unigenes from S. baicalensis roots, stems, leaves and flowers. More than 70% unigenes were annotated in at least one of the five public databases and 13,627 unigenes were assigned to 3,810 KEGG genes involved in 579 different pathways. 54 unigenes that encode 12 key enzymes involved in the pathway of flavonoid biosynthesis were discovered. One baicalinase and three baicalein 7-O-glucuronosyltransferases genes potentially involved in the transformation between baicalin/wogonoside and baicalein/wogonin were identified. Four candidate 6-hydroxylase genes for the formation of baicalin/baicalein and one candidate 8-O-methyltransferase gene for the biosynthesis of wogonoside/wogonin were also recognized. Our results further support the conclusion that, in S. baicalensis, 3,5,7-trihydroxyflavone was the precursor of the four above compounds. Then, the differential expression models and simple sequence repeats associated with these genes were carefully analyzed. All of these results not only enrich the gene resource but also benefit research into the molecular genetics and functional genomics in S. baicalensis.     

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.     

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.     

Liquid chromatography with tandem mass spectrometry for the simultaneous determination of baicalein, baicalin, oroxylin A and wogonin in rat plasma

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of baicalein, baicalin, oroxylin A and wogonin, Scutellaria baicalensis active components in rat plasma was developed. After liquid-liquid extraction with 2-(3,4-dimethoxy-phenyl)-5,7-dihydroxy-chromen-4-one as internal standard, baicalein, baicalin, oroxylin A and wogonin were eluted from an Atlantis C(18) column within 7 min with isocratic mobile phase consisting of methanol and 0.1% formic acid (60:40, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. The standard curves were linear (r=1.000) over the concentration ranges of 5-500 ng/ml for baicalein, wogonin and oroxylin A and 5-5000 ng/ml for baicalin. The coefficients of variation and relative errors of baicalein, wogonin, oroxylin A and baicalin for intra- and inter-assay at three or four quality control (QC) levels were 0.8-6.1% and -4.0 to 5.8%, respectively. The lower limits of quantification for baicalein, wogonin, oroxylin A and baicalin were 5ng/ml using 50 microl of plasma sample. This method was successfully applied to the pharmacokinetic study of baicalein, baicalin, wogonin and oroxylin A after an intravenous administration of Scutellariae radix extract to male Sprague-Dawley rats.     

Flavone-catalyzed apoptosis in Scutellaria baicalensis

In response to mechanical damage, roots of Scutellaria baicalensis undergo cell death within 24 h. The flavone baicalein was identified as the factor regulating apoptosis in the damaged roots of S. baicalensis. Plant apoptosis is known to be triggered by oxidative damage of DNA through oxidative bursts, whereas baicalein causes apoptosis in Scutellaria cells by a copper-dependent oxidation of nuclear DNA without inducing an oxidative burst. S. baicalensis possesses an interesting system for quickly producing this apoptosis-inducing flavone in its cells. Intact Scutellaria cells contain little baicalein but store a large amount of baicalin (baicalein 7-O-β-D-glucuronide). Stress treatment of Scutellaria cells immediately initiates hydrolysis of baicalin by endogenous β-glucuronidase, and the resulting baicalein is immediately translocated to the nucleus, leading to apoptosis. Thus, S. baicalensis possesses a unique apoptosis-inducing system that is linked with metabolism of baicalin.     

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.     

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.     

Selective fraction of Scutellaria baicalensis and its chemopreventive effects on MCF-7 human breast cancer cells

Based on our previous observation, the whole Scutellaria baicalensis extract (SbE) did not show significant breast cancer cell inhibitory effect. In this study, we isolated a baicalin-deprived-fraction (SbF1) of Scutellaria baicalensis, and baicalin-fraction (SbF3), and evaluated their anti-breast cancer properties using MCF-7 cells. The content of four flavonoids in extract/fractions were determined using high performance liquid chromatography. Analytical data showed that in SbF1, the major constituents are baicalein and wogonin, while SbF3 only contains baicalin. The antiproliferative effects of fractions and SbE were assayed using modified trichrome stain method. SbF1 showed significant antiproliferative effect. Treated with 100 μg/ml of SbF1 for 72 h inhibited MCF-7 cell growth by 81.6%, while in the same treatment concentration, SbF3 increased cell growth by 22.6%. SbF1 was recognized as an active fraction of SbE. The effects of four flavonoids in SbE, scutellarin, baicalin, baicalein and wogonin, were determined, and data showed that baicalein and wogonin significantly inhibited MCF-7 cell growth. In contrast, in certain concentrations, scutellarin and baicalin increased cancer cell growth. The effects of SbF1 on cell cycle and apoptosis were assayed using flow cytometry. SbF1 arrested MCF-7 cells in S- and G2/M-phases, and significantly increased induction of cell apoptosis. These combined phytochemical and biological data provide evidence for further chemopreventive studies of the baicalin-deprived SbE on breast cancer.     

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.     

Baicalin,baicalein and wogonin inhibits high glucose-induced vascular inflammation in vitro and in vivo

Vascular inflammatory process has been suggested to play a key role in initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Thus, in this study, we attempted to determine whether three structurally related polyphenols found in the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, can suppress vascular inflammatory processes induced by high glucose (HG) in human umbilical vein endothelial cells (HUVECs) and mice. Data showed that HG induced markedly increased vascular permeability, monocyte adhesion, expressions of cell adhesion molecules (CAMs), formation of reactive oxygen species (ROS) and activation of nuclear factor (NF)-κB. Remarkably, all of the above mentioned vascular inflammatory effects of HG were attenuated by pretreatment with baicalin, baicalein, and wogonin. Vascular inflammatory responses induced by HG are critical events underlying development of various diabetic complications, therefore, our results suggest that baicalin, baicalein, and wogonin may have significant therapeutic benefits against diabetic complications and atherosclerosis. [BMB Reports 2015; 48(9): 519-524]     

Enhanced production of flavonolignans in hairy root cultures of Silybum marianum by over-expression of chalcone synthase gene

In the present study, metabolic engineering approach was used through over-expressing the Petunia chalcone synthase (chsA) gene in order to enhance the silymarin production level in the hairy root cultures of Silybum marianum. Molecular analysis confirmed the presence and integration of chsA transgene in transgenic hairy roots. Chemical analysis indicated that the over-expression of chsA gene enhanced the silymarin production level in the transgenic line as much as 7-folds than the non-transgenic hairy roots. Moreover, the silybin content, the main active component of silymarin, was proved to be 10 times higher in transgenic hairy roots than those of the non-transgenic ones. Therefore, the over-expression of petunia chsA gene in S. marianum hairy roots did not result in gene silencing, but led to an enhanced biosynthesis of the flavonolignans.