Scutellaria baicalensis, the golden herb from the garden of Chinese medicinal plants

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.


Introduction
Scutellaria baicalensis Georgi is a species of flowering plant in the Lamiaceae family (Fig. 1a). It is indigenous to several East Asian countries and the Russian Federation and has been cultivated in many European countries [1,2]. Chinese people have used the dried root of this medicinal plant for more than 2000 years as a traditional medicine known as Huang-Qin (Fig. 1b) and it is now listed officially in the Chinese Pharmacopoeia. The dried root of Huang-Qin is often prepared by decoction (boiling) or as tinctures [3]. Huang (黄) means yellow. Qin (芩) is equivalent to Jin (菳), and means golden herb, as explained in Shuowen Jiezi, an early 2nd-century Chinese dictionary from the Han Dynasty [4,5]. Huang-Qin was first recorded in Shennong Bencaojing (The Classic of Herbal Medicine), written between about 200 and 250 AD, for treatment of bitter, cold, lung and liver problems [6]. The most authoritative book on traditional Chinese medicine, Bencao Gangmu (Compendium of Materia Medica) which was first published in 1593, reported that Scutellaria baicalensis (Fig. 1c) had been used in the treatment of diarrhea, dysentery, hypertension, hemorrhaging, insomnia, inflammation and respiratory infections. Its author, Li Shizhen, reported successful self-administration to treat a severe lung infection when he was 20 years old [4].

Clinical applications
Scutellaria baicalensis has been used as a medicine in several East Asian countries for more than 2000 years. Clinical data for this herb are accumulating and Huang-Qin alone has been reported to be useful for treating colds and bacterial pneumonia [7,8].
In many Eastern countries, Huang-Qin is prescribed as a part of a multi-herb formulation. Huang-Qin is an important ingredient of Xiaochai Hutang (Chinese) or Sho-saikoto (SST, Japanese) preparations, first described in Shanghan Lun (On Cold Damage), written by Zhang Zhongjing around 200 AD [9]. This formulation was described as having 'worked effectively in some instances where conventional Western therapies failed or proved to be insufficient to provide a palliative cure' by Xue and Roy in 2003 [10] and was subsequently taken up by the alternative medicine community in the USA [11]. A study of the effects of SST on hepatitis was reported by a Japanese group in 1994 [12]. Ninety-eight hepatitis patients were treated with SST and followed up for 5 years. Liver function was improved in 78 % of the hepatitis B patients and in 67 % patients with non-A non-B type hepatitis, with significantly reduced serum levels of aminotransferase AST, ALT, and rGTP [12]. SST is also effective in hepatitis C patients. Eighty hepatitis C patients who were interferon-resistant were treated with SST combined with a common unspecified medicine or the common medicine alone. These patients were studied for 7 years during which time, 5 patients on the SST treatment achieved fully normalized enzyme functions. Liver enzyme normalization was observed in only one control patient. Conversely, 5 control patients (common medicine alone) progressed to liver cancer compared to just one on the SST combination therapy [13].
Lung Fufang, another traditional prescription using Huang-Qin, can prolong the survival rate of patients with primary bronchial pulmonary squamous cell carcinoma [14], and it has a similar effect on NSCLC (non-small-cell lung cancer) patients. Elderly people suffering from NSCLC and treated with Lung Fufang Prescription showed improved indices for the clinical syndrome and improved quality of life compared to the control group who were treated with normal chemotherapy plus a TCM (Traditional Chinese Medicine) placebo [15]. Huang-Qin is also a major ingredient of Fuzheng anti-cancer prescription, which has been used in combination with chemotherapy and shown to have improved outcomes on NSCLC in middle and late stage patients, compared to conventional chemotherapy alone [16].

Antitumor effects
Many studies have shown that S. baicalensis extract is cytotoxic to a broad range of cancer cells from humans, including brain tumor cells [17], prostate cancer cells [18] and HNSCC (head and neck squamous cell carcinoma) cell lines [19]. Aqueous extracts of S. baicalensis roots induced apoptosis and therefore suppressed growth of lymphoma and myeloma cell lines, by changing the expression levels of Bcl genes, increasing cyclin-dependent kinase inhibitor p27 (KIP1) activity and decreasing expression of the c-myc oncogene [20]. Similarly, S. baicalensis extracts were selectively toxic to several human lung cancer cell lines, but not to normal human lung fibroblasts. Increases in p53 and Bax protein activities may be responsible for these effects [21].
The flavones baicalin, wogonoside and their aglycones baicalein and wogonin are the major bioactives in Scutellaria roots and the major bioactive constituents responsible for anti-cancer effects of Huang-Qin  induced DNA damage and resulted in cell death. Despite this genotoxic effect, baicalein did not induce mutations, a major problem of conventional anticancer drugs, suggesting that baicalein and related flavones are strong candidates for improved chemotherapeutic agents [28].

Hepatoprotection
Scutellaria baicalensis is the main component in the herbal remedy SST used for liver problems such as hepatitis, hepatic fibrosis and carcinoma [11,29,30]. Yang-Gan-Wan (YGW) is another prescription containing baicalin, which has long been known for its protective effects on the liver [31,32]. This herbal prescription prevents and reverses activation of hepatic stellate cells, (HSC; the major pathogenic cell type in fibrogenesis) by epigenetic derepression of PPARγ (Peroxisomal proliferator-activated receptor γ), so preventing liver fibrosis. Baicalin is a major active phytocompound in Yang-Gan-Wan (YGW) and suppresses the expression and signaling by canonical Wnts, which are involved in epigenetic repression of PPARγ [33].
Several studies have suggested that S. baicalensis can effectively inhibit fibrosis and lipid peroxidation in rat liver [34][35][36]. Consumption of the roots and shoots of S. baicalensis inhibits mutagenisis caused by the aflatoxin-B1 mycotoxin in rat liver cells [35]. The anti-fibrosis activity of S. baicalensis root extracts may be due to enhanced phosphorylation of the cAMP response element binding protein as proposed by Tan et al. [37], although extracts of Scutellaria baicalensis roots also arrest the cell cycle, activate the caspase system and activate ERK-p53 pathways resulting in apoptosis of HSC-T6 cells to prevent hepatic fibrosis [38].

Antibacterial and antiviral activities
Amongst 46 herb and spice extracts, S. baicalensis extracts have shown substantial antibacterial effects against Bacillus cereus, Escherichia coli, Listeria monocytogenes, Salmonella anatum and Staphylococcus aureus [39]. Aqueous extracts of S. baicalensis roots have antimycotic properties against Aspergillus fumigatus, Candida albicans, Geotrichum candidum and Rhodotorula rubra [40]. Baicalin, isolated from S. baicalensis, has been applied as a natural antibacterial agent against foodborne pathogens such as Salmonella and Staphylococcus spp. in homemade mayonnaise [41]. Extracts of S. baicalensis can also enhance the antimicrobial activity of several antibiotics such as ciprofloxacin, ceftriaxone, gentamicin and penicillin G, against Staphylococcus aureus [42].
Xiaochai Hutang or Sho-saiko-to (SST) is effective against hepatitis, and a reduction of viral load has been observed in some patients treated with SST [11], indicating an antiviral function of Scutellaria extracts [43]. Scutellaria root extracts can inhibit the replication of HCV-RNA significantly [44].
Baicalin has very good anti-HIV-1 activity as a nonnucleoside reverse transcriptase inhibitor [45]. Moreover, baicalin can prevent the entry of HIV-1 into animal cells by perturbing the interaction between HIV-1 Env and HIV-1 co-receptors on the cell surface [46]. Baicalin has been adopted as one of the popular lead natural products for preventing HIV infection [47]. Differences in the inhibitory activities of baicalein and baicalin against HIV-1 reverse transcriptase have been evaluated by Zhao et al. [48]. They found that baicalein has four times stronger inhibitory activity on HIV-1 reverse transcriptase than baicalin. However, baicalin can be deglycosylated to form baicalein in the human body [48].
Aqueous extracts of S. baicalensis elicit significant inhibition (91.1 %) of HIV-1 protease activity at concentrations of 200 g/ml [49]. Early in 1989, Ono et al. [50] reported baicalein could effectively inhibit reverse transcriptase activity of human immunodeficiency virus (HIV); 2 μg/mL baicalein inhibiting 90 % of the activity of HIV reverse transcriptases [50]. Baicalein is also an inhibitor of HIV-1 integrase, an essential enzyme in the life cycle of the virus, by binding to the hydrophobic region of the HIV-1 integrase catalytic core domain to induce a conformational change [51]. These effects of baicalein and baicalin on HIV have attracted considerable attention [52].

Other effects
In addition to the effects described above, preparations of S. baicalensis can also work as antioxidants, ROS scavengers [53,54] and anticonvulsants [55]. Recently, the neuroprotective effects of S. baicalensis and its component flavones, have been studied using both in vitro and in vivo models of neurodegenerative diseases. Results suggest that this medicinal plant may have promising applications in neuroprotection [56,57].

Biotechnology to enhance S. baicalensis synthesis
Given their established bioactivity, the possibility to enhance production of the flavones in this plant or alternatively produce them in common vegetables or fruits is attractive [58,59]. Understanding the regulation of production of bioactive flavones (baicalein, baibalin, wogonin and wogonoside) and their biosynthesis in S. baicalensis, and developing strategies to enhance their production are important objectives. However, like other members of the mint family, stable genetic transformation and regeneration of this plant are very difficult. Agrobacterium rhizogenes-mediated production of hairy roots of S. baicalensis has proved to be effective in this recalcitrant species [60,61] (Fig. 2). Hairy roots can be induced from either leaf or cotyledon explants [62,63] in an A. rhizogenes strain-dependant manner. Among the four strains (A4GUS, R1000 LBA 9402 and ATCC11325) tested by Tiwari et al.(2008), the A4 stain produced the most hairy roots, with an efficiency of 42.6 % [60]. 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).
Next-generation sequencing technologies have been employed to screen for candidate genes that may be responsible for biosynthesis of the flavones, and several structural genes including 6-hydroxylase, 8-O-methyltransferase, 7-O-glucuronosyltransferases have been suggested to be involved in their biosynthesis [69]. Yuan et al. [70,71] also screened RNA-sequencing databases and found that several MYB genes may be responsible for regulation of production of its flavonoids.
Flavones are present in aerial tissues of many flowering plants, with roles in co-pigmentation of flowers (they make anthoyanin pigments appear bluer) and in protection against UV irradiation [77,78]. Flavones are synthesized by the flavonoid pathway, which is part of phenylpropanoid metabolism [79,80]. Naringenin is a central intermediate in normal flavone biosynthesis [81] exemplified by the production of the flavones, scutellarin and scutellarein, derived from naringenin in the aerial parts (leaves and flowers) of Scutellaria baicalensis. Scutellarein and scutellarin are synthesised from phenylalanine by general phenyl propanoid metabolism; phenylalanine ammonia lyase (PAL), cinnamoyl 4 hydroxylase (C4H) and p-coumaroyl CoA ligase (4CL) followed by chalcone synthase (CHS) and chalcone isomerase (CHI) to form naringenin [82]. A flavone synthase (FNSII-1) then oxidises naringenin to form apigenin, which may be further hydroxylated, methylated and glycosylated to form scutellarein and scutellarin (Fig. 3a). Scutellaria roots however accumulate large amounts of specialized rootspecific flavones (RSFs), lacking a 4′-OH group on their B-rings (Fig. 3b) [83]. These RSFs, which include baicalein and wogonin, and their glycosides, are not synthesized from naringenin, but by an alternative pathway where cinnamic-acid is recruited by a specially-evolved cimmamoyl-CoA ligase (SbCLL-7) to form cinnamoyl CoA which is then condensed with malonyl CoA by a specialised isoform of chalcone synthase (SbCHS-2) to form a chalcone, which is then isomerized by the same chalcone isomerase (CHI) that acts in scutellarin biosynthesis, to form pinocembrin, a flavanone without a 4′-OH group. Pinocembrin is converted by a specialised isoform of flavone synthase (FNSII-2), to form chrysin, which serves as the founding 4′ deoxyflavone which may be decorated  (Fig. 4).
The evolution of this specialised pathway for 4′ deoxyflavone biosynthesis occurred relatively recently, following the divergence of the Laminaceae [64] and may have been facilitated by the recruitment of a CoA ligase activity from a gene encoding an enzyme of fatty acid metabolism, that is specific for cinnamate. Effective competition for cinnamate in the face of high level expression of C4H may have paved the way for effective production of 4′-deoxyflavones in roots of S. baicalensis. Production of 4′-deoxyRSFs in roots is induced by methyl jasmonate treatment, suggesting that RSFs are made as part of a defence mechanism or for plant-microbe signalling [85,86]. Understanding the regulation of this newlyevolved pathway may facilitate engineering of biosynthesis of these important bioactive metabolites. Their roles in defence in Scutellaria may also underpin some of their uses in traditional medicine, for example as anti-microbials.
The bioactive compounds baicalein, wogonin and their glysosides can be found in many species from the genus Scutellaria other than S. baicalensis [87]. As in traditional Chinese medicine, the roots of S. amoena and S. likiangensis have been used commonly as alternatives to S. baicalensis. To date, 4′-deoxyflavones have been found only in Oroxylum indicum vent [88] and Plantago major L. outside the genus Scutellaria but in the order Lamiales [89]. 4′-Deoxyflavones have also been reported in Anodendron affine and Cephalocereus senilis outside the order Lamiales [90,91]. The evolution of metabolic pathways  determining the taxa-specific distribution of these 4′deoxyflavones is fascinating, and we suspect that convergent evolution has most likely influenced the development of metabolic pathways responsible for producing these specialised bioactive flavones in widely diverged plant species [92,93].