A comprehensive review on phytochemistry, pharmacology, and flavonoid biosynthesis of Scutellaria baicalensis

Abstract Context:Scutellaria baicalensis Georgi (Lamiaceae) is a popular medicinal plant. Its roots are used as the famous traditional Chinese medicine Huang-Qin, which is recorded in Chinese Pharmacopoeia, European Pharmacopoeia, and British Pharmacopoeia. Objective: This review comprehensively summarizes research progress in phytochemistry, pharmacology, and flavonoid biosynthesis of S. baicalensis. Methods: English and Chinese literature from 1973 to March 2018 was collected from databases including Web of Science, SciFinder, PubMed, Elsevier, Baidu Scholar (Chinese), and CNKI (Chinese). Scutellaria baicalensis, chemical constituents, phytochemistry, biological activities, and biosynthesis were used as the key words. Results: A total of 126 small molecules (1–126) and 6 polysaccharides have been isolated from S. baicalensis. The small molecules can be classified into four structural types, namely, free flavonoids, flavonoid glycosides, phenylethanoid glycosides, and other small molecules. Extracts of S. baicalensis and its major chemical constituents have been reported to possess anti-viral, anti-tumor, anti-bacterial, antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective activities. Key steps in the biosynthetic pathways of Scutellaria flavonoids have also been summarized. Conclusions: This article could be helpful for researchers who are interested in the chemical constituents, bioactivities, biosynthesis, and clinical applications of S. baicalensis.


Introduction
The plants of genus Scutellaria L. (Lamiaceae) are perennial herbs with around 360 species in the world. Many of these species have medicinal uses (Cantor et al. 2009;Shang et al. 2010;Paton et al. 2016). Among them, the roots of Scutellaria baicalensis Georgi are used in China as Huang-Qin (Scutellariae Radix), one of the most popular traditional Chinese medicines ( Figure 1). Scutellaria baicalensis is widely distributed in North China, Japan, Korea, Mongolia, and Russia (Zhao et al. 2016a;Jiang et al. 2017). Due to its increasing demands in recent years, it is now cultivated on a large scale in Shandong, Hebei, Inner Mongonia, Shanxi, and Gansu provinces of China . It should be noted that the herb of an allied species, Scutellaria barbata D. Don, is used as the Chinese medicine Ban-Zhi-Lian.
In China, S. baicalensis has a medicinal history of at least 2000 years. Huang-Qin was first recorded in Shennong's Classic of Materia Medica (Shennong Bencao Jing in Chinese) in around 200 AD. In ancient Chinese language, the character 'Qin' means 'herb for hemostasis', and 'Huang' means yellow color . The Traditional Chinese Medicine (TCM) theory considers Huang-Qin has the functions of clearing heat, eliminating dampness, purging fire, detoxification, hemostasis, and preventing miscarriage. Huang-Qin is now listed officially in Chinese Pharmacopoeia (2015), European Pharmacopoeia (EP 9.0), and British Pharmacopoeia (BP 2018). It is the key component herb for many famous Chinese medicine patent drugs, such as Gegen Qinlian Pills (to treat diarrhea, dysentery, fever, and influenza), Lanqin Oral Liquid (to treat sore throat), Yinzhihuang Granules (to treat jaundice and hepatitis), and Xiongdan Huangqin Eye Drops (to treat conjunctivitis). Flavonoids are the major bioactive chemical constituents of Huang-Qin. Among them, baicalin has been developed into a new drug (Huangqingan Tablets, manufactured by a number of companies including Shanghai Hutchison Pharmaceuticals and Jingfukang Pharmaceutical Group Co. Ltd), and is used to treat acute and chronic hepatitis. The total flavonoids extract of the stems and leaves of S. baicalensis has also been developed into a new drug (Huangqin Jingye Jiedu Capsules), and is mainly used to treat sore throat.
Despite the popular clinical use of Huang-Qin, scientific evidences are not adequate to identify the effective chemical components responsible for the versatile biological activities. The quality control of Huang-Qin crude drugs and related patent drugs still needs to be improved, and the medicinal potential of many bioactive compounds of this plant has yet to be explored. A comprehensive review of S. baicalensis could be helpful for researchers, manufacturers, and policymakers to obtain a holistic view of this important herbal medicine.
Several review articles are available on the Scutellaria genus or S. baicalensis (Shang et al. 2010;Zhang et al. 2014;Zhao et al. 2016a;Karimov and Botirov 2017;Cheng et al. 2018). As an increasingly popular herbal medicine, important research progress has been made in recent years. Herein, we comprehensively summarized research literature on phytochemistry, pharmacology, and flavonoid biosynthesis of S. baicalensis. English and Chinese literature published during 1973 to March 2018 was collected from databases including Web of Science, PubMed, Elsevier, SciFinder, Baidu Scholar (Chinese), and CNKI (Chinese). Scutellaria baicalensis, chemical constituents, phytochemistry, biological activities, and biosynthesis were used as the key words.

Phytochemistry
To date, a total of 126 small molecule compounds (1-126) and 6 polysaccharides have been isolated from S. baicalensis Georgi ( Figure 2; Table 1). Most of these compounds were obtained from the roots (the Chinese medicine Huang-Qin). A few research groups studied chemical constituents of the aerial part (Ma 2013;Wang HW et al. 2016) and the hairy root cultures (Zhou et al. 1997). The small molecules can be classified into four structure types, i.e., free flavonoids, flavonoid glycosides, phenylethanoid glycosides, and other small molecules. Among them, flavonoids and their glycosides are the major compounds.
While glucuronides are not as prevalent as glucosides in plant secondary metabolites, S. baicalensis contains at least 17 O-glucuronides. Baicalin (74) and wogonoside (76) are the most abundant ones. For majority of these compounds, the glucuronyl group is linked to 7-OH, except for 79 (8-OH) and 80 (2 0 -OH).
The first two C-glycosides were reported from S. baicalensis in 1994 (Miyaichi and Tomimori 1994). Up to now, 11 C-glycosides have been isolated from this plant. Most of them are glycosides of chrysin, though it is not the most abundant free flavonoid in S. baicalensis. Aside from two mono-C-glucosides, majority of the other compounds are 6,8-di-C-glycosides, containing one glucosyl residue and one arabinosyl residue. Interestingly, the arabinosyl residue in these compounds occurred as both furano-and pyrano-forms, and in different configurations (a-L, b-L). Their structures were mainly determined by NMR spectroscopic analysis. Unlike O-glycosides, the sugar residues are not easily hydrolyzed to identify their forms and stereo-configurations. Structures for some C-glycosides need to be further confirmed.

Phenylethanoid glycosides (101-108)
A total of nine phenylethanoid glycosides have been reported from S. baicalensis. The aglycones are usually conjugated with a glucosyl group, which are further substituted with a rhamnosyl residue (Rha), or acylated with a caffeoyl (Caf) or feruloyl (Fer) group.

Qualitative and quantitative analyses
With the rapid development of mass spectrometry techniques, liquid chromatography coupled with mass spectrometry (LC/MS) has been widely used to characterize the chemical constituents in herbal extracts. A number of reports are available on chemical analysis of S. baicalensis to characterize tens of compounds within 1 h (Han et al. 2007;Liu GZ et al. 2009). Wang et al. (2013) depleted high-abundance flavonoids from an ethanol extract of S. baicalensis, and characterized 117 low-abundance compounds by LC/MS. Recently, our group established a targeted post-acquisition data processing strategy, key ion filtering (KIF), and tentatively characterized 132 compounds in Huang-Qin by ultra-high performance liquid chromatography coupled with hybrid quadrupole orbitrap mass spectrometry analysis (UHPLC/Orbitrap-MS) ). Among these compounds, 59 were reported in this herb for the first time.
The contents of bioactive compounds are critically important for quality control of herbal medicines. Chinese Pharmacopoeia requires the content of baicalin in Huang-Qin should be no less than 9% (Chinese Pharmacopoeia Commission 2015). A number of HPLC methods have been developed to determine the contents of baicalin and other bioactive compounds in Huang-Qin (Xie et al. 2002;Zg orka and Hajnos 2003;Horvath et al. 2005;Islam et al. 2012). We developed a simple and rapid UPLC/UV method, and simultaneously determined the contents of 12  Delphinidin Patuletin 7

Pharmacological activities of extracts and major compounds
In China, Huang-Qin is widely used for the treatment of influenza, pneumonia, dysentery, and cancer. A large number of investigations have been reported on the pharmacological activities of different extracts of S. baicalensis (including water extract, methanol extract, and ethanol extract) and its major compounds such as baicalin, baicalein, and wogonin. These results were reported by different research groups, the investigations were conducted using different experimental models, and thus the results were difficult to be compared or summarized. Recently, our group isolated 28 compounds from this herb, and evaluated their anti-H1N1 viral, cytotoxic, and Nrf2 activation activities (Ji et al. 2015). The results indicated that free flavones were more potent than the other types as anti-influenza, cytotoxic, and antioxidative compounds of S. baicalensis ( Figure 4). They may be key players in the clinical therapeutic effects of Huang-Qin.
In this section, we summarize literature reports on the antitumor, anti-viral, anti-microbial, anti-inflammatory, antioxidative, neuroprotective, and hepatoprotective activities of extracts and compounds of S. baicalensis, as well as their effects on cardiovascular and cerebrovascular diseases, and bones.

Anti-tumor activities
Scutellaria baicalensis extracts and compounds have been reported to show a wide spectrum of anti-tumor activities, both in vitro and in vivo (Table 2). These activities involve liver cancer, gastric cancer, lung cancer, breast cancer, prostate cancer, bladder cancer, brain cancer, squamous cell carcinoma, mucoepidermoid carcinoma, colorectal cancer, gallbladder carcinoma, oral cancer, leukemia, lymphoma, and myeloma.
The extracts of S. baicalensis could inhibit the proliferation of human myeloma, lung cancer, liver cancer, and prostate cancer cells in vitro, and suppress tumor growth in bladder, prostate, lung and head/neck squamous xenograft tumor models. In the head/neck squamous cell carcinoma (HNSCC) murine model, oral administration of a water extract (75 mg/kg, 5 times/week for 7 weeks) led to 66% reduction of xenograft tumor (Zhang et al. 2003). The anti-cancer activities of S. baicalensis could be related with its inhibitory effects on PGE 2 (prostaglandin E2) production via suppression of COX-2 (cyclooxygenase-2) expression and arachidonic acid release from cell membranes. The total free flavonoid extract (100 mg/kg for 30 d, p.o.) could also significantly reduce tumor size by 25.5% in A549 human lung cancer xenografted mice, via induction of growth arrest in S phase and inhibition of DNA synthesis .
Wogonin (10 mg/kg for 4 weeks, p.o.) could inhibit tumor growth of T47D and MDAMB-231 breast cancer xenografts by up to 88% without significant toxicity in athymic nude mice (Chung et al. 2008). The mechanism could be downregulation of the Aktdependent canonical Wnt signaling pathway and p27 kip pathway. Wogonin could also act as CDK (cyclin-dependent kinase) inhibitors to potentiate the activities of anti-tumor drugs, such as the Bcl-2 (B-cell lymphoma 2) family inhibitor ABT-263. The combination of wogonin (50 mg/kg for 10 d, i.p.) and ABT-263 remarkably promoted tumor regression in human T-cell leukemia xenografted mice, but wogonin did not exhibit significant effects when used alone (Polier et al. 2015).
Wogonoside exerted anti-proliferative properties, suppressing tumor growth by 41% and prolonging survival durations up to 2.3fold, in a U937 leucocythemia xenograft murine model (80 mg/kg/ 2 d for 14 d, i.p.) ). The anti-tumor effect of wogonoside was related to cell cycle arrest and differentiation via inhibition of PLSCR1 (phospholipid scramblase 1) expression and regulation of subcellular localization in the nucleus.
PHY906 is an herbal preparation derived from the traditional Chinese medicine formula Huang-Qin Decoction, a four-herb formula with Huang-Qin as the key component ). PHY906 could enhance the anti-tumor activities of sorafenib against HepG2 tumor both in vivo and in vitro. Among the four component herbs, S. baicalensis played an important role in increasing tumor apoptosis by multiple mechanisms targeting on the inflammatory state of microenvironment of tumor tissue (Lam et al. 2015). PHY906 could also decrease gastrointestinal toxicity caused by the chemotherapeutic drug irinotecan. In a murine MCA-38 allograft model, PHY906 remarkably increased the anti-tumor activities of irinotecan and decreased weight loss (Lam et al. 2010).
Baicalein (480 mg/kg for 4 d, p.o.) showed significant effects in preventing death, prolonging survival time, inhibiting lung consolidation, and reducing the viral titers in the lung in BALB/ c mice infected with the influenza A/FM1/1/47 (H1N1) virus. The effects were comparable to lamivudine. The mechanism could be inhibition of neuraminidase activity and modulation of the immune system (Xu et al. 2010  baicalein (400 mg/kg for 5 d, p.o.) and ribavirin (50 mg/kg) provided a higher survival rate and lower body weight loss than either treatment alone in ICR mice infected with H1N1 virus (protection rates, 100% vs 20% and 50%) ). Wogonin could suppress HBV antigen secretion with an IC 50 of 4 lg/mL for both HBsAg and HBeAg in the human HBVtransfected liver cell line HepG2.2.15, and was more potent than lamivudine. In vivo, wogonin (i.v. for 10 d) could reduce plasma duck hepatitis B virus (DHBV) DNA level in the liver of DHBVinfected ducks with an ED 50 of 5 mg/kg, via inhibition of DHBV DNA polymerase and thus reducing the relaxed circular and linear forms of DHBV DNA . 5,7,4 0 -Trihydroxy-8-methoxyflavone (21, 50lM) could remarkably inhibit influenza virus A/PR/8/34 (APR8) by reducing the replication of APR8 in MDCK cells, through inhibition of the fusion of the virus with endosome/lysosome membrane at  early stage and inhibition of the budding of the progeny virus from the cell surface (Nagai et al. 1995). Furthermore, baicalein, baicalin, and wogonin could also inhibit other types of viruses, including HIV, herpes simplex virus-1 (HSV-1), Moloney murine leukemia virus, and Rousassociated virus type 2 (Baylor et al. 1992;Li et al. 1993Li et al. , 2000aKitamura et al. 1998;Huang et al. 2000;Wang et al. 2004;Guo et al. 2007;Błach-Olszewska et al. 2008;Nayak et al. 2014). Recently, Lin et al. (2016) reported that S. baicalensis could be used to treat severe HFMD (Hand, Foot, and Mouth Disease) in patients aged >1 year, rapidly relieving fever, attenuating oral lesions and rashes, and improving nervous system involvement. This result was derived from a multi-center and retrospective analysis . It is reasonable to assume that S. baicalensis and its compounds possess a common, non-specific antiviral mechanism, based on its inhibitory effects on different types of viruses.
Baicalin (100 mg/kg, p.o.) could protect mice from staphylococcal pneumonia caused by Staphylococcus aureus, reducing mortality from 80% to 28% and protecting the lung from accumulation of cellular infiltrates (Qiu et al. 2012). This activity is associated with inhibition of the cytolytic activity of a-hemolysin, which is a self-assembling and channel-forming toxin secreted by S. aureus. Baicalein also showed potent synergistic effect with penicillin G/amoxicillin against 20 clinical penicillinase-producing S. aureus strains. Baicalin at 32 lg/mL could enhance the bacteriostatic effects, and decrease the MIC 50 values of penicillin and amoxicillin from 32-64 to 0.5-2 lg/mL (Qian et al. 2015).

Anti-inflammatory activities
An extract of S. baicalensis (750 mg/kg for 10 d, p.o.) showed potent anti-inflammatory activities in the zymosan-induced mice air-pouch, reducing NO production from 30 to 5 lM, through the down-regulation of IKKab (IjB kinase ab) and NF-jB activation via suppression of c-Raf-1/MEK1/2 and MAPK phosphorylation (Kim et al. 2009). The flavonoids extract (100 lg/mL) also exhibited significant anti-inflammatory activities through inhibiting the NF-jB signaling pathway via the MAPK (mitogenactivated protein kinase) signaling pathway in RAW264.7 cells (Hong et al. 2013).
Baicalein (50-100 lM) showed anti-inflammatory effects in double-stranded RNA (dsRNA)-induced macrophages by inhibiting NO, cytokines, chemokines, and growth factors via the endoplasmic reticulum stress-CHOP/STAT pathway (Kim et al. 2018). Baicalin (100 mg/kg for 7 d, i.p.) could relieve ankle swelling, and protect the joint against inflammatory destruction in a murine adjuvant-induced arthritis model, by inhibiting splenic Th17 cell expansion and IL-17 (interleukin 17A)-mediated inflammation in synoviocytes (Yang X et al. 2013). Furthermore, baicalin (200 mg/kg for 7 d, p.o.) could alleviate LPS-induced liver inflammation in chicks, reducing the cloacal temperature from 41.5 to 40.3 C and inhibiting NO production from 105 to 40 lM, via suppression of TLR4 (Toll-like receptor 4)-mediated NF-jB pathway (Cheng et al. 2017). Baicalin could also decrease inflammation by selective binding to chemokine ligands on CD4 and other leukocytes (Li et al. 2000b). Wogonoside (50 lM) could decrease the production of inflammatory mediators NO and PGE2, and inhibit the release of pro-inflammatory cytokines including TNF-a (tumor necrosis factor a) and IL-6 in LPS-induced RAW264.7 cells (Yang YZ et al. 2013). Wogonin treatment also regulated the production of inflammatory cytokines in mice with streptozotocin-induced vascular inflammation ).

Neuroprotective activities
The extract of S. baicalensis (200 mg/kg for 40 or 32 d, p.o.) could improve rat act in the Morris water assay, reducing search error to about 50% in the chronic cerebral hypoperfusion and the LPS infusion models (Hwang et al. 2011). Treatment with the extract attenuated the neuroinflammatory responses and reduced the spatial memory impairments, via mitigating alterations of hippocampal MAPK signaling (Table 3). The extact could also protect animals from global cerebral ischemia and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced Parkinson's disease, and protect cortical and neuronal cells from glutamate, NMDA (N-methyl-D-aspartic acid), and H 2 O 2 induced toxicity in vitro (Yang et al. 2014;Cao et al. 2016;Li et al. 2016).

Effects on cardiovascular and cerebrovascular diseases
Baicalin (6 lM) could protect against the hyperglycemia-induced cardiovascular malformation during chick embryo development, decreasing the high incidence of cardiac bifida from 32% to 16%, by reducing ROS production and regulating SOD (superoxide dismutase), GSH-Px (glutathione peroxidase), and GABA A (c-aminobutyric acid) levels . Baicalin also exerted angiogenesis and cardioprotective effects against chronic hypoxia-induced pulmonary hypertension and acute myocardial infarction in vivo, through mediation of MAPK cascades, the ERRa (estrogen-related receptor a) pathway, and the PI3K/AKT signaling Liu et al. 2013;Huang et al. 2017).
Baicalein could promote new blood vessel formation, attenuate cardiac remodeling and endothelium dysfunction against angiotensin II or myocardial ischemia reperfusion injury, via inhibition of AKT/mTOR, ERK1/2, NF-jB, and calcineurin sgnaling pathways in mice or chicks (Cho et al. 2008;Li et al. 2015;Wang AW et al. 2015).

Effects on bones
The extract of S. baicalensis (50 mg/kg for 42 d, p.o.) could significantly increase bone mineral density by 12-18%, and improve bone trabecula microstructure of weightlessness induced osteoporosis rats via the osteogenic differentiation enhancement effect (Zhang GW et al. 2017). A wogonin-rich fraction (50 lg/mL) exerted chondroprotective effects by inhibiting ROS production and suppressing catabolic markers (Khan et al. 2017). Baicalein and baicalin (10 lM) could significantly enhance the osteogenic differentiation of human periodontal ligament cells (hPDLCs) and rat bone marrow derived mesenchymal stem cells (rBMSC), reapectively, by increasing ALP (alkaline phosphatase) activities up to 1.5-2-fold and increasing the formation of mineralized nodules up to 2-fold Zhang GW et al. 2017). Arjmandi et al. (2014) reported that UP446 (a natural proprietary of S. baicalensis and Areca catechu L.) could reduce physical symptoms associated with knee osteoarthritis in patients after 500 mg/d treatment for 1 week.

Biosynthesis of Scutellaria flavonoids
The flavonoids in S. baicalensis Georgi possess various pharmacological activities. Their biosynthesis in the living plant has gained increasing attention in recent years. Zhao et al. systematically investigated the biosynthetic pathways of free flavones. The Scutellaria flavones are originally derived from phenylalanine, which is catalyzed by phenylalanine ammonia lyase (PAL) to form cinnamic acid. Interestingly, the subsequent biosynthetic steps were different for flavones in the aerial parts and in the roots (Figure 4). For the 4 0 -hydroxyl flavones, which are mainly distributed in the aerial parts, cinnamic acid is sequentially catalyzed by cinnamoyl 4 hydroxylase (C4H), p-coumaroyl CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavone synthase (FNSII-1) to form apigenin (Zhao et al. 2016a(Zhao et al. , 2016b. Then apigenin is hydroxylated by flavone 6-hydroxylase (F6H) to generate scutellarein, as shown in Figure 5 (Zhao Q et al. 2018). The flavones in the roots, however, usually lack a 4 0 -OH group on the B-ring. For their biosynthesis, cinnamic acid is catalyzed by cimmamoyl-CoA ligase (CLL-7), chalcone synthase (CHS-2), chalcone isomerase (CHI) to form pinocembrin. Pinocembrin is then converted by a specialized isoform of flavone synthase (FNSII-2) to form chrysin, which could be further hydroxylated by flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H) to produce baicalein and wogonin, respectively (Zhao et al. 2016a(Zhao et al. , 2016b. O-methyltransferases (OMTs) may participate in the biosynthesis of wogonin, though no OMT has been reported yet. Among the biosynthetic enzymes, SbCLL-7, SbCHS-2, FNSII-2 and F8H are expressed preferentially in the roots. Functions of these genes have been validated by RNAi in hairy roots of S. baicalensis and overexpression in transgenic Arabidopsis. Glycosyltransferases are responsible for the formation of glycosidic bonds of flavonoid O-glucuronides and O-glucosides of S. baicalensis. SbUBGAT showed O-glucuronyltransferase activities for various flavones, and may take part in the biosynthesis of glucuronides like baicalin and wogonoside (Nagashima et al. 2000;Yang et al. 2016). SbUBGAT also showed O-glycosyltransferase activities. Together with SbUBGT discovered from the hairy root cultures of S. baicalensis, they may contribute to the production of flavonoid-O-glycosides . Furthermore, the Arg residue (R) in the PSPG (Plant Secondary Product Glycosyltransferase) box plays a critical role in the recognition of UDP-glucuronic acid sugar donor, while the corresponding Trp residue (W) has better selectivity for UDP-glucose donor ( Figure 6). This was validated by homologymodeling and site-directed mutagenesis analysis (Noguchi et al. 2009). Scutellaria baicalensis also contains abundant flavonoiddi-C-glycosides, and the responsible C-glycosyltransferases have not been reported yet.

Conclusions and future prospects
Scutellaria baicalensis contains at least 126 small molecules and 6 polysaccharides. It possesses anti-tumor, anti-viral, anti-microbial, anti-inflammatory, antioxidative, and neuroprotective activities. Chemical compounds responsible for many of these activities are still unknown, though the bioactivities of a few major compounds (baicalin, baicalein, wogonoside, and wogonin) have been extensively studied. Recently, our group reported the comprehensive correlations of chemicals and bioactivities of another popular herbal medicine Gan-Cao (licorice, Glycyrrhiza uralensis Fisch), and discovered a number of promising bioactive natural products (Ji et al. 2016). Similar research strategy could be applied to Huang-Qin to discover potential new drugs. In fact, the clinical trial of wogonin as an anti-cancer drug candidate has recently been approved by the State Drug Administration of China. On the other hand, the identified major bioactive compounds could be used as chemical markers to improve quality control of Huang-Qin crude drugs and related patent drugs. Furthermore, biosynthetic studies could help large-scale production of the bioactive compounds by   metabolic engineering. Although enzymes involved in the biosynthesis of free flavones have been reported for S. baicalensis, many post-modification enzymes have yet to be characterized, including those responsible for the hydroxylation, methylation, and glycosylation reactions.

Disclosure statement
No potential conflict of interests was reported by the authors.

Funding
This work was supported by National Natural Science Foundation of China [No. 81470172, No. 81725023], and Young Elite Scientists Sponsorship Program by China Association for Science and Technology [2016QNRC001].