A systematic review of the active saikosaponins and extracts isolated from Radix Bupleuri and their applications

Abstract Context: Radix Bupleuri has been used in traditional Chinese medicine for over 2000 years with functions of relieving exterior syndrome, clearing heat, regulating liver-qi, and lifting yang-qi. More natural active compounds, especially saikosaponins, have been isolated from Radix Bupleuri, which possess various valuable pharmacological activities. Objective: To summarize the current knowledge on pharmacological activities, mechanisms and applications of extracts and saikosaponins isolated from Radix Bupleuri, and obtain new insights for further research and development of Radix Bupleuri. Methods: PubMed, Web of Science, Science Direct, Research Gate, Academic Journals and Google Scholar were used as information sources through the inclusion of the search terms ‘Radix Bupleuri’, ‘Bupleurum’, ‘saikosaponins’, ‘Radix Bupleuri preparation’, and their combinations, mainly from the year 2008 to 2016 without language restriction. Clinical preparations containing Radix Bupleuri were collected from official website of China Food and Drug Administration (CFDA). Results and conclusion: 296 papers were searched and 128 papers were reviewed. A broad spectrum of in vitro and in vivo research has proved that Radix Bupleuri extracts, saikosaponin a, saikosaponin d, saikosaponin c, and saikosaponin b2, exhibit evident anti-inflammatory, antitumor, antiviral, anti-allergic, immunoregulation, and neuroregulation activities mainly through NF-κB, MAPK or other pathways. 15 clinical preparations approved by CFDA remarkably broaden the application of Radix Bupleuri. The main side effect of Radix Bupleuri is liver damage when the dosage is excess, which indicates that the maximum tolerated dose is critical for clinical use of Radix Bupleuri extract and purified compounds.


Introduction
With a 2000-year medicinal history, Radix Bupleuri (Chai Hu in Chinese) is believed to be one of the most important herbal medicines in China. The earliest record about Radix Bupleuri in China appeared in Shen Nong Ben Cao Jing, the first Chinese medical book, since then, Radix Bupleuri has been widely used in traditional Chinese medicine (TCM) for its effects of relieving exterior syndrome, clearing heat, regulating the liver-qi, and lifting yang-qi (Sen 1959). It has been used in many traditional Chinese prescriptions, such as Xiao Chai Hu Tang and Chai Hu Shu Gan Yin to treat cold and liver diseases (Chen et al. 2011). The roots are usually the medicinal parts of Radix Bupleuri, and which is often processed into pieces for easy use (Figure 1).
Bupleurum chinense DC. (Apiaceae) and Bupleurum scorzonerifolium Willd. are defined as the original plants of Radix Bupleuri in Chinese Pharmacopeia (National Pharmacopoeia Committee 2010). In fact, many other Bupleurum species are also used as Radix Bupleuri in East Asia, such as Bupleurum falcatum L., which is officially listed in Japanese Pharmacopeia (Saiko in Japanese) (Japanese Pharmacopoeia Editorial Board 2011), and Bupleurum yinchowense Shan and Li, which is recorded in some provincial Pharmacopeia of China (The Inner Mongolia Autonomous Region Health Department 1988; Food and Drug Administration of Gansu Province 2008). These Bupleurum medicinal plants are widely distributed in the northern hemisphere (Judd 2008), and also commonly used in Eurasia and North Africa for their medicinal properties (Mabberley 2008). As shown in Figure 2, they are perennial herbs with compound umbels, yellowish or rarely purplish bisexual flowers, containing five stamens, cremocarps, and simple, long, slender leaves ( Figure 2).
With the development of modern pharmacology, many valuable and important activities of Radix Bupleuri have been discovered, such as anti-inflammatory , antitumor (Liu & Li 2014), antidepressant (Jin et al. 2013), antiviral (Chiang et al. 2003), hepatoprotection ), immunoregulation (Ying et al. 2014), and neuromodulation activities (Zhou et al. 2014). All of these potent effects are due to its various secondary metabolites, especially saikosaponins, the content of which is up to 7% of the total dry weight of Radix Bupleuri roots (Ashour & Wink 2011). To date, over 100 glycosylated oleananetype saponins have been isolated and identified from Radix Bupleuri (Pistelli et al. 1993;Ebata et al. 1996), and some of them have been demonstrated possessing bioactive properties both in vitro and in vivo. Therefore, reviewing and summarizing the pharmacological activities and mechanisms of saikosaponins from Radix Bupleuri is meaningful and important to obtain new insights for further research and development of Radix Bupleuri. In addition, since extracts are the main source of Chinese patent medicines containing Radix Bupleuri, their pharmacological properties and mechanisms are also summarized. Moreover, the applications and toxicity studies are discussed to provide a basis for further studies concerning the safety and efficacy of Radix Bupleuri.
In this paper, six main databases, PubMed, Web of Science, Science Direct, Research Gate, Academic Journals, and Google Scholar were used as information sources through the inclusion of the search terms 'Radix Bupleuri', 'Bupleurum', 'saikosaponins', 'Radix Bupleuri preparation', and their combinations, mainly from the year 2008 to 2016 without language restriction. As a result, we searched 296 papers and a total of 128 references were included in the present work.

Anti-inflammatory activity
Among all of the pharmacological activities of SSa, the most important one is anti-inflammatory activity. SSa develops its anti-inflammatory activity mainly by inhibiting some inflammation-associated cytokines, proteins and enzymes, and regulating inflammation-related signal pathways, such as nuclear factor-jB (NF-jB) pathway and mitogen-activated protein kinase (MAPK) pathway. In order to better explain the molecular mechanisms of the anti-inflammatory activity of SSa, Figures 4(a,b) are provided to describe its NF-jB pathway and MAPK pathway.
As shown in Figure 4(b), SSa also has an inhibiting effect on MAPK pathway. It downregulates the phosphorylation of three key kinase, p38 MAPK, c-JNK, and ERK 1/2, which are located in the downstream of MAPK pathway and marked by triangle symbol in Figure 4 For studying the anti-inflammatory activity of SSa, it has been applied to mouse macrophage cells RAW264.7 (Zhou et al. 2015), human umbilical vein endothelial cells (HUVECs) (Fu et al. 2015), mouse embryonic fibroblasts 3T3-L1 , hepatic stellate cells (HSCs) (Chen et al. 2013b), and human mast cells (HMCs) (Han et al. 2011) in vitro, and has been applied to the livers of Sprague-Dawley rats (Wu et al. 2010) and Wistar rats (Zhao et al. 2015a) in vivo.

Neuroregulation activity
SSa plays a significant role on neuroregulation. It exerts antiepileptic mainly by inhibiting N-methyl-D-aspartic acid (NMDA) receptor current, persistent sodium current  and inactivating K þ current (Xie et al. 2013). It inhibits the activation of p38 MAPK, NF-jB signaling pathways to attenuate neuropathic pain (Zhou et al. 2014), and activates c-aminobutyric acid (GABA) receptor B to attenuate cocaine-reinforced behavior (Yoon et al. 2012(Yoon et al. , 2013 and drug addiction (Maccioni et al. 2016). It also counteracts the inflammatory response and neurological function deficits via an anti-inflammatory response and inhibition of the MAPK signaling pathway to ease nerve injury (Mao et al. 2016). SSa has been applied to the hippocamp, CA1 neurons, and spinal cord tissues of Sprague-Dawley rats (Mao et al. 2016;Maccioni et al. 2016;Yu et al. 2012;Xie et al. 2013;Yoon et al. 2012Yoon et al. , 2013, and chronic constriction injury rats (Zhou et al. 2014) in vivo, which determined its potential application in epilepsy, chronic constriction injury, nerve injury, and drug addiction.

3T3-L1
In vitro SSa inhibits the expression of inflammatory associated genes and is a potent inhibitor of NF-jB activation.
Obesity-associated inflammation  Ileum

Male Wistar rats
In vivo SSa suppresses the production of TNF-a and IL-6 and inhibits the nucleotide-binding oligomerization domain 2 (NOD2)/NF-jB signalling pathway.

LX-2
In vitro SSa down-regulates BMP-4 expression and inhibits hepatic stellate cell activation.
Liver fibrosis (Wang et al. 2013b) Macrophages RAW 264.7 In vitro SSa regulates inflammatory mediators and suppresses the MAPK and NF-jB signalling pathways.

HSC-T6
In vitro SSa decreases the expressions of ERK1/2, PDGFR, TGF-b1R, a-smooth muscle actin, and connective tissue growth factor to inhibit proliferation and activation of HSCs.

HMC-1
In vitro SSa decreases the expression of IL-6, IL-1b and TNF-a and suppresses NF-jB signal pathway.

Sprague-Dawley rats
In vivo SSa inhibits the expression of hepatic proinflammatory cytokines and NF-jB signal pathway and increases the expression of anti-inflammatory cytokine IL-10.
Inhibition of liver injury (Wu et al. 2008(Wu et al. , 2010 Human monocytic leukemia cells

THP-1
In vitro SSa inhibits oxLDL-induced activation of AKT and NF-kappaB, assembly of NLRP3 inflammasome and production of pro-inflammatory cytokines.

Sprague-Dawley rats
In vivo SSa inhibits NMDA receptor current and persistent sodium current.

Sprague-Dawley rats
In vivo SSa exerts selectively enhancing effects on I A.

Chronic constriction injury rats
In vivo SSa inhibits the activation of p38 MAPK and NF-jB signalling pathways in spinal cord.

Sprague-Dawley rats
In vivo SSa attenuates cocaine-reinforced behaviour through activation of GABA(B) receptors.

Sprague-Dawley rats
In vivo SSa counteracts the inflammatory response and neurological function deficits via an anti-inflammatory response and inhibition of the MAPK signalling pathway.

Sprague-Dawley rats
In vivo SSa inhibits this addiction by regulating GABA(B) receptor system.
Drug addiction (Maccioni et al. 2016) Antitumor activity Different cancer cells

A549, SKOV3, HeLa and Siha
In vitro SSa sensitizes cancer cells to cisplatin through ROS -mediated apoptosis.

C6 glioma cells
In vitro SSa enhances the enzymatic activities of GS and CNP.

Human coronavirus 229E
In vitro SSa intervenes in the early stage of viral replication, such as absorption and penetration.

Influenza A virus infected A549
In vitro SSa attenuates viral replication, aberrant pro-inflammatory cytokine production and lung histopathology.
Pathological influenza virus infections  Immunoregulation Lymphoid tissue

Sprague-Dawley rats
In vivo SSa inhibits the proliferation and activation of T cells and causes the G0/G1 arrest as well as the induction of apoptosis via mitochondrial pathway. SSa exhibits antitumor activity in vitro by sensitizing cancer cells to cisplatin, such as human lung adenocarcinoma cells A549, ovarian cancer cells SKOV3, and cervix cancer cells Hela and Siha, through reactive oxygen species (ROS)-mediated apoptosis (Wang et al. 2010a) and enhancing the enzymatic activities of glutamine synthetase (GS) and 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphohydrolase (CNP) in rat C6 glioma cells (Tsai et al. 2002). Thus, the combination of SSa with cisplatin could be an effective therapeutic strategy against cancer.

Antiviral activity
SSa has generally inhibitory effects against human coronavirus 229E (Cheng et al. 2006) and influenza A virus . It exerts antiviral activity mainly through interference in the early stage of viral replication, such as absorption and penetration , and attenuating aberrant proinflammatory cytokine production (Cheng et al. 2006). These two viruses are cultured in human cells, human fetal lung fibroblasts MRC-5 and A549 cells, respectively.

Immunoregulation activity
SSa inhibits the proliferation and activation of T cells and causes the G0/G1 cells arrest as well as the induction of apoptosis via mitochondrial pathway to exhibit its immunoregulation effect in Sprague-Dawley rats ). This may herald a novel approach for further studies of SSa as a candidate for the treatment of autoimmune diseases.

SSd
SSd is the epimer of SSa, they have the same basal structure. So, it has some similar pharmacological activities with SSa, such as anti-inflammatory (Lu et al. 2012b), antitumor (Chen et al. 2013a), and immunoregulation activities Ying et al. 2014). However, SSd also possesses some specific pharmacological activities, such as anti-allergic (Hao et al. 2012) and  In vitro SSd promotes cell apoptosis and induced G1-phase cell cycle arrest.
Human hepatocellular carcinoma (He et al. 2014) Prostate carcinoma cells

DU145
In vitro SSd has effects on induction of apoptosis and cell cycle arrest at G0/G1 phase.
Prostate carcinoma (Yao et al. 2014) Different cancer cells

HeLa, HepG2
In vitro SSd suppresses TNF-a-induced NF-jB activation and its target genes expression to inhibit cancer cell proliferation, invasion, angiogenesis and survival.
As a combined adjuvant remedy with TNF-a for cancer patients (Wong et al. 2013a) Lung carcinoma

A549
In vitro SSd induces apoptosis and blocked cell cycle progression by activating Fas/FasL pathway in the G1 phase in A549 cells.
Human non-small cell lung cancer (Hsu et al. 2004a) Liver HepG2, 2.2.15 In vitro SSd induces the apoptosis through the activation of caspases-3 and caspases-7.
Human hepatocellular carcinoma (Chiang et al. 2003) Liver Hep3B In vitro SSd induces apoptosis in Hep3B cells through the caspase-3 -independent pathways.
Human hepatocellular carcinoma Zhou 2003 Breast carcinomas tissue

MCF-7
In vitro SSd activates oestrogen response element (ERE)-luciferase activity via the ER a-mediated pathway.
Acting as a weak phytoestrogen. (Wang et al. 2010a) Liver SMMC-7721, HepG2 In vitro SSd has a radiosensitizing effect on hepatoma cells under hypoxic conditions by inhibiting HIF-1a expression.
Radiotherapy sensitizer in hepatoma radiotherapy (Wang et al. 2014a(Wang et al. , 2014b Different cancer cells HeLa, MCF-7 In vitro SSd induces autophagy through the formation of autophagosomes by inhibiting SERCA. Apoptosis-resistant cancer cells (Wong et al. 2013b) Anti-inflammatory activity Inflammatory tissue

RAW264.7
In vitro SSd has inhibitory effects on NF-jB activation and iNOS, COX-2 and pro-inflammatory cytokines including TNF-a and IL-6.
Liver inflammation and fibrogenesis (Chen et al. 2013a) Human acute monocytic leukaemia cells

In vivo
SSd down-regulates NF-jB and STAT3-mediated inflammatory signal pathway.
Hepatotoxicity and liver injury  Liver

Hepatic fibrosis rats
In vivo SSd down-regulates liver TNF-a, IL-6 and NF-jB p65 expression and increases IjB-a activity.

LLC-PK1
In vitro SSd increases the activity and expression of anti-oxidant enzymes (SOD, CAT, GPx) and HSP72.
Oxidative damage in the kidney  Nervous tissue C6 rat glioma cells

In vitro
SSd possesses a dual effect: an inhibition of PGE2 production without a direct inhibition of cyclooxygenase activity and an elevation of [Ca 2þ ]i.

VILI rats
In vivo SSd decreases the expression of pro-inflammatory cytokines including MIP-2, IL-6 and TNF-a and elevates the expression of anti-inflammatory mediators, such as TGF-b1 and IL-10.
Lung injury  Renal tubular epithelial cells
High glucose induced kidney injury (Zhao et al. 2015b) Kidney

HK-2
In vitro SSd represses ROS-mediated activation of MAPK and NF-jB signal pathways.
DDP-induced kidney injury (Ma et al. 2015) (continued) anti-apoptosis activities (Li et al. 2014b). The various pharmacological activities, mechanisms, models and applications of SSd are listed in Table 2.

Antitumor activity
The most important pharmacological activity of SSd is antitumor activity. In order to better explain this important activity, Figure 5 is provided to describe its molecular mechanisms. SSd exhibits the antitumor activity mainly through activation and inhibition, which are marked by rectangle and triangle in Figure 5, respectively. First, SSd increases the expression of p53 and Bax (Liu & Li 2014;Wang et al. 2014aWang et al. , 2014bYao et al. 2014), activates caspases apoptosis pathway, including the activation of caspases-3 and caspases-7 (Chiang et al. 2003;Chou et al. 2003) and the Fas/FasL apoptotic system (Hsu et al. 2004a) in several cancer cell lines in vitro, which are marked by rectangle in Figure 5. Second, SSd decreases the expression of B cell lymphoma 2 (Bcl-2) family proteins (Liu & Li 2014;Wang et al. 2014aWang et al. , 2014bYao et al. 2014), suppresses the expression of COX-2, which has been shown to be involved in carcinogenesis (Lu et al. 2012b;He et al. 2014), and also potentiates TNF-a-mediated cell death via suppression of TNF-a-induced NF-jB activation (Wong et al. 2013a), which are marked by triangle in Figure 5. Besides, SSd also suppresses MCF-7 cells proliferation through the estrogenic effect of SSd by the estrogen receptor (Wang et al. 2010a(Wang et al. , 2010b, and induces autophagy of apoptosis-resistant cancer cells through the formation of autophagosomes by inhibiting sarcoplasmic/endoplasmic reticulum Ca 2þ ATPase pump (SERCA) (Wong et al. 2013b).

Anti-inflammatory activity
SSd also possesses an evident anti-inflammatory activity, and the mechanisms are similar to SSa, as shown in Figure 4(a). On the cytokines level, SSd suppresses pro-inflammatory cytokines including TNF-a, IL-6, macrophage inflammatory protein-2 (MIP-2), and elevates the expression of antiinflammatory cytokines, such as TGF-b1 and IL-10 (Lu et al. 2012a;Ma et al. 2015;Wang et al. 2015). On the level of proteins and enzymes, it inhibits the activity and expression of iNOS, COX-2, ERK1/2, PDGFR, a-smooth muscle actin, NF-jB, and signal transducer and activator of transcription 3 (STAT3) (Chen et al. 2013a;Liu et al. 2014a), and increases the activity and expression of inhibitor of nuclear factor of jB-a (IjB-a) (Dang et al. 2007), SirT3 (Zhao L et al. 2015), anti-oxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and heat shock protein (HSP) 72 . Furthermore, SSd also exhibits its particular anti-inflammatory pattern by inhibiting selectinmediated cell adhesion (Jang et al. 2014), and possessing a dual effect, an inhibition of prostaglandin E 2 (PGE 2 ) production without a direct inhibition of cyclooxygenase activity and an elevation of Ca 2þ (Kodama et al. 2003).

In vitro
SSd inhibits the T cell proliferation and activation through the NF-jB, NF-AT and AP-1 signal pathways, and it also inhibits the cytokine secretion and IL-2 receptor expression.

DCs
In vitro SSd reduces the differentiation of human DCs and promotes DCs maturation and increases the function of mature DCs.

In vitro
SSd suppresses the intracellular calcium mobilization and tyrosine phosphorylation, thereby prevents gene activation of Cdc42 and c-Fos.

PC12
In vitro SSd regulates mitochondrial and nuclear GR translocation, partial reversal of mitochondrial dysfunction, inhibition of the mitochondrial apoptotic pathway, and selective activation of the GR-dependent survival pathway.

PC12
In vitro SSD reduces PC12 cells apoptosis by removing ROS and blocking MAPK-dependent oxidative damage.
Neuronal oxidative stress  According to the above reports, SSa and SSd are very similar in mechanisms of anti-inflammation, however, there are still several different points, which are listed in Table 3. SSa is able to inhibit phosphorylation of three key kinase in MAPK pathway, which was not reported in researches of SSd. While SSd is able to restrain selectin-mediated cell adhesion, PGE 2 production, and elevate the Ca 2þ level intracellular, which were not reported in researches of SSa For a better understanding of SSd's anti-inflammatory activity, it has been applied to mouse leukaemic monocyte macrophage macroph RAW264.7 (Lu et al. 2012a (Ma et al. 2015) in vitro, and acetaminophen-induced hepatotoxicity C57/BL6 rats ), hepatic fibrosis model rats (Dang et al. 2007), and ventilator-induced lung injury (VILI) rats  in vivo, which determined its potential application for treating hepatitis, pneumonia, nephritis and other inflammation.

Immunoregulation activity
SSd plays its immunoregulation role by regulating the NF-jB, nuclear factor-AT (NF-AT), and activator protein 1 (AP-1) signal pathways to inhibit T cell proliferation and activation (Wong et al. 2009). It has been applied to condylomata acuminate, a disease caused by human papilloma virus (HPV), by reducing the differentiation of human monocyte-derived dendritic cells (DCs) and promoting DCs maturation and increasing the function of mature DCs (Ying et al. 2014).

Anti-allergic activity
b-Conglycinin has been identified as a potential diagnostic marker for severe basophil-dependent allergic reactions to soybean. SSd possesses anti-allergic activity by inhibiting b-conglycinin-induced rat basophilic leukemia-2H3 cell degranulation and suppressing critical incidents in the signal transduction pathway (Hao et al. 2012), Hence it could become an effective herbal therapy for alleviating soybean allergy.

Neuroregulation activity
Neuronal oxidative stress injury has been proven to be associated with many neurodegenerative diseases. SSd exerts neuroregulation activity on neuronal PC12 cells by inhibiting the translocation of the glucocorticoid receptor (GR) to the mitochondria, restoring mitochondrial function, down-regulating the expression of pro-apoptotic-related signalling events and up-regulating antiapoptotic-related signalling events (Li et al. 2014b). In H 2 O 2 -induced oxidative stress PC12 cells, SSd effectively decreases oxidative stress injury by blocking H 2 O 2 -induced phosphorylation of ERK, JNK, and p38MAPK to exert neuroregulation activity ). Thus, SSd treatment is an effective method for treating neurodegenerative diseases.

SSc
SSc has the same basal structure with SSa and SSd. They are epoxy-ether saikosaponins belonging to type I saikosaponins (Shin et al. 2015). However, the pharmacological activities of SSc are far weaker than SSa and SSd. To date, reports about pharmacological activities of SSc are very limited. SSc exerts anti-apoptotic effects on HUVECs by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase (FAK) and other cell adhesion signals, which is similar to SSa ). Thus, it will be a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction. Besides, SSc completely prevents the development of nephritis (Chen et al. 2008), but the mechanism of this activity is still unclear. In addition, SSc exhibits antiviral activity by inhibiting hepatitis B virus (HBV) DNA replication (Chiang et al. 2003).

SSb 2
SSb 2 has a different basic structure compared to SSa, SSd, and SSc. SSb 2 is a type II saikosaponin, and it is not considered as a main active compound in Radix Bupleuri. However, SSb 2 has fairly inhibitory effects against corona virus and hepatitis C virus (HCV). It mainly interferes with the early stages of viral replication, such as absorption and penetration of the virus (Cheng et al. 2006). SSb 2 potently inhibits HCV infection at non-cytotoxic concentrations through efficient inhibition on early HCV entry, including neutralization of virus particles, preventing viral attachment, and inhibiting viral entry/fusion (Lin et al. 2014).

The possible mechanisms of anti-inflammation SSa SSd
Inhibiting pro-inflammatory cytokines and promoting anti-inflammatory cytokines Inhibiting activity of enzymes associated with inflammation Inhibiting activation of NF-jB pathway Inhibiting activation of MAPK pathway Inhibiting selectin-mediated cell adhesion Inhibiting PGE2 production and elevating Ca 2þ level intracellular The neuroprotective mechanism relates with inhibiting the ER stress and the mitochondrial apoptotic pathways.  B. kaoi

Antitumor activity
The activity of the Fas/Fas ligand apoptotic system participates in the antiproliferative activity of TSS in A549 cells. (Hsu et al. 2004b) Methanol, reflux, 4 h Extracts from B. kaoi show potent antiproliferative effects on human A375.S2 melanoma cells. (Hu et al. 2016) and neuroregulation effects (Xie et al. 2006;Lee et al. 2009Lee et al. , 2012bLi et al. 2013;Liu et al. 2014b). Five kinds of extraction agents, water, methanol, ethanol, acetone and ethyl acetate, have been used to extract effective fractions from Radix Bupleuri. Aqueous extracts of Radix Bupleuri are obtained by boiling at 80 C for 3 h, and then evaporating and lyophilizing (Kang et al. 2008;Wen et al. 2011;Kim et al. 2012b;Chen et al. 2014). The method to obtain methanol, ethanol, acetone and ethyl acetate extracts is reflux extraction (You et al. 2002;Cheng et al. 2005;Lee et al. 2010;Liu et al. 2014a). To obtain methanol extracts, Radix Bupleuri is extracted twice by 100% methanol or 95% methanol with 5% pyridine at 70 C for 4 h (Xie et al. 2006;Kwon et al. 2010;Nakahara et al. 2011;Liu et al. 2014a;Ashour et al. 2014). To obtain ethanol extracts, Radix Bupleuri is extracted twice by 60% , 70%  or 80% ethanol (Lee et al. 2012a) at room temperature for 6 h. To obtain acetone and ethyl acetate extracts, Radix Bupleuri is extracted three times by 100% acetone and 100% ethyl acetate at room temperature for 4 h (You et al. 2002;Cheng et al. 2005).
The pharmacological activities of extracts from B. chinense and B. falcatum have relative in-depth studies. The aqueous extracts of B. chinense possess three activities, antitumor activity on HepG2 hepatoma cells (Kang et al. 2008), antiviral activity on H1N1-infected A549 cells (Wen et al. 2011), and an activity to affect drug distribution ). Methanol total saikosaponins (TSS) extracts of B. chinense have a neuroregulation effect (Xie et al. 2006;Liu et al. 2014a). In chronic kindling rats induced by pentetrazole (PTZ), TSS of B. chinense inhibit glial fibrillary acidic protein (GFAP) over-expression and suppress the abnormal activation of hippocampal astrocyte (Xie et al. 2006). Anti-depressant activity of TSS is investigated by tail suspension test, forced swimming test, and reserpine antagonism test in mice, which demonstrate that it shortens the immobility time of mice in the tail suspension test in a somewhat dose-dependent manner ).
Both ethanol extracts and methanol extracts of B. falcatum have an anti-inflammatory effect Nakahara et al. 2011) with similar mechanisms to SSa. They also possess an antidepressant activity possibly through central adrenergic mechanism (Kwon et al. 2010;Lee et al. 2012a). Besides, the ethanol extracts of B. falcatum has its specific memory improvement activity by attenuating immobilization (IMO) stress-induced loss of cholinergic immunoreactivity in the hippocampus (Lee et al. 2009). The aqueous extracts of B. falcatum has an anti-hyperthyroidism activity by attenuating leukotriene-4 (LT4)-induced hyperthyroidisms, normalizing LT4-induced liver oxidative stresses and reducing liver and epididymal fat pad changes (Kim et al. 2012b).
The acetone extracts of B. scorzonerifolium exerts stronger antitumor activity on A549 cells mainly through inducing tubulin polymerization , activating caspase-3 and caspase-9 , and inhibiting telomerase activity and activation of apoptosis (Cheng et al. 2003). Methanol extracts of B. marginatum and B. kaoi have an antitumor activity by inducing apoptosis (Ashour et al. 2014) and activating the Fas/Fas ligand apoptotic system respectively (Hsu et al. 2004b), and extracts of B. kaoi have antitumor activity on human A375.S2 melanoma cells by inhibiting phosphorylation of JNK, p38 and p53, decreasing level of cytochrome c (Hu et al. 2016). What's more, the ethanol TSS extracts of B. yinchowense show antidepressant activity by inhibiting the estrogen receptor (ER) stress and the mitochondrial apoptotic pathways , and the ethyl acetate extracts of B. longiradiatum exhibit an antiangiogenic activity by inhibiting the tube-like formation of HUVECs (You et al. 2002).

Applications of Radix Bupleuri in TCM
Radix Bupleuri has been used for more than 2000 years in China since its first record in Shen Nong Ben Cao Jing (Xie et al. 2009). And now, it is officially listed in Chinese Pharmacopeia. In TCM, Radix Bupleuri is mainly used to treat liver diseases, alleviate cold fever, chills, chest pain, regulate menstruation, and improve uterine prolapsed (Zhou 2003). In particular, Radix Bupleuri also plays a significant role in the treatment of malaria (Xue et al. 1996). Importantly, Radix Bupleuri is usually used as monarch drug in many traditional Chinese prescriptions.

Applications of Radix Bupleuri in modern Chinese medicine
With the development of TCM modernization, more Radix Bupleuri preparations have been developed, such as Xiao Chai Hu tablets, Chai Hu dripping pills, Chai Hu injection and Chai Hu Shu Gan pills (Li et al. 2014a). The preparations from Radix Bupleuri approved by CFDA from June 2010 to October 2015 are given in Table 5. Among them, Chai Hu injection is the first successful traditional Chinese medicine injection having been used in clinic since 1940s, which is widely used to treat fever caused by influenza or common cold and malaria (Zuo et al. 2013). Moreover, some new dosage forms of Radix Bupleuri have been prepared. A nasal temperature-sensitive in situ gel system is developed, which is more effective for the treatment of fever than the traditional nasal spray (Chen et al. 2010). Another benefit of this novel in situ gel is that it exhibits more noticeable antipyretic effects and remains much more time (Cao et al. 2007). Besides, the Radix Bupleuri suppositoria is very suitable for kids without pain (Wang & Chen 2003).

Side effects of Radix Bupleuri
Radix Bupleuri is not defined as a toxic medicine in many official pharmacopeias, such as Chinese Pharmacopeia and Japanese Pharmacopeia (National Pharmacopoeia Committee 2010; Japanese Pharmacopoeia Editorial Board 2011). However, in practical use, it exhibits liver, kidney, and blood system toxicity by taking a large dose for a long period, while it shows no side effect without over-dose (Liu et al. 2012). Chai Hu injection may cause a hypersensitivity-like response, hypokalemia and renal failure. And one case is reported to die from severe hypersensitivity shock (Wu et al. 2014). So, the safety of Radix Bupleuri preparations is of great concern to us.
Saikosaponins and essential oils are believed to be the main compounds responsible for side effects of Radix Bupleuri (Liu et al. 2012). Essential oils from B. chinense cause hepatic injury when the dosage is about 1.5-3.4 times of the clinical daily dosage of Radix Bupleuri oral liquid (Sun & Yang 2011). Saikosaponins from B. chinense induce the hepatoxicity by causing liver cell damage and necrosis administrating continuously to rats for 15 days (Huang et al. 2010). SSd stimulates mitochondrial apoptosis in hepatocytes to exhibit its hepatotoxicity (Chen et al. 2013a).
Extracts of Radix Bupleuri also show some side effects. Extracts of B. chinense induce hepatotoxicity damage through oxidative damage mechanism, and the hepatotoxicity damage caused by the alcohol extracts is more serious than that caused by aqueous extracts (Lv et al. 2009). Furthermore, LD 50 (50% lethal dose) of the aqueous extracts of Radix Bupleuri after single oral treatment in female and male mice are considered to be over 2000 mg/kg (Kim et al. 2012a). In Kampo (Japanese traditional herbal) medicines, studies of some potential interactions between Radix Bupleuri and other drugs are considered, especially in prescriptions containing Radix Bupleuri, such as Shosaikoto, Daisaikoto, Saikokeishito, Hochuekkito, Saibokuto and Saireito. They may lead to anorexia, slight fever, and nausea (Ikegami et al. 2006).
Among other Bupleurum species, B. longiradiatum is a toxic herb in Chinese Pharmacopeia (National Pharmacopoeia Committee 2010), and it cannot be used as Radix Bupleuri. The main toxic compounds in B. longiradiatum are acetyl-bupleurotoxin, bupleurotoxin (Zhao et al. 1987) and polyene acetylene compounds, which are able to cause neurotoxicity (Chen et al. 1981).

Discussion and perspective
Saikosaponins, especially SSa and SSd, are the main active compounds in Radix Bupleuri. They are also prescribed as the marker compounds to evaluate the quality of Radix Bupleuri in Chinese Pharmacopeia (National Pharmacopoeia Committee 2010). They possess evident anti-inflammatory, antitumor, neuroregulation, hepatoprotection, immunoregulation, antiviral, and antioxidative activities. And what need to emphasize is that SSa has a strongest anti-inflammatory effect, and SSd possesses a strongest antitumor effect compared with other saikosaponins, and both SSb 2 and SSc have a better antiviral activity than SSa and SSd, which proves that the activities of different saikosaponins have some extent tendency. Inspired by this feature, we speculate that purified saikosaponin has more concentrated pharmacological activities than extracts.
Recently, more preparations containing Radix Bupleuri have been developed, such as Xiao Chai Hu tablets, Chai Hu dripping pills, Chai Hu injection, and Chai Hu Shu Gan pills (Li et al. 2014a). In these preparations the extracts of Radix Bupleuri, especially saikosaponins (Hu et al. 2011), are the main composition. Although B. chinense and B. scorzonerifolium are the only two original plants of Radix Bupleuri in Chinese Pharmacopeia, many other Bupleurum species are often used as Radix Bupleuri in China. However, the extracts of B. chinenes, B. falcatum, B. marginatum, B. yinchowense, B. kaoi, B. scorzonerifolium, and B. longiradiatum possess different pharmacological activities, such as the antitumor and antiviral activities of B. chinenes extracts, and the anti-inflammatory, anti-hyperthyroidism and neuroregulation activities of B. falcatum extracts. Because the quality, botanic characteristic and property, and pharmacological activities of different Bupleurum species are different, the standardization of Bupleuri Radix extracts is vital for the safe use of Radix Bupleuri.
In addition, there are many other compounds in Radix Bupleuri, such as polysaccharides and essential oils. Polysaccharides in Radix Bupleuri usually exert hepatoprotective and immunoregulation activities. The hepatoprotective effect of Radix Bupleuri polysaccharides is evaluated by measuring aspartate transaminase (AST), alanine transaminase, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities in the plasma of mice (Zhao et al. 2012), and Radix Bupleuri polysaccharides inhibits complement activation on both the classical and alternative pathways (DI HY et al. 2013). The essential oils of Radix Bupleuri have strong antimicrobial (Ashour et al. 2009) and antifungal activities (Mohammadi et al. 2014). Besides, Radix Bupleuri also contains a little lignans, which exhibit antitumor (Ou et al. 2012) and hepatoprotective activities (Lee et al. 2011(Lee et al. , 2012. Since polysaccharides (Tong et al. 2013;Wu et al. 2013) and essential oils Yan et al. 2014) have been found to possess excellent pharmacological activities so far, we suppose that the quality evaluation method should be updated to meet the need of clinical therapy.
Radix Bupleuri also exhibits some security problems in the clinic. Since 'Xiao Chai Hu Decoction event' occurred in late 1980s in Japan, the clinical safety of Radix Bupleuri has been considered (Wu et al. 2014). The reasons of toxicity are complex and there is a great individual variation in the susceptibility to Radix Bupleuri. The current researches have shown that the toxicity of Radix Bupleuri mainly associated with dosage and drug administration time (Liu et al. 2012). For example, SSd exhibits antitumor activity on carcinoma cell lines with dose-dependence, but when the dosage of SSd increased to a high level it would exert cytotoxicity . Usually, Radix Bupleuri is believed to be safe in defined dose prescribed by pharmacopeia.

Disclosure statement
All authors declare that they have no competing interests.