Radix Bupleuri: A Review of Traditional Uses, Botany, Phytochemistry, Pharmacology, and Toxicology

Radix Bupleuri (Chaihu) has been used as a traditional medicine for more than 2000 years in China, Japan, Korea, and other Asian countries. Phytochemical studies demonstrated that this plant contains essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols. Crude extracts and pure compounds isolated from Radix Bupleuri exhibited various biological activities, such as anti-inflammatory, anticancer, antipyretic, antimicrobial, antiviral, hepatoprotective, neuroprotective, and immunomodulatory effects. However, Radix Bupleuri could also lead to hepatotoxicity, particularly in high doses and with long-term use. Pharmacokinetic studies have demonstrated that the major bioactive compounds (saikosaponins a, b2, c, and d) were absorbed rapidly in rats after oral administration of the extract of Radix Bupleuri. This review aims to comprehensively summarize the traditional uses, botany, phytochemistry, pharmacology, toxicology, and pharmacokinetics of Radix Bupleuri reported to date with an emphasis on its biological properties and mechanisms of action.


Introduction
Radix Bupleuri, also called "Chaihu" in Chinese, is derived from the dried roots of Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. [1]. As a traditional herbal medicine, Radix Bupleuri has been used widely for the treatments of influenza, fever, inflammation, malaria, menstrual disorders, and hepatitis in China, Japan, Korea, and other Asian countries [2,3]. According to ancient Chinese medical literatures, Radix Bupleuri is capable of regulating the exterior and interior metabolisms, dispersing evil heat from the superficies, soothing the liver, and promoting yang and qi (representing "life energy" or "life force" in TCM theories). In recent decades, investigations of Radix Bupleuri have focused on its biological activities, including its anti-inflammatory [4,5], anticancer [6,7], antipyretic [8], antimicrobial [9], antiviral [10], hepatoprotective [11], and immunomodulatory effects [12]. In addition, Radix Bupleuri also exhibited significant effects on membrane fluidity [13]. These studies have resulted in the isolation of essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols from this plant [14]. Among them, triterpenoid saponins are known to be the major bioactive compounds [15,16]. Saikosaponins a and d are commonly used as chemical standards for quality evaluation of Radix Bupleuri in the current Chinese Pharmacopoeia and recent publications. However, an increasing number of recently published studies have reported adverse effects of Radix Bupleuri. The purpose of this review is to provide updated, comprehensive information on the traditional uses, botany, phytochemistry, pharmacology, toxicology, and pharmacokinetics of Radix Bupleuri based on scientific literatures in the past few decades. This study will facilitate exploring the therapeutic potential of this plant and evaluate future research opportunities.

Traditional Uses
Radix Bupleuri, which is characterized by a wide spectrum of biological and pharmacological effects, has been used as a famous traditional Chinese medicinal herb with a history of medical use in China. According to TCM theory, Radix Bupleuri is thought to regulate the exterior and interior Curing rhinitis and nasosinusitis bioactive compounds owing to their antifungal and antiinflammatory activities [37,38]. In one study, the essential oils in Radix Bupleuri were extracted by steam distillation and solvent extraction and then analyzed by GC/MS; 78 peaks were identified. Among these peaks, the major volatile compounds were 3-methylbutanal (7.24%), pentanal (5.74%), hexanal (20.11%), furan-2-carbaldehyde (25.23%), and heptanal (12.07%) [39]. However, in another study, the results showed that E-2-heptanal, furan, 2-pentyl, and E-2-nonenal were some of the main compounds of the oil [40].

Pharmacological Effects
Radix Bupleuri exerts a great variety of pharmacological activities due to its complexbioactive compounds. An overview of the pharmacological studies on Radix Bupleuriis presented in detail in the following sections.

Anti-inflammatory Effects. Radix Bupleuri has been
widely used for the treatment of several types of chronic inflammatory diseases. The crude polysaccharides (80 mg/kg) isolated from the roots of Bupleurum chinense DC. significantly attenuated lung injury by inhibiting the level of myeloperoxidase (MPO), tumor necrosis factor-(TNF-), and serum nitric oxide (NO) [66]. Chun et al. reported that saikosaponins from Radix Bupleuri exhibited anti-inflammatory activity on inflammatory processes including inhibition of inflammatory exudation, capillary permeability, inflammatory mediators release, migration of white cells, connective tissue hyperplasia, and a variety of allergic inflammation [67]. Ma et al. was the first to show that saikosaponins exerted anti-inflammatory activity on paw edema mainly via regulating the nicotinate and nicotinamide metabolism and arachidonic acid metabolism [5]. Zhu et al. found that saikosaponin a (SSa) exhibited an inhibitory effect on proinflammatory cytokines in LPS-stimulated macrophages. The mechanism of these actions involved the regulation of MAPK and NF-B signals pathways [68]. In another study, SSa dose-dependently inhibited the production of ROS, TNF-, IL-8, COX-2, and iNOS in LPS-stimulated human umbilical endothelial cells (HUVECs) by upregulating of the LXR -ABCA1 signaling pathway [69]. Moreover, Lee et al. showed that saikosaponin c (SSc) was also shown to inhibited LPS-induced apoptosis in HUVECs via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation [70]. Zhao et al. showed that SSa also suppressed TNF-and IL-6 concentrations in the intestines of septic rats through the inhibition of the nucleotide-binding oligomerization domain 2 (NOD2)/NF-B signaling pathway [71]. Saikosaponin d (SSd) has been reported to inhibit PGE 2 production and intracellular free Ca 2+ concentration ([Ca 2+ ]i) in a concentration-dependent manner with an IC 50 value of 3 m in C6 rat glioma cells [72].
In addition, several studies showed that a wide range of Radix Bupleuri preparations also exhibited antiinflammatory effects in in vitro and in vivo model. Li et al. showed that Saireito and its active components (SSd) could suppress the proliferation of mesangial cells and expansion of the mesangial matrix in the rat glomerulonephritis model [73]. In experimental chronic pancreatitis rats model, "Chai-hu-shu-gan powder" exerted anti-inflammatory and antifibrotic effects by inhibiting the expression of nuclear factor-B (NF-B) and TNF-mRNA in the pancreas [74]. Furthermore, it also reduced the abnormally high plasma level of cholecystokinin, improved the gastric movement, and avoided nausea and flatulence [75]. In another experiment, a Chinese herbal formula called "RCM-101" (containing Flos magnoliae, Radix Bupleuri, Radix Glycyrrhizae, Radix Angelicae Sinensis, etc.) inhibited the NO production and iNOS protein expression in LPS-stimulated rat aorta and Raw 264.7 macrophages [76].

Anticancer Effects. The extracts and compounds of
Radix Bupleuri also possessed anticancer/antitumor effect. The acetone extract of Bupleurum scorzonerifolium could inhibit the proliferation of A549 human lung cancer cells in a dose-dependent manner via causing cell cycle arrest in the G2/M phase, increasing microtubule stabilization, suppressing telomerase activity, activating ERK 1/2 and caspase-3/9 in A549 cells [77][78][79]. Saponins isolated from Radix Bupleuri also exhibited significantly anti-proliferative activity in human non-small cell lung cancer A549 cells through Fas-dependent apoptotic pathway [80]. Su et al. found that the water extracts of Radix Bupleuri could enhance 5-fluorouracil-induced cytotoxicity in HepG2 hepatoma cells through cell arrest at the late G1/early S phase, while protecting normal blood lymphocytes [6]. SSd showed very potent activity against the HepG2 cell line with an IC 50 value of 12.5 mg/ml. The mechanism of cytotoxicity was  attributed to the induction of apoptosis through activation of caspase-3 and caspase-7, which subsequently resulted in poly-ADP-ribose polymerase (PARP) cleavage [81].  sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA), leading to the increase of intracellular calcium ion levels and activating the Ca 2+ /calmodulin-dependent kinase kinase--(CaMKK -) AMP-activated protein kinase-(AMPK-) mammalian target of rapamycin (mTOR) signaling cascade, endoplasmic reticulum (ER) stress, and unfolded protein responses (UPR) [43].
Several Chinese medicine preparations containing Radix Bupleuri also have been traditionally used in the treatment of tumors and cancer. The water extracts of "Long Dan Xie Gan Wan" exerted a significant growth inhibitory effect in HL60 and HT29 cancer cell lines, indicating that this formulation may possess some chemotherapeutic potential [83]. Treatment with "xiao-chai-hu decoction" exhibited a significantly lower incidence of hepatocellular carcinoma and reductions in cancer pain and tumor size. The underlying mechanism of the antitumor activities is based on stimulation of the reticuloendothelial system (RES) and is closely related of TNF production [84][85][86].

Antiviral Effects.
Wen et al. reported that the acetone extract of Radix Bupleuri possessed a significant antivirus effect on acute respiratory tract infections with H1N1 virus infection and suppressed influenza A virus-induced RANTES secretion in H1N1-infected A549 cells at a concentration of 100 and 200 g/ml, suggesting that Radix Bupleuri might be beneficial for the treatment of chronic inflammatory conditions followed by viral infection [10]. SSc has been reported to show effective anti-HBV activity through inhibiting DNA expression of HBsAg, HBeAg, and HBV [81]. Treatment with "xiao-chai-hu decoction" (20 g/ml, 3 days; 20 g/ml, 6 days) could inhibit the production of HBV ( < 0.0001) and the expression of HBeAg. Moreover, crude saponins of Bupleurum chinense DC. could inhibit the replication of HBV ( < 0.0001) [87]. Similarly, in another study, Yin et al. showed that SSd isolated from the MeOH extract of Bupleurum chinense DC. exhibited significant bioactivity in inhibiting DNA replication of HBV [88]. The antiviral activity of saikosaponins (a, b 2 , c, and d) and their mode of actin were examined. The results showed that all saikosaponins exerted antiviral activity on human coronavirus-229E at concentrations of 0.25-25 m, and the strongest activity was observed for saikosaponin b 2 with an IC 50 of 1.7 m. This mechanism might involve interference in the early stage of viral replication, such as absorption and penetration of the virus [89].

Antipyretic Effects.
The water extract of Radix Bupleuri was reported to exert its antipyretic effect on dry yeastinduced high fever rats. The mechanism is related to the adjustment of synthesis and exudation of cyclic adenosine monophosphate (cAMP) and arginine vasopressin (AVP) [90]. A novel in situ gel system for nasal delivery of the essential oil from Radix Bupleuri was prepared. The results suggested that Radix Bupleuri in situ gel can be more effective than the solution in the treatment of fever [91]. A similar study showed that the essential oil extracted from the herb exhibited dose-dependent antipyretic capacity on both fevered rabbits and rats [92].

Antibacterial Effects.
The ethanol extract of Bupleurum chinense DC. exerted a remarkable bacteriostatic effect on Gram-negative microorganism Helicobacter pylori. The bioactive minimum inhibitory concentration (MIC) value was 60 Mm [93]. Saikosaponins isolated from Radix Bupleuri have been reported to exhibit antibacterial activity, particularly against Pseudomonas aeruginosa and Listeria monocytogenes. The protective effect was attributed to the immunomodulatory action on macrophages [94]. "Chaihu injection" has also been tested for possible antimicrobial activity in vitro. The results demonstrated that mild inhibition of Staphylococcus aureus was observed but no effects were observed against Staphylococcus albus, Neisseria gonorrhoeae, Diplococcus pneumoniae, haemolytic Streptococcus, or Pseudomonas aeruginosa [95].

Hepatoprotective Effects.
The liver protective effects against CCl 4 induced liver injury were investigated after treatment of mice with raw and vinegar-baked Radix Bupleuri (5 g/kg/day) for 14 days. The results showed that both raw and processed Radix Bupleuri showed liver protective effects against CCl 4 induced liver injury, and the vinegar-baked Radix Bupleuri exerted better effects than that of raw Radix Bupleuri [96]. Pretreated with saikosaponins, especially SSa or SSd, showed remarkable inhibition of D-galactosamineinduced hepatic injury through decreasing the activity of glucose-6-phosphatase and NADPH-cytochrome C reductase and increasing 5 -nucleotidase activity [97]. Similarly, bupleurosides III, VI, IX, and XIII and saikosaponin b 3 isolated from Bupleurum scorzonerifolium Willd. were also found to exhibit protective effect on the D-galactosamineinduced cytotoxicity in primary cultured rat hepatocytes [98]. Further studies also demonstrated that the protective effects of saikosaponins isolated from Bupleurum chinense DC. could prevent hepatocyte injury through regulating intracellular calcium levels [99]. In a rat model with CCl 4 induced acute hepatic injury, the hepatic enzyme levels (GOT, GPT, and ALP) and the lipid peroxidation in the liver were significantly reduced by the administration of SSd [100]. Additionally, SSd significantly reduced collagen I deposition and alanine aminotransferase level on liver fibrosis rats and decreased the concentration of transforming growth factor 1 (TGF-1). Moreover, SSd was able to alleviate hepatocyte injury from oxidative stress. The effect of SSd on liver fibrosis may be related to its ability to reduce lipid peroxidation [101].

Immunomodulatory Effects. Yamakage et al. determined
the effects of Radix Bupleuri on spontaneous lymphatic vessel activity. The results indicated that Radix Bupleuri significantly increased the amplitude of spontaneous activity of lymphatic vessels in a concentration-dependent manner, and the mechanisms of this effect seem to be independent of endothelial function [102]. Eugenin ( ) and saikochrome A ( ) isolated from the MeOH extracts from Bupleurum scorzonerifolium possessed immunosuppressive effect on human peripheral blood T cells via inhibiting CD28-costimulated activation [62]. SSd (10 mg, intraperitoneally) significantly activated peritoneal macrophages in terms of enhancement of phagocytic activity, increased level of cellular lysosomal enzyme, and suppressed the response of plaque-forming cells to heterologous erythrocytes by stimulating T and B cells in a dose-dependent manner [103]. Moreover, SSd modulated lymphocyte activity through suppressing the T cell response and increasing the B cell response to different mitogens and the interleukin-(IL-) 2/IL-4 production through a receptor-bypassed pathway [41,104,105]. In another experiment, Wong et al. found that SSd was shown to inhibit OKT3/CD28-costimulated human T cell proliferation and PMA, PMA/ionomycin, and Con A-induced mouse T cell activation in vitro. The underlying mechanisms involved downregulation of NF-kB signaling by suppression of IKK and Akt activities [106].

Autophagic Effect.
Autophagy is a complex process in cells, which occurs through the formation of doublemembrane vesicles (autophagosomes), which are engulfed by cytoplasmic molecules. Then, the autophagosome fuses with the lysosomes, leading to degradation of long-lived proteins, aggregated proteins, and damaged organelles [107][108][109]. Moreover, autophagy might be triggered by hypoxia, nutritional deprivation, radiation, chemical drugs, and other stimulants [110]. Autophagy contributes to the pathogenesis of diverse diseases, such as neuronal degeneration, inflammatory bowel disease, aging, and cancer [111,112]. In the previous study, Law et al. demonstrated that the protective pharmacological effects of Radix Bupleuri might be attributed to its autophagy induction. The autophagic effect of Radix Bupleuri played an important role in relieving liver disease-related symptoms through anti-inflammatory, organ-protective, and aggregate removal functions. Furthermore, the anticancer effects of Radix Bupleuri could be attributed to its autophagy induction. Radix Bupleuri has been found to be an effective treatment against depression by regulating metabolite, hormone, and neurotransmitter levels via autophagy-mediated lipid metabolism [113].

Other Pharmacological Effects.
The effect of the ethanol extract from Radix Bupleuri on cytochrome 450 isoform activities using a six-drug cocktail approach was evaluated; the results demonstrated that Radix Bupleuri had strong induction activity on the CYP2E1, CYP2D6, and CYP3A4, which may lead to potential plant drug-drug interactions [114]. Radix Bupleuri was shown to be the inhibitor ofglucuronidase. The inhibition rate of Radix Bupleuri extracts RB1 (high molecular weight polysaccharides), RB2 (ethanol soluble/water insoluble component), RB3 (extracted by nbutanol, soluble in water), and RB4 (low molecular weight water soluble parts) on the activity of -glucuronidase was found to be 45.15%, 33.94%, 24.94%, and 34.54%, respectively [115]. In pentylenetetrazol (PTZ) induced epilepsy rats model, SSa isolated from Radix Bupleuri significantly reduced seizure severity and duration while it markedly elevated seizure latency and downregulated the cytokines expression of p-mTOR, p-70S6K, L-1 , and TNF-through inhibiting mTOR signaling pathway [116]. He et al. demonstrated that SSa obviously reduced lipoprotein uptake to block foam cell formation and the expression of LOX-1 and CD36, boosted cholesterol efflux, and the expression of ABCA1 and PPAR through inhibiting PI3K/Akt/NF-B/NLRP3 signaling pathway [117].
In another experiment, SSc exerted a potent effect on inducing human umbilical vein endothelial cells (HUVECs) viability and growth. Furthermore, SSc also induced endothelial cells migration and capillary tube formation. The underlying mechanisms might be related to the gene expression or activation of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and the p42/p44 mitogenactivated protein kinase (MAPK, ERK) [118]. In addition, SSc was shown to exhibit inhibitory activities against Alzheimer's disease (AD) via suppressing the secretion of A peptides and abnormal tau hyperphosphorylation-mediated microtubule depolymerization. Moreover, SSc suppressed A peptideinduced brain endothelial apoptosis, indicating that Ssc might be a novel therapeutic tool for treating human AD and other neurodegenerative diseases [119]. It was shown by Liu et al. for the first time that four polyacetylenes ( -) from Radix Bupleuri potently exhibited an antidepressant activity by inhibiting the reuptake of serotonin, norepinephrine, and dopamine. The mechanism might be mediated by increasing the level of monoamines, particularly 5-HT and NE [56]. Zhu et al. suggested that SSa and SSd exhibited the anthelmintic activity against Dactylogyrus spp. infecting goldfish. The effective concentration (EC 50 ) values for SSa and SSd were 1.46 and 0.74 mg −1 , respectively [120].

Summary of Pharmacological Effects. Radix
Bupleuri possesses a wide spectrum of pharmacological effects, including anti-inflammatory effect, anticancer effect, antiviral effect, antipyretic effect, antibacterial effect, hepatoprotective effect, and immunomodulatory effect (Table 3). Based on these pharmacological effects, we can conclude that the extracts and the compounds from this plant can prevent or treat certain diseases, such as cancer, fever, malaria, hepatitis, and AD. However, there is not enough systemic data of these chemical compounds and their pharmacological effects. Thus, in the future, the pharmacological effects and the possible molecular mechanisms of the pharmacological activities of Radix Bupleuri must be urgently explored on our modern understanding of these diseases' pathophysiologies.

Toxicology
Radix Bupleuri has been used for thousands of years as an important traditional herb in China. However, the toxic effects of Radix Bupleuri in clinical applications have been gradually reported. Several studies have found that the liver is the main organ affected by toxicity, particularly in longterm use. Major symptoms of liver injury induced by Radix Bupleuri included transaminase lifts, hepatitis, and jaundice. However, liver functions can return to normal levels after a specific period [121]. Radix Bupleuri has been reported to exhibit acute hepatitis and acute hepatic necrosis. The mean total daily dose was 18.0 ± 33.5 g, which was more than the Chinese Pharmacopoeia recommended range of 3 to 10 g [122]. Moreover, Radix Bupleuri had been implicated in multiple cases of acute hepatitis both as an ingredient alone and within a particular formulation "Xiao-Chai-Hu-Tang" (also known as Syo-Saiko-To in Japanese) [123]. Lee et al. demonstrated that two Chinese herbal products containing Radix Bupleuri might increase their risks of liver injury in HBV-infected patients. However, further mechanistic research on the hepatotoxicity of Radix Bupleuri in the presence of HBV infection is warranted [124]. In addition, the essential oil of Radix Bupleuri induced acute hepatotoxicity with asynchronous state, higher heart rate, and fast breathing [125]. The total saponins isolated from Radix Bupleuri could also cause evidently liver damage in dose-dependent manner manifested as hepatocyte organic lesion and liver function changes, as well as hepatocyte death [126].

Pharmacokinetics
A selective and sensitive LC-MS/MS method was developed and validated for simultaneous determination of SSa, b 2 , c, and d in rat plasma afteroral administration of the ethanolwater (50 : 50, v/v) extract of Radix Bupleuri for the first time. The results demonstrated that SSa, c, and d were absorbed rapidly with max less than 30 min [127]. In another pharmacokinetics experiment of rats, Liu et al. was the first to develop an UPLC-PDA-MS method to determine the pharmacokinetics of four polyacetylenes after i.g. administration of 95% ethanol extract of Radix Bupleuri. The results showed that compounds and were not detected in rat serum, whereas compounds and exerted a fast distribution phase followed by a relatively slow elimination phase ( 1/2 , 4-7 h) [56].

Future Perspectives and Conclusions
In traditional Chinese medicine, Radix Bupleuri has long been used regulate the exterior and interior metabolisms, disperse evil heat from superficies, sooth the liver, and promote yang and qi. It has been widely used to treat various diseases in China, Japan, Korea, and other Asian countries for many centuries. A total of 74 compounds including essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols have been isolated and identified from Radix Bupleuri . Pharmacological studies have revealed that Radix Bupleuri possesses a variety of biological effects, including anti-inflammatory, anticancer, antiviral, antipyretic, antibacterial, antiobesity, immunomodulatory, hepatoprotective, neuroprotective, and autophagic effects . However, there are some aspects that need to be further investigated.
Radix Bupleuri is an ingredient of many patent medicines or prescriptions. Although modern experiments have confirmed that this drug alone exhibits multiple pharmacological activities, it is important to investigate the molecular mechanisms of Radix Bupleuri combined with other herbs based on traditional uses. Furthermore, the pharmacological effects of only a few of the ingredients, such as saikosaponins, flavonoids, and the essential oils, have been investigated. Some polyacetylenes, lignans, and sterols have not been sufficiently researched in terms of their pharmacological effects. Radix Bupleuri shows both hepatoprotection and  In vitro [89] Antipyretic effect Dry yeast-induced high fever rats Adjusts the synthesis and exudation of cAMP and AVP

Water extracts
In vivo [90] Turpentine-induced fever rabbits Decreases body temperature The essential oil In vivo [91] Turpentine-induced fever rabbits and rats

Decreases body temperature
The essential oil In vivo [92] Antibacterial effect  In vivo [113] hepatotoxicity, which appears to be contradictory. This phenomenon is similar to that of Polygonum multiflorum Thunb. [128]. Based on the literature, the main reasons are likely the administration dosage and delivery time. High doses and long-term drug delivery are more likely to result in liver toxicity, whereas low doses and short-term drug delivery might result in liver protection. Therefore, this issue needs further study.
In conclusion, this review summarized the traditional uses, botany, phytochemistry, pharmacology, and toxicology of Radix Bupleuri. Moreover, it has provided a new foundation for further research on its mechanism of action and the development of better therapeutic agents employing Radix Bupleuri in the future. It is anticipated that the comprehensive and detailed research on toxicity, pharmacodynamics, pharmacokinetics, and molecular mechanism are necessary to be explored to develop its bioactive compounds as effective drugs.

Conflicts of Interest
The authors have declared that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Fude Yang and Xiaoxv Dong contributed equally to this work and share first authorship.