Highly sensitive indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody against saikosaponin b2 for quality control of Kampo medicines containing Bupleuri radix.

Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix-containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95-125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph-mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix.

Saikosaponin b2 inhibits tumor angiogenesis in liver cancer via down­regulation of VEGF/ERK/HIF­1α signaling.

Saikosaponin b2 (SSb2) is an active component of Radix Bupleuri, which is commonly used in traditional Chinese medicine for defervescence and liver protection. In the present study, it was demonstrated that SSb2 exhibited potent antitumor activity by inhibiting tumor angiogenesis in vivo and in vitro. As measured by tumor weight and measures of immune function such as thymus index, spleen index and white blood cell count, SSb2 inhibited tumor growth, with low immunotoxicity, in H22 tumor­bearing mice. Furthermore, proliferation and migration of HepG2 liver cancer cells was inhibited following SSb2 treatment, which demonstrated SSb2's antitumor effect. The angiogenesis marker CD34 was downregulated in the SSb2­treated tumor samples, which suggested the antiangiogenic activity of SSb2. Furthermore, the chick chorioallantoic membrane assay demonstrated the potent inhibitory effect of SSb2 on basic fibroblast growth factor­induced angiogenesis. In vitro, SSb2 significantly inhibited numerous stages of angiogenesis, including the proliferation, migration and invasion of human umbilical vein endothelial cells. Further mechanistic studies demonstrated that SSb2 treatment reduced the levels of key proteins involved in angiogenesis, including vascular endothelial growth factor (VEGF), phosphorylated ERK1/2, hypoxia­inducible factor (HIF)­1α, MMP2 and MMP9 in H22 tumor­bearing mice, which supported the HepG2 liver cancer cell results. Overall, SSb2 effectively inhibited angiogenesis via the VEGF/ERK/HIF­1α signal pathway and may serve as a promising natural agent for liver cancer treatment.

[Establishment and application of quality evaluation method for Xiaochaihu Granules based on calibrator samples].

With reference to the production process documented in Chinese Pharmacopoeia, this paper prepared the calibrator samples of Xiaochaihu Granules from multiple batches and established a method for fingerprint analysis and content determination that could be used to evaluate Xiaochaihu Granules available in market. Multiple batches of Chinese herbal pieces contained in Xiaochaihu Granules were collected for preparing the calibrator samples according to the process in Chinese Pharmacopoeia. Following the establishment of fingerprints for calibrator samples by UHPLC, the method for determining the contents of saikosaponin B2, saikosaponin B1, baicalin, wogonoside, baicalein, liquiritin, glycyrrhizin G2 and glycyrrhizic acid in Xiaochaihu Granules was established. The experimental results showed that the fingerprints of calibrator samples had 26 common peaks, covering the chemical compounds of main herbs Bupleuri Radix, Scutellariae Radix, Changii Radix, Glycyrrhizae Radix et Rhizoma, and Rhizoma Zingiberis Recens. The similarity of fingerprints for 47 batches of Xiaochaihu Granules from 31 companies with the calibrator sample fingerprint ranged from 0.74 to 0.99, indicating good applicability of the established fingerprint. The contents of main components baicalin, saikosaponin B2, and glycyrrhizic acid in Xiaochaihu Granules were within the ranges of 22.917-49.108 mg per bag(RSD 19%), 0.28-2.19 mg per bag(RSD 62%), and 0.897-6.541 mg per bag(RSD 41%), respectively. The quality difference in saikosaponin B2, and glycyrrhizic acid among different manufacturers was significant. The fingerprint analysis and content determination method for calibrator samples of Xiaochaihu Granules prepared according to the production process in Chinese Pharmacopoeia has been proved suitable for evaluating the quality of Xiaochaihu Granules from different manufacturers. Saikosaponin B2, glycyrrhizic acid, and liquiritin should be added as content control indicators for Xiaochaihu Granules, aiming to further improve the product quality.

Establishment and application of quality evaluation method for Xiaochaihu Granules based on calibrator samples / 中国中药杂志

With reference to the production process documented in Chinese Pharmacopoeia, this paper prepared the calibrator samples of Xiaochaihu Granules from multiple batches and established a method for fingerprint analysis and content determination that could be used to evaluate Xiaochaihu Granules available in market. Multiple batches of Chinese herbal pieces contained in Xiaochaihu Granules were collected for preparing the calibrator samples according to the process in Chinese Pharmacopoeia. Following the establishment of fingerprints for calibrator samples by UHPLC, the method for determining the contents of saikosaponin B2, saikosaponin B1, baicalin, wogonoside, baicalein, liquiritin, glycyrrhizin G2 and glycyrrhizic acid in Xiaochaihu Granules was established. The experimental results showed that the fingerprints of calibrator samples had 26 common peaks, covering the chemical compounds of main herbs Bupleuri Radix, Scutellariae Radix, Changii Radix, Glycyrrhizae Radix et Rhizoma, and Rhizoma Zingiberis Recens. The similarity of fingerprints for 47 batches of Xiaochaihu Granules from 31 companies with the calibrator sample fingerprint ranged from 0.74 to 0.99, indicating good applicability of the established fingerprint. The contents of main components baicalin, saikosaponin B2, and glycyrrhizic acid in Xiaochaihu Granules were within the ranges of 22.917-49.108 mg per bag(RSD 19%), 0.28-2.19 mg per bag(RSD 62%), and 0.897-6.541 mg per bag(RSD 41%), respectively. The quality difference in saikosaponin B2, and glycyrrhizic acid among different manufacturers was significant. The fingerprint analysis and content determination method for calibrator samples of Xiaochaihu Granules prepared according to the production process in Chinese Pharmacopoeia has been proved suitable for evaluating the quality of Xiaochaihu Granules from different manufacturers. Saikosaponin B2, glycyrrhizic acid, and liquiritin should be added as content control indicators for Xiaochaihu Granules, aiming to further improve the product quality.

The qualitative and quantitative assessment of xiaochaihu granules based on e-eye, e-nose, e-tongue and chemometrics.

Xiaochaihu granules (XCHG), a famous Chinese patent medicine with high sales, have more than 100 approved number by China Food and Drug Administration (CFDA). Therefore, it is important to evaluate the quality of XCHG from different pharmaceutical companies. The data fusion of electronic eye (e-eye), electronic nose (e-nose) and electronic tongue (e-tongue) combined with chemometrics were applied for qualitative identification and quantitative prediction of XCHG quality. Firstly, main chemical constituents, such as saikosaponin b2, baicalin and glycyrrhizin were quantified with ultra-high-performance liquid chromatography (UHPLC). Secondly, the characteristic features of odor, color, and taste of XCHG were measured by e-nose, e-eye and e-tongue, and the Pearson correlation between constituents and e-signals was analyzed. Thirdly, partial least squares discrimination analysis (PLS-DA) of e-eye, e-nose and e-tongue were classified by the hierarchical clustering analysis (HCA) results of the main constituents of XCHG separately. Finally, partial least-squares regression (PLSR) was used to build the prediction model between components and data fusion of e-eye, e-nose and e-tongue. The results showed that saikosaponin b2, baicalin and glycyrrhizin were the three main components in XCHG samples. in which saikosaponin b2 ranged from 0.280 to 2.186 mg (relative standard deviation (RSD), 62.10 %), baicalin range from 25.883 mg to 49.108 mg (RSD, 16.64 %), and glycyrrhizin ranged from 0.897 mg to 6.052 mg (RSD, 40.32 %) of 31 batches of XCHG in each bag. Pearson correlation results showed that the main constituents were related to the core e-signals of XCHG, such as Eab, bitterness and R2 (odor sensitive to nitrogen oxide). Data fusion of e-eye, e-nose and e-tongue with main constitutes of XCHG using the PLSR model showed that the root mean square error (RMSE) values were 0.320 and 0.090 for saikosaponin b2 and licoricesaponin G2 (P < 0.000). The saikosaponin b2 and licoricesaponin G2 contents in XCHG could be predicted with integrated data of e-nose, e-eye, and e-tongue using the PLSR model.

Saikosaponin B2 attenuates kidney fibrosis via inhibiting the Hedgehog Pathway.

BACKGROUND: Renal interstitial fibrosis is a common pathway through which chronic kidney disease progresses to end-stage renal disease. There are currently no effective drugs available to treat kidney fibrosis, so traditional medicine is likely to be a candidate. The therapeutic potential of saikosaponin B2 (SSB2), a biologically active ingredient of Radix Bupleuri, on renal fibrosis has not been reported. METHODS: A unilateral ureteral obstruction (UUO) model was conducted to induce renal interstitial fibrosis in mice. SSB2's effect was valuated by histological staining and exploring the changes in expression of relative proteins and mRNAs. A conditional medium containing sonic hedgehog variant protein stimulating normal rat kidney interstitial fibroblast cells (NRK-49F) was used in an in vitro model to determine the possible mechanism. The molecular target of SSB2 was verified using several mutation plasmids. RESULTS: SSB2 administration reduced kidney injury and alleviated interstitial fibrosis by decreasing excessive accumulation of extracellular matrix components in UUO mice. It could also reduce the expression of α-SMA, fibronectin and Gli1, a crucial molecule of the hedgehog (Hh) signaling pathway both in vivo and in vitro. In NIH-3T3 cells simulated by conditional medium containing sonic hedgehog variant protein, SSB2 showed the ability to decrease the expression of Gli1 and Ptch1 mRNA. Using a dual-luciferase reporter assay, SSB2 suppressed the Gli-luciferase reporter activity in NIH-3T3 cells, and the IC50 was 0.49 µM, but had no effect on the TNF-α/NF-κB and Wnt/ß-catenin signaling pathways, indicating the inhibition selectivity on the Hh signaling pathway. Furthermore, SSB2 failed to inhibit the Hh pathway activity evoked by ectopic expression of Gli2ΔN and Smo D473H, suggesting that SSB2 might potentially act on smoothened receptors. CONCLUSION: SSB2 could attenuate renal fibrosis and decrease fibroblast activation by inhibiting the Hh signaling pathway.

[Effects of saikosaponin b_2 on inflammation and energy metabolism in mice with acute liver injury induced by LPS/GalN].

To study the effects of saikosaponin b2( SS-b2) on inflammatory factors and energy metabolism against lipopolysaccharide/galactosamine( LPS/Gal N) induced acute liver injury in mice. Mice were randomly divided into normal group( equal amount of normal saline),model group( 100 g·kg~(-1) LPS and 400 mg·kg~(-1) Gal N),low,medium,high dose group of SS-b2( SS-b25,10,20 mg·kg~(-1)·d-1) and positive control group( dexamethasone,10 mg·kg~(-1)). All of the groups except for the normal group were treated with LPS/Gal N though intraperitoneally injection to establish the acute liver injury model. The organ indexes were calculated. The levels of serum transaminases( ALT and AST) and the activities of ATPase( Na+-K+-ATPase,Ca2+-Mg2+-ATPase) in liver were detected. The activity of tumor necrosis factor-α( TNF-α),interleukin-1ß( IL-1ß) and interleukin-6( IL-6) were determined by the enzyme-linked immunosorbent assay( ELISA). The contents of lactate dehydrogenase( LDH) in liver were determined by micro-enzyme method. HE staining was used to observe the histopathological changes of the liver. Histochemical method was used to investigate the protein expression of liver lactate dehydrogenase-A( LDH-A). The protein expressions of Sirt-6 and NF-κB in the liver were detected by Western blot. According to the results,compared with the model group,there were significant changes in organ indexes in the high-dose group of SS-b2( P<0. 05). The level of ALT,AST,TNF-α,IL-1ß,IL-6 and the activities of LDH in serum of mice with liver injury were significantly reduced in the medium and high dose groups of SS-b2( P<0. 01). With the increase of the concentration of SS-b2,the range of hepatic lesions and the damage in mice decreased. The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in liver of mice were significantly enhanced in each dose group( P<0. 01). The expression of NF-κB in liver tissues was significantly down-regulated in the medium and high dose group( P<0. 01). Meanwhile,the expression of Sirt-6 protein in the liver of mice with acute liver injury was significantly increased in each dose group( P<0. 01).In summary,SS-b2 has a significant protective effect on LPS/Gal N-induced acute liver injury in mice,which may be related to the down-regulation of NF-κB protein expression and up-regulation of Sirt-6 protein expression to improve inflammatory injury and energy metabolism.

Saikosaponin b2 enhances the hepatotargeting effect of anticancer drugs through inhibition of multidrug resistance-associated drug transporters.

AIMS: Vinegar-baked Radix Bupleuri (VBRB) potentiates the activity of anticancer drugs in the liver by increasing their hepatic distribution. However, this phenomenon may be associated with drug transporters. We investigated the effect of saikosaponin b2 (SSb2; the main component of VBRB) on the activity and expression of different drug transporters in both normal cells and those that overexpress the transporter. MAIN METHODS: The activities of transporters were analyzed by concentration of their cellular substrates. Concentrations of colchicine (substrate of Pgp and MRP1) and cisplatin (substrate of OCT2 and MRP2) were determined by high-performance liquid chromatography (HPLC). The concentration of rhodamine B was determined by flow cytometry. The expression of transporter gene and protein were determined by qRT-PCR and Western blotting analysis. KEY FINDINGS: SSb2 increased colchicine efflux in HEK293 cells by primarily increasing Mrp1 activity, independent of gene and protein expression. SSb2 enhanced Mrp2 function and increased cisplatin efflux in BRL3A cells by upregulating Mrp2 gene expression, with a marginal effect on Pgp in normal cells. SSb2 increased OCT2 activity in OCT2-HEK293 cells by increasing the expression of OCT2 protein and mRNA; however, SSb2 inhibited MRP2 activity in MRP2-HEK293 cells by decreasing MRP2 protein expression, and decreased Pgp and MRP1 activity in Pgp- and MRP1-HEK293 cells. SIGNIFICANCE: SSb2 might potentially be the key active component of VBRB that enhances the hepatotargeting of anticancer drugs through the inhibition of multidrug resistance-associated drug transporters (Pgp, MRP1, and MRP2) in an environment-dependent manner.