Roots Of Bupleurum Falcatum Research Articles

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 = 1.95-125ng/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.

DA-9805, a Herbal Mixture, Restores Motor Manifestations in 6-Hydroxydopamine-induced Parkinson's Disease Mouse Model by Regulating Striatal Dopamine and Acetylcholine Levels.

Loss of dopamine (DA) is one of the primary features of Parkinson’s disease (PD); however, imbalances of non-dopaminergic neurotransmitters significantly contribute to the disabilities noted in advanced PD patients. DA-9805 is the ethanolic extraction of the root bark of Paeonia × suffruticosa Andrews (Paeoniaceae), the root of Angelica dahurica (Hoffm.) Benth. and Hook.f. ex Franch. and Sav. (Apiaceae) and the root of Bupleurum falcatum L. (Apiaceae), which have been widely utilized as an enhancer of motor function in East Asia. This study aimed to investigate whether DA-9805 modified motor dysfunctions and imbalances associated with DA and other neurotransmitters in a 6-hydroxydopamine-induced PD mouse. We confirmed the expressions of proteins related with neurotransmissions in the striatum. In addition, we measured the striatal neurotransmitters using HPLC and analyzed their correlation. DA-9805 significantly improved motor impairments and restored the altered levels of neurotransmitters in the striatum. Moreover, DA-9805 improved the altered expressions of tyrosine hydroxylase (TH), DA transporter, and choline acetyltransferase (ChAT) in the ipsilateral part of mouse striatum or SNpc, which implies the neuroprotection. We also found that the level of striatal acetylcholine (Ach) has the moderate negative correlation with motor functions and TH expression in the SNpc. This study indicates that DA-9805 restores motor dysfunctions by normalizing the increased levels of striatal Ach via modulating DA transmission and ChAT expressions as well as its neuroprotective effects.

Antiviral effect of saikosaponin B2 in combination with daclatasvir on NS5A resistance-associated substitutions of hepatitis C virus.

Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The rapid progress in the development of direct-acting antivirals has greatly elevated the cure rate to ≥95% in recent years. However, the high cost of treatment is not affordable to patients in some countries, necessitating the development of less expensive treatment. We adopted a cell culture-derived HCV system to screen a library of the pure compounds extracted from herbs deposited in the chemical bank of the National Research Institute of Chinese Medicine, Taiwan. We found that saikosaponin B2 inhibited viral entry, replication, and translation. Saikosaponin B2 is a plant glycoside and a component of xiao-chai-hu-tang, a traditional Chinese herbal medicine extracted from the roots of Bupleurum falcatum. It also inhibited daclatasvir-resistant mutant strains of HCV, especially in combination with daclatasvir. Our results may aid the development of a new combination therapy useful for patients with HCV who are intolerant or refractory to the currently available medications, including pegylated interferon and direct-acting antiviral agents.

Pharmacokinetics and Brain Distribution of the Active Components of DA-9805, Saikosaponin A, Paeonol, and Imperatorin in Rats.

DA-9805 is a botanical anti-Parkinson’s drug candidate formulated from ethanol extracts of the root of Bupleurum falcatum, the root cortex of Paeonia suffruticosa, and the root of Angelica dahurica. The pharmacokinetics (PKs) and brain distribution of active/representative ingredients of DA-9805, Saikosaponin a (SSa; 1.1–4.6 mg/kg), Paeonol (PA; 14.8–59.2 mg/kg), and Imperatorin (IMP; 1.4–11.5 mg/kg) were evaluated following the intravenous or oral administration of each pure component and the equivalent dose of DA-9805 in rats. All three components had greater dose-normalized areas under the plasma concentration-time curve (AUC) and slower clearance with higher doses, following intravenous administration. By contrast, dose-proportional AUC values of SSa, PA, and IMP were observed following the oral administration of each pure component (with the exception of IMP at the highest dose) or DA-9805. Compared to oral administration of each pure compound, DA-9805 administration showed an increase in the AUC of SSa (by 96.1–163%) and PA (by 155–164%), possibly due to inhibition of their metabolism by IMP or other component(s) in DA-9805. A delay in the absorption of PA and IMP was observed when they were administered as DA-9805. All three components of DA-9805 showed greater binding values in brain homogenates than in plasma, possibly explaining why the brain-to-plasma ratios were greater than unity following multiple oral administrations of DA-9805. By contrast, their levels in cerebrospinal fluid were negligible. Our results further our understanding of the comprehensive PK characteristics of SSa, PA, and IMP in rats and the comparative PKs between each pure component and DA-9805.

Simultaneous determination of saikosaponin a, paeonol, and imperatorin, components of DA-9805, in rat plasma by LC–MS/MS and application to a pharmacokinetic study

DA-9805 is a new botanical antiparkinson drug candidate formulated using an ethanolic extract of the root of Bupleurum falcatum, the root cortex of Paeonia suffruticosa, and the root of Angelica dahurica. In this study, a sensitive and rapid LC-MS/MS method was developed to simultaneously determine, saikosaponin a, paeonol, and imperatorin, three active/representative ingredients of DA-9805, in rat plasma. Plasma was extracted by mixture of ethyl acetate and methyl tertiary butyl ether. Chromatographic separation was carried out using a C18 column and a gradient elution of mobile phases consisting of 5mM formic acid in water and acetonitrile. Total chromatographic run time was 10.5min. Multiple reaction monitoring mode was used for mass spectrometry; the transitions were m/z 779.5→617.2 for saikosaponin a in negative-ion mode, m/z 167→149 for paeonol and m/z 271.1→203 for imperatorin in positive-ion mode. Calibration curves were constructed in the range of 0.5-1000ng/mL for saikosaponin a, 20-10000ng/mL for paeonol, and 0.2-1000ng/mL for imperatorin. All the validation data, including the selectivity, linearity, precision, accuracy, recovery, matrix effect, and stability satisfied the acceptance requirements. The method was successfully applied in a pharmacokinetic study of saikosaponin a, paeonol, and imperatorin following oral administration of DA-9805.

성조숙증 치료에 대한 임상 연구 동향 -최근 중의학 저널을 중심으로-

Objectives The purpose of this study is to analyze recent clinical studies on treatment for precocious puberty. Methods 28 clinical studies from January 2009 to December 2016 about precocious puberty from the China Academic Journal (CAJ), China National Knowledge Infrastructure (CNKI), were selected and reviewed: 22 case control studies and 6 case series. Results The main traditional Chinese medicine (TCM) treatment was the herbal decoctions and granules. The frequently used herbs were root of Bupleurum falcatum (柴胡), fruit of Prunella vulgaris var. aleutica (夏枯草), root stem of Anemarrhena asphodeloides (知母), dried fungus nucleus of Poria cocos (茯苓), Rehmannia glutinosa var. purpurea (生地黃), peony Paeonia suffruticosa Andrews (牧丹皮), bark of Phellodendron amurense (黃柏), roots of a plant Paeoniae lactiflora (白芍藥). On the other hand, the main western medicine (WM) for precocious puberty was Gonadotropin-releasing hormone agonist (GnRHa). Total effective rate of the TCM group was 62.96-100%, that of the WM group was 36.6-93.3% and that of the TCM-WM group was 77.8-93.75%. Conclusions Traditional Chinese medicine has been shown as an effective treatment for precocious puberty. These research results can be utilized in other clinical studies and in treatment of precocious puberty.

Triterpene saponin constituents from roots of Bupleurum falcatum: Hepatoprotective effects on D-galactosamine-induced cell damage

Triterpene saponin constituents from roots of Bupleurum falcatum: Hepatoprotective effects on D-galactosamine-induced cell damage

섭식장애의 치료에 대한 임상연구 동향 -중의학 논문을 중심으로-

Objectives: This study was performed to review the research trends in the treatment of eating disorder in traditional Chinese medicine (TCM). Methods: The CNKI database (China National Knowledge Infrastructure) was searched under the key words &#x2018;Eating Disorder&#x2019;, &#x2018;Anorexia Nervosa&#x2019;, and &#x2018;Bulimia Nervosa&#x2019;. Results: Thirteen articles were selected. These included 2 studies on bulimia nervosa, and 11 studies on anorexia nervosa. According to study design, studies were classified into 1 randomized controlled trial, 2 single group before-and-after studies, and 8 case reports. According to therapy method, they included 11 herbal medicine studies, and 2 complex treatment studies. The most common pattern identification was &#x2018;liver qi depression&#x2019; (肝氣鬱結), and the most used herb was &#x2018;Root of Bupleurum falcatum&#x2019; (柴胡). Most of the studies showed effective results. Conclusions: The results of our study indicated that TCM clinical studies on eating disorder were being conducted more actively than in Korean medicine. Korean medical treatment might be effective to relieve eating disorder symptoms. In the future, more scientifically designed clinical studies should be performed to prove the effectiveness of traditional Korean medicine treatment on eating disorder.

A potential therapeutic effect of saikosaponin C as a novel dual-target anti-Alzheimer agent.

Alzheimer's disease (AD) is a chronic neurodegenerative disease and the risk of developing it increases with advancing age. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the main bioactive components produced by the traditional Chinese herb, radix bupleuri, the root of Bupleurum falcatum, against AD in various neuronal models. Interestingly, we found that SSc has dual effects on AD by targeting amyloid beta (Aβ) and tau, two key proteins in AD. SSc significantly suppressed the release of both Aβ peptides 1-40 and 1-42 into cell culture supernatants, though it does not affect BACE1 activity and expression. SSc also inhibited abnormal tau phosphorylation at multiple AD-related residues. Moreover, SSc seems to have beneficial effects on cellular tau function; it accelerated nerve growth factor-mediated neurite outgrowth and increased the assembly of microtubules. In addition, SSc increased synaptic marker proteins such as synaptophysin and PSD-95. Considering its various biological activities, our results suggest that SSc might be a novel therapeutic tool for treating human AD and other neurodegenerative diseases. Tau and amyloid beta are two key features in Alzheimer's disease. Saikosaponin C, an active component of Bupleuri Radix, inhibits abnormal tau phosphorylation and amyloid beta production, thereby promoting synaptic integrity. Saikosaponin C also prevents amyloid beta-induced apoptosis in brain vascular endothelial cells. Therefore, Saikosaponin C may provide a new therapeutic strategy for treatment of neurodegenerative diseases, including Alzheimer's disease.

Ethanol extract of Bupleurum falcatum and saikosaponins inhibit neuroinflammation via inhibition of NF-κB

Ethnopharmacological relevanceThe root of Bupleurum falcatum L. (BF) has been used in traditional Korean and Chinese medicines for over 2000 years to treat infections, fever, and chronic liver diseases. Among the many active compounds in BF ethanol extract (BFE), saikosaponins exert pharmacological activities including anti-inflammatory effects. Activated microglial cells release a variety of pro-inflammatory substances, leading to neuronal cell death and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The aim of the present study was to investigate the mechanism of the anti-neuroinflammatory effects of BFE using lipopolysaccharide (LPS)-stimulated microglial cells and LPS-intraperitoneal injected C57BL/6 mice. Materials and methodsDried roots of BF were extracted with 70% ethanol (tenfold volume) on a stirring plate for 24h at room temperature to prepare BFE. Pure saikosaponins (SB3, SB4, and SD) were prepared by solvent extraction and column chromatography fractionation. BV2 murine microglial cells were treated with BFE or saikosaponins for 4h and stimulated with LPS. Generation of nitric oxide (NO), inflammatory cytokines, and reactive oxygen species (ROS) from activated microglial cells were monitored. The effects of BFE on NF-κB activation were determined using RT-PCR, reporter assay, and immunostaining. The in vivo effects of BFE were also assessed by immunohistochemical staining of tissue sections from LPS-injected mouse brains. ResultsTreatment with BFE or saikosaponins dose-dependently attenuated LPS-induced production of NO, iNOS mRNA, and ROS by 30–50%. They reduced LPS-mediated increases in the mRNA levels of IL-6, IL-1β, and TNF-α by approximately 30–70% without affecting cell viability, and decreased LPS-mediated NF-κB activity via reducing p65/RELA mRNA, transcriptional activity, and nuclear localization of NF-κB. BFE also reduced LPS-induced activation of microglia and astrocytes in the hippocampus and substantia nigra of LPS-injected mice. ConclusionOur data suggest that BFE may be effective for reducing neuroinflammation-mediated neurodegeneration through suppressing NF-κB-mediated inflammatory pathways.

Saikosaponin D Isolated from Bupleurum falcatum Inhibits Selectin-Mediated Cell Adhesion

Three saikosaponins were isolated from the MeOH extract of the roots of Bupleurum falcatum L.: saikosaponins B3 (1); B4 (2); and D (3). Of the three, compound 3 inhibited the interaction of selectins (E, L, and P) and THP-1 cells with IC50 values of 1.8, 3.0 and 4.3 µM, respectively. Also, the aglycone structure 4 of compound 3 showed moderate inhibitory activity on L-selectin-mediated cell adhesion. From these results, we suspect that compound 3 isolated from Bupleurum falcatum roots would be a good candidate for therapeutic strategies to treat inflammation.

Saikosaponin-D attenuates heat stress-induced oxidative damage in LLC-PK1 cells by increasing the expression of anti-oxidant enzymes and HSP72.

Heat stress stimulates the production of reactive oxygen species (ROS), which cause oxidative damage in the kidney. This study clarifies the mechanism by which saikosaponin-d (SSd), which is extracted from the roots of Bupleurum falcatum L, protects heat-stressed pig kidney proximal tubular (LLC-PK1) cells against oxidative damage. SSd alone is not cytotoxic at concentrations of 1 or 3 μg/mL as demonstrated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To assess the effects of SSd on heat stress-induced cellular damage, LLC-PK1 cells were pretreated with various concentrations of SSd, heat stressed at 42°C for 1 h, and then returned to 37°C for 9 h. DNA ladder and MTT assays demonstrated that SSd helped to prevent heat stress-induced cellular damage when compared to untreated cells. Additionally, pretreatment with SSd increased the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) but decreased the concentration of malondialdehyde (MDA) in a dose-dependent manner when compared to controls. Furthermore, real-time PCR and Western blot analysis demonstrated that SSd significantly increased the expression of copper and zinc superoxide dismutase (SOD-1), CAT, GPx-1 and heat shock protein 72 (HSP72) at both the mRNA and protein levels. In conclusion, these results are the first to demonstrate that SSd ameliorates heat stress-induced oxidative damage by modulating the activity of anti-oxidant enzymes and HSP72 in LLC-PK1 cells.

Fast and Sensitive LC-DAD-ESI/MS Method for Analysis of Saikosaponins c, a, and d from the Roots of Bupleurum Falcatum (Sandaochaihu)

In the present study, we developed a liquid chromatography-diode array detector-electrospray ionization/mass spectrometric (LC-DAD-ESI/MS) method for analysis of saikosaponins in Bupleurum falcatum. The LC method employed a ZORBAX SB-Aq analytical column (150 × 4.6 mm i.d., 5 μm) at a flow rate of 0.8 mL/min coupled with a diode array detector at 204 nm. A step gradient of acetonitrile-water (v/v) containing 0.5% formic acid from 30 to 70% was applied, leading to a sample analysis time of 30 min. The ESI-MS was carried out in positive and negative modes from 500 to 1,500 m/z. Saikosaponins c, a, and d gave strong sodium adducts at m/z 949.6, 803.5 and 803.6, respectively, in positive mode. The data indicate that the present LC-DAD-ESI/MS assay is an effective method for the determination of saikosaponins c, a and d from the roots of Bupleurum falcatum.

Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in Bupleurum falcatum

The root of Bupleurum falcatum L. (Apiaceae) has long been one of the most important traditional herbal medicines in Asian countries. A group of triterpene saponins (saikosaponins) are the major constituents of this plant. Squalene synthase (SS) may play a regulatory role in directing triterpene intermediates and sterol pathways. Here, we investigated the regulatory role of the squalene synthase (BfSS1) gene in the biosynthesis of phytosterol and triterpene in B. falcatum. BfSS1 mRNA accumulated ubiquitously in plant organs and markedly increased in roots after treatment with methyl jasmonate (MeJA), ABA and ethephon. Transgenic B. falcatum constructs overexpressing BfSS1 in the sense and antisense orientations were assembled using the Agrobacterium-mediated method. Transgenic roots overexpressing BfSS1 in the sense orientation resulted in enhanced production of both phytosterol and saikosaponins. Overexpression of the BfSS1 gene in the sense orientation increased the mRNA accumulation of downstream genes such as squalene epoxidase and cycloartenol synthase but unexpectedly decreased the mRNA levels of β-amyrin synthase (β-AS), a triterpene synthase mRNA. MeJA treatment of wild-type roots strongly stimulated β-AS mRNA accumulation and saikosaponin production but suppressed phytosterol production. MeJA treatment of transgenic roots overexpressing BfSS1 in the sense orientation failed to stimulate β-AS mRNA accumulation but still enhanced saikosaponin and phytosterol production. These results indicate that overexpression of BfSS1 in B. falcatum regulates more powerfully the downstream genes than elicitor (MeJA) treatment in triterpene and phytosterol biosynthesis.

Inhibition of VLA-4/VCAM-1-mediated Cell Adhesion by Triterpenoid Saponins from Bupleurum falcatum L

Discovery and isolation of compounds capable of blocking the interactions between VCAM-1 and VLA-4, a major pair of adhesion molecules contributing to the different steps of leukocyte migration across the endothelium in inflammatory responses, has been a major goal of this lab. Through bioactivity-guided fractionation, five saikosaponins were subsequently isolated from the methanol extracts of the roots of Bupleurum falcatum L. Their structures were elucidated by spectroscopic analysis (<TEX>$^1H-$</TEX>, <TEX>$^{13}C$</TEX>-NMR and 2D-NMR), as follows, saikosaponins: A (1); D (2); C (3); B3 (4); B4 (5). Compounds 1 and 2 inhibited interaction of sVCAM-1 and VLA-4 of THP-1 cells with respective <TEX>$IC_{50}$</TEX> values of 7.8 and 1.7 <TEX>${\mu}M$</TEX>. The aglycone structure of 2 also showed cell adhesion inhibitory activity with an <TEX>$IC_{50}$</TEX> value of 21.1 <TEX>${\mu}M$</TEX>. With these results, we suspect these two saikosaponins from the Bupleurum falcatum L. roots to be prime candidates for therapeutic strategies towards inflammation.

Application of accelerated solvent extraction to the investigation of saikosaponins from the roots of Bupleurum falcatum

Accelerated solvent extraction (ASE) was applied to the extraction of saikosaponin a, saikosaponin c and saikosaponin d from the roots of Bupleurum falcatum. Main extraction parameters such as the extraction solvents, extraction temperature and static extraction time were investigated and optimized. The optimized procedure employed 70% methanol as extraction solvent, 120 degrees C of extraction temperature, 10 min of static extraction time, 60% of flush volume and the extraction recoveries of the three compounds were near to 100% with one extraction cycle. The extracted samples were analyzed by HPLC with UV detector. The HPLC conditions were as follows: Hypersil ODS2 (4.6 mmx250 mm, 5 microm) column, acetonitrile and water as mobile phase, flow rate of 1.0 mL/min, UV detection wavelength of 204 nm and injection volume of 20 microL. Compared with the traditional methods including heat-reflux extraction and ultrasonic-assisted extraction, the proposed ASE method was more efficient and faster to be operated. The results indicated that ASE was an alternative method for extracting saikosaponins from the roots of B. falcatum.

Comparison between evaporative light scattering detection and charged aerosol detection for the analysis of saikosaponins

Saikosaponins are triterpene saponins derived from the roots of Bupleurum falcatum L. (Umbelliferae), which has been traditionally used to treat fever, inflammation, liver diseases, and nephritis. It is difficult to analyze saikosaponins using HPLC-UV due to the lack of chromophores. Therefore, evaporative light scattering detection (ELSD) is used as a valuable alternative to UV detection. More recently, a charged aerosol detection (CAD) method has been developed to improve the sensitivity and reproducibility of ELSD. In this study, we compared CAD and ELSD methods in the simultaneous analysis of 10 saikosaponins, including saikosaponins-A, -B 1, -B 2, -B 3, -B 4, -C, -D, -G, -H and -I. A mixture of the 10 saikosaponins was injected into the Ascentis ® Express C18 column (100 mm × 4.6 mm, 2.7 μm) with gradient elution and detection with CAD and ELSD by splitting. We examined various factors that could affect the sensitivity of the detectors including various concentrations of additives, pH and flow rate of the mobile phase, purity of nitrogen gas and the CAD range. The sensitivity was determined based on the signal-to-noise ratio. The best sensitivity for CAD was achieved with 0.1 mM ammonium acetate at pH 4.0 in the mobile phase with a flow rate of 1.0 mL/min, and the CAD range at 100 pA, whereas that for ELSD was achieved with 0.01% acetic acid in the mobile phase with a flow rate at 0.8 mL/min. The purity of the nitrogen gas had only minor effects on the sensitivities of both detectors. Finally, the sensitivity for CAD was two to six times better than that of ELSD. Taken together, these results suggest that CAD provides a more sensitive analysis of the 10 saikosaponins than does ELSD.

Optimization of supercritical fluid extraction of saikosaponins fromBupleurum falcatumwith orthogonal array design

Supercritical fluid extraction (SFE) was used to extract saikosaponins a, c and d from the root of Bupleurum falcatum. An orthogonal array design L(9)(3)(4) was employed as a chemometric method for the optimization of the SFE conditions. The effects of four factors including pressure (30-40 MPa), temperature (40-50 degrees C), ethanol concentration (60-100%) and time (2.5-3.5 h) on the yields of saikosaponins were investigated by a preparative SFE system in the SFE mode. Under the optimized conditions, namely 35 MPa of pressure, 45 degrees C of temperature, 80% of ethanol concentration and 3.0 h of time, the yields of saikosaponin c, saikosaponin a, saikosaponin d, total saikosaponins and SFE extract were 0.16, 0.12, 0.96, 1.24 and 16.48 mg/g, respectively. Determinations of the saikosaponins were performed by HPLC.

Stimulatory effect of a pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L., on G-CSF secretion from intestinal epithelial cells

The inner surface of the intestinal tract possesses a large area of mucosal membranes, and the intestinal epithelial cells exist at the interface between an antigen-rich lumen and a lymphocyte-rich lamina propria. The crosstalk that occurs between these compartments serves to maintain intestinal homeostasis, and the cytokine network plays an important role in the crosstalk. In this study, the effect of a pectic polysaccharide, bupleuran 2IIc from Bupleurum falcatum L., on cytokine secretion of intestinal epithelial cells was investigated in vitro. When murine normal colonic epithelial cell line MCE301 cells were stimulated with bupleuran 2IIc, the contents of granulocyte colony-stimulating factor (G-CSF) in the conditioned medium were significantly increased in dose- and time-dependent manners. The enhanced G-CSF gene transcription in MCE301 cells by the stimulation of bupleuran 2IIc was observed by RT-PCR. The enhanced G-CSF secretion by bupleuran 2IIc was also observed in C3H/HeJ mice derived primary cultured colonic epithelial cells. Bupleuran 2IIc was digested with endo-(1→4)-α-d-polygalacturonase, and the resulting bupleuran 2IIc/PG-1 (“ramified” region) showed potent G-CSF secretion enhancing activity. The activity of bupleuran 2IIc/PG-1 disappeared after the removal of arabinosyl residues from bupleuran 2IIc/PG-1 by endo-(1→5)-α-l-arabinanase digestion. These results suggest that the “ramified” region (bupleuran 2IIc/PG-1) is the active site for the G-CSF secretion enhancing activity of bupleuran 2IIc, and the arabinan moiety of bupleuran 2IIc/PG-1 plays an important role in expression of the activity.

A Pectic Polysaccharide Isolated from the Roots of Bupleurum falcatum L. Stimulates the Tyrosine Phosphorylation of Lipid Rafts of Murine B Cells

Bupleuran 2IIc, a pectic polysaccharide isolated from the roots of Bupleurum falcatum L., was previously characterized as a T cell-independent B cell mitogen. The endo-(1-->4)-alpha-D-polygalacturonase-resistant moiety of bupleuran 2IIc (bupleuran 2IIc/PG-1) was the active site for expression of the activity, and expression of the cyclin D2 gene by bupleuran 2IIc/PG-1 may be mediated via activation of Src family tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C (PLC)-gamma followed by activation of protein kinase C (PKC) and calcium mobilization (Matsumoto et al., Int. Immunopharmacol., 5, 1373-1386 (2005)). Plasma membrane microdomains (lipid rafts) are enriched in signaling molecules and suggested to be involved in numerous cell functions, including membrane traffic and signaling. When B cells were stimulated with bupleuran 2IIc/PG-1, clustering of membrane lipid rafts was observed. To consider whether lipid rafts are implicated in bupleuran 2IIc/PG-1-mediated B cell proliferation, we analyzed the phosphorylation of tyrosine residues of proteins in lipid rafts. When murine B cells were stimulated with bupleuran 2IIc/PG-1, tyrosine phosphorylation of proteins in lipid rafts fraction was observed within 5 min. Tyrosine phosphorylation in lipid rafts fraction by bupleuran 2IIc/PG-1 was inhibited by the Src-family tyrosine kinase inhibitor, PP2. Together with previously published data, the results presented in this study suggest that activation of signaling molecules in lipid rafts by stimulation of bupleuran 2IIc/PG-1 contributes to B cell proliferation as the membrane-proximal signaling event.