Bupleurum Root Research Articles

Ulcer‐healing effects of jumihaidokuto extract in a mouse model of methicillin‐resistant Staphylococcus aureus causing skin infection

AbstractAimMethicillin‐resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes severe skin diseases with ulceration. Since MRSA are not susceptible to multiple antibiotics, novel therapeutic agents are desired. Jumihaidokuto, a combination of 10 crude drugs, is a Japanese traditional Kampo medicine that is used for the treatment of skin diseases. In this study, we investigated the effects of jumihaidokuto extract (JHT) on MRSA‐infected mice.MethodsMice infected subcutaneously with MRSA FPR3757 were administered JHT orally to analyze mouse survival, the condition of the mouse skin ulcer area, and transforming growth factor (TGF)‐β levels in the ulcer tissue. To evaluate the role of crude drug components, modified JHTs were prepared by removing one crude drug component at a time from the formula and the same experiment was conducted.ResultsJHT treatment significantly increased the survival rate. Ulcerated areas on the skin of MRSA‐infected mice were observed to be significantly reduced in size following JHT treatment. Furthermore, MRSA was not detected at the site of the ulcer in the JHT‐treated group. Additionally, TGF‐β levels decreased in the mouse skin ulcer tissues on treatment with JHT. All the crude drug components in JHT were found to partially contribute to the survival rate and healing of skin ulcers. Among the 10 crude drugs, Bupleurum root, cherry bark, Aralia rhizome, ginger, and Schizonepeta spike exhibited higher contribution than the other crude drugs.ConclusionOur findings indicate the efficacy of JHT in promoting the healing of MRSA‐induced murine skin ulcers.

Metabolomics Analysis Reveals Potential Mechanisms in Bupleurum L. (Apiaceae) Induced by Three Levels of Nitrogen Fertilization

Bupleurum (Apiaceae) is widely used in traditional Chinese medicine to treat inflammatory and infectious diseases. Although roots are the only used parts in China, other countries use the whole plant. The yield and quality of Bupleurum depend mainly on fertilizers, especially nitrogen. The current study aimed to assess the relationship between the nitrogen fertilization level and the quality and metabolomic response of different parts (flowers, main shoots, lateral shoots and roots) of Bupleurum to three nitrogen fertilization levels (control group: 0 kg·ha−1; low-nitrogen group: 55 kg·ha−1; high-nitrogen group: 110 kg·ha−1). The results showed that a high nitrogen level increases Bupleurum yield and quality parameters only in aerial parts, especially flowers, but has no significant effect on roots. The HPLC method was exploited for simultaneous quantification of three saikosaponins (A, C and D), which are the main bioactive components in the plant. It was found that the total content of saikosaponins decreased with high nitrogen fertilization in roots but significantly increased in flowers. Moreover, nitrogen fertilizer promoted the content of saikosaponin A but inhibited saikosaponins C and saikosaponins D in most parts of the plant. To study the response of primary metabolites, we adopted gas chromatography–mass spectrometry (GC−MS) analysis; 84 metabolites were identified that were mostly up-regulated with a high nitrogen level in flowers but down-regulated in roots. Four differential metabolites—D-fructose, lactose, ether and glycerol—were recognized as key metabolites in Bupleurum under nitrogen fertilization. Meanwhile, The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results explained that the impact of nitrogen fertilization on Bupleurum was attributed to the C-metabolism, N-metabolism, and lipids metabolism. This research put forward new insights into potential mechanisms and the relationship between the quality and yield of Bupleurum and nitrogen fertilization.

Cheminformatics modeling of the correlation between Bupleurum Root-formula medicines and Excess and Deficiency pattern in the diagnostic criteria of Sho in Kampo (traditional Japanese medicine) by non-targeted direct infusion mass spectrometry with machine learning.

Kampo is a form of traditional Japanese medicine, and its therapeutic strategy has been validated empirically over millennia, mainly in Asia. Kampo therapy aims to holistically prevent and treat disease based on the specific diagnosis Sho (in Japanese), in contrast with modern medical treatment which focuses on a patient's affected parts and local conditions. The medicines formulated using crude drugs derived from natural sources (Kampo formulas) are prescribed for patients according to their specific Sho, and thus the Kampo medication system is very complex. However, our previous study strongly suggested that Kampo medication theory could be explained by chemometrics and informatic approaches [Okada et al. in J Nat Med 70:107-114, 2016]. Here, we studied a group of seven formulas with Bupleurum Root and Scutellaria Root as the principal crude drugs. First, decoctions of the formulas were prepared and their supernatants were analyzed by non-targeted direct infusion mass spectrometry (MS) and principal component analysis, which is a type of unsupervised machine learning. Next, supervised machine learning was used to perform partial least squares modeling of the MS data matrix trained on the patients' constitution of Excess, Deficiency, or Medium between these two states (EDM) in Sho. The results showed that the correlation between the chemical fingerprints obtained by MS analysis and EDM could be modeled well using this approach. This cheminformatics modeling approach successfully interpreted part of the complex Kampo medication system studied using the fingerprints of formulas obtained by MS analysis and was consistent with the predicted Sho.

Differential Expression of Genes Involved in Saikosaponin Biosynthesis Between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd.

Radix Bupleuri (roots of Bupleurum spp.) is an important medicinal herb. Triterpenoid saponins of saikosaponins generally constitute the main class of secondary metabolites of plants in the Bupleurum genus. However, the molecular regulatory mechanism underlying their biosynthesis remains elusive. In this study, we observed significantly different saikosaponin biosynthesis between Bupleurum chinense and Bupleurum scorzonerifolium at the seedling stage. The sequential and expression characterization of 232 genes in the triterpenoid saponin biosynthetic pathway, which includes the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway, between B. chinense and B. scorzonerifolium was also investigated. Sixty of these genes may be involved in saikosaponin biosynthesis. Manipulation of these genes, especially those of the β-AS, P450, and UGT families, may improve saikosaponin production.

Triple herbal extract DA-9805 exerts a neuroprotective effect via amelioration of mitochondrial damage in experimental models of Parkinson\u2019s disease

Moutan cortex, Angelica Dahurica root, and Bupleurum root are traditional herbal medicines used in Asian countries to treat various diseases caused by oxidative stress or inflammation. Parkinson’s disease (PD) has been associated with mitochondrial dysfunction, but no effective treatment for mitochondrial dysfunction has yet been identified. In this study we investigated the neuroprotective effects of the triple herbal extract DA-9805 in experimental models of PD. DA-9805 was prepared by extracting three dried plant materials (Moutan cortex, Angelica Dahurica root, and Bupleurum root in a 1:1:1 mixture) with 90% ethanol on a stirring plate for 24 h at room temperature and fingerprinted using high-performance liquid chromatography. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+), which both exert neurotoxic effects on dopaminergic neurons by inhibiting mitochondrial oxidative phosphorylation (OXPHOS) complex I, were used to make experimental models of PD. In MPP+-treated SH-SY5Y cells, DA-9805 ameliorated the suppression of tyrosine hydroxylase expression and mitochondrial damage on OXPHOS complex 1 activity, mitochondrial membrane potential, reactive oxygen species (ROS) generation, and oxygen consumption rate. In the MPTP-induced subacute PD model mice, oral administration of DA-9805 recovered dopamine content as well as bradykinesia, as determined by the rotarod test. DA-9805 protected against neuronal damage in the substantia nigra pars compacta (SNpc) and striatum. In both in vitro and in vivo models of PD, DA-9805 normalized the phosphorylation of AKT at S473 and T308 on the insulin signaling pathway and the expression of mitochondria-related genes. These results demonstrate that the triple herbal extract DA-9805 showed neuroprotective effects via alleviating mitochondria damage in experimental models of PD. We propose that DA-9805 may be a suitable candidate for disease-modifying therapeutics for PD.

Inhibitory Activity of Yokukansankachimpihange against Nerve Growth Factor-Induced Neurite Growth in Cultured Rat Dorsal Root Ganglion Neurons.

Chronic pruritus is a major and distressing symptom of many cutaneous diseases, however, the treatment remains a challenge in the clinic. The traditional Chinese-Japanese medicine (Kampo medicine) is a conservative and increasingly popular approach to treat chronic pruritus for both patients and medical providers. Yokukansankachimpihange (YKH), a Kampo formula has been demonstrated to be effective in the treatment of itching of atopic dermatitis in Japan although its pharmacological mechanism is unknown clearly. In an attempt to clarify its pharmacological actions, in this study, we focused on the inhibitory activity of YKH against neurite growth induced with nerve growth factor (NGF) in cultured rat dorsal root ganglion (DRG) neurons because epidermal hyperinnervation is deeply related to itch sensitization. YKH showed approximately 200-fold inhibitory activity against NGF-induced neurite growth than that of neurotropin (positive control), a drug used clinically for treatment of chronic pruritus. Moreover, it also found that Uncaria hook, Bupleurum root and their chemical constituents rhynchophylline, hirsutine, and saikosaponin a, d showed inhibitory activities against NGF-induced neurite growth, suggesting they should mainly contribute to the inhibitory activity of YKH. Further study on the effects of YKH against epidermal nerve density in “itch-scratch” animal models is under investigation.

Yokukansan, a traditional Japanese medicine, decreases head-twitch behaviors and serotonin 2A receptors in the prefrontal cortex of isolation-stressed mice

Ethnopharmacological relevanceYokukansan, a traditional Japanese (Kampo) medicine, has recently been used to treat the behavioral and psychological symptoms of dementia (BPSD), including aggressiveness, excitability, and hallucination. The present study was designed to investigate the mechanisms underlying the ameliorative effects of yokukansan on BPSD using animals exhibiting hallucination-like behaviors. For this purpose, we initially examined whether chronic isolation stress increases the frequency of hallucination in response to a psychedelic drug. Using this animal model, we next examined the effects of yokukansan on drug-induced hallucination-like behaviors. Finally, we examined the density and mRNA levels of serotonin 2A (5-HT2A) receptors. Materials and methodsMale mice were subjected to isolation stress for six weeks. Yokukansan was incorporated into food pellets, and administered to the mice for six weeks. In some experiments, yokukansan and each of seven constituent herbs were administered orally to the mice for the last two weeks during the six-week period of isolation stress. A 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI, 2.5mg/kg), was injected into the mice, and head-twitch behaviors were quantified. The binding sites of 5-HT2A receptors on the plasma membrane of the prefrontal cortex (PFC) were assessed by a receptor-binding assay using tritium-labeled ketanserin, and the density and affinity were calculated from a Scatchard plot. The level of mRNAs was measured by PCR analyses. ResultsIsolation stress enhanced the frequency of the DOI-induced head-twitch response, and yokukansan treatment by feeding significantly reduced this enhancement. Isolation stress significantly increased the 5-HT2A receptor density in the PFC, and yokukansan treatment by feeding as well as administration significantly down-regulated this increase. Isolation stress and yokukansan did not affect the affinity. Among seven constituent herbs, Bupleurum Root, Uncaria Hook, Japanese Angelica Root, and Glycyrrhiza down-regulated the increase, but statistically not significant, in which their efficacies were over 50% relative to yokukansan. Neither isolation stress nor yokukansan affected mRNA levels of 5-HT2A receptors. ConclusionYokukansan attenuated drug-induced hallucination-like behaviors in isolated mice, which is suggested to be mediated by 5-HT2A receptor down-regulation in the PFC. This mechanism may underlie the ameliorative effects of yokukansan on hallucination.

Study on composition of essential oil in above-ground and root of Bupleurum malconense and root of B. chinense by AMDIS and retention index

Chemical constituents of the essential oil in above-ground and root of Bupleurum malconense and root of B. chinense were investigated by GC-MS compiled with automated mass spectral deconvolution and identification system (AMDIS) and retention index. The results showd that the components of essential oil in B. malconense have some similarities with the one in B. chinense, and both of them have the higher content of caryophyllene oxide which is an active component of anti-inflammatory and analgesic. These results suggested that as a local substitute, B. malconense has a certain scientific basis of the treatment for cold fever.

Effect of Dai-Saiko-To (Da-Chai-Hu-Tang) on LDL-Receptor Gene Expression in Human Hepatoma Cell Line (HepG2)

We previously reported that Dai-saiko-to (Da-Chai-Hu-Tang), a traditional Japanese kampo medicine, increased LDL receptor mRNA expression in the liver of the hypercholesterolemic rabbits. In this study, we focused on LDL receptor gene expression in a human hepatoma cell line (HepG2) treated with Dai-saiko-to extract and the extracts of eight herbs presented in Dai-saiko-to. Dai-saiko-to extract significantly increased LDL receptor gene and SREBP2 gene expression compared with the control. The extracts of four herbs, Bupleurum root, Pinellia tuber, Scutellaria root and Peony root significantly increased the LDL receptor gene expression. Whereas, Jujube, Immature orange, Ginger and Rhubarb extracts did not change the gene expression. These results suggest that Dai-saiko-to increased the expression of the cholesterol transport gene (LDL receptor) regulated by SREBP2 gene in the human hepatoma cell line. The pharmacological activity of Dai-saiko-to against hypercholesterolemia and atheromatous lesions related for these four herbal components.

Determination of saikosaponin, phenolic and podophyllotoxin contents of five endemic Bupleurum root extracts and their effects on MCF-7 cells

Bupleurum species are among the plants used in Chinese medicine for phytotherapy. Various compounds obtained from plants have been found to exhibit anticancer activity. In this study, phenolic compounds, saikosaponins and podohyllotoxin contents of total root extracts from endemic Bupleurum species (Bupleurum sulphureum, Bupleurum lycaonicum, Bupleurum turcicum, Bupleurum heldreichii, Bupleurum pauciradiatum) were determined by high-performance liquid chromatography (HPLC). Total phenol content and free radical scavenging activities of total extracts were also identified. Finally, the effects of plant root extracts on viability of P-gp overexpressing paclitaxel resistant (MCF-7/Pac) and vincristine resistant (MCF-7/Vinc) MCF-7 mammary carcinoma cell lines and their parental line (MCF-7/S) were evaluated by cytotoxicity tests. Results showed that saikosaponin A, saikosaponin D and isoquercitrin contents of the root extracts were higher compared to podophyllotoxin, catechin and quercetin. While, B. lycaonicum root extract has about 1.5 fold more total phenol content with respect to others, B. turcium root extract has the highest free radical scavenging activity. According to cytotoxicity tests, B. turcicum and B. pauciradiatum root extracts were more toxic to the MCF-7/Pac cell line than the root extracts of other three species. In addition, B. heldrechii extract has been found to be the most toxic to the MCF-7/Vinc cell line among the others. Key words: Bupleurum species, plant phenolics, saikosaponin, podophyllotoxin, high-performance liquid chromatography (HPLC), MCF-7.

Beneficial Effect ofBupleurumPolysaccharides on Autoimmune-Prone MRL-lpr Mice

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease leading to inflammatory tissue damage in multiple organs. The crude polysaccharides (BPs) isolated from the roots of Bupleurum smithii var. parvifolium have anticomplementary activity and immunomodulatory functions on macrophages. To study its potential benefit on SLE, we examined effects of BPs on MRL-lpr mice, which have similar disease features to human SLE. MRL-lpr mice were treated orally with BPs 15, 30, or 60 mg kg−1 day−1 for 12 weeks and their SLE characteristics were evaluated. The results revealed that BPs elongated life span, improved kidney function, delayed lymphadenopathy, and reduced autoantibodies. It seemed to be mediated by inhibition of complement and macrophages activation and suppression of interferon-γ (IFN-γ) and interleukin-6 (IL-6) gene expression in the kidney. These results implicate that BPs may be an immunomodulator for the treatment of autoimmune diseases like SLE.

Antioxidant and Protective Effects ofBupleurum falcatumon the L-Thyroxine-Induced Hyperthyroidism in Rats

Bupleuri Radix (BR), the dried roots of Bupleurum falcatum L., has been used in folk medicine as an antiinflammatory and antioxidative agent. The aqueous extract of BR was evaluated for its possible ameliorative effect in the regulation of hyperthyroidism in l-thyroxine- (LT4-) induced rat model. After oral administration of 300, 150, and 75 mg/kg of BR extracts, once a day for 15 days from 12th LT4 treatments, changes on the body, thyroid gland, liver, and epididymal fat pad weights, serum triiodothyronine, thyroxine, thyroid-stimulating hormone, asparte aminotransferase and alanine aminotransferase concentrations, hepatic lipid peroxidation, glutathione contents, superoxide dismutase, and catalase activities were investigated with thyroid gland, liver, and epididymal fat histopathological changes. The effects of BR extracts were compared with that of propylthiouracil, a standard antithyroid drug 10 mg/kg (intraperitoneally). In this experiment, BR extracts dose dependently reversed LT4-induced hyperthyroidisms, and these effects indicating their potential in the regulation of hyperthyroidism. Further, the BR extract normalized LT4-induced liver oxidative stresses, and also reduced liver and epididymal fat pad changes. BR extracts 150 mg/kg showed comparable effects on the LT4-induced rat hyperthyroidism as compared with PTU 10 mg/kg. These effects of BR may help the improvement of hyperthyroidisms and accompanied various organ damages.

UP-03.004 The Use of Kampo Formula (Bupleurum Root Drug Group) in Mentally Retarded Patients with Enuresis

Diurnal enuresis and nocturnal enuresis can occur at the same time, however, many children with nighttime wetting will not have wetting during the day. Children with daytime wetting may have frequent urination or urgent urination. They may also dribble after urination. Diurnal enuresis is encountered in about 10% of children between 4 and 6 years old, and this figure decreases steadily to reach 5% in 6 to 12 years olds, 4% in adolescents, and 2% in adults. Otherwise, approximate nocturnal eneuresis rates are: 20% at age 5, 10 to15% at age 6, 7% at age 7, 5% at age 10, 1 to 2% age 15 and 0.5 to 1% between age 18 to 64.

Sho-saiko-to-ka-kikyo-sekko as an alternative treatment for chronic tonsillitis to avoid surgery

Sho-saiko-to-ka-kikyo-sekko (TJ-109) is composed of 9 herbs (gypsum, Bupleurum root, Pinellia tuber, Scutellaria root, Platycodon root, jujube fruit, ginseng root, Glycyrrhiza root, and ginger rhizome). It is a folk medicine that has been used to treat pharyngitis or acute tonsillitis. The efficacy of TJ-109 for treating patients with chronic tonsillitis was investigated. Ten outpatients who experienced chronic tonsillitis for more than 2 years were recruited. TJ-109 was prescribed, and after one year of daily treatment the incidence of acute tonsillitis before and after the treatment was compared. The incidence of acute tonsillitis due to chronic tonsillitis decreased in all 7 patients who were followed up. No adverse events were observed in any of the patients. In conclusion, the herbal medicine TJ-109 effectively reduced the incidence of acute tonsillitis. In some cases, planned tonsillectomy was avoided.

Essential oil of Bupleurum rotundifolium L. flowers

Bupleurum is a genus of the family Umbelliferae (Apiaceae), comprising about 50 taxon, including 20 endemic in Turkey. Bupleurum root is widely used in traditional medicine for the treatment of fever, pain and inflammation associated with influenza or the common cold [1,2].

Fast determination of saikosaponins in Bupleurum by rapid resolution liquid chromatography with evaporative light scattering detection

A rapid resolution liquid chromatography coupled with evaporative light scattering detection method was established for simultaneous determination of six saikosaponins, namely saikosaponin a, saikosaponin c, saikosaponin d, 6″- O-acetylsaikosaponin a, 3″- O-acetylsaikosaponin d and 6″- O-acetylsaikosaponin d in Bupleurum. The analysis was performed by using an Agilent Zorbax SB-C18 column (1.8 μm, 3.0 mm × 50 mm i.d.) at gradient elution of water and acetonitrile, and the saikosaponins were well separated within 12 min, which provided about a fourfold reduction in analysis time by comparing a conventional high performance liquid chromatography method. Owing to their low ultraviolet absorption, the saikosaponins were detected by evaporative light scattering. The standard curves to quantify the saikosaponins were constructed by the log–log plot, which showed good linearity with the correlation coefficients exceeding 0.9954. The detection limits and quantification limits ranged in 8.38–25.00 μg/mL and 25.13–45.00 μg/mL, respectively. Satisfactory intra-day and inter-day precisions were achieved with the relative standard deviation (R.S.D.) less than 6.58%, and the average recoveries obtained were in the range of 96.9–100.4%. In addition, MeOH–1.0% (v/v) pyridine was found to be the best the extraction solvent when compared to MeOH and MeOH–1.0% (v/v) ammonia water. A total of 23 samples of roots of Bupleurum from different species or locations were examined with this analytical method, and their chemical profiles provided information for the chemotaxonomic investigation. The results demonstrated that the analytical method is highly effective for the quality evaluation of Bupleurum species.

Growth and saikosaponin production of the medicinal herb Bupleurum chinense DC. under different levels of nitrogen and phosphorus

Dried roots of Bupleurum spp. have been used medicinally in China for over 2000 years. The roots, which are called Bupleuri Radix, are used in at least 66% of the formulations/prescriptions in traditional Chinese medicine and Kampo medicine. Demand for Bupleuri Radix is increasing and B. chinense is one of the most important Bupleurum spp. in China. We conducted a 2-year pot experiment to investigate the effect of nitrogen (N) and phosphorus (P) fertilizer on biomass production and root saikosaponin a (SSa) and saikosaponin d (SSd) content of B. chinense. The experiment included seven combinations of N and P fertilizer. The results showed that medium levels of N and P fertilizer significantly increased the biomass and SSa content of B. chinense roots, but had no significant effect on root SSd content. The application of moderate amounts of N or P fertilizer increased total SSa and SSd yield significantly compared to the unfertilized control, but the greatest increase in total SSa and SSd yield occurred when N and P were applied together. This suggests that N and P have a synergistic effect on B. chinense growth and saikosaponin production. When high amounts of N and P fertilizer were used total SSa and SSd yields declined. This implied that there was a nutrient supply threshold for the biosynthesis of saikosaponins. Total SSa and SSd yields were greater in treatments which received only P fertilizer compared to treatments that received only N fertilizer. Our results reinforce the importance of soil testing and the application of recommended amounts N and P fertilizers for the cultivation of B. chinense.

A novel method of protein extraction from perennial Bupleurum root for 2‐DE

The perennial Bupleurum root is thick and woody and contains high levels of interfering compounds. Common protein extraction methods have proved refractory towards the isolation of proteins suitable for 2-DE, due to the presence of interfering compounds. A novel method for extracting proteins suitable for 2-DE was established to overcome these problems. The main characteristic of this protocol is the partitioning of the proteins into the aqueous (fraction A-2), chloroform and isoamyl alcohol phases (A-3), and the interphase (A-1). The proteins are then extracted from each of these phases. From A-1, 85% (extracted protein against total proteins) proteins could be extracted and purified. For fraction A-2, a novel phenol extraction step is employed for the extraction of proteins. Based on the well-resolved 2-DE patterns, our protein preparation is free of interfering compounds. Using these methods (A-1, A-2, and A-3-3), a total of 3662 (1526 + 1128 + 1008) spots could be separated, and a protein yield of about 1.41 mg per 1.0 g fresh root material was obtained. To our knowledge, this is the first time that a protocol for protein extraction from perennial Bupleurum root has been reported that gives reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues as well.

Pharmacology and Toxicology of Bupleurum Root-Containing Kampo Medicines in Clinical Use

Kampo (Japanese traditional herbal) medicines have been produced by combining multiple crude drugs, almost all of plant origin but with some of animal or mineral origin, and contain a great many substances. Their effect is a combination of the various interactions of the constituent substances, whether they are enhancing, synergistic or suppressive. Kampo medicine has an overall effect that is different from the combined effects of individual crude drugs, and several side effects such as anorexia, slight fever and nausea have been reported in the treatment of certain disorders and disease states with Kampo medicines. Among 210 medical formulations used in Japan, some relevant information on the clinical uses, pharmacology and toxicology of six manufactured Kampo medical formulations, Shosaikoto, Daisaikoto, Saikokeishito, Hochuekkito, Saibokuto and Saireito, containing Bupleurum root are reviewed. Studies of some potential interactions between Kampo medicine and western drugs are also considered.

Pharmacological evaluation of several major ingredients of Chinese herbal medicines in human hepatoma Hep3B cells.

Long-dan-tan (Chinese name) is one of the most common herbal medicines used by Chinese people with chronic liver disease. Accumulated anecdotal evidence suggests that Long-dan-tan may show a beneficial effect in patients with hepatocellular carcinoma. Long-dan-tan is made from five plants: Gentiana root, Scutellaria root, Gardenia fruit, Alisma rhizome, and Bupleurum root. In this study, we have examined the cytotoxic effects of the five major ingredients isolated from the above plants, i.e. gentiopicroside, baicalein, geniposide, alisol B acetate and saikosaponin-d, respectively, on human hepatoma Hep3B cells. Annexin V immunofluorescence detection, DNA fragmentation assays and FACScan analysis of propidium iodide-staining cells showed that gentiopicroside, baicalein, and geniposide had little effect, whereas alisol B acetate and saikosaponin-d profoundly induced apoptosis in Hep3B cells. Alisol B acetate, but not saikosaponin-d, induced G2/M arrest of the cell cycle as well as a significant increase in caspase-3 activity. Interestingly, baicalein by itself induced an increase in H(2)O(2) generation and the subsequent NF-kappaB activation; furthermore, it effectively inhibited the transforming growth factor-beta(1) (TGF-beta(1))-induced caspase-3 activation and cell apoptosis. We suggest that alisol B acetate and saikosaponin-d induced cell apoptosis through the caspase-3-dependent and -independent pathways, respectively. Instead of inducing apoptosis, baicalein inhibits TGF-beta(1)-induced apoptosis via increase in cellular H(2)O(2) formation and NF-kappaB activation in human hepatoma Hep3B cells.