Angiotensin I-Converting Enzyme-Inhibitory Activity and Phytochemical Profile of Constituents of the Leaves of Rehmannia glutinosa f. hueichingensis.

The root of Rehmannia glutinosa Liboschitz forma hueichingensis HSIAO has been used as a tonic and treatment for urinary and skin disorders in Japanese Kampo medicine. Phytochemical investigation of the root has been well reported, but that of the leaves is limited. To explore the potential value of R. glutinosa leaves, we focused on the angiotensin I-converting enzyme (ACE)-inhibitory activity. The leaf extract exhibited ACE-inhibitory activity, and the inhibitory potency of leaves was stronger than that of roots. Using this activity as an indicator, we isolated linaride (1), 6-O-hydroxybenzoyl ajugol (2), acteoside (3), leucosceptoside A (4), martynoside (5), luteolin (6), apigenin (7), and chrysoeriol (8) by separating and purifying the extract. We then examined the ACE-inhibitory activities of 1-8, catalpol (9), aucubin (10), ajugol (11), and echinacoside (12). Among them, 3, 6, and 12 displayed the most potent inhibitory activity. A simultaneous analytical method was also developed using compounds contained in R. glutinosa leaves and roots, and their contents were compared. The method consisted of extraction with 50% aqueous methanol under sonication for 60 min and LC/MS measurement. R. glutinosa leaves tended to have higher levels of majority of the analytes than the roots, including 3 and 6, which had higher ACE-inhibitory activity. These results suggest that 3 and 6 contribute to the ACE-inhibitory activity of R. glutinosa leaves, which may represent a useful medicinal resource for hypertension.

(+)-Magnolin Enhances Melanogenesis in Melanoma Cells and Three-Dimensional Human Skin Equivalent; Involvement of PKA and p38 MAPK Signaling Pathways.

Magnoliae Flos is a traditional herbal medicine used to treat nasal congestion associated with headache, empyema, and allergic rhinitis. In our preliminary screening of crude drugs used in Japanese Kampo formulas for melanin synthesis, the methanol extract of Magnoliae Flos was found to exhibit strong melanin synthesis activity. However, there have been no studies evaluating the effects of Magnoliae Flos or its constituents on melanogenesis. The present study aimed to isolate the active compounds from Magnoliae Flos that activate melanin synthesis in melanoma cells and three-dimensional human skin equivalent, and to investigate the molecular mechanism underlying melanin induction. The methanol extract of Magnoliae Flos induced an increase of melanin content in both B16-F1 and HMV-II cells. A comparison of melanin induction by three fractions prepared from the extract showed that the ethyl acetate fraction markedly induced melanin synthesis. Bioassay-guided separation of the ethyl acetate fraction resulted in the isolation of seven lignans (1:  - 7: ). Among them, (+)-magnolin (5: ) strongly induced melanin synthesis and intracellular tyrosinase activity. Furthermore, the ethyl acetate fraction and 5: clearly induced melanin content in a three-dimensional human skin equivalent. Molecular analysis revealed that 5: triggered the protein expression of tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. Further analysis of transcriptional factors and signaling pathways demonstrated that 5: induces the protein expression of tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2 activated by the protein kinase A- and p38 mitogen-activated protein kinase-dependent pathways, leading to cAMP-responsive element-binding protein phosphorylation and microphthalmia-associated transcription factor expression. These findings demonstrate the potential of 5: as a potent therapeutic agent for hypopigmentation.

Development of Highly Sensitive Chemiluminescence Enzyme Immunostaining Assay to Determine Glycyrrhizin Content Using Anti-glycyrrhizin Monoclonal Antibody.

Licorice, the root of Glycyrrhiza spp., is used in a large number of herbal medicines, such as traditional Chinese medicines, Japanese Kampo medicines, and therapeutic drugs. Since glycyrrhizin (GL) is among the main components in licorice and exhibits numerous beneficial pharmacological activities, the content of GL directly affects biological activity. The quality control based on GL content is an important factor in ensuring biological activity; however, the content of GL in licorice varies depending on plant cultivation environment, genetic factors, and species type. Previously, we prepared an anti-GL monoclonal antibody (anti-GL mAb) and employed it in various immunochemical assays for quality control of licorice and licorice-based products. In this study, we employed the anti-GL mAb in chemiluminescence enzyme immunostaining (CLEIS) to develop a very simple, rapid, specific, and sensitive quality control assay for licorice products, with a limit of detection of 3.9 ng. Furthermore, the CLEIS assay enabled semiquantitative analysis of GL in Kampo medicines. Our results showed that multiple samples can be simultaneously analyzed using CLEIS, and it is a useful tool for determining GL content, as well as ensuring chemical quality control of licorice-containing products and herbal medicines.

In vitro fertilization using sperm activated by ML-2-3 isolated from Morinda lucida Bentham leaves.

Purpose: ML-2-3 is a novel tetracyclic iridoid derived from Morinda lucida Bentham leaves. This compound has anti-trypanosomal and anti-leishmanial effects. In this study, the authors investigated effects of ML-2-3 on in vitro fertilization (IVF) rates, motility, and acrosome reaction of the mouse sperm. Methods: IVF was performed using sperm from BALB/cByJJcl mice treated with ML-2-3. Computer-assisted sperm analysis (CASA) was performed on the sperm of C57BL/6J mice to investigate sperm motility. The effect of ML-2-3 on the acrosome reaction was examined by observing the fluorescence of sperm labeled with the acrosin-EGFP transgene. Results: ML-2-3 improved IVF in BALB/cByJJcl mice with low fertilization rates. The optimum concentration of ML-2-3 in sperm pre-culture medium was 20 µM, and no significant toxicity of ML-2-3 was observed in developing embryos at this concentration. ML-2-3 affected sperm motility but not the acrosome reaction. ML-2-3 increased the IVF rate of mouse sperm that had been refrigerated for 3 days. Conclusions: ML-2-3 can improve the outcome of IVF and motility without inducing the acrosome reaction in mice. These effects of ML-2-3 on sperm behaviors are different from those of the similar drugs.

Antiproliferative activity and apoptosis induction by trijuganone C isolated from the root of Salvia miltiorrhiza Bunge (Danshen).

In this study, we found that the hexane fraction of Danshen, the dried root of Salvia miltiorrhiza (Lamiaceae), exerted antiproliferative effects on human leukemia cells. Phytochemical investigation of the hexane fraction achieved the isolation of the tanshinone diterpenes: dihydrotanshinone I (1), trijuganone C (2), trijuganone B (3), cryptotanshinone (4), tanshinone IIA (5), and tanshinone I (6). Compound 2 showed significant antiproliferative activities against human leukemia cells HL-60, Jurkat, and U937. The antiproliferative activities of 2 against human cancer and normal cells indicated that 2 exhibited potent antiproliferative activities with IC50 values less than 10 µM against HL-60 and Jurkat cells as well as on the colon cancer cells DLD-1, COLO 205, and Caco-2. Compound 2 induced chromatin condensation, DNA fragmentation, activation of caspase-3, -8, and -9, and the cleavage of poly (ADP-ribose) polymerase (PARP) in HL-60 cells. Moreover, 2 activated Bid and Bax, leading to the loss of mitochondrial membrane potential, and 2 induced the cytochrome c release from mitochondria into cytosol. In contrast, Bcl-2 and Bcl-xL were unaffected by 2. These results suggest that 2 exerts antiproliferative effects via apoptosis induction mediated by mitochondrial dysfunction and caspase activation. Compound 2 may serve as a candidate of potential chemotherapeutic agent for human leukemia.

In vitro antiprotozoan activity and mechanisms of action of selected Ghanaian medicinal plants against Trypanosoma, Leishmania, and Plasmodium parasites.

Trypanosomiasis, leishmaniasis, and malaria are protozoan infections of public health importance with thousands of new cases recorded annually. Control of these infection(s) with existing chemotherapy is limited by drug toxicity, lengthy parenteral treatment, affordability, and/or the emergence of resistant strains. Medicinal plants on the other hand are used in the treatment of various infectious diseases although their chemical properties are not fully evaluated. In this study, we screened 112 crude extracts from 72 selected Ghanaian medicinal plants for anti-Trypanosoma, anti-Leishmania, and anti-Plasmodium activities in vitro and investigated their mechanisms of action. Twenty-three extracts from 20 plants showed significant antiprotozoan activity against at least 1 of 3 protozoan parasites screened with IC50 values less than 20 µg/ml. Eleven extracts showed high anti-Trypanosoma activity with Bidens pilosa whole plant and Morinda lucida leaf extracts recording the highest activities. Their IC50 (selectivity index [SI]) values were 5.51 µg/ml (35.00) and 5.96 µg/ml (13.09), respectively. Nine extracts had high anti-Leishmania activity with Annona senegalensis and Cassia alata leaf extracts as the most active. Their IC50 (SI) values were 10.8 µg/ml (1.50) and 10.1 µg/ml (0.37), respectively. Six extracts had high anti-Plasmodium activity with the leaf and stem-bark extracts of Terminalia ivorensis recording the highest activity. Their IC50 (SI) values were 7.26 µg/ml (129.36) and 17.45 µg/ml (17.17), respectively. Only M. lucida at 25 µg/ml induced significant apoptosis-like cell death in Trypanosoma parasites. Anti-Leishmania active extracts induced varying morphological changes in Leishmania parasites such as multiple nuclei and/or kinetoplast, incomplete flagella division, or nuclear fragmentation. Active extracts may be potential sources for developing new chemotherapy against these infections.

Bioactive Triterpenes from the Root of Salvia miltiorrhiza Bunge.

Danshen (Salvia miltiorrhiza) is a well-known medicinal herb in the oriental medicine. The current study on bioactive triterpenoid in the root of S. miltiorrhiza led to the isolation of a new highly hydroxylated ursane-type triterpene, urs-12-ene-2α,3ß,7ß,16α-tetraol (1) and five known ones including 2ß-hydroxypomolic acid (2), maslinic acid (3), asiatic acid (4), ursolic acid (5), and oleanolic acid (6). Their structures were elucidated on the basis of extensive spectroscopic analyses and comparison with literature data. The antiproliferative testing against HL-60 cells revealed that the new compound 1 and ursolic acid (5) showed weak and moderate activities with IC50 values of 42.2 and 11.7 µM. In addition, compounds 1-3 showed inhibitory effect on ghrelin activity. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous determination of glycyrrhizin and liquiritin in licorice roots and Kampo medicines by combination enzyme-linked immunosorbent assay using anti-glycyrrhizin and anti-liquiritin monoclonal antibodies.

Immunoassay systems using monoclonal antibodies (mAbs) are one of the most useful techniques in the analytical, biochemical, and clinical fields. In this study, a combination enzyme-linked immunosorbent assay (ELISA) using both anti-glycyrrhizin and anti-liquiritin mAbs (anti-GL/Liq mixture mAbs) was developed for quality control of licorice and its products. The combination ELISA demonstrated high sensitivity, reproducibility, and specificity for the total content of GL and Liq by a single assay. The developed ELISA was effective and useful as the first screening method in the selection of high-quality licorice from the Glycyrrhiza species and in confirming the quality of licorice-containing Kampo medicines.

Effect of glycyrrhetinic acid on lipid raft model at the air/water interface.

To investigate an interfacial behavior of the aglycon of glycyrrhizin (GC), glycyrrhetinic acid (GA), with a lipid raft model consisting of equimolar ternary mixtures of N-palmitoyl sphingomyelin (PSM), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL), Langmuir monolayer techniques were systematically conducted. Surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms showed that the adsorbed GA at the air/water interface was desorbed into the bulk upon compression of the lipid monolayer. In situ morphological analysis by Brewster angle microscopy and fluorescence microscopy revealed that the raft domains became smaller as the concentrations of GA in the subphase (CGA) increased, suggesting that GA promotes the formation of fluid networks related to various cellular processes via lipid rafts. In addition, ex situ morphological analysis by atomic force microscopy revealed that GA interacts with lipid raft by lying down at the surface. Interestingly, the distinctive striped regions were formed at CGA=5.0 µM. This phenomenon was observed to be induced by the interaction of CHOL with adsorbed GA and is involved in the membrane-disrupting activity of saponin and its aglycon. A quantitative comparison of GA with GC (Sakamoto et al., 2013) revealed that GA interacts more strongly with the raft model than GC in the monolayer state. Various biological activities of GA are known to be stronger than those of GC. This fact allows us to hypothesize that differences in the interactions of GA/GC with the model monolayer correlate to their degree of exertion for numerous activities.

Antitrypanosomal Activities and Mechanisms of Action of Novel Tetracyclic Iridoids from Morinda lucida Benth.

Trypanosoma brucei parasites are kinetoplastid protozoa that devastate the health and economic well-being of millions of people in Africa through the disease human African trypanosomiasis (HAT). New chemotherapy has been eagerly awaited due to severe side effects and the drug resistance issues plaguing current drugs. Recently, there has been an emphasis on the use of medicinal plants worldwide. Morinda lucida Benth. is a popular medicinal plant widely distributed in Africa, and several research groups have reported on the antiprotozoal activities of this plant. In this study, we identified three novel tetracyclic iridoids, molucidin, ML-2-3, and ML-F52, from the CHCl3 fraction of M. lucida leaves, which possess activity against the GUTat 3.1 strain of T. brucei brucei The 50% inhibitory concentrations (IC50) of molucidin, ML-2-3, and ML-F52 were 1.27 µM, 3.75 µM, and 0.43 µM, respectively. ML-2-3 and ML-F52 suppressed the expression of paraflagellum rod protein subunit 2, PFR-2, and caused cell cycle alteration, which preceded apoptosis induction in the bloodstream form of Trypanosoma parasites. Novel tetracyclic iridoids may be promising lead compounds for the development of new chemotherapies for African trypanosomal infections in humans and animals.

Procyanidin trimer C1 derived from Theobroma cacao reactivates latent human immunodeficiency virus type 1 provirus.

Despite remarkable advances in combination antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) infection remains incurable due to the incomplete elimination of the replication-competent virus, which persists in latent reservoirs. Strategies for targeting HIV reservoirs for eradication that involves reactivation of latent proviruses while protecting uninfected cells by cART are urgently needed for cure of HIV infection. We screened medicinal plant extracts for compounds that could reactivate the latent HIV-1 provirus and identified a procyanidin trimer C1 derived from Theobroma cacao as a potent activator of the provirus in human T cells latently infected with HIV-1. This reactivation largely depends on the NF-κB and MAPK signaling pathways because either overexpression of a super-repressor form of IκBα or pretreatment with a MEK inhibitor U0126 diminished provirus reactivation by C1. A pan-PKC inhibitor significantly blocked the phorbol ester-induced but not the C1-induced HIV-1 reactivation. Although C1-induced viral gene expression persisted for as long as 48 h post-stimulation, NF-κB-dependent transcription peaked at 12 h post-stimulation and then quickly declined, suggesting Tat-mediated self-sustainment of HIV-1 expression. These results suggest that procyanidin C1 trimer is a potential compound for reactivation of latent HIV-1 reservoirs.

New anti-trypanosomal active tetracyclic iridoid isolated from Morinda lucida Benth.

Human African trypanosomiasis (HAT), commonly known as sleeping sickness has remained a serious health problem in many African countries with thousands of new infected cases annually. Chemotherapy, which is the main form of control against HAT has been characterized lately by the viewpoints of toxicity and drug resistance issues. Recently, there have been a lot of emphases on the use of medicinal plants world-wide. Morinda lucida Benth. is one of the most popular medicinal plants widely distributed in Africa and several groups have reported on its anti-protozoa activities. In this study, we have isolated one novel tetracyclic iridoid, named as molucidin, from the CHCl3 fraction of the M. lucida leaves by bioassay-guided fractionation and purification. Molucidin was structurally elucidated by (1)H and (13)C NMR including HMQC, HMBC, H-H COSY and NOESY resulting in tetracyclic iridoid skeleton, and its absolute configuration was determined. We have further demonstrated that molucidin presented a strong anti-trypanosomal activity, indicating an IC50 value of 1.27 µM. The cytotoxicity study using human normal and cancer cell lines indicated that molucidin exhibited selectivity index (SI) against two normal fibroblasts greater than 4.73. Furthermore, structure-activity relationship (SAR) study was undertaken with molucidin and oregonin, which is identical to anti-trypanosomal active components of Alnus japonica. Overlapping analysis of the lowest energy conformation of molucidin with oregonin suggested a certain similarities of aromatic rings of both oregonin and molucidin. These results contribute to the future drug design studies for HAT.

Generation of an anti-Dabigatran Monoclonal Antibody and Its Use in a Highly Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Serum Dabigatran.

BACKGROUND: Dabigatran (DT) is a direct thrombin inhibitor used to prevent venous and arterial thromboembolism due to atrial fibrillation. DT is the active form of the commercially available prodrug DT etexilate. Although DT has many clinical advantages over warfarin, it increases the incidence of bleeding in patients with renal dysfunction. Circulating levels of DT are increased in such patients because it is mainly eliminated by renal excretion. Therapeutic drug monitoring may therefore help to prevent adverse DT effects, but no method for measuring circulating DT levels has been reported, except for an analysis by liquid chromatography-tandem mass spectrometry. This study sought to develop a novel enzyme-linked immunosorbent assay (ELISA) to measure DT concentrations. METHODS: Mice were immunized with a DT-keyhole limpet hemocyanin conjugate to generate an anti-DT antibody. Immunized mouse splenocytes and myeloma cells (SP2/0) were fused to obtain an anti-DT monoclonal antibody (DT-mAb). DT-mAb and DT solutions were added to microplate wells coated with a DT-human serum albumin conjugate. DT concentrations were determined based on the principles of ELISA. RESULTS: DT-mAb was successfully purified from a hybridoma, and the competitive ELISA developed using this DT-mAb could evaluate DT concentrations ranging from 7.8 to 125 ng/mL. The ELISA signal was not linear using DT-spiked serum; however, it was linear when serum ultrafiltrate was used. Weak cross-reactivity with DT etexilate was detected, but no cross-reactivity was observed with other structurally related drugs or drugs commonly used for the treatment of atrial fibrillation. CONCLUSIONS: The developed competitive ELISA is a valuable and specific tool to analyze free DT in serum ultrafiltrate for therapeutic drug monitoring and pharmacokinetic studies.

Anti-inflammatory Activity of Constituents Isolated from Aerial Part of Angelica acutiloba Kitagawa.

Recently, the resources of medicinal plants have been exhausting. The root of Angelica acutiloba is one of the most important ingredients in Japanese Kampo medicine for the treatment of gynecological diseases. In our search for alternative medicinal plant resources of the root of A. acutiloba, we found that its aerial part has the anti-inflammatory potency as well as the root. Phytochemical investigation of the aerial part resulted in the isolation of four compounds including a new dimeric phthalide, namely tokiaerialide (2), along with Z-ligustilide (1), falcarindiol (3), and bergaptol (4). Next, we investigated the in vitro anti-inflammatory activity of 1-4 in lipopolysaccharide-stimulated RAW264 macrophages. Among the isolated compounds, 1 exhibited the most potent inhibition against lipopolysaccharide-induced production of prostaglandin E2 , nitric oxide, and pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α). Compounds 3 and 4 also inhibited all inflammatory mediators, but their inhibitory abilities were weaker than those of 1. Furthermore, 1, 3, and 4 strongly also induced heme oxygenase-1. These results suggest that 1, 3, and 4 potentially exert anti-inflammatory activity, and the aerial part of A. acutiloba may be considered to be a useful medicinal resource for inflammatory diseases.

Investigation of interfacial behavior of glycyrrhizin with a lipid raft model via a Langmuir monolayer study.

An interaction of glycyrrhizin (GC) with a lipid raft biomembrane model that consisted of N-palmitoyl-d-erythro-sphingosylphosphorylcholine (PSM), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (CHOL) was systematically studied using the Langmuir monolayer technique. To construct the lipid raft model, the surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms for three-component (PSM/DOPC/CHOL) systems on 0.02M Tris buffer with 0.13M NaCl (pH7.4) were primarily measured by changing their compositions. Thermodynamic and interaction parameters for binary PSM/DOPC and PSM/CHOL systems revealed that PSM interacts more strongly with CHOL than with DOPC. In addition, a morphological analysis performed with Brewster angle microscopy (BAM) and fluorescence microscopy (FM) revealed an optimal ratio of PSM/DOPC/CHOL (1/1/1, by mole) as a model of lipid rafts. Second, the interaction of GC with the ternary PSM/DOPC/CHOL monolayers was investigated on Tris buffer solutions containing different GC concentrations (1, 5, 10, 25, and 50µM). In BAM and FM images, microdomains were found to become smaller by increasing the GC concentration in the subphase, suggesting that GC regulates the size of raft domains, which provide dynamic scaffolding for numerous cellular processes. More interestingly, the distinctive GC striped regions were formed at the interface at 50µM, which shows that GC divides the ternary monolayer into pieces. This phenomenon was observed only in the presence of CHOL in the monolayer. These results suggest that CHOL plays an essential role in the interaction with GC, which results in one of the major activities associated with saponins' membrane disruption.

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.

Antiproliferative and apoptotic effects of compounds from the flower of Mammea siamensis (Miq.) T. Anders. on human cancer cell lines.

On the search for anti-cancer compounds from Thai traditional herb medicines, a bioassay-guided fractionation and chemical investigation of the methanol extract of Mammea siamensis flower resulted in the isolation and identification of eight compounds (1-8) including a novel geranylated coumarin, namely mammeanoyl (2), and seven known compounds (1 and 3-8). The structure of new compound 2 was elucidated based on the extensive spectroscopic and chemical methods. Among the isolated compounds, three structurally related coumarins 3, 4, and 5 showed significant antiproliferative activities against human leukemia and stomach cancer cell lines. However, these compounds did not affect the cell viabilities of colon cancer, hepatoma, and normal skin fibroblast cell lines. Further analysis demonstrated that the morphological features of apoptosis including DNA fragmentation and chromatin condensation were observed in human leukemia HL-60 cells treated with compounds 3, 4, and 5. In addition, compound 3 led to caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP), and compound 3-induced DNA fragmentation was inhibited by caspase-specific inhibitors. These results suggest that compound 3, 4, and 5 exert antiproliferative actions through apoptotic cell death in leukemia cells and these compounds may have the potential to be developed into new anti-cancer drug candidates.

Detection of paeoniflorin and albiflorin by immunostaining technique using anti-paeoniflorin monoclonal antibody.

INTRODUCTION: Paeoniae radix is one of the most important crude drugs used in Kampo medicines (KMs). A part of its pharmaceutical properties is due to the presence of paeoniflorin (PF) and albiflorin (AF). OBJECTIVE: For the specific and easy identification of PF and AF, an immunostaining technique was developed using anti-PF monoclonal antibody (MAb). METHODOLOGY: PF and AF were treated with a NaIO4 solution and reacted with bovine serum albumin (BSA) preparing PF- and AF-BSA conjugates on the polyethersulphone (PES) membrane. Anti-PF MAb was bound and then antibody labelled with peroxidase directed against anti-PF MAb. Finally, a substrate was added and then PF and AF were detected. RESULTS: Anti-PF MAb recognised not only PF but also AF when 10 µg was present on the PES membrane. As little as 0.5 µg of PF and AF were still detected under immunostaining. Various Paeoniae radix samples and KMs were qualitatively analysed, and total amounts of PF and AF were visually detected by immunostaining technique. This method was applied to investigate the distribution of PF and AF in fresh peony root using immunoblotting by transferred from peony root to the PES membrane. CONCLUSION: The technique permitted the visualisation of PF and AF on PES membrane using immunostaining. The immunostaining technique established would be a powerful tool for probing the sources of PF and AF in plant extracts.

Development of an Eastern blotting technique for the visual detection of aristolochic acids in Aristolochia and Asarum species by using a monoclonal antibody against aristolochic acids I and II.

INTRODUCTION: Aristolochic acids (AAs) are naturally occurring nephrotoxicants and human carcinogens. Aristolochic acid I (AA-I) and aristolochic acid II (AA-II) are two important AAs with clear toxicity. OBJECTIVE: To obtain a monoclonal antibody (MAb) recognising AA-I and AA-II and develop an Eastern blotting technique for the specific visualisation and easy determination of AA-I and AA-II in plant extracts or tissues of Aristolochia and Asarum species. METHODS: A hybridoma secreting MAb against AAs was prepared by cell fusion with splenocytes derived from a mouse immunised with AA-I-keyhole limpet haemocyanin (KLH) conjugate and the myeloma cell line SP2/0-Ag14. AA-I and AA-II were separated by thin-layer chromatography (TLC) and then blotted onto a positively charged polyethersulphone (PES) membrane using a modified carbodiimide method. The resulting membrane-bound AA-protein conjugates were linked to the newly prepared MAb and then to the secondary antibody labelled with peroxidase. 4-Chloro-1-naphthol was then added as the peroxidase substrate for staining. RESULTS: MAb 2A10-10B showed a high specificity for AA-I (100%) and AA-II (69.3%) and low cross reactivity (≤ 2.2%) toward analogues that may disrupt detection of AA-I and AA-II in plants. An established Eastern blotting method was applied to the immunohistolocalisation of AA-I and AA-II in dry plant tissues, and this analysis showed that the phelloderm, cortex and phloem of Aristolochia manshuriensis stem may contain higher amounts of total AA-I and AA-II as compared with the pith and xylem. CONCLUSION: This method was extremely useful for the visual screening of AA-I and AA-II among easily mistaken herbal medicines.

Anti-Proliferative Activities and Apoptosis Induction by Triterpenes Derived from Eriobotrya japonica in Human Leukemia Cell Lines.

Eriobotrya japonica leaf is a traditional herbal medicine that contains numerous triterpenes, which have various pharmacological properties. In this study, we investigated the anti-proliferative activity of four triterpenes derived from E. japonica, including corosolic acid (CA), ursolic acid (UA), maslinic acid (MA) and oleanolic acid (OA), in human leukemia cell lines. CA showed the strongest anti-proliferative activity in all of the leukemia cell lines tested, but not in normal human skin fibroblast cell lines. To determine the mechanism underlying the anti-proliferative effect of CA, we examined the effect of CA on molecular events known as apoptosis induction. CA induced chromatin condensation, DNA fragmentation, sub-G(1) phase DNA, activation of caspase-3, -8 and -9 and the cleavage of PARP in HL-60. CA also activated Bid and Bax, leading to the loss of mitochondrial membrane potential (∆ψ(m)) and cytochrome c release into the cytosol, whereas Bcl-2 and Bcl-xL were unaffected by CA. These results suggest that CA has an anti-proliferative effect on leukemia cells via the induction of apoptosis mediated by mitochondrial dysfunction and caspase activation. CA may be a potential chemotherapeutic agent for the treatment of human leukemia.