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Monoclonal antibodies (Mabs) are widely used in a variety of fields, including protein identification, life sciences, medicine, and natural product chemistry. This review focuses on Mabs against naturally occurring active compounds. The preparation of Mabs against various active compounds began in the 1980s, and now there are fewer than 50 types. Eastern blotting, which was developed as an antibody staining method for low-molecular-weight compounds, is useful for its ability to visually represent specific components. In this method, a mixture of lower-molecular-weight compounds, particularly glycosides, are separated by thin-layer chromatography (TLC). The compounds are then transferred to a membrane by heating, followed by treatment with potassium periodate (KIO4) to open the sugar moiety of the glycoside on the membrane to form an aldehyde group. Proteins are then added to form Schiff base bonds to enable adsorption on the membrane. A Mab is bound to the glycoside moiety on the membrane and reacts with a secondary antibody to produce color. Double Eastern blotting, which enables the simultaneous coloration of two glycosides, can be used to evaluate quality and estimate pharmacological effects. An example of staining by Eastern blotting and a component search based on the results will also be presented. A Mab-associated affinity column is a method for isolating antigen molecules in a single step. However, the usefulness of the wash fractions that are not bound to the affinity column is unknown. Therefore, we designated the wash fraction the “knockout extract”. Comparing the nitric oxide (NO) production of a glycyrrhizin (GL)-knockout extract of licorice with a licorice extract revealed that the licorice extract is stronger. Therefore, the addition of GL to the GL-knockout extract of licorice increased NO production. This indicates that GL has synergic activity with the knockout extract. The GL-knockout extract of licorice inhibited high-glucose-induced epithelial–mesenchymal transition in NRK-52E cells, primarily by suppressing the Notch2 pathway. The real active constituent in licorice may be constituents other than GL, which is the causative agent of pseudohyperaldosteronism. This suggests that a GL-knockout extract of licorice may be useful for the treatment of diabetic nephritis.

The objective of this review is to evaluate the anti-dementia activities of saffron and its combination with Kampo medicine. The Kampo formula Kamiuntanto composed of 13 crude drugs is well known for its anti-dementia activity. A significant increase in choline acetyltransferase activity and mRNA levels were observed. Polygala radix was identified as the most essential component drug in Kamiuntanto, probably due to the saponins, tenuifolin, and sinapinic acid. Ginseng was also identified as an essential Kamiuntanto component in terms of its synergistic functions with Polygala radix. Saffron, which was recommended in the Bencao Gangmu for memory and dementia, and is used as an anti-spasmodic, anti-catarrhal, and sedative herbal drug. Saffron and its major constituent, crocin were shown to enhance learning-memory, non-rapid eye movement (rem) sleep, and inhibit depression and neuronal cell death due to strong anti-oxidant and anti-inflammation activities. In addition based on the epidemiological studies such as the treatment of sleeping disorders and the clinical trials of saffron for Alzheimer patients, we demonstrated the indirect and direct anti-dementia activities of crocin and saffron.

Cannabis sativa L. is an annual herb oldest cultivated plants as a source of fiber since about 5000 B.C. On the other hand, the cannabis flower and seed are listed in Shennong’s classic Materia Medica approximately 2000 years ago. The formulas prescribed with cannabis in Kampo medicine have been summarized. Cannabidiol (CBD) and tetrahydrocannabinol (THC) are the major neurological and psychiatric cannabinoids, and develop to drugs. It becomes evident that the therapeutic CBD and/or THC are the important candidate of anti-dementia drugs having different mechanism for Alzheimer’s patients. Two receptors and endocannabinoids are also discussed for underlying mechanism of action. In order to promote the breeding of cannabis plant containing higher concentration of target cannabinoid the biosynthetic enzymes were isolated, cloning and the tertiary structure of THCA synthase determined by x-ray analysis resulting in the possibility of molecular breeding for cannabinoids.

Abstract 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.

Abstract Purpose The direct effects of Lepidium meyenii (Maca) on sperm remain unclear. Herein, we examined the direct effect of Maca on in vitro fertilization. Methods We examined the fertilization rate in a mouse model and the rate of acrosome reaction in sperm from transgenic mice expressing enhanced green fluorescent protein (EGFP) in a Maca extract‐containing human tubal fluid (HTF) medium. Using human sperm, we assessed acrosome status via fluorescein isothiocyanate‐conjugated peanut agglutinin (FITC‐PNA) staining and performed detailed analysis using a sperm motility analysis system (SMAS). Results In the mouse model, the fertilization rate in the Maca extract‐containing HTF was significantly higher than that in the control medium. The acrosome reaction rate in sperm from transgenic mice expressing EGFP was also significantly higher in the Maca extract‐containing HTF than that in the control medium. Similarly, a high acrosome reaction rate, identified via FITC‐PNA staining of human sperm samples, was found in the Maca extract‐containing HTF compared with that in the control medium. Human sperm motility in the Maca extract‐containing HTF was also increased compared with that in the control medium as measured using an SMAS. Conclusions Maca improved in vitro fertilization rates by inducing an acrosome reaction and increasing sperm motility.

Hirsutanone (Hir) and oregonin (Ore) are diarylheptanoids isolated from the bark of Alnus japonica. In this study, we investigated the anti-melanogenic activity of Hir and Ore in B16-F1 murine melanoma and normal human epidermal melanocytes (HEMn-DP) and elucidated the mechanisms of action. In B16-F1 cells, Hir and Ore suppressed melanin synthesis induced by α-melanocyte-stimulating hormone (α-MSH) without cytotoxicity. The inhibitory effect of Hir on melanin synthesis was much stronger than that of Ore. In addition, Hir reduced melanin content in HEMn-DP cells. As tyrosinase is a key enzyme in melanin synthesis, the effect of Hir on tyrosinase activity was assessed. The results demonstrated that Hir partially decreased tyrosinase activity and intracellular tyrosinase activity. Moreover, Hir suppressed the protein expression of melanogenic enzymes, including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2, leading to reduced melanin biosynthesis. Hir also led to the suppression of cAMP response element-binding protein (CREB) phosphorylation and microphthalmia-associated transcription factor (MITF) expression, which control the expression of melanogenic enzymes. These results suggest that Hir suppressed melanin synthesis by dual inhibition of tyrosinase activity and the CREB/MITF pathway leading to the expression of melanogenic enzymes and may be a potent cosmetic and therapeutic agent for hyperpigmentation disorders.

In this study, we present a review on a useful approach, namely, immunoaffinity column coupled with monoclonal antibodies (MAbs), to separate natural compounds and its application for cell-based studies. The immunoaffinity column aids in separating the specific target compound from the crude extract. The column capacity was stable even after more than 10 purification cycles of use under the same conditions. After applying the crude extract to the column, the column was washed with washing buffer and eluted with elution buffer. The elution fraction contained the target compound bound to MAb, whereas the washing fraction was the crude extract, which contained all compounds except a group of target compounds; therefore, the washing fraction was referred to as a knockout (KO) crude extract. Cell-based studies using the KO extract revealed the actual effects of the natural compounds in the crude extract. One-step separation of natural compounds using the immunoaffinity column coupled with MAbs may help in determining the potential functions of natural compounds in crude extracts.

An immunoblotting system (“eastern blotting”) was developed for small-molecule herbal medicines like glycosides, with no conjugation function to the membrane. Briefly, the crude extracts of herb medicines were developed by thin-layer chromatography (TLC). The small-molecule herbal medicines on TLC plates were transferred to polyvinylidene fluoride (PVDF) or polyethersulfone (PES) membranes by heating. Antigen components were divided into two categories based on their function, i.e., their membrane recognizing (aglycone part) and fixing (sugar moiety) abilities. This procedure allows for the staining of only target glycosides. Double eastern blotting was developed as a further staining system for two herb medicines using a set of MAbs and substrates.

Our preliminary screening identified an extract from the rhizome of Dioscorea tokoro, which strongly suppressed the proliferation of HepG2 hepatocellular carcinoma cells and inhibited autophagy. This study aimed to isolate active compounds from the rhizome of D. tokoro that exert antiproliferative effects and inhibit autophagy. The bioassay-guided fractionation of the active fraction led to the isolation of two spirostan-type steroidal saponins, dioscin (1) and yamogenin 3-O-α-l-rhamnopyranosyl (1→4)-O-α-l-rhamnopyranosyl(1→2)-β-d-glucopyranoside (2), and the frostane-type steroidal saponin protodioscin (3) from the n-BuOH fraction. Furthermore, acid hydrolysis of 1 and 2 produced the aglycones diosgenin (4) and yamogenin (5), respectively. Compounds 1–5 suppressed proliferation of HepG2 cells. The analysis of structure-activity relationships indicated that the 25(R)-conformation, structures with a sugar moiety, and the spirostan-type aglycone moiety contributed to antiproliferative activity. Analysis of autophagy-related proteins demonstrated that 1–3 clearly increased the levels of both LC3-II and p62, implying that 1–3 deregulate the autophagic pathway by blocking autophagic flux, which results in p62 and LC3-II accumulation. In contrast, 1–3 did not significantly affect caspase-3 activation and PARP cleavage, suggesting that the antiproliferative activity of 1–3 occurred independently of caspase-3-mediated apoptosis. In summary, our study showed that 1–3, active compounds in the rhizome of D. tokoro, suppressed cell proliferation and autophagy, and might be potential agents for autophagy research and cancer chemoprevention.

Monoclonal antibodies (MAbs) against natural products with low-molecular weights have become an important tool when combined with other analytical systems. Eastern blotting involves a typical staining system wherein, for example, glycosides can be blotted to a membrane and cross-linked and stained using MAbs. An immunoaffinity column combined with a monoclonal antibody allows a one-step purification of hapten compounds or preparation of a knockout extract that removes only the target hapten molecules from a crude extract. Here, we discuss the application of these extracts. Single-chain variable fragment (scFv) proteins have led to novel assay systems such as fluobodies or antibodies coupled with green fluorescent protein for natural products. A typical novel application of a scFv gene would be in the context of plant breeding; this is designated “missile-type” molecular breeding, intended to increase hapten molecule concentrations in transgenic plants.

Tubulointerstitial fibrosis is a major pathological hallmark of diabetic nephropathy. Increasing evidence has shown that epithelial-to-mesenchymal transition (EMT) of renal proximal tubular cells plays a crucial role in tubulointerstitial fibrosis. Herein, we aimed to elucidate the detailed mechanism of EMT in renal tubular cells under high glucose (HG) conditions, and to investigate the potential of licorice, a medicinal herb, to inhibit HG-induced EMT. Our results showed that renal tubular epithelial cells (normal rat kidney cell clone 52E; NRK-52E) exposed to HG resulted in EMT induction characterized by increased fibronectin and α-SMA (alpha-smooth muscle actin) but decreased E-cadherin. Elevated levels of cleaved Notch2, MAML-1 (mastermind-like transcriptional coactivator 1), nicastrin, Jagged-1 and Delta-like 1 were also concomitantly detected in HG-cultured cells. Importantly, pharmacological inhibition, small interfering RNA (siRNA)-mediated depletion or overexpression of the key components of Notch2 signaling in NRK-52E cells supported that the activated Notch2 pathway is essential for tubular EMT. Moreover, we found that licorice extract (LE) with or without glycyrrhizin, one of bioactive components in licorice, effectively blocked HG-triggered EMT in NRK-52E cells, mainly through suppressing the Notch2 pathway. Our findings therefore suggest that Notch2-mediated renal tubular EMT could be a therapeutic target in diabetic nephropathy, and both LE and de-glycyrrhizinated LE could have therapeutic potential to attenuate renal tubular EMT and fibrosis.

Background: Liquiritin (LQ) and its aglycone, liquiritigenin (LQG), are major flavonoids in licorice root (Glycyrrhiza spp.). Our preliminary screening identified LQ and LQG, which promote melanin synthesis in the melanoma cells. In this study, we investigated the molecular mechanism of melanin synthesis activated by LQ and LQG. Methods: Murine (B16-F1) and human (HMVII) melanoma cell lines were treated with LQ or LQG. After incubation, melanin contents, intracellular tyrosinase activity, and cell viability were evaluated. Protein levels were determined using Western blotting. Results: LQ and LQG activated melanin synthesis and intracellular tyrosinase activity. The induction of melanin and intracellular tyrosinase activity by LQG was higher than that by LQ. LQ and LQG induced the expression of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. LQ and LQG also enhanced microphthalmia-associated transcription factor (MITF) expression, and cyclic AMP-responsive element-binding protein (CREB) phosphorylation. The phosphorylation of p38 and extracellular signal-regulated kinase (ERK), but not Akt, was significantly increased by LQ or LQG. Furthermore, LQ- or LQG-mediated melanin synthesis was partially blocked by p38 inhibitor (SB203580) and protein kinase A (PKA) inhibitor (H-89); however, ERK kinase (MEK) inhibitor (U0126) and phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002) had no effect. Conclusions: The results suggest that LQ and LQG enhance melanin synthesis by upregulating the expression of melanogenic enzymes, which were activated by p38 and PKA signaling pathways, leading to MITF expression and CREB phosphorylation.

Background: Autophagy is a catabolic process through which dysfunctional proteins and organelles are degraded, and that is associated with the proliferation of cancer cells. The aim of this study was to screen approximately 130 kinds of crude drugs used in Japanese Kampo formulas to identify crude drugs that would regulate the proliferation through autophagy of human hepatocellular carcinoma HepG2 cells. Methods: Extracts of each crude drug were prepared using methanol. Protein levels were determined using Western blotting. Cell viability was measured by MTT assay. Results: Among the 130 crude extracts, 24 of them increased LC3-II expression. Among these, Goboshi (burdock fruit), Soboku (sappan wood), Mokko (saussurea root), Rengyo (forsythia fruit), and Hikai (dioscorea) notably suppressed the proliferation of HepG2 cells and increased p62 expression levels, which suggested that these five extracts downregulate the autophagic activity resulting in the accumulation of p62. On the other hand, Hishinomi (water chestnut), Biwayo (loquat leaf), and Binroji (areca) induced cell growth and decreased or were uninvolved with p62 expression levels, which implied that these three extracts might induce autophagy modulators for cell growth. Conclusions: The results suggest that the compounds contained in the crude drugs selected for this study could control cell viability by regulating autophagic activity in HepG2 cells. The isolation and identification of the active compounds in these drugs might lead to the development of agents for autophagy research and cancer chemoprevention.

The aim of this study is to establish a time-resolved fluoroimmunoassay (TRFIA) system for quantitative analysis of saikosaponin a (SSa) in the crude drug of Chaihu (Bupleuri Radix). A 96-well microplate coated with rabbit anti-mouse IgG was incubated with the methanol extracts of Chaihu samples and a mouse anti-SSa monoclonal antibody, and a Eu3+-labeled SSa-human serum albumin conjugate was used as the tracer. The established competitive TRFIA showed a good fourth order polynomial fitting from 0.01 to 10.0 μg/mL for standard SSa sample with a detection limit of 0.006 μg/mL. The intra- and inter-assay coefficients of variation of the assay were 7.3% and 8.9%, respectively, and the average SSa recovery was 119.2%. For samples of Chaihu extract, the results of this assay showed a good correlation with those by enzyme-linked immunosorbent assay established previously. This TRFIA system is ultrasensitive for detecting SSa with a wide detection range and a good stability and represents the first attempt of using TRFIA for quality evaluation of the crude drug of Chaihu.

Importance of herbal medicines have recently increased owing to rising interest in their health benefits. However, medicinal plant extracts are complex mixtures of phytochemicals that act synergistically or additively on specific and/or multiple molecular and cellular targets. Thus, it is difficult to examine the actual pharmacological roles of active compounds in plant extracts. This review describes a new strategy for isolating target compounds from plant extracts using immunoaffinity columns coupled with monoclonal antibodies (mAbs) against natural compounds. Through one-step purification using mAb-coupled immunoaffinity columns, we succeeded in preparing a knockout (KO) extract, which contains all components except the target compound. Furthermore, we investigated the pharmacological effects of the KO extract to reveal the actual effects of a bioactive compound in the crude extract. This approach may help determine the potential function of target compounds in herbal medicines.

We prepared a monoclonal antibody (mAb 1C1) showing specificity for artemisinin (AM) and artesunate (AS), and we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) using this novel mAb. Moreover, we prepared a recombinant antibody derived from mAb 1C1 in order to overcome insufficient mAb production by hybridoma culture. A recombinant antigen-binding fragment (Fab) was easily constructed using antibody manipulation technologies and was produced by microorganisms in high yield. We herein review immunochemical approaches for analysis of the antimalarial drugs AM and AS that were able to yield analysis results for multiple samples in a short period of time using simple and reliable protocols.

Scutellariae radix (S. radix) is one of the most important crude drugs used in Kampo medicines (KMs). A part of its pharmaceutical properties is due to flavone glycoside, baicalin (BI). A technique named eastern blotting was developed for the specific and easy identification of BI in the extracts of crude drugs and KMs using anti-BI monoclonal antibody (MAb). BI separated by silica gel thin-layer chromatography (TLC) transferred to a polyethersulfone (PES) membrane was treated with a NaIO4 solution and reacted with bovine serum albumin (BSA) preparing BI-BSA conjugate on the PES membrane. Anti-BI MAb was bound and then antibody labeled with peroxidase directed against anti-BI MAb. Finally, a substrate was added and then BI was detected. As little as 1 mg of BI was still detected on the PES membrane under immunostaining method. Various samples of S. radix and KMs which contain S. radix were qualitatively analyzed, and BI was visually detected by eastern blotting technique. Furthermore, this method was applied for the immunohistochemical study to investigate the distribution of BI in the fresh root of Scutellaria baicalensis using immunoblotting by transferred from fresh root to the PES membrane.

An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb), Fab antibody, and single-chain variable fragment (scFv) antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP) with a scFv antibody, named fluobody, was proposed as a probe for an alternative immunosorbent assay; i.e., fluorescence-linked immunosorbent assay (FLISA). In this FLISA, an even more sensitive, simple, and rapid immunoassay can be performed by detecting the highly sensitive fluorophore of GFP that is genetically and directly fused to the scFv antibody. In addition, the time- and cost-consuming secondary antibody reaction and the following enzyme-substrate reaction, necessary for conventional ELISA, can be avoided, making it possible to complete the assay more rapidly. Focusing on naturally occurring bioactive products, fluobody recognizing 1,4-naphthoquinone, plumbagin and triterpenoid saponin, ginsenosides were successfully expressed in Escherichia coli (E. coli) and applied to FLISA. The construction, the expression, and the potential use of fluobody in quantitative/qualitative analysis of bioactive natural products are reviewed in this article.

To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs) were prepared. Ginsenosides were developed on thin layer chromatography (TLC) plates which were covered by a polyvinylidene difluoride (PVDF) membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

This study showed the effective microanalytic methods for detection of saikosaponin by a competitive enzyme-linked immunosorbent assay (ELISA) using anti-saikosaponin a (SSa) monoclonal antibody (MAb). The results showed that the competitive ELISA had higher sensitivity than high performance liquid chromatography (HPLC) for the detection of saikosaponin. ELISA showed a highly significant difference in SSa concentrations between the roots of Bupleurum falcatum with different origins. In addition, a difference in SSa concentrations was observed between cytologically different cultivars originating from Japan (2n = 26) and Korea (2n = 20). It also showed excellent performance in quantitative analysis of SSa in the small samples from regenerated plantlets and calli. In addition, Eastern blotting using anti-SSa MAb 3G10 multispecific to saikosaponins was applied to analyze the distribution of saikosaponins in Bupleurum falcatum roots and leaves. The localized distribution of saikosaponins in whole leaves and cork layer of the roots in B. falcatum was successfully demonstrated by this novel immunoassay.

To prepare the antiglycyrrhizin (GC) monoclonal antibody (MAb), GC was treated with NaIO4 resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated by NaIO4 and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4 treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

Matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was used for the confirmation of hapten number in synthesized antigen. As application of MAb, the MAbs against ginsenosides and glycyrrhizin have been prepared resulting in the development of two new techniques that we named the eastern blotting method and the knockout extract preparation. In eastern blotting technique, glycosides like ginsenosides and glycyrrhizin separated by silica gel TLC were blotted to PVDF membrane that was treated with a NaIO4 solution followed by BSA resulted in glycoside-BSA conjugate on a PVDF membrane. The blotted spots were stained by MAb. Double staining of eastern blotting for ginsenosides using antiginsenoside Rb1 and Rg1 MAbs promoted complete identification of ginsenosides in Panax species. The immunoaffinity concentration of glycyrrhizin was determined by immunoaffinity column conjugated with antiglycyrrhizin MAb resulting in the glycyrrhizin-knockout extract, which was determined by the synergic effect with glycyrrhizin on NO production using the cell line.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a major bioactive compound in wasabi (Wasabia japonica), which is a typical Japanese pungent spice. Recently, in vivo and in vitro studies demonstrated that 6-MSITC has several biological properties, including anti-inflammatory, antimicrobial, antiplatelet, and anticancer effects. We previously reported that 6-MSITC strongly suppresses cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and cytokines, which are important factors that mediate inflammatory processes. Moreover, molecular analysis demonstrated that 6-MSITC blocks the expressions of these factors by suppressing multiple signal transduction pathways to attenuate the activation of transcriptional factors. Structure-activity relationships of 6-MSITC and its analogues containing an isothiocyanate group revealed that methylsulfinyl group and the length of alkyl chain of 6-MSITC might be related to high inhibitory potency. In this paper, we review the anti-inflammatory properties of 6-MSITC and discuss potential molecular mechanisms focusing on inflammatory responses by macrophages.