Best Practice in the chemical characterisation of extracts used in pharmacological and toxicological research-The ConPhyMP-Guidelines.

Background: Research on medicinal plants and extracts derived from them differs from studies performed with single compounds. Extracts obtained from plants, algae, fungi, lichens or animals pose some unique challenges: they are multicomponent mixtures of active, partially active and inactive substances, and the activity is often not exerted on a single target. Their composition varies depending on the method of preparation and the plant materials used. This complexity and variability impact the reproducibility and interpretation of pharmacological, toxicological and clinical research. Objectives: This project develops best practice guidelines to ensure reproducibility and accurate interpretations of studies using medicinal plant extracts. The focus is on herbal extracts used in pharmacological, toxicological, and clinical/intervention research. Specifically, the consensus-based statement focuses on defining requirements for: 1) Describing the plant material/herbal substances, herbal extracts and herbal medicinal products used in these studies, and 2) Conducting and reporting the phytochemical analysis of the plant extracts used in these studies in a reproducible and transparent way. The process and methods: We developed the guidelines through the following process: 1) The distinction between the three main types of extracts (extract types A, B, and C), initially conceptualised by the lead author (MH), led the development of the project as such; 2) A survey among researchers of medicinal plants to gather global perspectives, opportunities, and overarching challenges faced in characterising medicinal plant extracts under different laboratory infrastructures. The survey responses were central to developing the guidelines and were reviewed by the core group; 3) A core group of 9 experts met monthly to develop the guidelines through a Delphi process; and. 4) The final draft guidelines, endorsed by the core group, were also distributed for feedback and approval to an extended advisory group of 20 experts, including many journal editors. Outcome: The primary outcome is the "Consensus statement on the Phytochemical Characterisation of Medicinal Plant extracts" (ConPhyMP) which defines the best practice for reporting the starting plant materials and the chemical methods recommended for defining the chemical compositions of the plant extracts used in such studies. The checklist is intended to be an orientation for authors in medicinal plant research as well as peer reviewers and editors assessing such research for publication.

Investigation of Chemopreventive and Antiproliferative Potential of Dicliptera roxburghiana.

CONTEXT: Carcinogenesis causes much human misery. It is a process involving multistage alterations. Medicinal plants are candidates for beneficial anticancer agents. OBJECTIVES: Investigation of anticancer proficiencies of the plant Dicliptera roxburghiana. MATERIAL AND METHODS: Crude extract and derived fractions were inspected for their inhibitory potential against nuclear factor KB (NFκB), nitric oxide synthase inhibition, aromatase inhibition and induction of quinone reductase 1 (QR 1). Antiproliferative activity was determined by using various cancer cell lines for example hormone responsive breast cancer cell line MCF-7, estrogen receptor negative breast cancer cell line MDA-MB-231, murine hepatoma cells Hepa 1c1c7, human neuroblastoma cells SK-N-SH and neuroblastoma cells MYCN-2. RESULTS: Ethyl acetate and n-butanol fractions of D. roxburghiana were strongly active against NFκB with IC50 of 16.6 ± 1.3 and 8.4 ± 0.7 µg/ml respectively with 100% survival. Chloroform fraction of the plant exhibited an induction ratio of 2.4 ± 0.09 with CD value of 17.7 µg/ml. Regarding the nitrite assay, the n-hexane fraction exhibited significant inhibition of NO activity with IC50 of 17.8 ± 1.25 µg/ml. The n-butanol fraction exhibited strong antiproliferative activity against IcIc-7 cell lines with IC50 values of 13.6 ± 1.91 µg/ml; against MYCN-2 a cytotoxic effect developed with dose dependence, with IC50 of 12.6 ± 1.24 µg/ml. In antiproliferative activity against SK-N-SH cell lines, chloroform, ethyl acetate and n-butanol fractions were efficiently active with IC50 values of 11.2 ± 0.84, 14.6 ± 1.71 and 16.3 ± 1.57 respectively. DISCUSSION AND CONCLUSION: It was demonstrated that various fractions of D. roxburghiana displayed appreciable anticancer characteristics and could be a potent source for the development of anticancer leads.

Best practice in research - Overcoming common challenges in phytopharmacological research.

BACKGROUND: The pharmacology, toxicology and pharmacokinetics of bioactive preparations derived from natural sources has become a flourishing field of research. However, researching complex extracts and natural products faces numerous challenges. More broadly in recent years the critique of pharmacological research, and specifically its design, the methods used and reporting has intensified. AIMS: This consensus document provides a perspective on what constitutes best practice in pharmacological research on bioactive preparations derived from natural sources, providing a perspective of what the leading specialist journals in the field consider as the core characteristics of good research. APPROACH ('METHODS'): The editors-in-chief of seven journals developed this best practice statement in an iterative process. A first draft of the guidelines (prepared by MH) was then discussed and amended by the other editors. OUTCOMES: Core to this contribution is a table which provides detailed advice including simple points like a use of appropriate controls and the full taxonomic validity of the material under investigation (see also below), to the relevance of the model for the question being researched (e.g., can specific in silico or in vitro models really address the species anti-inflammatory activity?). Therefore, obviously, researchers must pay detailed attention to reporting and discussing such studies. This information must be discussed critically (as much as it is possible based on the published papers) in terms of their scientific quality and validity. While these points are obvious, as editors, we are aware that they are often not properly implemented. CONCLUSION: We call for an approach which incorporates a careful design, meticulous execution and a detailed reporting of studies focusing on the pharmacology/bioactivity of bioactive preparations. Clearly testable research questions must be developed and investigated experimentally. As the founder of pharmacology Claude Bernard put it already in 1865: '…. either the experimenter's hypothesis will be disproved or it will be proved by experiment. When experiment disproves its preconceived ideas, the experimenter must discard or modify it.'

Induction of NAD (P)H: Quinone reductase 1 (QR1) and antioxidant activities in vitro of 'Toranja Burarama' (Citrus maxima [Burm.] Merr.).

Toranja 'Burarama', Citrus maxima (Burm.) Merr. (Citrus grandis), is a new citrus discovered in the State of Espírito Santo, Brazil. As several varieties of citrus are known to possess antioxidant and cancer chemopreventive properties, the aim of the study was to evaluate in vitro if this Toranja possess these properties. The antioxidant activity, the potential to induce quinone reductase 1, and the influence on cell viability were measured. ESI(-)FT-ICR MS analysis was also performed and identified flavonoids, coumarins, and fatty acids in the extract. The ethyl acetate and methanolic extracts of the peels presented the highest antioxidant activity in vitro by DPPH (IC50  = 298.3 ± 2.6 µg/ml and 303.8 ± 0.4 µg/ml), ABTS assay (IC50  = 298.2 ± 6.4 µg/ml and 296.4 ± 2.5 µg/ml), and FRAP (IC50  = 234.6 ± 1.8 µg/ml and 398.1 ± 3.8 µg/ml). The ethyl acetate extract of the peel induced quinone reductase 1 activity in Hepa1c1c7 cells, indicating that C. maxima exhibited cancer chemopreventive properties.

Heliotropiumides A and B, new phenolamides with N-carbamoyl putrescine moiety from Heliotropium foertherianum collected in Hawaii and their biological activities.

Two new compounds heliotropiumides A (1) and B (2), phenolamides each with an uncommon carbamoyl putrescine moiety, were isolated from the seeds of a naturalized Hawaiian higher plant, Heliotropium foertherianum Diane & Hilger in the borage family, which is widely used for the treatment of ciguatera fish poisoning. The structures of compounds 1 and 2 were characterized based on MS spectroscopic and NMR analysis, and DP4+ calculations. The absolute configuration (AC) of compound 1 was determined by comparison of its optical rotation with those reported in literature. Compound 2 showed inhibition against NF-κB with an IC50 value of 36µM.

The Daniel K. Inouye College of Pharmacy Scripts: Poha Berry (Physalis peruviana) with Potential Anti-inflammatory and Cancer Prevention Activities.

The Daniel K. Inouye College of Pharmacy, during a historic event in Spring 2016, graduated the first two students in the Pacific region to earn a PhD in pharmaceutical sciences at the University of Hawai'i at Hilo. The college offers PhD programs in these five disciplines: Cancer Biology, Medicinal Chemistry, Pharmaceutics, Pharmacognosy, and Pharmacology. One of the Pharmacognosy dissertations focused on plant-derived natural products with potential anti-inflammatory and cancer chemopreventive activities. Physalis peruviana (Pp) L. originated in tropical South America. It has become naturalized and is found readily on the Island of Hawai'i. The edible fruits are commonly known as cape gooseberry or poha in Hawai'i. In part of our study, three new withanolides, physaperuvin G (1), physaperuvins I-J (2-3), along with four known withanolides, namely, 4ß-hydroxywithanolide E (4), withaperuvin C (5), and physalactone (6), coagulin (7) were isolated from the aerial parts of P. peruviana. In addition, two known compounds, phyperunolide F (8), and withanolide S (9), were isolated and identified from the poha berry fruits. The structures and absolute stereochemistry of new compounds from poha were elucidated by several spectroscopy methods: Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray diffraction, and mass spectrometry analyses. All isolated poha compounds (aerial parts and fruits) were evaluated for their anti-inflammatory activity with lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells, and tumor necrosis factor alpha (TNF-α)-activated nuclear factor-kappa B (NF-κB) with transfected human embryonic kidney cells 293. Most of the isolated natural compounds showed activity with these assays. Additional studies were performed with models of colon cancer. Specifically, 4ß-hydroxywithanolide E (4HWE) inhibited the growth of colon cancer monolayer and spheroid cultures. The compound induced cell cycle arrest at low concentrations and apoptosis at higher concentrations. These data suggest the ingestion of poha berries may have some effect on the prevalence of colon cancer. Additionally, poha isolates compounds were evaluated for their growth inhibitory effects with U251MG glioblastoma and MDA-MB-231 breast cancer cells that harbor aberrantly-active signal transducer and activation of transcription 3 (STAT3), compared to normal NIH-3T3 mouse fibroblasts. This work has led to the filing of three provisional patents with the University of Hawai'i Office of Technology Transfer and Economic Development.

Anti-inflammatory triterpenes from the apical bud of Gardenia sootepensis.

Bioassay-guided fractionation of the dichloromethane extract from the apical bud of Gardenia sootepenesis Hutch. (Rubiaceae) led to the isolation of four new cycloartane triterpenes, sootepins F-I (1-4), along with four known derivatives (5-8). The structures of the new compounds were determined by 1D and 2D NMR experiments and by comparison of the physicochemical data with published values. The isolates were evaluated for cancer chemopreventive potential based on their ability to inhibit nitric oxide (NO) production and tumor necrosis factor-alpha (TNF-α)-induced nuclear factor kappa (NF-κB) activity. Compounds 6-8 inhibited TNF-α-induced NF-κB activity with half maximal inhibitory concentration (IC50) values of 8.3, 5.6, and 6.0µM, respectively; compounds 7 and 8 showed significant NO-inhibitory activity with IC50 values of 3.2 and 2.0µM, respectively.

Litsea Species as Potential Antiviral Plant Sources.

Litsea verticillata Hance (Lauraceae), a Chinese medicine used to treat swelling caused by injury or by snake bites, was the first plant identified by our National Institutes of Health (NIH)-funded International Cooperative Biodiversity Group (ICBG) project to exhibit anti-HIV activities. From this plant, we discovered a class of 8 novel litseane compounds, prototypic sesquiterpenes, all of which demonstrated anti-HIV activities. In subsequent studies, 26 additional compounds of different structural types were identified. During our continuing investigation of this plant species, we identified two new litseanes, litseaverticillols L and M, and a new sesquiterpene butenolide, litseasesquibutenolide. Litseaverticillols L and M were found to inhibit HIV-1 replication, with an IC[Formula: see text] value of 49.6[Formula: see text][Formula: see text]M. To further determine the antiviral properties of this plant, several relatively abundant isolates, including a litseane compound, two eudesmane sesquiterpenes and three lignans, were evaluated against an additional 21 viral targets. Lignans 8 and 9 were shown to be active against the Epstein-Barr Virus (EBV), with EC[Formula: see text] values of 22.0[Formula: see text][Formula: see text]M ([Formula: see text]) and 16.2[Formula: see text][Formula: see text]M ([Formula: see text]), respectively. Since many antiviral compounds have been discovered in L. verticillata, we further prepared 38 plant extracts made from the different plant parts of 9 additional Litsea species. These extracts were evaluated for their anti-HIV and cytotoxic activities, and four of the extracts, which ranged across three different species, displayed 97-100% inhibitory effects against HIV replication without showing cytotoxicity to a panel of human cell lines at a concentration of 20 µg/mL.

Induction of cell cycle arrest and apoptosis with downregulation of Hsp90 client proteins and histone modification by 4ß-hydroxywithanolide E isolated from Physalis peruviana.

SCOPE: Physalis peruviana (Solanaceae) is used for culinary and medicinal purposes. We currently report withanolides, isolated from P. peruviana, inhibit the growth of colon cancer monolayer and spheroid cultures. A detailed mechanistic evaluation was performed with 4ß-hydroxywithanolide E (4HWE). METHODS AND RESULTS: Treatment of HT-29 cells with low concentrations of 4HWE inhibited growth while enhancing levels of p21(Waf1/Cip1) and reducing levels of several cell cycle-related proteins. Apoptosis was induced at higher concentrations. In addition, 4HWE treatment downregulated the levels of Hsp90 client proteins. Nuclear sirtuin 1 (SIRT1) was increased and histone H3 acetylated at lysine 9 was decreased. An additional consequence of SIRT1 elevation in the nucleus may be inhibition of c-Jun activity. The expression of 21 genes was altered, including downregulation of PTGS2, and this correlated with reduced protein levels of cyclooxygenase-2 (COX-2). Overall, efficacious induction of G0/G1 cell cycle arrest at low concentrations, and induction of apoptosis at higher concentrations are interesting 4HWE-mediated phenomena that are accompanied by a complex array of molecular events. CONCLUSION: Considering the worldwide prevalence of colon cancer, and the unique mode of action mediated by 4HWE, it is reasonable to investigate additional mechanistic details and the potential utility of this compound.

Natural Products as a Vital Source for the Discovery of Cancer Chemotherapeutic and Chemopreventive Agents.

Throughout history, natural products have played a dominant role in the treatment of human ailments. For example, the legendary discovery of penicillin transformed global existence. Presently, natural products comprise a large portion of current-day pharmaceutical agents, most notably in the area of cancer therapy. Examples include Taxol, vinblastine, and camptothecin. These structurally unique agents function by novel mechanisms of action; isolation from natural sources is the only plausible method that could have led to their discovery. In addition to terrestrial plants as sources for starting materials, the marine environment (e.g., ecteinascidin 743, halichondrin B, and dolastatins), microbes (e.g., bleomycin, doxorubicin, and staurosporin), and slime molds (e.g., epothilone B) have yielded remarkable cancer chemotherapeutic agents. Irrespective of these advances, cancer remains a leading cause of death worldwide. Undoubtedly, the prevention of human cancer is highly preferable to treatment. Cancer chemoprevention, the use of vaccines or pharmaceutical agents to inhibit, retard, or reverse the process of carcinogenesis, is another important approach for easing this formidable public health burden. Similar to cancer chemotherapeutic agents, natural products play an important role in this field. There are many examples, including dietary phytochemicals such as sulforaphane and phenethyl isothiocyanate (cruciferous vegetables) and resveratrol (grapes and grape products). Overall, natural product research is a powerful approach for discovering biologically active compounds with unique structures and mechanisms of action. Given the unfathomable diversity of nature, it is reasonable to suggest that chemical leads can be generated that are capable of interacting with most or possibly all therapeutic targets.

Hedera nepalensis K. Koch: A Novel Source of Natural Cancer Chemopreventive and Anticancerous Compounds.

Traditional medicinal plants are often used for both the prevention and the treatment of local diseases. Taking into consideration the medicinal importance of Hedera nepalensis within local Pakistani traditions, the present study was undertaken to analyze the in vitro cancer chemopreventive and cytotoxic properties of the plant. The in vitro cancer chemopreventive testing was performed using nitrite assay, NFκB assay, aromatase assay, and quinone reductase 1 (QR1) assay. The cytotoxic potential was evaluated on three cancer-cell lines: MCF-7, MDA-MB-231, and HeLa using sulforhodamine B (SRB) assay. The results of cancer chemopreventive assays show that n-hexane and ethyl acetate fractions of tested plant have promising cancer chemopreventive potential. Lupeol isolated from n-hexane as well as ethyl acetate fraction showed lowest IC50 (0.20 ± 1.9 µM) in NFκB assay. Crude extract and its fractions inhibited the growth of three cancer cell lines by more than 60%, IC50 value of lupeol varied from 2.32 to 10.2 µM. HPLC-DAD-based quantification of lupeol in different plant tissues demonstrated that leaves of H. nepalensis are a rich source of lupeol (0.196 mg/100 mg dry weight). Our data have shown that H. nepalensis harbors cancer chemopreventive and cytotoxic agents.

Cancer chemoprevention activity of labdane diterpenes from rhizomes of Hedychium coronarium

Hedychium coronarium J. Koenig, Zingiberaceae, is a medicinal plant popularly used to treat inflammatory conditions in different countries. Three labdane diterpenes [isocoronarin D (1), methoxycoronarin D (2), ethoxycoronarin D (3)] and benzoyl eugenol (4) were isolated from rhizomes and their chemopreventive potential was evaluated using in vitro assays, namely the inhibition of NF-κB, COX-1 and -2, the induction of antioxidant response element (ARE), and the inhibition of cell proliferation. Diterpene 1 activated ARE (EC50 57.6 ± 2.4 µM), while 2, 3 and 4 significantly inhibited NF-κB (IC50 of 7.3 ± 0.3, 3.2 ± 0.3 and 32.5 ± 4.9 µM, respectively). In addition, 2 and 3 selectively inhibited COX-1 (IC50 values of 0.9 ± 0.0 and 3.8 ± 0.0 µM, respectively). These data support the potential chemopreventive activity of constituents from H. coronarium rhizomes.

Resveratrol derivatives: a patent review (2009 - 2012).

INTRODUCTION: There is currently a wealth of information on the effects of resveratrol and its derivatives in therapeutic, cosmetic and nutraceutical patent applications. Structure-activity studies of the resveratrol scaffold provide a foundation for the development of new analogs with potent activity or other beneficial properties. Ongoing research has yielded promising results and potential use in the treatment of various diseases. AREAS COVERED: This review provides analysis of patents published from January 2009 to April 2013. There is a focus on different approaches for the production of resveratrol derivatives, combinations of new derivatives with old drugs, and applications in therapeutic areas, nutraceutical compositions and cosmetics. EXPERT OPINION: The ability of resveratrol to interact with a disparate array of subcellular targets is uncanny. Nonetheless, even though limited or no toxicity is apparent, the molecule is not a panacea due to lack of potency and issues with bioavailability. Thus, as witnessed by a number of patents, a large assortment of derivatives have been synthesized under the guise of having superior characteristics for treating or preventing various diseases or for use as neutraceutics and cosmetics. Some of these suppositions are probably correct, but evidence-based applications are essentially nil due to a lack of commitment in terms of investing the resources necessary for the conduct of obligatory clinical trials. Current usage is largely based on anecdotes and publicity. Hopefully, at some point in time, it will be possible to follow a standard protocol with a predicable outcome.

Preliminary evaluation for cancer chemopreventive and cytotoxic potential of naturally growing ethnobotanically selected plants of Pakistan.

CONTEXT: Natural products are a very productive source of leads for the development of medicines. Six Pakistani plants were chosen for study based on ethnobotanical data. OBJECTIVE: Exploration of important medicinal plants of Pakistan for cancer treatment. MATERIALS AND METHODS: The crude extracts of the six plants and their fractions were tested for inhibition of nuclear factor κB (NFκB), aromatase, and nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells, induction of quinone reductase 1 (QR1), agonism of retinoid X receptor, and growth inhibition with MCF-7, LU-1 and MDA-MB-231 cancer cells. RESULTS: Two samples of Withania coagulans (Stocks) Dunal (Solanaceae) demonstrated inhibition of TNF-α induced activity of NFκB with IC50 values of 2.6 and 4.3 µg/mL, respectively. Two fractions from W. coagulans and Euphorbia wallichii Hook F. (Euphorbiaceae) aerial parts inhibited aromatase with IC50 values of 17.0 and 17.7 µg/mL, respectively. A total of 13 samples (five from E. wallichii, one from Acer oblongifolium Hort. ex Dippel (Aceraceae), one from Aster thomsonii C. B. Clarke (Asteraceae) and six from W. coagulans aerial parts with fruits) inhibited NO production with IC50 values ranging from 1.3 to 15.6 µg/mL. Fourteen samples demonstrated induction of QR1 with CD ranging from 1.0 to 20.6 µg/mL, and a total of eight extracts and fractions inhibited the proliferation of cancer cells in culture with IC50 values ranging from 1.2 to 7.8 µg/mL. DISCUSSION AND CONCLUSION: Selected plants can be a valuable source of chemopreventive and anticancer products. W. coagulans aerial parts showed the strongest activity.

Anti-inflammatory sesquiterpene lactones from the flower of Vernonia cinerea.

Bioassay-guided fractionation of the hexane extract from the flowers of Vernonia cinerea (Asteraceae) led to the isolation of a new sesquiterpene lactone, 8α-hydroxyhirsutinolide (2), and a new naturally occurring derivative, 8α-hydroxyl-1-O-methylhirsutinolide (3), along with seven known compounds (1 and 4-9). The structures of the new compounds were determined by 1D and 2D NMR experiments and by comparison with the structure of compound 1, whose relative stereochemistry was determined by X-ray analysis. The isolated compounds were evaluated for their cancer chemopreventive potential based on their ability to inhibit nitric oxide (NO) production and tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Compounds 1, 2, 4, 5, and 9 inhibited TNF-α-induced NF-κB activity with IC(50) values of 3.1, 1.9, 0.6, 5.2, and 1.6 µM, respectively; compounds 4 and 6-9 exhibited significant NO inhibitory activity with IC(50) values of 2.0, 1.5, 1.2, 2.7, and 2.4 µM, respectively.

Suppression of nitric oxide synthase by thienodolin in lipopolysaccharide-stimulated RAW 264.7 murine macrophage cells.

The measurement of nitric oxide in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells is used as a model for evaluating the anti-inflammatory or chemopreventive potential of substances. Thienodolin, isolated from a Streptomyces sp. derived from Chilean marine sediment, inhibited nitric oxide production in LPS-stimulated RAW 264.7 cells (IC50 = 17.2 +/- 1.2 microM). At both the mRNA and protein levels, inducible nitric oxide synthase (iNOS) was suppressed in a dose-dependent manner. Mitogen-activated protein kinases (MAPKs), one major upstream signaling pathway involved in the transcription of iNOS, were not affected by treatment of thienodolin. However, the compound blocked the degradation of IkappaBa resulting in inhibition of NF-kappaB p65 nuclear translocation, and inhibited the phosphorylation of signal transducers and activators of transcription 1 (STAT1) at Tyr701. This study supports further exploration of thienodolin as a potential therapeutic agent with a unique mechanistic activity.

Strawberry fields forever?

On the basis of copious preclinical data supporting the preventive efficacy of small fruits such as berries and grapes, Chen and colleagues conducted a randomized (noncomparative) phase II trial evaluating two doses of strawberry powder (60 g/d or 30 g/d for six months) to prevent esophageal cancer in China (reported in this issue of the journal, beginning on page 41); 60 g/d reduced the histologic grade of dysplastic lesions and reduced localized biomarkers, whereas 30 g/d was not effective. Fundamental questions remain such as the best formulation of strawberry powder, the active components associated with powder, and the actual mechanism of action, and standardized preparations will be required to permit the widespread use of strawberry powder with a predicable outcome. Clearly, however, this work is a good example of proof-of-principle and highlights the important role of diet, nutrition, and natural products in cancer prevention.

Pharmaceutical Biology: a retrospective.

A historical perspective of the growth and development of Pharmaceutical Biology.

Inhibition of lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression by 4-[(2'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate from Moringa oleifera.

Moringa oleifera Lamarck is commonly consumed for nutritional or medicinal properties. We recently reported the isolation and structure elucidation of novel bioactive phenolic glycosides, including 4-[(2'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate (RBITC), which was found to suppress inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 mouse macrophage cells. Inhibitors of proteins such as cyclooxygenase-2 (COX-2) and iNOS are potential antiinflammatory and cancer chemopreventive agents. The inhibitory activity of RBITC on NO production (IC(50) = 0.96 ± 0.23 µM) was greater than that mediated by other well-known isothiocyanates such as sulforaphane (IC(50) = 2.86 ± 0.39 µM) and benzyl isothiocyanate (IC(50) = 2.08 ± 0.28 µM). RBITC inhibited expression of COX-2 and iNOS at both the protein and mRNA levels. Major upstream signaling pathways involved mitogen-activated protein kinases and nuclear factor-κB (NF-κB). RBITC inhibited phosphorylation of extracellular signal-regulated kinase and stress-activated protein kinase, as well as ubiquitin-dependent degradation of inhibitor κBα (IκBα). In accordance with IκBα degradation, nuclear accumulation of NF-κB and subsequent binding to NF-κB cis-acting element was attenuated by treatment with RBITC. These data suggest RBITC should be included in the dietary armamentarium of isothiocyanates potentially capable of mediating antiinflammatory or cancer chemopreventive activity.

Suppression of tumor necrosis factor-α-induced nuclear factor κB activation and aromatase activity by capsaicin and its analog capsazepine.

Target-specific drugs, including natural products, offer promise for the amelioration of cancer and other human ailments. Capsaicin, the pungent ingredient present in chilies (Capsicum annuum L.), and capsazepine, a synthetic analog of capsaicin (collectively referred to as vanilloids), are known to possess a variety of pharmacological and physiological properties. In our continuous effort to discover and characterize cancer chemopreventive agents from natural products, we investigated the effect of vanilloids on nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activation using stably transfected 293/NFκB-Luc human embryonic kidney cells induced by treatment with tumor necrosis factor-α (TNFα) and on aromatase activity. Capsaicin and capsazepine blocked TNFα-induced NFκB activation in a dose-dependent manner with 50% inhibitory concentration (IC(50)) values of 0.68 and 4.2 µM, respectively. No significant cytotoxicity was observed at the highest concentrations tested (53.1 µM for capsazepine and 65.5 µM for capsaicin). In addition, these vanilloids inhibited aromatase activity with IC(50) values of 13.6 and 8.8 µM, respectively. Computer-aided molecular docking studies showed docking scores indicative of good binding affinity of vanilloids with aromatase and NFκB. The highly conserved residues for capsaicin and capsazepine binding with NFκB p50 were Ser299 and Ile278 (H-bond 2.81Å) and with NFκB p100 were Ser6, Arg82, Val86, Arg90 (H-bond 2.89Å), Gly4, and Ser2 (H-bond 2.81Å). The amino acids Trp224, Arg435, and Val373 (H-bond 2.80Å) were found to be important for the binding of capsaicin and capsazepine with aromatase. Based on these findings, aromatase and NFκB are suggested as valid targets for these compounds; additional investigation of chemopreventive or chemotherapeutic potential is required.