iso-Guttiferone J and Structure Revision of Guttiferone J from Garcinia gummi-gutta: A Combined Experimental and Integrated QM/NMR Approach.

Many polyprenylated acylphloroglucinols with fascinating chemical structures and intriguing biological activities have been identified as key to phytochemicals isolated from Garcinia, Hypericum, and related genera. In the present work, two chiral, tautomeric, highly-oxygenated polyprenylated acylphloroglucinols tethered with acyl and prenyl moieties on a bicyclo[3.3.1]nonanetrione core were isolated from the 95% ethanolic extract of Garcinia gummi-gutta fruit. The structures of both compounds were elucidated based on the NMR and MS data with ambiguity in the exact position of the enol and keto functions at C-1 and C-3 of the core structure. The structures of both polyprenylated acylphloroglucinols were established as a structurally revised guttiferone J and the new iso-guttiferone J with the aid of gauge-independent atomic orbital NMR calculations, CP3 probability analyses, specific rotation calculations, and electronic circular dichroism calculations in combination with the experimental data. The structures of both compounds resemble hyperforin, a potent activator of the human pregnane X receptor. As expected, both compounds showed strong pregnane X receptor activation at 10 µM [7.1-fold (guttiferone J) and 5.0-fold (iso-guttiferone J)], explained by a molecular docking study, necessitating further in-depth investigation to substantiate the herb-drug interaction potential of G. gummi-gutta upon co-administration with pharmaceutical drugs.

Bulbine natalensis (currently Bulbine latifolia) and select bulbine knipholones modulate the activity of AhR, CYP1A2, CYP2B6, and P-gp.

Bulbine natalensis, an emerging medicinal herb on the global market with androgenic properties, is often formulated in dietary supplements that promote perceived sexual enhancement. However, to date, comprehensive safety studies of B. natalensis are lacking, particularly those related to its herb-drug interaction potential. The purpose of this study was to assess the inductive and inhibitory effects of extracts and pure compounds of B. natalensis on human cytochrome P-450 isozymes in vitro. Our findings demonstrated that both water and methanolic extracts of B. natalensis as well as knipholone, bulbine-knipholone, and 6'-O-methylknipholone dose-dependently increased mRNA expression encoded by CYP2B6, CYP1A2, and ABCB1 genes. Functional analyses showed that water (60 to 2.20 µg/mL) and methanolic (30 to 3.75 µg/mL) extracts and knipholones (10 to 0.33 µM) increased CYP2B6 and CYP1A2 activity in a dose-dependent manner. Additionally, water extract (60 µg/mL), methanolic extract (30 µg/mL), and knipholone (10 µM) caused activation of the aryl hydrocarbon receptor up to 11.1 ± 0.7, 8.9 ± 0.6, and 7.1 ± 2.0-fold, respectively. Furthermore, inhibition studies revealed that methanolic extract attenuated the activity of metabolically active CYP1A2 (IC50, 22.6 ± 0.4 µg/mL) and CYP2B6 (IC50, 34.2 ± 6.6 µg/mL) proteins, whereas water extracts had no inhibitory effect on either isoform. These findings suggest that chronic consumption of B. natalensis may affect normal homeostasis of select CYPs with subsequent risks for HDIs when concomitantly ingested with conventional medications that are substrates of CYP2B6 and CYP1A2. However, more in-depth translational studies are required to validate our current findings and their clinical relevance.

Screening of medicinal plants for possible herb-drug interactions through modulating nuclear receptors, drug-metabolizing enzymes and transporters.

ETHNOPHARMACOLOGICAL RELEVANCE: The last three decades have witnessed a surge in popularity and consumption of herbal products. An unintended consequence of such popularity is that chronic consumption of these products can often modulate the functions of various proteins involved in drug disposition and may, in turn, impose risks for herb-drug interactions (HDIs), leading to serious adverse health outcomes. Identifying plants that may give rise to clinically relevant HDIs is essential, and proactive dissemination of such research outcomes is necessary for researchers, clinicians, and average consumers. AIM OF THE STUDY: The main objective of this study was to evaluate the HDI potential of plants commonly used as ingredients in many herbal products, including BDS. MATERIALS AND METHODS: The dried material of 123 plants selected from the NCNPR repository was extracted with 95% ethanol. The extracts were screened for agonistic effects on nuclear receptors (PXR and AhR) by reporter gene assays in PXR-transfected HepG2 and AhR-reporter cells. For cytochrome P450 enzyme (CYP) inhibition studies, CYP450 baculosomes were incubated with enzyme-specific probe substrates by varying concentrations of extracts. The inhibitory effect on the efflux transporter P-glycoprotein (P-gp) was investigated via rhodamine (Rh-123) uptake assay in P-gp overexpressing MDR1-MDCK cells. RESULTS: Out of 123 plants, 16 increased transcriptional activity of human PXR up to 4 to 7-fold at 60 µg/mL, while 18 plants were able to increase AhR activity up to 10 to 40-fold at 30 µg/mL. Thirteen plants inhibited the activity of CYP3A4, while 10 plants inhibited CYP1A2 activity with IC50 values in the range of 1.3-10 µg/mL. Eighteen plants (at 50 µg/mL) increased intracellular accumulation of Rh-123 (>150%) in MDR1-MDCK cells. Additionally, other plants tested in this study were able to activate PXR, AhR, or both to lesser extents, and several inhibited the catalytic activity of CYPs at higher concentrations (IC50 >10 µg/mL). CONCLUSIONS: The results indicate that prolonged or excessive consumption of herbal preparations rich in such plants (presented in Figs. 1a, 2a, 3a, 4a, and 5a) may pose a risk for CYP- and P-gp-mediated HDIs, leading to unwanted side effects due to the altered pharmacokinetics of concomitantly ingested medications.

Evaluation of the Herb-Drug Interaction (HDI) Potential of Zingiber officinale and Its Major Phytoconstituents.

Ginger is currently one of the most popular herbs commonly added to diverse foods, beverages, and dietary supplements. We evaluated the ability of a well-characterized ginger extract, and several of its phytoconstituents, to activate select nuclear receptors as well as modulate the activity of various cytochrome P450s and ATP-binding cassette (ABC) transporters because phytochemical-mediated modulation of these proteins underlies many clinically relevant herb-drug interactions (HDI). Our results revealed ginger extract activated the aryl hydrocarbon receptor (AhR) in AhR-reporter cells and pregnane X receptor (PXR) in intestinal and hepatic cells. Among the phytochemicals investigated, (S)-6-gingerol, dehydro-6-gingerdione, and (6S,8S)-6-gingerdiol activated AhR, while 6-shogaol, 6-paradol, and dehydro-6-gingerdione activated PXR. Enzyme assays showed that ginger extract and its phytochemicals dramatically inhibited the catalytic activity of CYP3A4, 2C9, 1A2, and 2B6, and efflux transport capabilities of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). Dissolution studies with ginger extract conducted in biorelevant simulated intestinal fluid yielded (S)-6-gingerol and 6-shogaol concentrations that could conceivably exceed cytochrome P450 (CYP) IC50 values when consumed in recommended doses. In summary, overconsumption of ginger may disturb the normal homeostasis of CYPs and ABC transporters, which in turn, may elevate the risk for HDIs when consumed concomitantly with conventional medications.

Modulation of CYP3A4 and CYP2C9 activity by Bulbine natalensis and its constituents: An assessment of HDI risk of B. natalensis containing supplements.

BACKGROUND: Bulbine natalensis is an African-folk medicinal plant used as a dietary supplement for enhancing sexual function and muscle strength in males by presumably boosting testosterone levels, but no scientific information is available about the possible herb-drug interaction (HDI) risk when bulbine-containing supplements are concomitantly taken with prescription drugs. PURPOSE: This study was aimed to investigate the HDI potential of B. natalensis in terms of the pregnane X receptor (PXR)-mediated induction of major drug-metabolizing cytochrome P450 enzyme isoforms (i.e., CYP3A4 and CYP2C9) as well as inhibition of their catalytic activity. RESULTS: We found that a methanolic extract of B. natalensis activated PXR (EC50 6.2 ± 0.6 µg/ml) in HepG2 cells resulting in increased mRNA expression of CYP3A4 (2.40 ± 0.01 fold) and CYP2C9 (3.37 ± 0.3 fold) at 30 µg/ml which was reflected in increased activites of the two enzymes. Among the constituents of B. natalensis, knipholone was the most potent PXR activator (EC50 0.3 ± 0.1 µM) followed by bulbine-knipholone (EC50 2.0 ± 0.5 µM), and 6'-methylknipholone (EC50 4.0 ± 0.5 µM). Knipholone was also the most effective in increasing the expression of CYP3A4 (8.47 ± 2.5 fold) and CYP2C9 (2.64 ± 0.3 fold) at 10 µM. Docking studies further confirmed the unique structural features associated with knipholones for their superior inductive potentials in the activation of PXR compared to other anthraquinones. In a CYP inhibition assay, the methanolic extract as well as the anthraquinones strongly inhibited the catalytic activity of CYP2C9 while, inhibition of CYP3A4 was weak. CONCLUSIONS: These results suggest that consumption of B. natalensis may pose a potential risk for HDI if taken with conventional medications that are substrates of CYP3A4 and CYP2C9 and may contribute to unanticipated adverse reactions or therapeutic failures. Further studies are warranted to validate these findings and establish their clinical relevancy.

Arginine deaminase from Pseudomonas aeruginosa PS2: purification, biochemical characterization and in-vitro evaluation of anticancer activity.

In the present study, arginine deaminase (ADI) was purified from Pseudomonas aeruginosa PS2 which showed relative molecular mass of 70 ± 3 kDa on native-PAGE and 36 ± 0.5 kDa on SDS-PAGE. Purified ADI exhibited optimum activity at pH 6.5 and temperature 40 ºC. Metal ions, K+ and Mg2+ had positive, while Mn2+, Cr2+, Co2+, Fe3+, Ni2+, Cu2+, Cd2+ and Hg2+ had negative effects on catalytic activity of ADI. Purified enzyme showed high substrate specificity towards natural substrate L-arginine and did not hydrolyse its structural analogues. In-vitro serum half-life of purified ADI was 40 h, whereas proteolytic half-life was 28, 27, and 32 min against trypsin, elastase-I and proteinase-K, respectively. Anticancer activity of ADI has been evaluated against panel of human cancer cell lines (LS-180, HCT-116, MCF-7, BT-549, T47D, HL-60, MOLT-4, K-562, and PC-3) but lowest IC50 1.2 IU ml-1 was recorded with MCF-7 cells. Colony forming assay, wound-healing migration assay, phase contrast microscopy, DAPI staining, cell cycle analysis and DNA laddering assay revealed that ADI treatment induced apoptotic cell death in dose dependent manner. Increased level of MMP loss, ROS generation and decreased level of SOD, CAT, GPx and GSH displayed ADI treatment induced mitochondrial dysfunctioning. Furthermore, purified ADI had no substantial toxicity against human normal cell lines and blood erythrocytes. These findings suggesting that purified ADI could be developed as an anticancer agent but more in depth studies are warranted.

Insight into hydroxyl radical-mediated cleavage of caged methylene blue: the role of Fenton's catalyst for antimalarial hybrid drug activation.

Methylene blue with a 10-N carbamoyl linkage was reported to be a hydroxyl radical triggered cleavable ligand. Probed by this platform, hemoproteins were demonstrated to be a much more efficient Fenton's catalyst than commonly used inorganic Fe(ii) salts. The applicability of this ligand was demonstrated through the capability of being triggered by elevated reactive oxygen species levels at diseased tissue, with malaria-parasitized erythrocytes as an in vitro model.

Methylene blue as a far-red light-mediated photocleavable multifunctional ligand.

Methylene blue (MB) with a 10-N-carbamoyl linkage was discovered and developed as a multifunctional far-red (660 nm) photocleavable ligand capable of rendering a series of MB-conjugated compounds with off-to-on fluorescence switch properties through the controlled release of MB.

Arginase purified from endophytic Pseudomonas aeruginosa IH2: Induce apoptosis through both cell cycle arrest and MMP loss in human leukemic HL-60 cells.

Arginase is a therapeutic enzyme for arginine-auxotrophic cancers but their low anticancer activity, less proteolytic tolerance and shorter serum half-life are the major shortcomings. In this study, arginase from Pseudomonas aeruginosa IH2 was purified to homogeneity and estimated as 75 kDa on native-PAGE and 37 kDa on SDS-PAGE. Arginase showed optimum activity at pH 8 and temperature 35 °C. Mn2+ and Mg2+ ions enhanced arginase activity while, Li+, Cu2+, and Al3+ ions reduced arginase activity. In-vitro serum half-life of arginase was 36 h and proteolytic half-life against trypsin and proteinase-K was 25 and 29 min, respectively. Anticancer activity of arginase was evaluated against colon, breast, leukemia, and prostate cancer cell lines and lowest IC50 (0.8 IU ml-1) was found against leukemia cell line HL-60. Microscopic studies and flow cytometric analysis of Annexin V/PI staining of HL-60 cells revealed that arginase induced apoptosis in dose-dependent manner. Cell cycle analysis suggested that arginase induced cell cycle arrest in G0/G1 phase. The increasing level of MMP loss, ROS generation and decreasing level of SOD, CAT, GPx and GSH suggested that arginase treatment triggered dysfunctioning of mitochondria. The cleavage of caspase-3, PARP-1, activations of caspase-8, 9 and high expression of proapoptotic protein Bax, low expression of anti-apoptotic protein Bcl-2 indicated that arginase treatment activates mitochondrial pathway of apoptosis. Purified arginase did not exert cytotoxic effects on human noncancer cells. Our study strongly supports that arginase could be used as potent anticancer agent but further studies are required which are underway in our lab.

Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells.

Asparaginase is an important antileukemic agent extensively used worldwide but the intrinsic glutaminase activity of this enzymatic drug is responsible for serious life threatening side effects. Hence, glutaminase free asparaginase is much needed for upgradation of therapeutic index of asparaginase therapy. In the present study, glutaminase free asparaginase produced from Enterobacter cloacae was purified to apparent homogeneity. The purified enzyme was found to be homodimer of approximately 106 kDa with monomeric size of approximately 52 kDa and pI 4.5. Purified enzyme showed optimum activity between pH 7-8 and temperature 35-40°C, which is close to the internal environment of human body. Monovalent cations such as Na+ and K+ enhanced asparaginase activity whereas divalent and trivalent cations, Ca2+, Mg2+, Zn2+, Mn2+, and Fe3+ inhibited the enzyme activity. Kinetic parameters Km, Vmax and Kcat of purified enzyme were found to be 1.58×10-3 M, 2.22 IU µg-1 and 5.3 × 104 S-1, respectively. Purified enzyme showed prolonged in vitro serum (T1/2 = ~ 39 h) and trypsin (T1/2 = ~ 32 min) half life, which is therapeutically remarkable feature. The cytotoxic activity of enzyme was examined against a panel of human cancer cell lines, HL-60, MOLT-4, MDA-MB-231 and T47D, and highest cytotoxicity observed against HL-60 cells (IC50 ~ 3.1 IU ml-1), which was comparable to commercial asparaginase. Cell and nuclear morphological studies of HL-60 cells showed that on treatment with purified asparaginase symptoms of apoptosis were increased in dose dependent manner. Cell cycle progression analysis indicates that enzyme induces apoptosis by cell cycle arrest in G0/G1 phase. Mitochondrial membrane potential loss showed that enzyme also triggers the mitochondrial pathway of apoptosis. Furthermore, the enzyme was found to be nontoxic for human noncancerous cells FR-2 and nonhemolytic for human erythrocytes.

Purification and characterization of glutaminase free asparaginase from Pseudomonas otitidis: Induce apoptosis in human leukemia MOLT-4 cells.

Asparaginase is an important antineoplastic drug extensively used for the treatment of acute lymphoblastic leukemia, but the intrinsic glutaminase activity of this enzymatic drug is responsible for several life threatening side effects. This study describes the purification and characterization of glutaminase free asparaginase from Pseudomonas otitidis. The purified enzyme exhibited molecular mass of approximately 205±3 kDa on native-PAGE and Ì´34±1 kDa on SDS-PAGE, revealing that the enzyme is homohexamer. The isoelectric point of enzyme was 5.5, calculated by 2D-PAGE. Optimum activity of asparaginase was achieved at 40 °C and pH 7.5, which is close to the internal environment of the human body. Monovalent cations (Na(+) and K(+)) and reducing agents (2-mercaptoethanol and glutathione) has enhanced asparaginase activity. Whereas, divalent (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)), trivalent (Fe(3+)) cations and thiol group blocking agent (iodoacetamide) inhibited the enzyme activity significantly. In vitro serum and trypsin half life of asparaginase is almost 2 and 1.5 fold respectively, which is higher than commercial asparaginase. MTT assay results showed that the anticancer activity of purified asparaginase was comparable or higher than commercial E. coli asparaginase. Microscopic studies and cell cycle analysis suggested that purified enzyme induced apoptotic cell death in dose-dependent manner. Loss of mitochondrial membrane potential suggests that enzyme induces cell death via activation of intrinsic apoptotic pathway. Purified asparaginase was found to be nontoxic for human noncancerous FR-2 cells and human blood lymphocytes, which is a remarkable therapeutic feature.