Combination of metformin/efavirenz/fluoxetine exhibits profound anticancer activity via a cancer cell-specific ROS amplification.

The possible anticancer activity of combination (M + E + F) of metformin (M), efavirenz (E), and fluoxetine (F) was investigated in normal HDF cells and HCT116 human colon cancer cells. Metformin increased cellular FOXO3a, p-FOXO3a, AMPK, p-AMPK, and MnSOD levels in HDFs but not in HCT116 cells. Cellular ATP level was decreased only in HDFs by metformin. Metformin increased ROS level only in HCT116 cells. Transfection of si-FOXO3a into HCT116 reversed the metformin-induced cellular ROS induction, indicating that FOXO3a/MnSOD is the key regulator for cellular ROS level. Viability readout with M, E, and F alone decreased slightly, but the combination of three drugs dramatically decreased cell survival in HCT116, A549, and SK-Hep-1 cancer cells but not in HDF cells. ROS levels in HCT116 cells were massively increased by M + E + F combination, but not in HDF cells. Cell cycle analysis showed that of M + E + F combination caused cell death only in HCT116 cells. The combination of M + E + F reduced synergistically mitochondrial membrane potential and mitochondrial electron transport chain complex I and III activities in HCT116 cells when compared with individual treatments. Western blot analysis indicated that DNA damage, apoptosis, autophagy, and necroptosis-realated factors increased in M + E + F-treated HCT116 cells. Oral administration with M + E + F combination for 3 weeks caused dramatic reductions in tumor volume and weight in HCT116 xenograft model of nude mice when compared with untreated ones. Our results suggest that M + E + F have profound anticancer activity both in vitro and in vivo via a cancer cell-specific ROS amplification (CASRA) through ROS-induced DNA damage, apoptosis, autophagy, and necroptosis.

Purple corn extract alleviates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like phenotypes in BALB/c mice.

Zea mays L. (Poaceae), also known as purple corn, is an annual herbaceous plant that is grown as food for human consumption in a variety of forms, including cooking oils and sweeteners in processed food and beverage products. The purpose of this study was to determine whether a novel purple corn extract, FB801, might have an anti-atopic dermatitis (AD) effect on AD-like skin lesions induced by 2,4-dinitrochlorobenzene (DNCB) in BALB/c mice. Topical sensitization (1%) and challenge (0.3%) by DNCB were performed on the dorsal skin and right ear of BALB/c mice to induce AD. Following FB801 and dexamethasone administered orally, the severity of skin lesions was examined macroscopically and histologically. Serum levels of immunoglobulin E (IgE) and various cytokines were determined by enzyme-linked immunosorbent assay. Oral administration of FB801 significantly reduced typical symptoms of AD (erythema/bleeding, swelling, molting/erosion and scaling/drying), scratching frequencies, and the recruitment of inflammatory and mast cells. In addition, FB801 suppressed serum levels of IgE and T helper (Th)2 type cytokines such as interleukin (IL)-4 and IL-10 in DNCB-treated BALB/c mice. Furthermore, FB801 reduced the degradation of inhibitor of nuclear factor-κB proteins (NF-κB) in tumor necrosis factor (TNF)-α-stimulated human keratinocyte (HaCaT) cells. These results suggest that FB801 inhibited the development of the AD-like skin symptoms by regulating Th1 and Th2 responses in the skin lesions in mice and suppressing TNF-α induced NF-κB activation in HaCaT cells, suggesting that FB801 has potential application as an effective alternative therapy for the prevention and management of AD.

Anti-Nociceptive Effect and Standardization from Mixture of Agrimonia pilosa Ledeb and Salvia miltiorrhiza Bunge Extracts.

Agrimonia pilosa Ledeb has been previously reported to produce an anti-nociceptive effect in ICR mice in both tail-flick and hot-plate tests. Studies have shown that Salvia miltiorrhiza Bunge, also renowned in traditional Chinese medicine, is an effective anti-inflammatory treatment. Among the extraction solvents investigated, a 50% ethanol (EtOH) extract of A. pilosa produced the highest anti-nociceptive effect in monosodium uric acid-induced gout pain models and the greatest yield. The 80% EtOH extract of S. miltiorrhiza moderately inhibited lipopolysaccharide-induced nitric oxide release from RAW 264.7 murine macrophages and exhibited outstanding yield. The mixture of optimized A. pilosa and S. miltiorrhiza extracts were evaluated for enhanced anti-nociceptive effects in gout arthritis treatment. To control extract quality, four marker compounds were determined using an HPLC-DAD method. A 1:1 mixture of A. pilosa 50 and S. miltiorrhiza 80% EtOH extracts of produced better results than when the extracts were administered individually.

An in vitro affinity-based method for studying herb-drug interactions for direct identification of cytochrome P450 1A2, 3A4, and 2C9 specific ligands from herbal extracts using ultrafiltration-high performance liquid chromatography.

A novel in vitro strategy for affinity-based ultrafiltration-high performance liquid chromatography (HPLC) was developed for the direct identification of cytochrome P450 (CYP) 1A2, 3A4, and 2C9 specific ligands from Danshen extracts, in which human liver microsome (HLM) was used as the source of CYP enzymes. The Danshen extract was incubated without HLM, with HLM, or with HLM where the active site of the target enzyme was blocked with a specific competitive probe before ultrafiltration to isolate ligand-enzyme complexes from unbound compounds. Subsequently, HPLC analysis was performed on the released ligands from the complexes. α-Naphthoflavone, ketoconazole, and sulfaphenazole were used as specific competitive probes for CYP1A2, 3A4, and 2C9, respectively. The signal-to-noise ratio (S/N) and specific-signal-to-noise ratio (S-S/N) of each compound were calculated using the obtained peak areas. Finally, two criteria were applied to select putative ligands for each specific target: (1) S/N > 1; (2) S-S/N > 0. Finally, dihydrotanshinone was identified as a specific ligand for CYP1A2 and tanshinone I, cryptotanshinone, and tanshinone IIA were identified as specific ligands for CYP1A2, 2C9, and 3A4. It was demonstrated that the newly developed method can be used to rapidly and directly detect specific ligands from natural product extracts in multi-target systems.

Rapid Identification and Isolation of Inhibitors of Rat Lens Aldose Reductase and Antioxidant in Maackia amurensis.

Oxidative stress and aldose reductase activity have been implicated in the development of diabetic complications. In this study, the antioxidant and aldose reductase (AR) inhibitory effects of Maackia amurensis (MA) were investigated. The ethyl acetate fraction of the MA extract showed the highest inhibitory activity in antioxidant and rat lens AR (RLAR). To identify and isolate the active components in the ethyl acetate fraction of the MA extract, high-speed countercurrent chromatography and Sephadex LH-20 column chromatography were performed and guided by an offline HPLC-ABTS assay and HPLC microfractionation AR assay. Four antioxidants, namely, piceatannol (IC50 = 6.73 µM), resveratrol (IC50 = 11.05 µM), trans-ferulic acid (IC50 = 13.51 µM), and chlorogenic acid (IC50 = 27.23 µM), and six AR inhibitors, namely, chlorogenic acid (IC50 = 4.2 µM), tectoridin (IC50 = 50.4 µM), genistein (IC50 = 57.1 µM), formononetin (IC50 = 69.2 µM), resveratrol (IC50 = 117.6 µM), and daidzein (IC50 = 151.9 µM), were isolated and identified. The screening results of the offline HPLC-ABTS assay and HPLC microfractionation AR assay matched the activity of isolated compounds. Thus, MA is potentially valuable for antioxidant and AR inhibitor discovery and efficient drug design for the prevention and treatment of diabetic complications.

Inhibitory Activities of Stauntonia hexaphylla Leaf Constituents on Rat Lens Aldose Reductase and Formation of Advanced Glycation End Products and Antioxidant.

Stauntonia hexaphylla (Thunb.) Decne. (Lardizabalaceae) leaves (SHL) have been used traditionally as analgesics, sedatives, diuretics, and so on, in China. To date, no data have been reported on the inhibitory effect of SHL and its constituents on rat lens aldose reductase (RLAR) and advanced glycation end products (AGEs). Therefore, the inhibitory effect of compounds isolated from SHL extract on RLAR and AGEs was investigated to evaluate potential treatments of diabetic complications. The ethyl acetate (EtOAC) fraction of SHL extract showed strong inhibitory activity on RLAR and AGEs; therefore, EtOAc fraction (3.0 g) was subjected to Sephadex LH-20 column chromatography, for further fractionation, with 100% MeOH solvent system to investigate its effect on RLAR and AGEs. Phytochemical investigation of SHL led to the isolation of seven compounds. Among the isolated compounds, chlorogenic acid, calceolarioside B, luteolin-3'-O-ß-D-glucopyranoside, quercetin-3-O-ß-D-glucopyranoside, and luteolin-7-O-ß-D-glucopyranoside exhibited significant inhibitory activity against RLAR with IC50 in the range of 7.34-23.99 µM. In addition, 3-(3,4-dihydroxyphenyl) propionic acid, neochlorogenic acid, and luteolin-3'-O-ß-D-glucopyranoside exhibited the most potent inhibitory activity against formation of AGEs, with an IC50 value of 115.07-184.06 µM, compared to the positive control aminoguanidine (820.44 µM). Based on these findings, SHL dietary supplements could be considered for the prevention and/or treatment of diabetes complication.

Enzymatic Synthesis of a Novel Kaempferol-3-O-ß-d-glucopyranosyl-(1→4)-O-α-d-glucopyranoside Using Cyclodextrin Glucanotransferase and Its Inhibitory Effects on Aldose Reductase, Inflammation, and Oxidative Stress.

Kaempferol-3-O-ß-d-glucopyranoside (astragalin, AS), a major flavonoid that exists in various plants, exerts antioxidant, antitumor, anti-human immunodeficiency virus (HIV), and anti-inflammatory effects. However, the low water solubility of AS limits its use. In this study, we used cyclodextrin glucanotransferase (CGTase) with maltose (G2) as a donor molecule to enzymatically modify AS to improve its water solubility and physiochemical properties. We isolated the glycosylated astragalin (G1-AS) and identified the structure of G1-AS as kaempferol-3-O-ß-d-glucopyranosyl-(1→4)-O-α-d-glucopyranoside, where one glucose residue was transferred to AS. G1-AS retained the antioxidative activity of the original AS compound; however, the solubility of G1-AS was 65-fold higher than that of AS. In addition, G1-AS showed enhanced anti-inflammatory effects and aldose reductase inhibitory activity compared to AS when applied to rat lenses.

Phytochemical Analysis of Agrimonia pilosa Ledeb, Its Antioxidant Activity and Aldose Reductase Inhibitory Potential.

The aim of this study was to determine aldose reductase (AR) inhibitory activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of compounds from Agrimonia pilosa Ledeb (AP). We isolated agrimoniin (AM), four flavonoid glucosides and two flavonoid glucuronides from the n-butanol fraction of AP 50% methanol extract. In addition to isolated compounds, the AR-inhibitory activity and the DPPH free radical scavenging activity of catechin, 5-flavonoids, and 4-flavonoid glucosides (known components of AP) against rat lens AR (RLAR) and DPPH assay were measured. AM showed IC50 values of 1.6 and 13.0 µM against RLAR and DPPH scavenging activity, respectively. Additionally, AM, luteolin-7-O-glucuronide (LGN), quercitrin (QU), luteolin (LT) and afzelin (AZ) showed high inhibitory activity against AR and were first observed to decrease sorbitol accumulation in the rat lens under high-sorbitol conditions ex vivo with inhibitory values of 47.6%, 91.8%, 76.9%, 91.8% and 93.2%, respectively. Inhibition of recombinant human AR by AM, LGN and AZ exhibited a noncompetitive inhibition pattern. Based on our results, AP and its constituents may play partial roles in RLAR and oxidative radical inhibition. Our results suggest that AM, LGN, QU, LT and AZ may potentially be used as natural drugs for treating diabetic complications.