The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Preventative effects of alpha-naphtho flavone in vascular dementia.

Vascular dementia (VaD) refers to the loss of reasoning, planning, judgment, and memory as a result of reduced blood flow, inflammation, activation of immune cells, and neurodegenerative processes. Here, we tested the preventive effects of alpha-naphthoflavone (ANF) on a model of vascular dementia induced by four-vessel occlusion (4VO) in rats. Animals were treated orally for 5 consecutive days with 10 mg/kg of donepezil (a traditional therapeutic drug) and 40 and 80 mg/kg ANF. The treatment with ANF exerted effects that were similar to those induced by donepezil. This included prevention of cognitive impairment, alterations in the plasma levels of homocysteine and nitrate and the activity of acetylcholinesterase and myeloperoxidase and the tissue level of glutathione, and thiobarbituric acid reactive substances in the brain tissues. These findings suggest that herbal-derived ANF is as effective as traditional drugs in vascular dementia.

Alpha-naphthoflavone attenuates non-alcoholic fatty liver disease in oleic acid-treated HepG2 hepatocytes and in high fat diet-fed mice.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease. The literature suggests that the aryl hydrocarbon receptor (AHR) may be a key player in the pathogenesis of NAFLD, and it can modulate the synthesis of cytochrome P450 1A1 (CYP1A1) and tumor necrosis factor-α (TNF-α). Previous studies have shown that CYP1A1 is a key enzyme of oxidative stress, TNF-α is involved in the formation of insulin resistance (IR), oxidative stress and insulin resistance are the key factors for the formation of NAFLD. Therefore, it can be said that AHR may participate in contributing to NAFLD by regulating CYP1A1 and TNF-α. Alpha-naphthoflavone (ANF) is an effective AHR inhibitor. The present study was designed to explore the hepatoprotective effect of ANF in high fat diet (HFD)-induced NAFLD mice and oleic acid (OA)-treated HepG2 hepatocytes. Mice were fed HFD to induce NAFLD, HepG2 cells were exposed to OA to induce hepatocyte injury, and ANF significantly reduced mouse and cellular liver damage compared to the HFD-induced NAFLD and OA-treated HepG2 hepatocytes. ANF treatment reduces liver damage by reducing ROS and IR, the data show that ANF inhibits the expression of AHR, CYP1A1 and TNF-α in NAFLD. Taken together, these findings show that ANF alleviate NAFLD via regulation of AHR/CYP1A1 and AHR/TNF-α pathways, which may have potential for further development as novel therapeutic agents for NAFLD.

A cell-based, microplate colorimetric screen identifies 7,8-benzoflavone and green tea gallate catechins as inhibitors of the hepatitis C virus.

We describe a cell-based, microplate colorimetric screen for anti-hepatitis C virus (HCV) drugs that exploits the HCV-JFH1 viral culture system. Antiviral activity was assessed by measuring protection against the HCV-JFH1-induced cytopathic effect (CPE) in Huh7.5.1 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay. The use of serum-free medium substantially sensitized Huh7.5.1 cells to HCV-induced CPE, causing sufficient cell death to perform colorimetric assays for anti-HCV activity in 96-well plates. As a proof of concept, we carried out a pilot screen of an inhibitor library and identified cyclosporin A and tamoxifen, two compounds with reported anti-HCV activity. Using the assay, we discovered the anti-HCV properties of the plant flavonoids epigallocatechin gallate (EGCG) and 7,8-benzoflavone (α-naphthoflavone). Other gallate-type catechins and flavones also displayed anti-HCV activity, but 5,6-benzoflavone (ß-naphthoflavone), flavanone, and non-gallate catechins were inactive. EGCG apparently acted mainly on HCV entry, although it may also block other steps. In contrast, 7,8-benzoflavone was presumed to inhibit later stages of the HCV life cycle. This assay is simple, reliable and cost-effective; does not require any specially engineered cell lines or viruses; and should be useful in the identification of compounds with anti-HCV activity.

Vitamin C and alpha-naphthoflavone prevent estrogen-induced mammary tumors and decrease oxidative stress in female ACI rats.

The mechanisms underlying the pathogenesis of estrogen-induced breast carcinogenesis remain unclear. The present study investigated the roles of estrogen metabolism and oxidative stress in estrogen-mediated mammary carcinogenesis in vivo. Female August Copenhagen Irish (ACI) rats were treated with 17beta-estradiol (E(2)), the antioxidant vitamin C, the estrogen metabolic inhibitor alpha-naphthoflavone (ANF), or cotreated with E(2) + vitamin C or E(2) + ANF for up to 8 months. E(2) (3 mg) was administered as an subcutaneous implant, ANF was given via diet (0.2%) and vitamin C (1%) was added to drinking water. At necropsy, breast tumor incidence in the E(2), E(2) + vitamin C and E(2) + ANF groups was 82, 29 and 0%, respectively. Vitamin C and ANF attenuated E(2)-induced alterations in oxidative stress markers in breast tissue, including 8-iso-prostane F(2alpha) formation and changes in the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase. Quantification of 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)) formation in breast tissue confirmed that ANF inhibited 4-hydroxylation of E(2) and decreased formation of the highly carcinogenic 4-OHE(2). These results demonstrate that antioxidant vitamin C reduces the incidence of estrogen-induced mammary tumors, increases tumor latency and decreases oxidative stress in vivo. Further, our data indicate that ANF completely abrogates breast cancer development in ACI rats. The present study is the first to demonstrate the inhibition of breast carcinogenesis by antioxidant vitamin C or the estrogen metabolic inhibitor ANF in an animal model of estrogen-induced mammary carcinogenesis. Taken together, these results suggest that E(2) metabolism and oxidant stress are critically involved in estrogen-induced breast carcinogenesis.

Drug/substance reversal effects of a novel tri-substituted benzoflavone moiety (BZF) isolated from Passiflora incarnata Linn.--a brief perspective.

The present work is a mini-review of the author's original work on the plant Passiflora incarnata Linn., which is used in several parts of the world as a traditional medicine for the management of anxiety, insomnia, epilepsy and morphine addiction. A tri-substituted benzoflavone moiety (BZF) has been isolated from the bioactive methanol extract of this plant, which has been proposed in the author's earlier work to be responsible for the biological activities of this plant. The BZF moiety has exhibited significantly encouraging results in the reversal of tolerance and dependence of several addiction-prone psychotropic drugs, including morphine, nicotine, ethanol, diazepam and delta-9-tetrahydrocannabinol, during earlier pharmacological studies conducted by the author. In addition to this, the BZF moiety has exhibited aphrodisiac, libido-enhancing and virility-enhancing properties in 2-year-old male rats. When administered concomitantly with nicotine, ethanol and delta-9-tetrahydrocannabinol for 30 days in male rats, the BZF also prevented the drug-induced decline in sexuality in male rats. Because the BZF moiety isolated from P. incarnata is a tri-substituted derivative of alpha-naphthoflavone (7,8-benzoflavone), a well-known aromatase-enzyme inhibitor, the mode of action of BZF has been postulated to be a neurosteroidal mechanism vide in which the BZF moiety prevents the metabolic degradation of testosterone and upregulates blood - testosterone levels in the body. As several flavonoids (e.g. chrysin, apigenin) and other phytoconstituents also possess aromatase-inhibiting properties, and the IC50 value of such phytomoieties is the main factor determining their biochemical efficacy, by altering their chemical structures to attain a desirable IC50 value new insights in medical therapeutics can be attained, keeping in view the menace of drug abuse worldwide.

Prevention of chronic alcohol and nicotine-induced azospermia, sterility and decreased libido, by a novel tri-substituted benzoflavone moiety from Passiflora incarnata Linneaus in healthy male rats.

Excessive long term consumption of alcohol and nicotine have serious detrimental effects upon the libido, fertility, and sperm count in male species. The present work describes the beneficial effects of a novel tri-substituted benzoflavone moiety (BZF) isolated from Passiflora incarnata Linneaus, the phyto-chemical isolation, spectroscopic elucidation, and multifarious biological activities of which have recently been reported by the authors. The BZF moiety has been reported to increase libido, sperm count, and sexual fertility in 2 years old male rats at 10 mg/kg, po dose, in the one of our previous studies. Presently, the BZF moiety has been evaluated against chronic ethanol- and nicotine-induced decrease in libido, sexual fertility and mating efficiency in healthy male rats. The male rats were given ethanol (3 g/kg, po) A, nicotine (2 mg/kg, sc) N, alcohol-nicotine combinations (AN) alone, and also with 10 mg/kg po dose of BZF (concurrent administrations). These treatments were given for 30 days. At the end of treatments, it was observed that rat groups A, N, and AN had no libido (evaluated by mounting behaviour), declined sperm count, and consequently no mating efficiency or fertility (upon pairing with pro-estrus female rats). However, the rats which were given 10 mg/kg BZF along-with nicotine (NP group), alcohol (AP group), and alcohol-nicotine combination (ANP) exhibited significant libido-oriented mounting behaviour, increased sperm count (significantly comparable to the control group), and increased fertilization potential. The rats having decreased sperm count, libido and fertilization potential due to chronic administration of alcohol, nicotine and alcohol-nicotine combinations, i.e., rats of A, N, and AN groups were again subdivided and were given 10 mg/kg BZF for 7 days. This treatment confirmed that BZF speeds up the restoration of sexuality in rats upon cessation of the administration of substances like alcohol, nicotine and alcohol-nicotine combinations, which have severe detrimental effects upon male sexuality, fertility and vigour. BZF, the strongest inhibitor of aromatase enzyme, when administered concurrently with substances like alcohol and nicotine restores sexual virility, libido and vigour in male rats by maintaining the blood-testosterone levels to be high.

Suppression of alcohol-cessation-oriented hyper-anxiety by the benzoflavone moiety of Passiflora incarnata Linneaus in mice.

A benzoflavone moiety has been reported recently to be responsible for the multifarious CNS effects of Passiflora incarnata Linneaus. In the light of the established usefulness of the benzoflavone moiety in counteracting the withdrawal effects of substances like morphine, cannabinoids and nicotine by the authors, the bioactive benzoflavone moiety (BZF) has been tested in mice treated with an addictive dose (2 g/kg, bid for 6 days) of ethyl alcohol, in order to evaluate its effectiveness in countering alcohol dependence. In a 7-day regimen, different groups of mice were administered vehicle, alcohol and alcohol+three doses (10, 20 and 50 mg/kg of the benzoflavone moiety) of P. incarnata; all treatments (chronic) being administered orally, twice daily for 6 days. Similarly, three other groups of mice were rendered addicts upon alcohol by administration of the addictive dose (2 g/kg, bid for 6 days) of ethyl alcohol, and a single acute administration of 10, 20 and 50 mg/kg dose of benzoflavone moiety was given on the 7th day. In both, chronic and acute administrations, the benzoflavone moiety prevented significantly the expression of withdrawal effects of alcohol as there was a significant decrease in anxiety oriented behavior in mice that received benzoflavone moiety of P. incarnata. The chronic administration of P. incarnata with alcohol had better preventive effects than the single acute treatment with P. incarnata in alcohol-dependent mice.

Nicotine reversal effects of the benzoflavone moiety from Passiflora incarnata Linneaus in mice.

A benzoflavone moiety (BZF) has recently been reported to be liable for many of the biological effects of the plant Passiflora incarnata Linneaus. In light of various reports mentioning the usefulness of P. incarnata in tobacco addiction, studies have been performed using four doses (1, 5, 10 and 20 mg/kg) of the bioactive BZF moiety isolated from the aerial parts of P. incarnata. In a 7-day experimental regimen, mice (n = 5) were given nicotine hydrogen tartrate (2 mg/kg), and combinations of nicotine with four doses of BZF (NnP-1, NnP-5, NnP-10 and NnP-20) q.i.d. by the s.c. route. At the end of the 7 days of treatment, naloxone was given to the mice from all groups to induce a nicotine withdrawal syndrome. The mice that had been treated with 10 and 20 mg/kg dose of BZF concurrently with nicotine showed a significantly fewer number of withdrawal jumps relative to the group treated with nicotine alone (Nn group). Separately, in a 14-day treatment regimen, mice (n = 10; for the N group, n = 12) were administered nicotine (2 mg/kg) and combinations of nicotine with four doses of BZF (NP-1, NP-5, NP-10, NP-20 groups) q.i.d. by the s.c. route. Spontaneous physical and behavioural signs of nicotine dependence were observed 3 hours after cessation of treatments on the 14th day. Mice administered with combinations of nicotine and 5, 10 and 20 mg/kg doses of BZF (i.e. NP-5, NP10 and NP-20 groups), exhibited less intensity and severity of withdrawal effects compared to the mice treated with nicotine alone. Those mice treated with the two highest doses of BZF,in combination with nicotine (NP-10 and NP-20), showed significantly fewer nicotine-abstinence withdrawal jumps and normal ambulatory behaviour. BZF treatment prevented weight loss and resulted in normal performance in the swimming endurance test, which may be a measure of stress and/or depression. Similarly, acute administration of a single 20 mg/kg dose of BZF prevented some of the nicotine-withdrawal effects; lower doses were almost inert. These studies, although preliminary, suggest that the BZF may have value in treating nicotine addiction.

Beneficial effects of chrysin and benzoflavone on virility in 2-year-old male rats.

This work describes the potential usefulness of bioflavonoids for countering the deleterious effects of aging on male sexuality in 2-year-old rats. A flavone chrysin from Passiflora caerulea Linn. and a benzoflavone moiety (BZF) recently isolated from Passiflora incarnata Linn. were administered to 2-year-old male rats for a period of 30 days. After cessation of these treatments, there was a significant improvement in overall sexual functions in the rats given bioflavonoids, compared with control rats. The rats receiving chrysin (1 mg/kg) and BZF (10 mg/kg) exhibited increased libido when they were allowed to interact with nonestrous female rats. Additionally, both treated groups had increased sperm count, greater fertilization potential, and greater litter size when they were allowed to interact with proven proestrous female rats of a similar strain. BZF was more potent than chrysin as an antiaromatase agent and exhibited better effects on the sexual system of the 2-year-old male rats. Plant flavonoids have great potential for clinical and therapeutic applications against the physiological and biochemical effects of aging.

Chemoprevention of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine-induced mammary carcinogenesis in rats.

Modifying effects of dietary exposure of diallyl disulfide (DAD), aspirin, DL-alpha-difluoromethylomithine (DFMO), beta-naphthoflavone (beta-NF), alpha-naphthoflavone (alpha-NF), indole-3-carbinol (I3C) and protocatechuic acid (PCA) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were examined in two experiments with female rats. For both experiments, PhIP in corn oil at a concentration of 85 mg/kg was given to animals via an intragastric tube for eight doses for an initial 4 weeks, and test chemicals were given in the diet (Experiment 1: DAD, 200 ppm; aspirin, 400 ppm; DFMO, 400 ppm; beta-NF, 1000 ppm; Experiment 2: alpha-NF, 1000 ppm; I3C, 1000 ppm; PCA, 2000 ppm) for an initial 4 weeks. The experiments were terminated after 25 weeks. In Experiment 1, exposure of beta-NF decreased the incidence and multiplicity of total mammary tumors (fibroadenoma, intraductal carcinoma and invasive ductal carcinoma) (P < 0.001 and P < 0.0001), and lowered the incidence of ductal carcinoma (P < 0.0001). DAD lowered the incidence of ductal carcinoma and decreased the multiplicity of the total tumors (P < 0.01 and P < 0.005). Furthermore, aspirin decreased the total tumors (P < 0.05). In Experiment 2, alpha-NF decreased the multiplicity of ductal carcinoma (P < 0.05). These results suggest that alpha-NF, beta-NF, DAD or aspirin could be chemopreventing agents for mammary neoplasia.

[Protection of the pulmonary toxic effects of normobaric oxygen by inducers of cytochrome P450-linked monooxygenases]. / Protection des effets toxiques pulmonaires de l'oxygène normobare par les inducteurs enzymatiques des mono-oxygénases liés au cytochrome P450.

Inducers of cytochrome P450-linked mono-oxygenases increase the normobaric oxygen tolerance of the adult rat. Pulmonary inducers, as 3-methylcholanthrene and beta-naphthoflavone permit the rat survival and simultaneously a decrease of pulmonary edema. Phenobarbital, an hepatic inducer had lesser effects both on survival rate and on pulmonary and lymphoïd oxygen toxicity.

Effect of enzyme inducers and inhibitors and glutathione precursor and depleter on induced acute aflatoxicosis in rabbits.

Male New Zealand White rabbits were treated with microsomal enzyme inducers, inhibitors of hemoprotein synthesis or action, and glutathione precursor and depletor before they were orally given the median lethal dose (LD50) of aflatoxin B1 (AFB1; 0.4 mg/kg) at the start of a 7-day experimental period. The drugs administered, mean duration of illness (hours), and survival percentage were as follows: controls (saline solution)-85, 50%; phenobarbital (PB)-100, 100%; phenylbutazone-115, 67%; benzoflavone-39, 17%; stanozolol-67, 67%; cobaltous chloride (CoCl2)-46, 67%; piperonyl butoxide (PBO)-88, 100% cysteine (CYS)-68, 100%; ethyl maleate-71, 83%. Signs of toxicosis included decreased feed and water consumption, weight loss, dehydration, lethargy, and emaciation; some rabbits died or were euthanatized. Clinico-pathologic changes included increased serum aspartate aminotransferase (AST) activity by 24 hours and bilirubin concentration by 48 to 72 hours after AFB1 was given. Grossly, livers were pale or tan and friable, with prominent lobular architecture. Kidneys of affected rabbits were pale to dark red. Microscopically, livers were normal or had lesions as great as extensive necrosis, hemorrhage, mineralization, and bile duct proliferation. Treatment of rabbits with PB, CoCl2, PBO, and CYS protected against AFB1 hepatic pathology, and PB, PBO, and CYS also had protective effect against lethality. Ethyl maleate provided some protection against lethality and increased serum AST activity and bilirubin concentration. Toxicosis was enhanced by benzoflavone; phenylbutazone and stanozolol had litte influence.

Effect of some enzyme inducers, fluids, and methionine-thiosulfate on induced acute aflatoxicosis in goats.

Groups of four 6- to 12-month-old male goats were injected intraruminally with a lethal dose (3 mg/kg of body weight) of aflatoxin B1 (AFB1). Drugs were administered parenterally before (pretreatment) or beginning 8 hours after goats were doses with AFB1. These drugs were phenobarbital (PB), phenylbutazone (PBZ), piperonyl butoxide (PRO), benzoflavones, water, and 5% glucose solution (D5W). Most groups given the drugs after AFB1 was administered also were given intraperitoneal injections of methionine-sodium thiosulfate (MET-TS) solution. Clinical signs of toxicosis, serum aspartate aminotransferase activities, serum bilirubin concentrations, duration of illness, mortality, and gross and microscopic pathologic findings taken together indicated that toxicosis was increased with MET-TS + PB therapy, PBZ pretreatment, PBZ therapy, benzoflavone pretreatment, benzoflavone therapy, MET-TS + benzoflavone therapy, and MET-tS + water therapy. Toxicosis was not altered appreciably by MET-TS + PBO therapy. Beneficial effects (less severe toxicosis) were produced by PB pretreatment; these effects were prolonged maintenance of strength, vigor, and appetite and (in 1 goat that recovered) absence of pathologic changes or serum bilirubin increase. Therapy with MET-TS + D5W (but not MET-TS alone) also lengthened maintenance of strength, vigor, and appetite, but did not prevent pathologic changes. The beneficial effect of MET-TS therapy reported in a previous study (AFB2 dosage of 4 mg/kg) was not observed with the 3 mg/kg lethal dose. In conclusion, therapy for acute aflatoxicosis with inducers of hepatic microsomal enzymes is ineffective (PBO) or contraindicated (PB, PBZ, benzoflavones). Therapy with D5W may be a useful adjunct to other therapeutic drugs, but multiple intraperitoneal injections of D5W may decrease survival time because of stress.

Toxicologic study of carboxyatractyloside (active principle in cocklebur--Xanthium strumarium) in rats treated with enzyme inducers and inhibitors and glutathione precursor and depletor.

Male rats (10 rats/group) were treated with phenobarbital (PB), phenylbutazone (PBZ), stanozolol (3 inducers of cytochrome P450-dependent enzymes), piperonyl butoxide (PBO; a P450 inhibitor), cobaltous chloride (CoCl2; an inhibitor of hemoprotein synthesis), 5,6-benzoflavone (BNF; an inducer of cytochrome P448 dependent enzymes), cysteine [CYS; a glutathione (GSH) precursor], or ethyl maleate (EM; a GSH depletor). The rats were then given a calculated LD50 dosage (13.5 mg/kg of body weight) of carboxyatractyloside (CAT) intraperitoneally. Clinical signs of toxicosis, duration of illness, lethality, gross lesions, and hepatic and renal histopathologic lesions were recorded. Seemingly, (i) CAT toxicosis has independent lethal and cytotoxic components (PBZ decreased lethality and cytotoxicity; CoCl2 decreased cytotoxicity but not lethality; BNF decreased duration of illness, and perhaps lethality, but not cytotoxicity); (ii) CAT cytotoxicity could be partly due to an active metabolite formed by de novo-synthesized, P450-/P448-independent hemoprotein (PBZ and CoCl2 had anticytotoxic effects, but PB, stanozolol, PBO, and BNF did not); (iii) CAT detoxification may occur partly through a hemoprotein-independent, PBZ-inducible enzyme, and partly through a P448-dependent (BNF-inducible) enzyme; and (iv) CAT detoxification apparently is not P450 or GSH-dependent because PB, stanozolol, and CYS had no beneficial effects, and PBO, CoCl2, and EM did not enhance toxicosis. Metabolism of CAT may have a role in its cytotoxic and lethal effects.