De novo sequencing of peptides from the parotid secretion of the cane toad, Bufo marinus (Rhinella marina)

Amphibian skin secretions are well known as a rich source of bioactive peptides. However, little is known about the presence or role of peptides in the highly toxic, parotid secretion of the cane toad or giant toad, Bufo marinus (Rhinella marina), though small molecule bufadienolides, which act as potent cardiotoxins, have been described. In the current study we used RP-HPLC, MALDI-TOF mass spectrometry and tandem mass spectrometry to analyze and determine the first sequences of peptides from the parotid secretion of B. marinus. We show that peptides in the range of 900-2500 Da are indeed present, however in extremely low abundance. Despite the low abundance, the sequences of 14 peptides were determined, several of which match fragments of larger cellular proteins, yet none share substantial homology with defensive or anti-microbial peptides reported from frog skin secretions. We conclude that peptides are present in the parotid glands of B. marinus only in very low quantities and that they are likely to be breakdown products of proteins involved in cell maintenance. Given these results, we conclude that peptides are unlikely to contribute directly to the high toxicity of the cane toad.     

Differential cytotoxic effects of denitroaristolochic acid II and aristolochic acids on renal epithelial cells

Aristolochic acids (AAs), naturally occurring nephrotoxins and rodent carcinogens, are commonly found in medicinal plants such as Radix aristolochiae. The toxicity of AAs is believed to be associated with the formation of promutagenic AA-DNA adducts, and it has also been suggested that the nitro group in AAs might be important in the process. In order to investigate the role of the nitro group in AA-mediated cytotoxicity, the effects of denitroaristolochic acid II (dN-AAII), aristolochic acid II (AAII) and aristolochic acid I (AAI) on renal tubular epithelial Madin–Darby canine kidney (MDCK) cells were examined and compared. The cytotoxicity of AAI, AAII and dN-AAII was found to be time- and concentration-dependent. As determined by MTT assay, AAI was found to be most toxic in MDCK cells upon exposure for 24, 48 and 72 h, followed by AAII, and dN-AAII showed the least cytotoxicity. The effect of AAI and AAII on the integrity of cell membrane was found to be similar and appeared to be more prominent than that of dN-AAII. Based on the results obtained from the flow cytometric analysis, significant apoptosis in MDCK cells was observed with AAI and AAII at as low as 25 μmol/L following exposure for 24 h, whereas significant apoptosis was induced by dN-AAII at a much higher concentration, 300 μmol/L, suggesting that both AAI and AAII were significantly more cytotoxic than dN-AAII. In addition, the levels of reactive oxygen species (ROS) were increased following treatment with AAI, AAII and dN-AAII at concentrations of 5, 25 and 25 μmol/L, respectively, for 4 h. The results suggest that the nitro group plays an important role in AA-mediated cytotoxicity in MDCK cells and increased intracellular ROS levels may be associated, at least in part, with the cell injury observed in MDCK cells.     

In vitro and in vivo studies on some toxic effects of the venom from Hemiscorpious lepturus scorpion

The aim of this laboratory-based study was to investigate some of the toxic effects induced by the venom from Hemiscorpious lepturus (H. lepturus). For this aim, pharmacological, histological, biochemical methods as well as complete blood cell count were used to assess these toxic actions. In addition, in vitro haemolysis studies on human washed blood suspension and cytotoxicity on cultured fibroblasts were also undertaken. In vitro pharmacological test was made on rat isolated ileal segment. To this end, the effects of the venom on the contractile responsiveness to acetylcholine were recorded using F30 transducer and Darco chart recorder. For assessment of the haemolytic potency, varying concentrations (2, 10, 20 and 40 μg/ml) of the venom were added to 0.5 ml of 5% washed human blood and after 30 min, 2, 4, 8, 12 and 24 h of exposure, the degree of lysis (extent of redness developed in the supernatant solution after centrifugation) were measured by ELISA method. Cytotoxicity potential of the venom was assessed by trypan blue exclusion test. The venom (0.1, 1 and 10 μg/ml) was mixed with confluent fibroblast cell culture and the extent cytotoxicity was assessed microscopically. In vivo studies were conducted by a subcutaneous administration of sub-lethal dose (10 μg) of the venom and after 7 days the skin, at the site of injection, and kidney samples were stained by H & E method and examined microscopically. In addition, biochemical assessments including measurement of serum aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and amylase levels and urine analysis were made. The results showed that the venom prevented the relaxation phase of the acetylcholine-induced contractions on the isolated ileal segments and finally produced sustained spasmodic contractions. This spasmodic action was abolished by 1 μM atropine. The venom produced haemolysis of red blood cells in a concentration-dependent and duration-of-exposure manner, with 100% of haemolysis produced after 24 h following exposure to 40 μg/ml of venom. While cultured fibroblasts cells were more sensitive and disintegrated after 15 min of exposure to 1 μg/ml of the venom. Histological findings showed evidences of excessive inflammatory responses accompanied with signs of necrosis in the skin at the site of injection as well as structural damage in the nephrones. There was a significant rise in the serum enzymes. In addition, the number of the RBCs were reduced. The urine showed positive readings for proteinuria, blood and intact RBCs. The overall results suggest that the venom from H. lepturus primarily is a cytotoxic agent and has haemolytic, nephrotoxic and to some extent hepatotoxic activity.     

Toxic dinoflagellates (Dinophyceae) from Rarotonga, Cook Islands

Dinoflagellate species isolated from the green calcareous seaweed, Halimeda sp. J.V. Lamouroux, growing in Rarotongan lagoons, included Gambierdiscus australes Faust & Chinain, Coolia monotis Meunier, Amphidinium carterae Hulburth, Prorocentrum lima (Ehrenberg) Dodge, P. cf. maculosum Faust and species in the genus Ostreopsis Schmidt. Isolates were identified to species level by scanning electron microscopy and/or DNA sequence analysis. Culture extracts of G. australes isolate CAWD149 gave a response of 0.04 pg P-CTX-1 equiv. per cell by an N2A cytotoxicity assay (equivalent to ca 0.4 pg CTX-3C cell⁻¹). However, ciguatoxins were not detected by LC-MS/MS. Partitioned fractions of the cell extracts potentially containing maitotoxin were found to be very toxic to mice after intraperitoneal (i.p.) injection. A. carterae was also of interest as extracts of mass cultures caused respiratory paralysis in mice at high doses, both by i.p. injection and by oral administration. The Rarotongan isolate fell into a different clade to New Zealand A. carterae isolates, based on DNA sequence analysis, and also had a different toxin profile. As A. carterae co-occurred with G. australes, it may contribute to human poisonings attributed to CTX and warrants further investigation. A crude extract of C. monotis was of low toxicity to mice by i.p. injection, and an extract of Ostreopsis sp. was negative in the palytoxin haemolysis neutralisation assay.     

Inhibitory effects of methanol extract of plum (Prunus salicina L., cv. ‘Soldam’) fruits against benzo(α)pyrene-induced toxicity in mice

This study was carried out to investigate the chemopreventive effects of immature plum extracts. The methanol extract of immature plums (plum 1), that are picked at 20–40 days before final harvest, has remarkably inhibited the growth of hepatoma HepG2 cells. The effects of immature plum extracts on hepatotoxicity in benzo(α)pyrene (B(α)P, carcinogen)-treated mice were investigated. Male ICR mice were pretreated with immature plum extracts (2.5 or 5 g/kg bw/day, for 5 days, i.p.) before treatment with B(α)P(0.5 mg/kg bw, i.p., single dose). The activities of serum aminotransferase, cytochrome P450 (CYPs) and the hepatic content of lipid peroxide were increased on B(α)P-treatment group than control, but those levels were significantly decreased by the pretreatment of immature plum extracts. The primary CYPs involved in the metabolism and bioactivation of B(α)P are CYP1A1. The pretreatment of immature plum extracts inhibited the induction of CYP1A1 expression. The activities of glutathione peroxidase, superoxide dismutase and catalase were decreased by the pretreatment of immature plum extracts more than with B(α)P alone. Whereas, the hepatic content of glutathione and glutathione S-transferase activity depleted by B(α)P was significantly increased (p > 0.05). These results suggest that immature plum extracts may counteract toxic effects of carcinogens, such as B(α)P, and therefore possess the chemopreventive efficacy.     

Inhibition of inflammatory mediators by polyphenolic plant extracts in human intestinal Caco-2 cells

The mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) are involved in transduction cascades that play a key role in inflammatory response. We tested the ability of preselected natural polyphenolic extracts (grape seed, cocoa, sugar cane, oak, mangosteen and pomegranate) to modulate intestinal inflammation using human intestinal Caco-2 cells treated for 4 h with these extracts and then stimulated by cytokines for 24 or 48 h. The effect of polyphenolic extracts, at 50 μmol of gallic acid equivalent/l, was investigated on inflammation-related cellular events: (i) NF-κB activity (cells transfected with a NF-κB-luciferase construct), (ii) activation of Erk1/2 and JNK (western blotting), (iii) secretion of interleukin 8 (IL-8) (ELISA), (iv) secretion of prostaglandin (PG) E₂ (ELISA), (v) production of NO (Griess method). Results show that: (i) sugar cane, oak and pomegranate extracts inhibited NF-κB activity (from 1.6 to 1.9-fold) (P < 0.001); (ii) pomegranate slightly inhibited Erk1/2 activation (1.3-fold) (P = 0.008); (iii) oak and pomegranate decreased NO synthesis by 1.5-fold (P < 0.001) and that of IL-8 by 10.3 and 6.7-fold respectively; (iv) pomegranate and cocoa decreased PGE₂ synthesis by 4.6 (P < 0.0001) and 2.2-fold (P = 0.001), respectively. We suggest that pomegranate extract could be particularly promising in dietary prevention of intestinal inflammation.     

Effects of Aspergillus niger-fermented Terminalia catappa seed meal-based diet on selected enzymes of some tissues of broiler chicks

Effects of Aspergillus niger-fermented Terminalia catappa seed meal-based diet on the activities of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and gamma-glutamate transferase (γ-GT) in the crop, small intestine, gizzard, heart, liver and serum of broiler chicks were investigated. Milled T. catappa seed was inoculated with spores of A.niger (2.21 × 10⁴ spores per ml) for 3 weeks. Forty-five day-old broiler chicks weighing between 27.62 and 36.21 g, were divided into three groups. The first group was fed soybean-based (control) diet; the second on raw T. catappa seed meal-based diet; and the third on A. niger-fermented T. catappa seed meal-based diet for 7 weeks. The results revealed a significantly increased (p < 0.05) activity of ALP in the tissues. Contrarily, there were significant reductions (p < 0.05) in the activities of ALP, ALT, AST and γ-GT in the liver and heart of the broilers fed the raw T. catappa seed meal-based diet while there were significant increase (p < 0.05) in the activities of these enzymes in the serum of the broilers in this group. The data obtained showed that A. niger-fermented T. catappa seed meal reduced the toxic effects of the raw seed meal on the tissues of broiler chicks.     

The antioxidative effect of Iranian Mentha pulegium extracts and essential oil in sunflower oil

The aim of the present study was to evaluate antioxidative activities of the essential oil, methanol and water extracts of Iranian pennyroyal in vegetable oil during storage. Different concentrations (0, 200, 400, 600, 800 and 1000 ppm) of essential oil, water and methanol extracts and beta-hydroxy toluene (BHT; 200 ppm) were added to sunflower oil emulsion in the presence of cupric ions and incubated for 7 days at 60 °C. Peroxide values (PVs) and thiobarbituric acid reacting substances (TBARS) levels were measured in each day up to day of seven. Furthermore, antioxidant capacity of the essential oil and extracts were determined using DPPH and β-carotene–linoleic acid methods. Values were compared among groups in each incubation time points using ANOVA. Results showed that DPPH and β-carotene–linoleic acid assay findings on the Mentha pulegium extracts were comparable to those found on BHT. Furthermore, in all incubation time points, M. pulegium extracts lowered PVs and TBARS levels when compared to the control (p < 0.001). In this respect, water extract was more potent than the methanol extract. Essential oil did not show considerable antioxidative effect. It seems that water extract of M. pulegium is a potent antioxidant which makes it as a potential antioxidant for oil and oily products during storage.     

Comparative antihemolytic and radical scavenging activities of strawberry tree (Arbutus unedo L.) leaf and fruit

The present study reports the antioxidant properties of Arbutus unedo L. leaf and fruit extracts using different in vitro assays including (i) reducing power, (ii) scavenging effect on DPPH free radicals, and (iii) inhibitory effect on AAPH-induced hemolysis and lipid peroxidation in human erythrocytes. All assays demonstrated antioxidant efficiency for A. unedo L. aqueous extracts, being consistently higher in the leaf. EC₅₀ values for reducing power and DPPH radical scavenging activities were, respectively, 0.318 ± 0.007 and 0.087 ± 0.007 mg/mL for leaf, and 2.894 ± 0.049 and 0.790 ± 0.016 mg/mL for fruit extracts. Under the oxidative action of AAPH, A. unedo leaf and fruit extracts protected the erythrocyte membrane from hemolysis (IC₅₀ of 0.062 ± 0.002 and 0.430 ± 0.091 mg/mL, respectively) and decreased the levels of malondialdehyde, a breakdown product of lipid peroxidation (IC₅₀ of 0.075 ± 0.014 and 0.732 ± 0.452 mg/mL, respectively). In accordance with antioxidant activity, phenolic content was found to be significantly higher in leaf extract. To our knowledge, this is the first time that the antioxidant activity of A. unedo species is evaluated using human biological membranes. Overall, our results suggest that A. unedo leaves are a promising source of natural antioxidants with potential application in diseases mediated by free radicals.     

Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin

A protein (BMP1) was purified from common Indian toad (Bufo melanostictus, Schneider) skin through DEAE cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the BMP1 was found to be 79 kDa. BMP1 (0.5 and 1 mg/kg/day, i.p.) significantly decreased the number of viable Ehrlich ascites carcinoma (EAC) cells, thereby increased the lifespan of EAC bearing mice (p < 0.001). MTT values reduced significantly with the treatment of BMP1 (0.5 and 1.0 mg/kg/day, i.p. for 3 days) on EAC cells indicated its antiproliferative activity. This was also supported by flow-cytometric data on the cell cycle arrest at G1 in EAC cells. BMP1 (1 mg/kg) reduced the solid tumor weight and volume of about three times further support the antiproliferative nature. Fluorescence and confocal microscopic study on EAC cells after BMP1 (0.5 mg/kg/day, i.p. for 3 days) treatment indicated certain features of apoptosis, like nuclear fragmentation, membrane blebbing, and vacuolization of cells. DNA fragmentation was clearly observed in alkaline comet assay. Apoptosis induced by BMP1 was further confirmed through flow-cytometric analysis of annexin-V binding study, sub-G1 arrest in the cell cycle and found to be mediated through caspase 3 dependent pathway. LD50 of BMP1 was found to be 12.2 mg/kg, i.p. in male Swiss albino mice. BMP1 treatment at 0.5 mg/kg and 1.0 mg/kg for 10 days did not alter any hematological and biochemical parameters in mice, but after 30 days of treatment produce significant rise in total leucocyte count, neutrophil percentage, serum urea, creatinine, GOT, LDH and decrease in lymphocyte percentage as compared to respective control. In conclusion, BMP1, a protein molecule isolated from Indian toad (B. melanostictus, Schneider) skin, showed antiproliferative and apoptogenic activity on EAC cancer cell with limited toxicity.     

Effects of Thai Musa species on prevention of UVB-induced skin damage in mice

The effects of oral administration of Musa sapientum and Musa suerier on prevention of UVB induced skin damages were investigated in male ICR mice. Animals were orally administered 50 mg/day ascorbic acid, or M. sapientum or M. suerier’s fruit pulps at dose of 0.5, 1 or 1.5 mg/g body weight/day for 12 weeks. Concurrently, the shaved backs of animals were irradiated with UVB for 12 weeks. The intensity of irradiation was progressively increased, from 54 mJ/cm² per exposure at week 1–126 mJ/cm² at week 11. A significant decrease (p < 0.05) in skin elasticity (from 0.82 ± 0.02 to 0.42 ± 0.09) and total glutathione (from (193.6 ± 18.7 to 152.7 ± 7.8 ng/mg protein) as compared with the control group (water-administered UVB-irradiated mice) was observed after 12 weeks of UVB exposure. When l-ascorbic acid (0.72 ± 0.01) or 1 mg/g body weight/day M. suerier (0.84 ± 0.06) were administered to UVB-irradiated mice, the reduction in skin elasticity was significantly inhibited (p < 0.05). Moreover, the significant increase (p < 0.05) in level of total glutathione was found in these groups (220.8 ± 13.3 ng/mg protein for l-ascorbic acid and 224.9 ± 20.1 ng/mg protein for M. suerier). These findings suggest the potential effect of daily consumption of M. suerier on prevention of skin damage from repeated UVB exposure.     

Biological potentials and cytotoxicity of various extracts from endemic Origanum minutiflorum O. Schwarz & P.H. Davis

The aim of the present study was to investigate antioxidative, antimicrobial and cytotoxic effects of endemic Origanum minutiflorum (O. Schwarz & P.H. Davis). Antioxidant activities of the extracts from O. minutiflorum were evaluated by using DPPH radical scavenging, β-carotene bleaching and metal chelating activity assays. In addition, the amounts of total phenol components in the plant extracts were determined. In the β-carotene bleaching test, the extracts exhibited in the range of 58.1 ± 0.2% – 98.2 ± 0.3% inhibition against linoleic acid oxidation. The antimicrobial efficiency of the plant was evaluated according to agar well diffusion and microdilution broth methods. The n-hexane extract of O. minutiflorum having an inhibition zone of 20.2 ± 0.2 mm, had the maximum antibacterial efficiency against Shigella sonnei RSKK 878. Cytotoxic effects of the extracts were determined by MTT assay. O. minutiflorum extracts (at concentration of 10–100 μg/ml) did not show any cytotoxic effect on baby hamster kidney fibroblast cell line. The results showed that O. minutiflorum could be used as a natural source in food industry.     

Growth and toxin production in the ciguatera-causing dinoflagellate Gambierdiscus polynesiensis (Dinophyceae) in culture

The growth and toxin production in a clonal strain of Gambierdiscus polynesiensis, TB-92, was examined in batch culture conditions. The mean growth rate at exponential phase was (0.13 ± 0.03) division day⁻¹. Regardless of the age of cultures, all mice injected with dichloromethanolic and methanolic extracts showed symptoms specific to ciguatoxin (CTX) and maitotoxin (MTX) bioactivity, respectively. The highest total toxicity assessed in TB-92 cultures was 10.4 × 10⁻⁴ mouse unit cell⁻¹. The toxin production pattern reveals an enhanced cellular toxin content with the age of the culture. CTX- and MTX-like compounds each accounted for approx. 50% of the total toxicity of TB-92 cultures, except in aged cells where CTXs were dominant. The high ciguatoxic activity of TB-92 was further confirmed in dichloromethanolic extracts by means of the receptor-binding assay. The highest CTX level monitored at late stationary phase was (11.9 ± 0.4) pg P-CTX-3C equiv cell⁻¹. Further HPLC and LC-MS analysis revealed the presence of five CTXs congeners in lipid-soluble extracts, i.e. CTX-3C, -3B, -4A, -4B and M-seco-CTX-3C, and of new CTX congeners. Toxin composition comparison between two G. polynesiensis strains suggests that the toxin profile is a stable characteristic in this species. G. polynesiensis clones also proved inherently more toxic than other Gambierdiscus species isolated from other geographical areas.     

Essential oil composition and antioxidant activities of Curcuma aromatica Salisb.

The chemical composition of the hydro-distilled essential oil from leaves of Curcuma aromatica Salisb. was analysed by GC–MS. Twenty-three compounds representing 94.29% of the total oil were identified. The antioxidant activities of the oil and various extracts of C. aromatica were evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical-scavenging assays. The oil and methanol extract showed potent DPPH radical-scavenging activities (IC₅₀ = 14.45 and 16.58 μg/ml, respectively), which were higher than butylated hydroxyanisole (IC₅₀ = 18.27 μg/ml). The extracts also exhibited remarkable superoxide radical-scavenging activities (IC₅₀ = 22.6–45.27 μg/ml) and the activity in the methanol extract was superior to all other extracts (IC₅₀ = 22.6 μg/ml). Furthermore, the amount of total phenolic compounds was determined and its content in ethyl acetate extract was the highest as compared to other extracts. The results indicate that the oil and extracts of C. aromatica could serve as an important bio-resource of antioxidants for using in the food industries.     

Inhibition of hepatic δ-aminolevulinate dehydratase activity induced by mercuric chloride is potentiated by N-acetylcysteine in vitro

Mercuric chloride (HgCl)₂ is a toxic metal that causes oxidative damage in several tissues. N-acetylcysteine (NAC) is a sulfhydryl compound with antioxidant activity. In the present study, we investigated the in vitro effects of the association between HgCl₂ and NAC in tissues of mice. For this purpose, we evaluated the in vitro effect of HgCl₂ + NAC association on δ-aminolevulinate dehydratase (δ-ALA-D) activity and on thiobarbituric acid reactive substances (TBARS) levels in liver and kidney of mice. The results demonstrate that HgCl₂ inhibited δ-ALA-D activity in both tissues. Hepatic δ-ALA-D activity inhibited by HgCl₂ was potentiated by the highest concentration of NAC. The inhibition of hepatic δ-ALA-D activity seems to be related to sulfhydryl groups oxidation of the enzyme. We observed also that HgCl₂ increased TBARS levels in kidney and liver. Hepatic TBARS levels were reduced by NAC, at higher concentration. In contrast, NAC, at higher concentration, increased renal TBARS levels. In conclusion, the inhibition of hepatic δ-aminolevulinate dehydratase activity induced by HgCl₂ is potentiated by NAC in vitro, and this effect is not related to hepatic lipid peroxidation.     

Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines

There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r² = 0.47, P = 0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r² = 0.56, P = 0.03 and r² = 0.62, P = 0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA.LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.     

Phytochemical profile, antioxidant, anti-inflammatory and hypoglycemic potential of hydroalcoholic extracts from Citrus medica L. cv Diamante flowers, leaves and fruits at two maturity stages

Since the past decade consumption of certain foods has been reported to have a positive effect on health. The object of the study was to determine for the first time the chemical composition and the antioxidant, anti-inflammatory and hypoglycemic potential of Citrus medica L. cv Diamante flowers, leaves and fruits (endocarp and mesocarp) at two maturity stages. Flowers and leaves were characterized by the highest total phenols and flavonoids content. A declining trend was observed during maturity of fruits for both phenols and flavonoids. The antioxidant activity evaluated by the β-carotene bleaching test showed a strong activity for flowers and endocarp of mature fruits with IC₅₀ values of 2.8 μg/mL and 3.5 μg/mL, respectively, after 30 min of incubation. Interestingly, the mature fruits endocarp (IC₅₀ value of 426.0 μg/mL) could inhibit α-amylase with an IC₅₀ value 2-fold higher than immature fruits. None of the tested extracts affected the proliferation of human skin fibroblasts 142BR. The obtained results suggest a potential use of C. medica L. cv Diamante as new valuable Citrus species with functional properties for food or nutraceutical product on the basis of high content of phytochemicals.     

Antioxidant and antidermatophytic activities of essential oil and extracts of Metasequoia glyptostroboides Miki ex Hu

This study was undertaken to assess the antioxidant and antidermatophytic potential of the essential oil and extracts (hexane, chloroform, ethyl acetate and methanol) of Metasequoia glyptostroboides Miki ex Hu. Antioxidant activity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the oil and ethyl acetate extract were found to be superior (IC₅₀ = 9.1 and 14.24 μg/ml, respectively) as compared to butylatedhydroxyanisole (BHA), (IC₅₀ = 18.27 μg/ml). Also the ethyl acetate extract revealed the highest phenolic contents (93.26 mg/g of dry wt) as compared to the other extracts. Further, oil (1250 μg/disc) and extracts (1750 μg/disc) revealed 35.33–67.66 and 18.0–53.3% antidermatophytic effect, respectively, along with their respective MIC values (62.5–500 and 250–4000 μg/ml) against Trichophyton rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, T. mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, Microsporum canis KCTC 6591, M. canis KCTC 6348 and M. canis KCTC 6349. The oil also had a strong detrimental effect on spore germination as well as concentration and time-dependent kinetic inhibition of M. canis KCTC 6591.