Antimicrobial and antioxidant activities of Melissa officinalis L. (Lamiaceae) essential oil

The present study describes antimicrobial and free radical scavenging capacity (RSC) together with the effects on lipid peroxidation (LP) of Melissa officinalis essential oil. The chemical profile of essential oil was evaluated by the means of gas chromatography-mass spectrometry (GC-MS) and thin-layer chromatography (TLC). RSC was assessed measuring the scavenging activity of essential oil on the 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) and OH(*) radicals. The effect on LP was evaluated following the activities on Fe(2+)/ascorbate and Fe(2+)/H(2)O(2) systems of induction. The antimicrobial activity was tested against 13 bacterial strains and six fungi. The examined essential oil exhibited very strong RSC, reducing the DPPH radical formation (IC(50) = 7.58 microg/mL) and OH radical generation (IC(50) = 1.74 microg/mL) in a dose-dependent manner. According to the GC-MS and TLC (dot-blot techniques), the most powerful scavenging compounds were monoterpene aldehydes and ketones (neral/geranial, citronellal, isomenthone, and menthone) and mono- and sesquiterpene hydrocarbons (E-caryophyllene). Very strong inhibition of LP, particularly in the Fe(2+)/H(2)O(2) system of induction (94.59% for 2.13 microg/mL), was observed in both cases, also in a dose-dependent manner. The most effective antibacterial activity was expressed on a multiresistant strain of Shigella sonei. A significant rate of antifungal activity was exhibited on Trichophyton species.     

Antimicrobial and antioxidant properties of rosemary and sage (Rosmarinus officinalis L. and Salvia officinalis L., Lamiaceae) essential oils

The essential oils of rosemary ( Rosmarinus officinalis L.) and sage ( Salvia officinalis L.) were analyzed by means of gas chromatography-mass spectrometry and assayed for their antimicrobial and antioxidant activities. Antimicrobial activity was tested against 13 bacterial strains and 6 fungi, including Candida albicans and 5 dermatomycetes. The most important antibacterial activity of both essential oils was expressed on Escherichia coli, Salmonella typhi, S. enteritidis, and Shigella sonei. A significant rate of antifungal activity, especially of essential oil of rosemary, was also exhibited. Antioxidant activity was evaluated as a free radical scavenging capacity (RSC), together with the effect on lipid peroxidation (LP). RSC was assessed by measuring the scavenging activity of essential oils on 2,2-diphenyl-1-picrylhydrazil (DPPH) and hydroxyl radicals. Effects on LP were evaluated following the activities of essential oils in Fe(2+)/ascorbate and Fe(2+)/H2O2 systems of induction. Investigated essential oils reduced the DPPH radical formation (IC50 = 3.82 microg/mL for rosemary and 1.78 microg/mL for sage) in a dose-dependent manner. Strong inhibition of LP in both systems of induction was especially observed for the essential oil of rosemary.     

Characterization of the volatile composition of essential oils of some lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils

The essential oils of Ocimum basilicum L., Origanum vulgare L., and Thymus vulgaris L. were analyzed by means of gas chromatography-mass spectrometry and assayed for their antioxidant and antimicrobial activities. The antioxidant activity was evaluated as a free radical scavenging capacity (RSC), together with effects on lipid peroxidation (LP). RSC was assessed measuring the scavenging activity of the essential oils on 2,2-diphenyl-1-picrylhydrazil (DPPH(*)) and OH(*) radicals. Effects on LP were evaluated following the activities of essential oils in Fe(2+)/ascorbate and Fe(2+)/H(2)O(2) systems of induction. Essential oils exhibited very strong RSCs, reducing the DPPH radical formation (IC(50)) in the range from 0.17 (oregano) to 0.39 microg/mL (basil). The essential oil of T. vulgaris exhibited the highest OH radical scavenging activity, although none of the examined essential oils reached 50% of neutralization (IC(50)). All of the tested essential oils strongly inhibited LP, induced either by Fe(2+)/ascorbate or by Fe(2+)/H(2)O(2). The antimicrobial activity was tested against 13 bacterial strains and six fungi. The most effective antibacterial activity was expressed by the essential oil of oregano, even on multiresistant strains of Pseudomonas aeruginosa and Escherichia coli. A significant rate of antifungal activity of all of the examined essential oils was also exhibited.     

Inhibitory effect of hot-water extract from dried fruit of Crataegus pinnatifida on low-density lipoprotein (LDL) oxidation in cell and cell-free systems

The dried fruit of Crataegus pinnatifida, a local soft drink material and medical herb, was found to possess potential against oxidative stress. In the preliminary study, the antioxidant potential of a hot-water extract obtained from the dried fruit of C. pinnatifida (CF-H) was evaluated in terms of its capacity of quenching 1,1-diphenyl-2-picrylhydrazyl free radicals (EC(50) = 0.118 mg/mL). After content analysis, it was found that CF-H is mainly composed of polyphenols including flavonoids (6.9%), procyanidins (2.2%), (+)-catechin (0.5%), and (-)-epicatechin (0.2%). The antioxidative bioactivity of CF-H had been assess previously using the models of CuSO(4) as cell-free system and sodium nitroprusside (SNP) plus macrophage RAW 264.7 cells as cell system to induce human low-density lipoprotein oxidation. CF-H was found to inhibit relative electrophoretic mobility and thiobarbituric acid reactive substances at the concentration of 0.5-1.0 mg/mL in the cell-free system and at 0.01-0.10 mg/mL in the cell system. Furthermore, it was found that CF-H decreased the SNP-induced cell lipid peroxidation and reduced glutathione depletion.     

Inhibition of reactive nitrogen species in vitro and ex vivo by trypsin inhibitor from sweet potato 'Tainong 57' storage roots

Peroxynitrite (ONOO-), formed from a reaction of superoxide and nitric oxide, is one of the most potent cytotoxic species known to oxidize cellular constituents including essential proteins, lipids, and DNA. ONOO- induces cellular and tissue injury, resulting in several human diseases such as Alzheimer's disease, atherosclerosis, and stroke. Due to the lack of endogenous enzymes responsible for ONOO- scavenging activity, finding a specific ONOO- scavenger is of considerable importance. In this study, the ability of trypsin inhibitor (TI), isolated from sweet potato storage roots (SPTI), to scavenge *ON and ONOO- was investigated. The data obtained show that TI generated a dose-dependent inhibition on production of nitrite and superoxide radicals. The IC50 value of TI on superoxide radical was 143.2 +/- 4.29 microg/mL. SOD activity staining was used to confirm SOD activity of SPTI. SPTI also caused a dose-dependent inhibition of the oxidation of dihydrorhodamine 123 (DHR) by peroxynitrite. A calculated IC50 value of 809.1 +/- 32.36 microg/mL was obtained on the inhibition of peroxynitrite radical. Spectrophotometric analyses revealed that TI suppressed the formation of ONOO--mediated tyrosine nitration through an electron donation mechanism. In further studies, TI also showed a significant ability to inhibit nitration of bovine serum albumin (BSA) in a dose-dependent manner. In vivo TI inhibited lipopolysaccharide-induced nitrite production in macrophages in a concentration-dependent manner with an IC50 value of 932.8 +/- 29.85 microg/mL. The present study suggested that TI had an efficient reactive nitrogen species scavenging ability. TI might be a potential effective NO and ONOO- scavenger useful for the prevention of NO- and ONOO--involved diseases.     

Radical scavenging capacity and antimutagenic properties of purified proteins from Solanum betaceum fruits and Solanum tuberosum tubers

In this study, antioxidant activities in free-radical-mediated oxidative systems and the genotoxic/antigenotoxic effects of two proteins with molecular mass around 17 kDa, purified from Solanum betaceum fruits (cyphomine) and Solanum tuberosum tubers (solamarine), were investigated. Both proteins inhibited uric acid formation with IC(50) values between 55 and 60 μg/mL, and both proteins were able to reduce oxidative damage by scavenging hydroxyl radicals and superoxide anion in a dose-dependent manner. Furthermore, the DPPH? reduction assay showed SC(50) values of 55-73 μg/mL. Cyphomine and solamarine were able to retain their antioxidant activity after heat treatment at 80 °C for 15 min. Allium cepa and Salmonella /microsome assays showed no genotoxic and mutagenic effects. Solamarine showed an antimutagenic effect against a direct mutagen (4-nitro-o-phenylenediamine). Consequently, the present study showed that the investigated proteins are promising ingredients for the development of functional foods with a beneficial impact on human health and an important source for the production of bioactive peptides.     

Antioxidant property of an ethanol extract of the stem of Opuntia ficus-indica var. saboten

An ethanol extract of the stem of Opuntia ficus-indica var. saboten (OFS) was assessed to determine the mechanism(s) of its antioxidant activity. The ethanol extract exhibited a concentration-dependent inhibition of linoleic acid oxidation in a thiocyanate assay system. In addition, the OFS extract showed dose-dependent free-radical scavenging activity, including DPPH radicals, superoxide anions (O(2)(*-)), and hydroxyl radicals (*OH), using different assay systems. The OFS ethanol extract was also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals in a Fenton's reaction mixture. Furthermore, the extract showed significant (p < 0.01) dose-dependent protection of mouse splenocytes against glucose oxidase-mediated cytotoxicity. Finally, the OFS extract was characterized as containing a high amount of phenolics (180.3 mg/g), which might be the active compounds responsible for the antioxidant properties of the OFS extract.     

Potential chemopreventive properties of anthocyanin-rich aqueous extracts from in vitro produced tissue of sweetpotato (Ipomoea batatas L.)

Anthocyanin-rich aqueous extracts from cell suspension cultures of a high anthocyanin-producing sweetpotato PL (purple line) cell line grown under two different media conditions, MM (multiplication medium) and APM (high anthocyanin-producing medium) and from the cell line's donor tissue, field-grown storage root (SR) of sweetpotato, cv. Ayamurasaki, were evaluated for antioxidative (DPPH test), antimutagenic (Salmonella/reversion assay; mutagen, Trp-P-1), and antiproliferative (human promyelocytic leukaemia cells HL-60) activities. Both cell line extracts MM and APM exhibited higher radical scavenging activities (RSA), 3.8- and 1.4-fold, respectively, than the SR extract. The antimutagenic activity of all extracts was found to be dose-dependent. At a dose of 1 mg/plate, the highest activity exhibited APM (73% inhibition of Trp-P-1-induced reverse mutation of Salmonella typhimurium TA98), followed by MM (54% inhibition) and SR (36% inhibition). The MM extract was the strongest inhibitor of the proliferation of human promyelocytic leukemia cells. At a concentration of 1.6 mg/mL medium during 24 h, it suppressed the growth of 47% of HL-60 cells. A significantly lower growth suppression effect displayed APM and SR extracts (21 and 25%, respectively). Total anthocyanin levels and anthocyanin composition in evaluated samples seem to be related to their activities. The MM extract, which exhibited the highest RSA and antiproliferation activities, contained the highest level of anthocyanins. Among them, nonacylated cyanidin 3-sophoroside-5-glucoside dominated. It is speculated that the presence of this anthocyanin contributed toward enhanced activities of MM extract.     

Radical scavenging and anti-lipoperoxidative activities of Smallanthus sonchifolius leaf extracts

Radical scavenging and anti-lipoperoxidative effects of two organic fractions and two aqueous extracts from the leaves of a neglected Andean crop-yacon (Smallanthus sonchifolius Poepp. & Endl., Asteraceae) were determined using various in vitro models. The extracts' total phenolic content was 10.7-24.6%. They exhibited DPPH (IC50 16.14-33.39 microg/mL) and HO* scavenging activities (4.49-6.51 mg/mL). The extracts did not scavenge phenylglyoxylic ketyl radicals, but they retarded their formation. In the xanthine/xanthine oxidase superoxide radical generating system, the extracts' activities were 26.10-37.67 superoxide dismutase equivalents/mg. As one of the extracts displayed xanthine oxidase inhibitory activity, the effect of the extracts on a nonenzymatically generated superoxide was determined (IC50 7.36-21.01 microg/mL). The extracts inhibited t-butyl hydroperoxide-induced lipoperoxidation of microsomal and mitochondrial membranes (IC50 22.15-465.3 microg/mL). These results make yacon leaves a good candidate for use as a food supplement in the prevention of chronic diseases involving oxidative stress.     

Antioxidative and hepatoprotective effects of Antrodia camphorata extract

Antrodia camphorata (A. camphorata) is well-known in Taiwan as a traditional Chinese medicine. The purpose of this study was to evaluate the ability of A. camphorata extracts to protect against oxidative stress in vitro and against carbon tetrachloride (CCl(4))-induced hepatic injury in vivo. An extract of A. camphorata inhibited nonenzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC(50) value about 3.1 mg/mL. It also scavenged the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). The dose of the A. camphorata extract resulting in a decrease of 0.20 in the absorbance of DPPH was about 31 +/- 0.7 microg/mL. Furthermore, an A. camphorata extract dose-dependently (250-1250 mg/kg) ameliorated the increase in plasma aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) levels caused by chronic repeated CCl(4) intoxication in mice. Moreover, A. camphorata extract significantly improved the CCl(4)-induced increase in hepatic glutathione peroxidase, reductase, and CCl(4)-induced decrease in superoxide dismutase activities. It also restored the decrement in the glutathione content and catalase activity of hepatic tissues in CCl(4)-intoxicated mice. Furthermore, it also dose-dependently inhibited the formation of lipid peroxidative products during CCl(4) treatment. Histopathological changes of hepatic lesions induced by CCl(4) were significantly ameliorated by treatment with an A. camphorata extract in a dose-dependent manner. These results suggest that A. camphorata extract exerts effective protection against chronic chemical-induced hepatic injury in vivo, by mediating antioxidative and free radical scavenging activities.     

Antioxidant mechanisms of caseinophosphopeptides and casein hydrolysates and their application in ground beef

Caseinophosphopeptides (CPP) and casein hydrolysates have been shown to bind prooxidant metals such as iron, but their effectiveness as metal chelators to inhibit lipid oxidation in foods has still not been fully investigated. Thus, the antioxidant activity of CPP and casein hydrolysates was studied in phosphatidylcholine liposome model systems. CPP (< 1.0 mg/mL) and casein hydrolysates (0.3-1.7 mg/mL) were effective inhibitors of TBARS development when oxidation was promoted by ferric/ascorbate. High amounts of CPP (> 1.0 mg/mL) were prooxidant, whereas casein hydrolysates were observed to be only antioxidative. In the presence of peroxyl radicals, casein hydrolysates were more effective scavengers than enriched CPP (3-15 mM). In cooked ground beef, TBARS formation was inhibited 75, 39, and 17% by 0.5% enriched CPP, casein hydrolysates, and low molecular weight casein hydrolysates, respectively, after 4 days of storage. The results show that CPP and casein hydrolysates are promising sources of natural antioxidants for foods.     

Antimutagenicity of supercritical CO2 extracts of Terminalia catappa leaves and cytotoxicity of the extracts to human hepatoma cells

Natural antimutagens may prevent cancer and are therefore of great interest to oncologists and the public at large. Phytochemicals are potent antimutagen candidates. When the Ames test was applied to examine the antimutagenic potency of supercritical carbon dioxide (SC-CO(2)) extracts of Terminalia catappa leaves at a dose of 0.5 mg/plate, toxicity and mutagenicity were not detected. The antimutagenic activity of SC-CO(2) extracts increased with decreases of temperature (60, 50, and 40 degrees C) and pressure (4000, 3000, and 2000 psi) used for extraction. The most potent antimutagenicity was observed in extracts obtained at 40 degrees C and 2000 psi. At a dose of 0.5 mg of extract/plate, approximately 80% of the mutagenicity of benzo[a]pyrene (B[a]P, with S-9) and 46% of the mutagenicity of N-methyl-N '-nitroguanidine (MNNG, without S-9) were inhibited. Media supplemented with SC-CO(2) extracts at a range of 0-500 microg/mL were used to cultivate human hepatoma (Huh 7) and normal liver (Chang liver) cells. The viability of the cells was assayed by measuring cellular acid phosphatase activity. A dose-dependent growth inhibition of both types of cells was observed. The SC-CO(2) extracts were more cytotoxic to Huh 7 cells than to Chang liver cells. The observation that SC-CO(2) extracts of T. catappa leaves did not induce mutagenicity at the doses tested while exhibiting potent antimutagenicity and were more cytotoxic to human hepatoma cells than to normal liver cells is of merit and warrants further investigation.     

Antimutagenic and antioxidant properties of milk-kefir and soymilk-kefir

This study was aimed at evaluating the antimutagenic and antioxidant properties of milk-kefir and soymilk-kefir. Such antimutagenic activity was determined by means of the Salmonella mutagenicity assay, whereas the antioxidant properties of kefir were evaluated by assessing the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, lipid peroxidation inhibition activity, ferrous ion chelating ability, reducing power, and antioxidative enzyme activity. Both milk-kefir and soymilk-kefir demonstrated significantly greater antimutagenic activity than milk and soymilk. Milk-kefir and soymilk-kefir also displayed significantly greater scavenging activity upon DPPH radicals, an inhibition effect upon linoleic acid peroxidation, and more substantial reducing power but displayed a reduced glutathione peroxidase activity than was the case for milk and soymilk. Milk and soymilk fermented by kefir grains did not alter the ferrous ion chelating ability and superoxide dismutase activity of the original materials. These findings have demonstrated that milk-kefir and soymilk-kefir possess significant antimutagenic and antioxidant activity and suggest that milk-kefir and soymilk-kefir may be considered among the more promising food components in terms of preventing mutagenic and oxidative damage.     

山楂黄酮提取物对辐照肉糜脂肪氧化的抑制能力

试验研究山楂黄酮的抗氧化性以及对辐照肉糜的抗氧化作用.结果表明,山楂黄酮提取物对DPPH·具有一定清除作用,每110g肉糜中添加0.525mg山楂黄酮粗提物对DPPH自由基的清除率可达到56%.山楂黄酮提取物可有效降低辐照肉糜的脂肪氧化程度,且山楂黄酮提取物与Vc复配添加使用效果明显优于山楂黄酮提取物或茶多酚单独处理....     

Gastroprotective effect of red pigments in black chokeberry fruit (Aronia melanocarpa Elliot) on acute gastric hemorrhagic lesions in rats

It has been reported that the fruits and leaves of berries such as the blackberry, raspberry, and strawberry contain a high level of scavenging activity for chemically generated active oxygen species. This study investigated the antioxidative activities of black chokeberry fruit (Aronia melanocarpa Elliot) both in vitro and in vivo using the DPPH stable radical and rats with ethanol-induced gastric injury, respectively. The red pigment fraction of the black chokeberry contained three main components, one of which was identified as cyanidin 3-O-beta-glucoside by HPLC analysis and (1)H NMR. The black chokeberry red pigment fraction scavenged >44% of DPPH radicals at a concentration of 25 microg/mL compared to the control solution. The black chokeberry extract and its hydrolysate administrated at 2 g/kg of body weight each had nearly the same protective effect as quercetin administrated at 100 mg/kg of body weight in suppressing the area of gastric mucosal damage caused by the subsequent application of ethanol to <30% compared to the control group. The black chokeberry red pigment fraction had a similarly significant protective effect on gastric mucosa in a dose-dependent manner when administered at 30-300 mg/kg of body weight, and the administration of 30 mg/kg of body weight could suppress ethanol-induced gastric mucosal damage by approximately 50% (ID(50) = 30 mg/kg of body weight).     

Antimutagenic effect of various honeys and sugars against Trp-p-1

Honey has been used since ancient times as a flavorful sweetener and for its therapeutic and medicinal effects. Consumers' demand for natural, healthy products has driven renewed interest in honey's health benefits. The commonly encountered food mutagen, Trp-p-1, has been demonstrated to be mutagenic in bacteria and carcinogenic in animals. Chemically, honey is quite complex. Honey is comprised primarily of sugars; however, it contains many other potentially biologically active components, such as antioxidants. Sugars have been reported to display both mutagenic and antimutagenic effects in different systems; antioxidants often display antimutagenic activity. Little information exists about potential antimutagenic effects of honey. Antimutagenicity of honeys from seven different floral sources against Trp-p-1 was tested via the Ames assay and compared to that of a sugar analogue and to individually tested simple sugars. All honeys exhibited significant inhibition of Trp-p-1 mutagenicity; most demonstrated a linear correlation between percentage inhibition and log transformed honey concentration from 10 microg/mL to 20 mg/mL. Each displayed significant degrees of inhibition of mutagenicity above concentrations of 1 mg/mL, with individual variations in degree of effectiveness. Buckwheat honey displayed the greatest inhibition at 1 mg/mL, with slightly less effectiveness at higher concentrations. A sugar analogue demonstrated a pattern of inhibition similar to that of the honeys, with enhanced antimutagenicity at concentrations greater than 1 mg/mL. Glucose and fructose were also similar to honeys and were more antimutagenic than maltose and sucrose.     

Antioxidative activity of urate in bovine milk

The antioxidative effects of urate on peroxidase-induced protein oxidation and light-induced riboflavin degradation and lipid oxidation in whole milk were studied. In addition, experiments using ascorbate were conducted to directly compare the antioxidative activity of urate and ascorbate. The presence of urate and/or ascorbate (10-30 mg/L) lowered peroxidase-induced formation of dityrosine by 44-96% in unpasteurized whole milk. No synergistic effect of urate and ascorbate on peroxidase-induced dityrosine formation was registered, but merely an additive effect. Light exposure of pasteurized whole milk showed that ascorbate was oxidized at the expense of urate, which indicated ascorbate-mediated recycling of the urate radical. Moreover, both urate and ascorbate (30 mg/L) retarded light-induced lipid oxidation in pasteurized whole milk as measured by formation of lipid hydroperoxides with urate being the most effective (28% reduction in lipid hydroperoxides) compared with ascorbate (14%). Finally, addition of urate or ascorbate (300 mg/L) to pasteurized whole milk showed a slight protective effect against light-induced degradation of riboflavin with urate being the most effective.     

Antimutagenic effect of fruit and vegetable ethanolic extracts against N-nitrosamines evaluated by the Ames test.

The inhibitory effect of nine fruit and vegetable ethanolic extracts against the mutagenicity of N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA), and N-nitrosopiperidine (NPIP) was evaluated by means of the Ames test. Licorice ethanolic extract was the only one that showed an inhibitory effect (ranging from moderate to strong) against mutagenicity of all N-nitrosamines tested. This ethanolic extract showed the greatest inhibition effect against NPIP (72%), NDMA (45%), and NPYR (39%). The greatest inhibition effect (51%) of the mutagenicity of NDBA was shown by kiwi ethanolic extract. Vegetable and fruit ethanolic extracts that exhibited an antimutagenic effect (at the range 50-2000 microg/plate), in decreasing order, against NDMA and NPYR were as follows: licorice > kiwi > carrot and licorice > broccoli > pineapple > kiwi, respectively. Decreasing orders against NDBA and NPIP were, respectively, kiwi > onion > licorice = garlic > green pepper > carrot and licorice > garlic > pineapple > carrot.     

Free radical scavengers and antioxidants from Lemongrass (Cymbopogon citratus (DC.) Stapf.)

Methanol, MeOH/water extracts, infusion, and decoction of Cymbopogon citratus were assessed for free radical scavenging effects measured by the bleaching of the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical, scavenging of the superoxide anion, and inhibition of the enzyme xanthine oxidase (XO) and lipid peroxidation in human erythrocytes. The extracts presented effect in the DPPH and superoxide anion assay, with values ranging between 40 and 68% and 15-32% at 33 and 50 microg/mL, respectively, inhibited lipid peroxidation in erythrocytes by 19-71% at 500 microg/mL and were inactive toward the XO at 50 microg/mL. Isoorientin, isoscoparin, swertiajaponin, isoorientin 2' '-O-rhamnoside, orientin, chlorogenic acid, and caffeic acid were isolated and identified by spectroscopic methods. Isoorientin and orientin presented similar activities toward the DPPH (IC(50): 9-10 microM) and inhibited lipid peroxidation by 70% at 100 microg/mL. Caffeic and chlorogenic acid were active superoxide anion scavengers with IC(50) values of 68.8 and 54.2 microM, respectively, and a strong effect toward DPPH. Caffeic acid inhibited lipid peroxidation by 85% at 100 microg/mL.