In vitro anti‐enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers

Aims: The anti‐enterovirus 71 (EV71) activity of six Nepalese plants’ extracts and gallic acid (GA) isolated from Woodfordia fruticosa Kurz (family; Lythaceae) flowers were evaluated in Vero cells. Methods and Results: The anti‐EV71 activity of tested compounds was evaluated by a cytopathic effect reduction method. Our results demonstrated that flowers’ extracts of W. fruticosa exerted strong anti‐EV71 activity, with a 50% inhibitory concentration (IC₅₀) of 1·2 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived therapeutic index (TI) was more than 83·33. Rivabirin showed no antiviral activity against EV71. Furthermore, GA isolated from W. fruticosa flowers exhibited a higher anti‐EV71 activity than the extract of W. fruticosa flowers, with an IC₅₀ of 0·76 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived TI was 99·57. Conclusions: This study demonstrated that flower extracts of W. fruticosa possessed anti‐EV71 activity and GA isolated from these flowers showed stronger anti‐EV71 activity than that the extracts. Significance and Impact of the Study: Our results suggest that the GA from W. fruticosa flowers may be used as a potential antiviral agent.     

Polyphenol Characterization,Antioxidant and Skin Whitening Properties of Alnus cordata Stem Bark

In this study, we investigated the phenolic composition of the crude extract (MeOH 80 %) of Alnus cordata (Loisel .) Duby stem bark (ACE) and its antioxidant and skin whitening properties. RP‐LC‐DAD analysis showed a high content of hydroxycinnamic acids (47.64 %), flavanones (26.74 %) and diarylheptanoids (17.69 %). Furthermore, ACE exhibited a dose‐dependent antioxidant and free‐radical scavenging activity, expressed as half‐maximal inhibitory concentration (IC₅₀): Oxygen radical absorbance capacity (ORAC, IC₅₀ 1.78 μg mL^?1)>Trolox equivalent antioxidant capacity (TEAC, IC₅₀ 3.47 μg mL^?1)>2,2‐Diphenyl‐1‐picrylhydrazyl (DPPH, IC₅₀ 5.83 μg mL^?1)>β‐carotene bleaching (IC₅₀ 11.58 μg mL^?1)>Ferric reducing antioxidant power (FRAP, IC₅₀ 17.28 μg mL^?1). Moreover, ACE was able to inhibit in vitro tyrosinase activity (IC₅₀ 77.44 μg mL^?1), l ‐DOPA auto‐oxidation (IC₅₀ 39.58 μg mL^?1) and in an in vivo model it exhibited bleaching effects on the pigmentation of zebrafish embryos (72 h post fertilization) without affecting their development and survival. In conclusion, results show that A. cordata stem bark may be considered a potential source of agents for the treatment of skin disorders due to its bleaching properties and favorable safety profiles, associated to a good antioxidant power.     

Diterpenoids from Euphorbia neriifolia and Their Related Anti‐HIV and Cytotoxic Activity

Fifteen diterpenoids ( 1 – 15 ), including three undescribed ones with ent‐atisane skeleton, eupnerias G–I ( 1 – 3 ), were obtained from Euphorbia neriifolia. Compounds 1 – 3 were established through comprehensive spectroscopic analysis. Compounds 4 and 5 exhibited obvious anti‐HIV‐1 effect, and their EC₅₀ were 6.6±3.2 and 6.4±2.5 μg mL^?1, respectively. Compound 6 exhibited moderate cytotoxicity on HepG2 and HepG2/Adr cells with IC₅₀ at 13.70 and 15.57 μm , respectively. In addition, compound 15 exhibited significant cytotoxicity on HepG2 cell lines (IC₅₀=0.01 μm ), while it did not show any cytotoxicity against HepG2/Adr cell lines.     

Synthesis and Fungicidal Activities of New 1,2,4‐Triazolo[1,5‐a]pyrimidines

A series of new acetohydrazone‐containing 1,2,4‐triazolo[1,5‐a]pyrimidine derivatives were designed and synthesized for the purpose of searching for novel agrochemicals with higher fungicidal activity. Their in vitro fungicidal activities against Rhizoctonia solani were evaluated, and the most promising compound, 2‐[(5,7‐dimethyl[1,2,4]triazolo[1,5‐a]pyrimidin‐2‐yl)sulfanyl]‐2′‐[(2‐hydroxyphenyl)methylidene]acetohydrazide ( 2‐17 ), showed a lower EC₅₀ value (5.34 μg ml^?1) than that of commercial carbendazim (EC₅₀=7.62 μg ml^?1). Additionally, compound 2‐17 was also found to display broad‐spectrum fungicidal activities, and its EC₅₀ value (4.56 μg ml^?1) against Botrytis cinereapers was very similar to that of carbendazim. Qualitative structure–activity relationships (QSARs) of the synthesized compounds were also discussed.     

Anti-barnacle activity of novel simple alkyl isocyanides derived from citronellol

Twenty novel simple alkyl isocyanides derived from citronellol were synthesized and evaluated for their antifouling activity and toxicity against cypris larvae of the barnacle, Balanus amphitrite. The anti-barnacle activity of the synthesized isocyanides was in the EC₅₀ range of 0.08–1.49 μg ml^?1. Simple isocyanides containing a benzoate and chloro group showed the most potent anti-barnacle activity. In addition, none of the synthesized compounds showed significant toxicity and LC₅₀ values were <10 μg ml^?1. The LC₅₀/EC₅₀ ratios of almost all of the synthesized compounds were >10². The results indicate that these simple isocyanides are promising low-toxicity antifouling agents.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Effect of garlic and allium-derived products on the growth and metabolism of Spironucleus vortens

Spironucleus is a genus of small, flagellated parasites, many of which can infect a wide range of vertebrates and are a significant problem in aquaculture. Following the ban on the use of metronidazole in food fish due to toxicity problems, no satisfactory chemotherapies for the treatment of spironucleosis are currently available. Using membrane inlet mass spectrometry and automated optical density monitoring of growth, we investigated in vitro the effect of Allium sativum (garlic), a herbal remedy known for its antimicrobial properties, on the growth and metabolism of Spironucleus vortens, a parasite of tropical fish and putative agent of hole-in-the-head disease. The allium-derived thiosulfinate compounds allicin and ajoene, as well as an ajoene-free mixture of thiosulfinates and vinyl-dithiins were also tested. Whole, freeze-dried garlic and allium-derived compounds had an inhibitory effect on gas metabolism, exponential growth rate and final growth yield of S. vortens in Keister’s modified, TY-I-S33 culture medium. Of all the allium-derived compounds tested, the ajoene-free mixture of dithiins and thiosulfinates was the most effective with a minimum inhibitory concentration (MIC) of 107 μg ml⁻¹ and an inhibitory concentration at 50% (IC_50%) of 58 μg ml⁻¹. It was followed by ajoene (MIC = 83 μg ml⁻¹, IC_50% = 56 μg ml⁻¹) and raw garlic (MIC >20 mg ml⁻¹, IC_50% = 7.9 mg ml⁻¹); allicin being significantly less potent with an MIC and IC_50% above 160 μg ml⁻¹. All these concentrations are much higher than those reported to be required for the inhibition of most bacteria, protozoa and fungi previously investigated, indicating an unusual level of tolerance for allium-derived products in S. vortens. However, chemically synthesized derivatives of garlic constituents might prove a useful avenue for future research.     

Endophytic fungi with anti-microbial,anti-cancer and anti-malarial activities isolated from Thai medicinal plants

A total of 81 Thai medicinal plant species collected from forests in four geographical regions of Thailand were examined for the presence of endophytic fungi with biological activity. Of 582 pure isolates obtained, 360 morphologically distinct fungi were selected for cultivation on malt Czapek broth and yeast extract sucrose broth, from which extracts were tested for biological activity. Extracts of 92 isolates could inhibit Mycobacterium tuberculosis (MIC 0.0625–200 μg ml⁻¹) when tested by the microplate Alamar blue assay, while extracts of six inhibited Plasmodium falciparum (IC₅₀ of 1.2–9.1 μg ml⁻¹) as determined by the [³H]hypoxanthine incorporation method. Strong anti-viral activity against Herpes simplex virus type 1 was observed in 40 isolates (IC₅₀ of 0.28–50 μg ml⁻¹). The sulphorhodamine B assay for activity against cancer cell lines revealed that 60 were active against human oral epidermoid carcinoma cells (EC₅₀ 0.42–20 μg ml⁻¹) and 48 against breast cancer cells (EC₅₀ 0.18–20 μg ml⁻¹). Bioactivity profile was affected by the type of culture medium. Given the high incidence of bioactive extracts and the fact that most of the isolated fungi could not be identified due to lack of spore formation, the results suggested that Thai medicinal plants can provide a wide variety of endophytes that might be a potential source of novel bioactive compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biological Activities of Phenolics from the Fruits of Phyllanthus emblica L. (Euphorbiaceae)

Seven phenolic compounds, 1 – 7 , including a new organic acid gallate, mucic acid 1‐ethyl 6‐methyl ester 2‐O‐gallate ( 7 ), were isolated from the MeOH extract of the fruits of Phyllanthus emblica L. (Euphorbiaceae). The structures were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. Upon evaluated for their antioxidant abilities by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. The inhibitory activities against melanogenesis in B16 melanoma cells induced by α‐MSH, as well as cytotoxic activities against four human cancer cell lines were also evaluated. All phenolic compounds, 1 – 7 , exhibited potent antioxidant abilities (DPPH: IC₅₀ 5.6 – 12.9 μm ; ABTS: 0.87 – 8.43 μm Trolox/μm ; FRAP: 1.01 – 5.79 μm Fe²⁺/μm , respectively). Besides, 5 – 7 , also exhibited moderate inhibitory activities against melanogenesis (80.7 – 86.8% melanin content), even with no or low toxicity to the cells (93.5 – 101.6% cell viability) at a high concentration of 100 μm . Compounds 1 – 3 exhibited cytotoxic activity against one or more cell lines (IC₅₀ 13.9 – 68.4%), and compound 1 with high tumor selectivity for A549 (SI 3.2).     

7,8-secolignans from Schisandra wilsoniana and their anti-HIV-1 activities

Two new 7,8‐secolignans, marphenols A and B ( 1 and 2 , resp.), together with a known related derivative, 7,8‐secoholostylone B ( 3 ), were isolated from the stems of Schisandra wilsoniana. The structures of 1 and 2 were elucidated by spectroscopic methods, including extensive 1D‐ and 2D‐NMR techniques. The anti‐HIV‐1 activities of 1 – 3 were evaluated. Compound 1 inhibited HIV‐1_IIIB‐induced syncytia formation with an EC₅₀ value of 0.55 μg ml^?1. It reduced p24 antigen expression in acutely HIV‐1_IIIB‐infected C8166 cells and primary isolate HIV‐1_TC‐2‐infected peripheral blood mononuclear cells (PBMCs), with EC₅₀ values of 3.34 and 0.52 μg ml^?1, respectively. It showed no effects on the HIV‐1_IIIB replication in chronically infected H9 cells as well as fusion inhibition.     

Two New Limonoids from the Fruits of Melia azedarach (Meliaceae)

Two new limonoids, trichilinin M ( 1 ) and ohchinin benzoate ( 2 ), along with two known limonoids, 12-hydroxyamoorastatone ( 3 ) and mesendanin H ( 4 ), were isolated from the fruits of Melia azedarach Linn. The structures of new limonoids were determined by analyses of HR-ESI-MS, 1D and 2D NMR (HSQC, HMBC and NOESY) data. All compounds were evaluated against human pancreatic cancer PANC1 cells and the results showed that compounds 3 – 4 exhibited substantial cytotoxic activity ( 3 : IC₅₀=4.55 μM; 4 : IC₅₀=7.54 μM), and compounds 1 – 2 exhibited moderate cytotoxicity ( 1 : IC₅₀=27.06 μM; 2 : IC₅₀=21.17 μM).     

Cytotoxic and Nitric Oxide Production‐Inhibitory Activities of Limonoids and Other Compounds from the Leaves and Bark of Melia azedarach

Nine limonoids, 1 – 9 , one apocarotenoid, 11 , one alkaloid, 12 , and one steroid, 13 , from the leaf extract; and one triterpenoid, 10 , five steroids, 14 – 18 , and two flavonoids, 19 and 20 , from the bark extract of Melia azedarach L. (Chinaberry tree; Meliaceae) were isolated. Among these compounds, three compounds, 4 – 6 , were new, and their structures were established as 3‐deacetyl‐28‐oxosalannolactone, 3‐deacetyl‐28‐oxosalanninolide, and 3‐deacetyl‐17‐defurano‐17,28‐dioxosalannin, respectively, on the basis of extensive spectroscopic analyses and comparison with literature data. All of the isolated compounds were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines. 3‐Deacetyl‐4′‐demethyl‐28‐oxosalannin ( 3 ) against HL60 and AZ521 cells, and methyl kulonate ( 10 ) against HL60 cells exhibited potent cytotoxicities with IC₅₀ values in the range of 2.8–5.8 μM . In addition, upon evaluation of compounds 1 – 13 against production of nitric oxide (NO) in mouse macrophage RAW 264.7 cells induced by lipopolysaccharide (LPS), seven, i.e., trichilinin B ( 1 ), 4 , ohchinin ( 7 ), 23‐hydroxyohchininolide ( 8 ), 21‐hydroxyisoohchininolide ( 9 ), 10 , and methyl indole 3‐carboxylate ( 12 ), inhibited production of NO with IC₅₀ values in the range of 4.6–87.3 μM with no, or almost no, toxicity to the cells (IC₅₀ 93.2–100 μM ). Western blot analysis revealed that compound 7 reduced the expression levels of the inducible NO synthase (iNOS) and COX‐2 proteins in a concentration‐dependent manner. Furthermore, compounds 5, 6, 13 , and 18 – 20 exhibited potent inhibitory effects (IC₅₀ 299–381 molar ratio/32 pmol TPA) against Epstein? Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cell line.     

Bioactive Steroid Derivatives and Butyrolactone Derivatives from a Gorgonian‐Derived Aspergillus sp. Fungus

Six steroid derivatives, 1 – 6 , and five butyrolactone derivatives, 7 – 11 , were isolated from the fermentation broth of a gorgonian‐derived Aspergillus sp. fungus. Their structures were elucidated on the basis of NMR and MS spectral data. Compound 1 is a new, highly conjugated steroid. The NMR and MS data of 7 and 8 are reported for the first time, as their structures were listed in SciFinder Scholar with no associated reference. Compounds 1, 4, 5 , and 8 – 11 inhibited the larval settlement of barnacle Balanus amphitrite with EC₅₀ values ranging from 0.63 to 18.4 μg ml^?1. Butyrolactone derivatives 7 and 8 showed pronounced antibacterial activities against Staphylococcus aureus with the same MIC values as the positive control ciprofloxacin (MIC 1.56 μM for all three compounds).     

Variation in Essential Oil and Bioactive Compounds of Curcuma kwangsiensis Collected from Natural Habitats

The chemical compositions of essential oils (EOs) extracted from Curcuma kwangsiensis rhizomes collected from six natural habitats in P. R. China were evaluated using gas chromatography/mass spectrometry (GC/MS). Fifty‐seven components were identified from the six EOs, and their main constituents were 8,9‐dehydro‐9‐formyl‐cycloisolongifolene (2.37 – 42.59%), germacrone (6.53 – 22.20%), and l ‐camphor (0.19 – 6.12%). The six EOs exhibited different DPPH radical‐scavenging activities (IC₅₀, 2.24 – 31.03 μg/ml), with the activity of most of EOs being much higher than that of Trolox C (IC₅₀, 10.49 μg/ml) and BHT (IC₅₀, 54.13 μg/ml). Most EOs had potent antimicrobial effects against the tested bacteria and fungus. They also exhibited cytotoxicity against B16 (IC₅₀, 4.44 – 147.4 μg/ml) and LNCaP cells (IC₅₀, 73.94 – 429.25 μg/ml). The EOs showed excellent anti‐inflammatory action by significantly downregulating expression of pro‐inflammatory cytokines, cyclooxygenase‐2, and tumor necrosis factor‐α. This study provides insight into the interrelation among growth location, phytoconstituents, and bioactivities, and the results indicate the potential of C. kwangsiensis as natural nutrients, medicines, and others additives.     

Inhibitory effect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite amphitrite darwin

This study examined the inhibitory effect of 3 phenolic compounds and 12 strains of marine bacteria on the larval settlement of Balanus amphitrite amphitrite. The phenolic compounds used were phlorotannins, phloroglucinol and tannic acid. Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene) known only from brown algae. Tannic acid, which exists in terrestrial plants, is composed of oligomers of phloroglucinol attached to a sugar molecule. The bacterial strains used were isolated from a natural biofilm. The following were investigated: 1) the toxicity of the phenolic compounds to B. a. amphirite in three different larval stages, viz. nauplius II, nauplius V and cyprid; 2) the potency of the compounds as inhibitors of larval settlement and the possible mechanism involved in settlement inhibition; and 3) the effects of the bacteria on larval settlement. The level of toxicity of the phenolic compounds varied widely for the larvae. Phlorotannins were most toxic, having LC₅₀ values ranging from 9.47 to 40.35 μg ml^‐1; phloroglucinol was least toxic, having LC₅₀ values of 235.12 to 368.28 μg ml^‐1. In general, nauplii were more sensitive to the toxicity of the phenolic compounds than cyprids. The greater sensitivity of nauplii may be due to their active feeding behavior, which exposes the interior of their bodies to the compounds by active intake. Phloroglucinol was the most potent settlement inhibitor, having an EC₅₀ value of 0.02 μg ml^‐1. Phlorotannins and tannic acid had EC₅₀ values of 1.90 μg ml^‐1 and 14.05 μg ml^‐1, respectively. Phloroglucinol appeared to inhibit larval settlement through a relatively non‐toxic mechanism as its LC₅₀ value was four orders of magnitude higher than its EC₅₀ value. The high potency of phloroglucinol indicates that a simple constituent of a complex natural compound can be more effective than the natural compound itself. Larval settlement bioassays with monospecies bacterial films indicated that some of the bacterial species were inhibitory to larval settlement while the others showed no effect. None of the bacterial strains in this study induced larval settlement.     

Naphthalene Derivatives and Quinones from Ventilago denticulata and Their Nitric Oxide Radical Scavenging,Antioxidant, Cytotoxic,Antibacterial, and Phosphodiesterase Inhibitory Activities

New naphthalene derivatives ( 1 and 2 ) and a new isomer ( 3 ) of ventilagolin, together with known anthraquinones, chrysophanol ( 4 ), physcion or emodin 3‐methyl ether ( 5 ), and emodin ( 6 ), were isolated from vines of Ventilago denticulata. The isolated compounds exhibited cytotoxic activity with IC₅₀ values of 1.15 – 40.54 μg/ml. Compounds 1 – 3 selectively exhibited weak antibacterial activity (MIC values of 200.0 – 400.0 μg/ml), while emodin ( 6 ) displayed moderate antibacterial activity with MIC value of 25.0 μg/ml. The isolated compounds showed nitric oxide and DPPH radical scavenging activities. Compounds 1 – 3 and 6 exhibited weak xanthine oxidase inhibitory activity, while emodin ( 6 ) acted as an aromatase inhibitor with the IC₅₀ value of 10.1 μm . Compounds 1 and 2 exhibited phosphodiesterase 5 inhibitory activity with IC₅₀ values of 8.28 μm and 6.48 μm , respectively.     

Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil

Aims: To investigate the in vitro antiviral activity of Distictella elongata (Vahl) Urb. ethanol extracts from leaves (LEE), fruits (FEE), stems and their main components. Methods and Results: The antiviral activity was evaluated against human herpesvirus type 1 (HSV‐1), murine encephalomyocarditis virus (EMCV), vaccinia virus Western Reserve (VACV‐WR) and dengue virus 2 (DENV‐2) by the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay. LEE presented anti‐HSV‐1 [EC₅₀ 142·8 ± 5·3 μg ml^?1; selectivity index (SI) 2·0] and anti‐DENV‐2 activity (EC₅₀ 9·8 ± 1·3 μg ml^?1; SI 1·5). The pectolinarin ( 1 ) isolated from LEE was less active against HSV‐1 and DENV‐2. A mixture of the triterpenoids ursolic, pomolic and oleanolic acids was also obtained. Ursolic and oleanolic acids have shown antiviral activity against HSV‐1. A mixture of pectolinarin ( 1 ) and acacetin‐7‐O‐rutinoside ( 2 ) was isolated from FEE and has presented anti‐DENV‐2 activity (EC₅₀ 11·1 ± 1·6 μg ml^?1; SI > 45). Besides the antiviral activity, D. elongata has disclosed antioxidant effect. Conclusions: These data shows that D. elongata has antiviral activity mainly against HSV‐1 and DENV‐2, besides antioxidant activity. These effects might be principally attributed to flavonoids isolated. Significance and Impact of the Study: Distictella elongata might be considered a promising source of anti‐dengue fever phytochemicals.     

Synthesis,Characterization, and Anti‐Amoebic Activity of N‐(Pyrimidin‐2‐yl)benzenesulfonamide Derivatives

A new series of N‐(pyrimidin‐2‐yl)benzenesulfonamide derivatives, 3a – 3i and 4a – 4i , was synthesized from pyrimidin‐2‐amines, 2a – 2i , with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis, FT‐IR, ¹H‐ and ¹³C‐NMR, and ESI mass‐spectral data. In vitro anti‐amoebic activity was evaluated against HM1 : IMSS strain of Entamoeba histolytica. The IC₅₀ values were calculated by using the double dilution method. The results were compared with the IC₅₀ value of the standard drug ‘metronidazole’. The selected compounds were tested for their cytotoxic activities by cell‐viability assay using H9C2 cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80% viabilities to a concentration of 100 μg/ml.