The calcium channel blocking and phosphodiesterase inhibitory activities of the extract of Andropogon muricatus explains its medicinal use in airways disorders

The present work was carried out to provide a pharmacological base for the medicinal use of Andropogon muricatus in airways disorders, such as asthma. In isolated guinea‐pig tracheal strips, the crude extract of Andropogon muricatus exhibited a non‐specific relaxant effect against carbachol (1 µ m ) and high K⁺ precontractions, with EC₅₀ values of 0.10 (0.07–0.11) and 0.15 mg/mL (0.11–0.18), respectively, similar to papaverine, while verapamil was more potent against high K⁺. This suggests the involvement of a non‐specific relaxant effect, mediated possibly through Ca⁺⁺ channel blockade and phosphodiesterase inhibition. The functional nature of the relaxant effect was further confirmed through indirect evidence when pretreatment of the tissues with the plant extract caused potentiation of the isoprenaline inhibitory response curves, similar to papaverine, while the effect of verapamil remained unchanged. These data indicate that the crude extract of Andropogon muricatus contains constituent(s) that mediate the tracheal relaxant effect, possibly through dual inhibition of Ca⁺⁺ channels and phosphodiesterase and provide pharmacological evidence for its medicinal use in airways disorders, particularly asthma. Copyright © 2011 John Wiley & Sons, Ltd.     

Herbal medicine Catuama induces endothelium-dependent and -independent vasorelaxant action on isolated vessels from rats,guinea-pigs and rabbits

The present study was designed to examine the vasorelaxant action of the herbal medicine Catuama and the hydroalcoholic extracts (HE) of each plant present in this product and to compare their effects with that caused by acetylcholine (ACh) in intact (⁺E) or in endothelium-rubbed (⁻E) rings of rat thoracic aorta (RA), guinea-pig pulmonary artery (GPPA), guinea-pig mesenteric artery (GPMA), rabbit pulmonary artery (RPA), rabbit mesenteric artery (RMA) precontracted with noradrenaline (NA) or phenylephrine (PE). The extract of Catuama (1-3000 μg/mL) produced graded relaxation of RA, ⁺E or ⁻E, with mean EC₅₀ of 430 μg/mL and ≊ 3000 μg/mL and E_max of 81 % ± 15 % and 47% ± 4 %, respectively. The nitric oxide (NO) synthase inhibitor, N^ω-nitro-L -arginine (L -NOARG, 100 μM ), inhibited in vasorelaxant action (p < 0.05) in RA (⁺E), while indomethacin (3 μM ), propranolol (1 μM ), glibenclamide (1 μM ), methylene blue (10 μM ) and apamin (0.1 μM ) had no significant effect. ACh (1-1000 μM ) caused graded relaxation in RA with ⁺E, these effects being markedly antagonized by L -NOARG (100 μM ), methylene blue (10 μM ) and partially by apamin (0.1 μM ), but not by indomethacin (3 μM ), glibenclamide (1 μM ) or propranolol (1 μM ). The Catuama extract (1-3000 μg/mL) produces partial relaxations in rings of RMA (mean EC₅₀ of 1073 μg7/ml and E_max of 56 % ± 13 %), an effect which was antagonized by L -NOARG (100 μM ). In RPA (⁺E) the extract produces partial relaxation followed by contraction (E_max 28 % ± 6 %), an effect which was abolished by L -NOARG (100 μM ) or methylene blue (10 μM ). The extract caused graded relaxation in rings of GPPA and GPMA with mean EC₅₀ values of 60 μg/mL and 1148 μg/mL and E_max 96% ± 2% and 88% ± 12%, respectively. L -NOARG (100 μM ) blocked the Catuama extract vasorelaxation in GPPA and only partially in GPMA, but markedly antagonized the vasorelaxations caused by ACh in both GPPA andRMA. The HE Paullinea cupana, Zinziber officinalis and Trichilia catigua (1-3000 μg/mL) caused a graded vasorelaxant effect ⁺E of RA with a mean EC₅₀ of 22, 55 and 1793 μg/mL and E_max 100%, 86% ± 7% 70% ± 2%, respectively. In addition the HE of P. cupana also caused graded relaxation in ⁻E of RA with EC₅₀ and E_max of 233 μg/mL and 100%, respectively, while T. catigua and Z. officinalis produced partial relaxation in RA ⁺E. In contrast the HE of Ptychopetalum olacoides caused little contraction (46% ± 14%). These results demonstrate that the medicinal herb Catuama produces significant vasorelaxation responses in vessels from different animal species, and show that its effects are in great part dependent on the release of NO or NO-derived substances. Our results also demonstrate that the vasorelaxant action of the product Catuama seems to be due to the action of the active principles present mainly in P. cupana; T. catigua and, to a lesser extent, in Z. officinalis. Such results may contribute to the explanation of its beneficial effect of Catuama herbal medicine in the management of cardiovascular disturbances. © 1997 John Wiley & Sons, Ltd.     

Pharmacological studies of Striga senegalensis Benth (Scrophulariaceae) as an abortifacient

The methanol extract of Striga senegalensis Benth (Scrophulariaceae) was investigated on isolated rat uterus. Acetylcholine and the methanol extract of the plant produced dose related contractions of smooth muscle of the isolated rat uterus in vitro. Atropine in doses of 2 × 10⁻² to 32 × 10⁻² μg/mL antagonized dose dependently the contraction of the isolated rat uterus produced by both acetylcholine (1.6 × 10⁻¹ μg/mL) and the methanol extract (160 μg/mL). © 1998 John Wiley & Sons, Ltd.     

Vasodilator Effects of the Extract of the Leaves of Cistus populifolius on Rat Thoracic Aorta

The effects of different extracts of Cistus populifolius L. on smooth muscle were studied. The aqueous extract inhibited in a dose dependent manner the noradrenaline (NA) (10⁻⁹–1.03×10⁻⁶ MSC ) and CaCl₂ (2×10⁻⁴–1.28×10⁻² MSC )-induced contractions in rat thoracic aorta. Furthermore, this extract induced relaxant effects in rat aorta precontracted with NA (10⁻⁶ MSC ) and with K⁺ (80 mMSC ). Two different vasodilator responses were observed on NA-induced contractions: one an endothelium-dependent response abolished by methylene blue but not by indomethacin, and the other an endothelium-independent response unaffected by methylene blue and indomethacin similar to that obtained with a flavone, luteolin (10⁻⁵ MSC ). The search for active constituents included a bioactivity-directed fractionation of the lyophilized aqueous extract (LAE) using vascular reactivity experiments. Four different fractions (Cl₂CH₂, Me₂CO, MeOH, H₂O) were analysed at two different concentrations (1 and 2 mg (dry plant)/mL). The methanol fraction was the most active on NA precontracted vascular segments with endothelium, whereas the water fraction was the most active on segments without endothelium. These relaxant effects were less on K⁺ (80 mMSC )-induced contractions. Due to these results we suggest that this plant contains interesting hydrophilic compounds with strong pharmacological action. The endothelium-independent relaxant effect is probably related to flavonoid constituents. However we cannot relate the endothelium-dependent relaxation to known natural compounds in Cistus species. The presence of these constituents with relaxing properties on smooth muscle could account for the use of Cistus plant in traditional medicine.     

Antioxidant activity of Osbeckia aspera

Plants of the Osbeckia family have been shown to possess hepatoprotective properties, which could be due to the presence of antioxidant compounds. The plant extract was shown to inhibit significantly in a dose-dependent manner, the activities of the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical (EC₅₀ of 27.5 μg/mL), xanthine oxidase (EC₅₀ of 1.16 mg/mL) and demonstrate a scavenging effect on hydroxyl radical mediated damage to deoxyribose (EC₅₀ of 140 μg/mL). The plant extract possessed some prooxidant activity from the effect on bleomycin-induced DNA damage, but this was less than that shown by comparable concentrations of (+)-catechin or silymarin. © 1998 John Wiley & Sons, Ltd.     

Antiviral activity of chilean medicinal plant extracts

Chilean flora is a potential source of bioactive compounds, including some with antiviral activity. Ninety aqueous and hydroaloholic extracts from 36 native and introduced plant species were screened for antiviral activity on herpes (HSV-1 and HSV-2) and HIV viruses. Furthermore, the samples were assayed for antimicrobial effect on pathogenic bacteria and a yeast. Plants were selected according to their indication of use for treating symptomatology of possible viral aetiology in Chilean folk medicine. The hydroaloholic extracts of Cassia stipulacea and Escallonia illintia exhibited detectable antiviral effects towards HSV-1 with IC₅₀ values of 80 and 40 μg crude extract/mL, respectively. Samples belonging to Aristotelia chilensis (IC₅₀ of 40 μg/mL), Drymis winteri (IC₅₀ values of 35 and 80μg/mL), Elytropus chilensis and Luma apiculata, with an IC₅₀ value of 100 μg/mL showed activity against HSV-2. None of the extracts showed activity against HIV at extract concentrations which were nontoxic for cells.     

Evaluation of pharmacological activity of a crude hydroalcoholic extract from Jatropha elliptica

The pharmacological effect of the hydroalcoholic extract of Jatropha elliptica was analysed in in vivo and in vitro models. When given orally in mice, the extract showed a low acute toxicity (LD₅₀ 5 g/kg). In a dose of 0.5 or 1 g/kg p.o. the extract did not interfere with diuresis in the rat, but was found to be effective in blocking rat paw oedema induced by carrageenan and partially, serotonin-induced oedema. In the same dose, the extract failed to inhibit rat paw oedema induced by dextran and the increase of rat cutaneous vascular permeability caused by Bothrops Jararaca venom, dextran, histamine, PAF-acether and serotonin. Pre-incubation of the isolated rat uterus and guinea-pig ileum with the extract (0.2–0.8 mg/mL), produced a concentration-related and non-competitive inhibition of contractions induced by acetylcholine and bradykinin. However, the extract was about 2-fold more potent in inhibiting the contraction of both agonists in guinea-pig ileum than in rat uterine muscle. In rat aorta, the extract (50–100 μg/mL) caused a concentration-dependent inhibition of noradrenaline-evoked contractions, being about 5-fold more potent when compared to the IC₅₀ values obtained in rat uterus.     

Antioxidant activity of yellow dock (Rumex crispus L., Polygonaceae) fruit extract

The methanol extract of ripe Rumex crispus L. fruits was evaluated for its antioxidant potential by assays for ferric‐reducing antioxidant power (FRAP), DPPH‐free radical scavenging activity (DPPH) and the influence on lipid peroxidation in liposomes (LP). Considerable activity was observed in all test systems (FRAP: 9.9 mmol Fe²⁺/g; DPPH IC₅₀: 3.7 μg/mL; LP IC₅₀: 4.9 μg/mL), comparable to that of BHT (FRAP: 8.0 μg/mL; DPPH IC₅₀: 19.4 μg/mL; LP IC₅₀: 3.5 μg/mL), but lower than the activity of ascorbic acid, rutin and quercetin, used as positive control substances. The in vivo effects were evaluated in several hepatic antioxidant systems (activities of LPx, GSH‐Px, Px, CAT and XOD, as well as GSH content), after treatment with the studied yellow dock extract in different doses, or in combination with carbon tetrachloride (CCl₄). Pretreatment with the R. crispus extract inhibited CCl₄‐induced oxidative stress by decreasing LPx and increasing GSH content in a dose dependent manner, bringing the levels of antioxidant enzymes to near control values. Copyright © 2010 John Wiley & Sons, Ltd.     

Screening of Indonesian plant extracts for anti-human immunodeficiency virus—type 1 (HIV-1) activity

Of 30 Indonesian plant extracts tested for their human immunodeficiency virus type-1 (HIV-1) inhibitory activities, six were shown to be effective by assays using HIV-1-infected MT-4 cells: a methanol extract of mahoni (bark of Swietenia mahagoni) and water extracts of benalu teh (stems and branches of Loranthus parasiticus), kiules (fruit of Helicteres isora), supratul (fruits of Sindora sumatrana), sambiloto (leaves of Andrographis paniculata) and temu ireng (rhizoma of Curcuma aeruginosa). Their ED₅₀ values ranged from 4.2 to 175 μg/mL. The samples also suppressed the formation of syncytia in co-cultures of MOLT-4 and MOLT-4/HIV-1 cells. The most potent inhibitor was a methanol extract of mahoni, which also showed a significant inhibition of HIV-1 protease.     

Tracheal relaxation of five Ivorian anti-asthmatic plants: role of epithelium and K⁺ channels in the effect of the aqueous-alcoholic extract of Dichrostachys cinerea root bark

Ethnopharmacological relevance

Leaves of Boerhavia diffusa (Nyctaginaceae), Baphia nitida, Cassia occidentalis, Desmodium adscendens (Fabaceae), and root bark of Dichrostachys cinerea (Fabaceae) are used in Ivory Coast for the treatment of asthma. The aim of this study was to evaluate the potential airway relaxant activity of different extracts of these plants.

Materials and methods

Extracts of different polarities (H₂O, EtOH/H₂O, MeOH and CH₂Cl₂) were obtained from these five plants. Their ex vivo relaxant activity was tested in mice isolated trachea precontracted with carbachol (1 μM).

Results

Cumulative concentrations of most extracts induced moderate to strong relaxation, the methanolic extracts being the most potent and the polar extracts the most active at the concentrations used, supporting the traditional use of these five plants as anti-asthmatic remedies. We further investigated the molecular and cellular mechanisms of the mouse trachea relaxant effect of the aqueous-alcoholic extract of Dichrostachys cinerea root bark, the most potent extract. Its effect was not modified in the presence of β-adrenoceptor antagonists (propranolol or ICI 118,551) or a PKA inhibitor (H89). By contrast, it was decreased after depolarization-induced precontraction (with 80 mM KCl), in the presence of some K⁺ channels blockers [4-aminopyridine as voltage-dependent K⁺ (K_v) channel blocker and tetraethylammonium chloride as large conductance Ca²⁺-activated K⁺ (BK_Ca) channel blocker, but not with glibenclamide, an ATP-sensitive K⁺ (K_ATP) channel blocker] or after epithelium removal.

Conclusions

The mouse tracheal relaxant effect of Dichrostachys cinerea EtOH/H₂O extract was independent of β₂-adrenoceptors activation and cAMP/PKA pathway, but dependent on epithelium and K⁺ channels, namely K_v and BK_Ca channels. Further investigation will be required to identify the component(s) responsible for this airways relaxant activity.     

Catechin‐based Procyanidins from Peumus boldus Mol. Aqueous Extract Inhibit Helicobacter pylori Urease and Adherence to Adenocarcinoma Gastric Cells

In this work, the anti‐Helicobacter pylori effect of an aqueous extract from dried leaves of Peumus boldus Mol. (Monimiaceae) was evaluated. This extract displayed high inhibitory activity against H. pylori urease. Therefore, in order to clarify the type of substances responsible for such effect, a bioassay‐guided fractionation strategy was carried out. The active compounds in the fractions were characterized through different chromatographic methods (RP‐HPLC; HILIC‐HPLC). The fraction named F5 (mDP = 7.8) from aqueous extract was the most active against H. pylori urease with an IC₅₀ = 15.9 µg gallic acid equivalents (GAE)/mL. HPLC analysis evidenced that F5 was composed mainly by catechin‐derived proanthocyanidins (LC‐MS and phloroglucinolysis). The anti‐adherent effect of boldo was assessed by co‐culture of H. pylori and AGS cells. Both the aqueous extract and F5 showed an anti‐adherent effect in a concentration‐dependent manner. An 89.3% of inhibition was reached at 2.0 mg GAE/mL of boldo extract. In conjunction, our results suggest that boldo extract has a potent anti‐urease activity and anti‐adherent effect against H. pylori, properties directly linked with the presence of catechin‐derived proanthocyanidins. Copyright © 2014 John Wiley & Sons, Ltd.     

Vasoconstrictor and cardiotonic actions of Haloxylon recurvum extract

Intravenous administration of an ethanolic extract of Haloxylon recurvum (30–300 μg/kg) caused a rise in arterial blood pressure in normotensive anaesthetized rats. Pretreatment of animals with phentolamine (1 mg/kg) abolished the vasoconstrictor response of the plant extract in a way similar to that of norepinephrine (NE). In an isolated rabbit aorta preparation, both plant extract and NE produced contractile responses which were blocked by phentolamine (0.3 μg/mL). In isolated guinea-pig atria, the plant extract caused positive inotropic and chronotropic responses similar to that of NE and was blocked by propranolol (0.3 μg/mL). These results suggest that vasoconstrictor and cardiac stimulatory effects of the plant extract are mediated through mechanisms similar to those of NE (stimulation of α-adrenergic receptors in the blood vessels and cardiac β-receptors respectively) and hence this plant contains sympathomimetic constituent(s).     

Medicinal and edible lignicolous fungi as natural sources of antioxidative and antibacterial agents

The antioxidant activity of organic extracts of eight fungal species, Ganoderma lucidum, Ganoderma applanatum, Meripilus giganteus, Laetiporus sulphureus, Flammulina velutipes, Coriolus versicolor, Pleurotus ostreatus and Panus tigrinus, was evaluated for free radical (DPPH^· and OH^·) scavenging capacity and an effect on lipid peroxidation, and the antibacterial activity was tested by the agar well diffusion method. The highest DPPH^· scavenging activity was found in the methanol extract of G. applanatum (12.5 μg/mL, 82.80%) and the chloroform extract of G. lucidum (510.2 μg/mL, 69.12%). The same extracts also showed the highest LP inhibition (91.83%, 85.09%) at 500 μg/mL, while the methanol extracts of G. applanatum and L. sulphureus showed the highest scavenging effect on OH^· radicals (68.47%, 57.06%, respectively) at 400 μg/mL. A strong antibacterial activity against Gram‐positive bacteria was also manifested. The antioxidative potencies correlated generally with the total phenol content (0.19–9.98 mg/g). The HPLC determination showed that the majority of analysed species contained gallic and protocatechic acids. Consequently, these fungi are shown to be potential sources of antioxidative and antibacterial agents. Copyright © 2010 John Wiley & Sons, Ltd.     

Preliminary studies on the hydroalcoholic extract of the root of Cissampelos sympodialis Eichl in guinea-pig tracheal strips and bronchoalveolar leucocytes

The aqueous fraction of the ethanol extract of the root of Cissampelos sympodialis, reduced the spontaneous tone, and inhibited the contractions induced by submaximal concentrations of carbachol, histamine, prostaglandin F_2α and substance P, in guinea-pig tracheal preparations. The fraction was approximately equipotent to aminophyl-line and the IC₅₀ values of the above agents varied from 13.9 to 95.5 μg/mL, depending on the agonist used. In concentrations ranging from 1–100 μg/mL, the fraction also increased the intracellular levels of cyclic adenosine monophosphate in guinea-pig bronchoalveolar leucocytes. It is proposed that the combined effects on smooth muscles and on leucocytes indicate a pharmacological basis for the use of the plant in asthma.     

In vitro pharmacological evaluation of the dichloromethanol extract from Schinus molle l.

The pharmacological activity of the dichloromethanol extract of Schinus molle L. (SM-DCM) was analysed in in vitro models. Preincubation of the isolated guinea-pig ileum or rat uterus preparations with the extract (100 μg/mL) abolished the contractile effects of histamine and serotonin respectively. At the same dose, the extract partially reduced the contractile effects of acetylcholine on the isolated rat duodenum. A 10 μg/mL dose showed an inhibitory effect on histamine and serotonin, but not on acetylcholine-induced contractions (NS). No significant effect was found with a 1 μg/mL dose. © 1998 John Wiley & Sons, Ltd.     

Evaluation of Bioactive Potential of an Aloe vera Sterol Extract

We prepared a crude gel material from Aloe vera succulent leaf tissues. The ethanolic extract of lyophilized A. vera gel was used for the GC‐MS analysis. Hexadecanoic acid (22.22%) was identified as major compound. Sitosterol and stigmasterol were found to be 2.89% and 2.1% in the extract. HPLC analysis was carried out to confirm the presence of stigmasterol. The concentration of sterol extract needed to scavenge DPPH free radical by 50% was calculated as 5.2 mg mL^?1. In the FRAP assay, the sterol extract showed significant hydroxyl radical scavenging in a dose‐dependent manner (IC₅₀ value 1.17 µg mL^?1). Concentration of the sample required to reduce lipid peroxidation was found to be 4.18 µg mL^?1, and the extract also possessed acetylcholinesterase activity (IC₅₀ ‐ 5.26 µg mL^?1). Catalase activity was 0.196 μM H₂O₂ decomposed min^?1 µg^?1 protein, whereas the peroxidase activity was 17.01 μM of pyragallol oxidized min^?1 µg^?1 protein. The extract recorded higher activity against growth of S. greseus and C. albicans in the experiments carried out to determine antibacterial and antifungal activity, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

The Immunosuppressive Effects of Globularia alypum Extracts

Responder lymphocytes were cultured with an equal number of irradiated stimulator lymphocytes from another donor for 6 days in RPMI 1640 (a modified Mc Coh's 5 A Medium). After 48 h incubation with PMA (phorbol 12-myristate), PHA (phytohaematogglutin) and 120 h with MLR (mixed lymphocytes reaction), the cells were labelled with ³H-thymidine. Cells were harvested in a scintillation counter Globularia alypum L. extracts were dissolved in HPLC quality ethanol or water and diluted in RPMI to concentrations ranging from 0.156–10 μg/mL. Extract solutions were added immediately after cell stimulation in the cell walls. Cyclosporin A was used as a control. Both the water and ethanol extracts of Globularia alypum L. demonstrated a dose response effect in all three systems of MLR, PHA and PMA with no statistical difference in water or ethanol extracts. The IC₅₀ were: PHA 1.14 μg/mL, PMA 1.05 μg/mL and MLR 2.06 μg/mL. The PHA and MLR IC₅₀ are approximately double the IC₅₀ for cyclosporin A (0.5 μg/mL). The effects of Glubularia alypum L. in PMA stimulated lymphocytes suggests it may suppress T cell function through a pathway that cyclosporin A does not effect.     

In vitro inhibition of mycobacteria by Rwandese medicinal plants

A series of 15 Rwandese medicinal plants used by traditional healers to treat pulmonary diseases were screened for anti-Mycobacterium tuberculosis activity. Three plant extracts showed activity at 1000 μg/mL: Bidens pilosa (leaves), Pentas longiflora (roots) and Tetradenia riparia (leaves). These plant extracts were studied further against several mycobacterial species: M. tuberculosis, M. avium complex, M. simiae and a new simiae like Mycobacterium species (SLM). None of the plant extracts showed activity against M. avium and SLM at 1000 μg/mL. M. simiae was sensitive to T. riparia and P. longiflora extracts (1000 μg/mL) and M. tuberculosis towards T. riparia (500 μg/mL) and B. pilosa (100 μg/mL). The active principle of T. riparia, 8(14), 15-sandaracopimaradiene-7α, 18-diol, was also evaluated for its antimycobacterial activity and the concentration required for inhibiting M. tuberculosis ranged from 25 μg/mL to 100 μg/mL. The extract of the leaves of T. riparia can be used as a differentiation test (at 1000 μg/mL in the medium) for M. simiae and SLM.     

Effects of crinum glaucum aqueous extract on intestinal smooth muscle activity

Crinum glaucum aqueous extract (1–8 mg/mL) produced a concentration dependent, non-competitive inhibition of contractions induced by acetylcholine (1.1 × 10⁻⁸–3.3 × 10⁻⁷M ) and calcium chloride (CaCl₂, 0.05–2 mM ) on the rat duodenum. Contractions of the guinea-pig ileum induced by acetylcholine (1.1 × 10⁻⁸–3.3 × 10⁻⁷M ) and histamine (1.1 × 10⁻⁸–3.3 × 10⁻⁷M ) were inhibited by the extract (1–4 mg/mL). The extract (0.125–2.0 mg/mL) also, produced a concentration dependent relaxation of the guinea-pig taenia coli, precontracted with potassium chloride (40 mM ). It is concluded that the extract is a non-specific relaxant of the gastrointestinal smooth muscles used. © 1998 John Wiley & Sons, Ltd.     

Antifilarial activity of Argyria speciosa against Setaria cervi in vitro

The effect of aqueous and alcoholic extracts of Argyria speciosa on the spontaneous movements of both the whole worm and a nerve/muscle preparation of Setaria cervi, and on the survival of microfilariae in vitro, was studied. Both extracts caused the inhibition of spontaneous movements of the whole worm and the nerve/muscle preparation of S. cervi, characterized by decrease in tone, amplitude and rate of contractions. The concentration required to inhibit the movements of the whole worm preparation was 150 μg/mL for the aqueous, and 75 μg/mL for the alcoholic extract. The concentration of A. speciosa extract required to produce an equivalent effect on the nerve/muscle preparation was 25 μg/mL for aqueous, and 50 ng/mL for the alcoholic extract.