Bioassay-guided Fractionation Procedures Research Articles

Effects of Naphthoquinones from the Roots of Onosma Armeniacum Klokov on Wound Healing.

In Turkish folk medicine, the roots of Onosma armeniacum Klokov are used to heal wounds, burns, hemorrhoids, hoarseness, dyspnea, stomach ulcers, and abdominal aches. The objective was to evaluate the plant's ethnopharmacological applications using in vivo pharmacological experimental models. In vivo linear incision and circular excision the wound models were used to assess the wound healing activity along with histopathological investigation. The active component(s) were isolated and identified after being exposed to several chromatographic separation procedures on the primary extract. The n-hexane-dichloromethane mixture extract was subjected to chromatographic separation after the wound-healing activity was confirmed. Deoxyshikonin (1), β,β-dimethylacrylshikonin (2), α-methyl-n-butylshikonin (3), isovalerylshikonin (4), acetylshikonin (5), β-hydroxyisovalerylshikonin (6), and 5,8-O-dimethylacetylshikonin (7) were identified as the structures of the isolated compounds. The efficacy of O. armeniacum to heal wounds was investigated in this study. Shikonin derivatives were identified as the primary active components of the roots by bioassay-guided fractionation and isolation procedures.

Ethyl gallate isolated from phenol-enriched fraction of Caesalpinia mimosoides Lam. Promotes cutaneous wound healing: a scientific validation through bioassay-guided fractionation.

The tender shoots of Caesalpinia mimosoides Lam. are used ethnomedically by the traditional healers of Uttara Kannada district, Karnataka (India) for the treatment of wounds. The current study was aimed at exploring phenol-enriched fraction (PEF) of crude ethanol extract of tender shoots to isolate and characterize the most active bio-constituent through bioassay-guided fractionation procedure. The successive fractionation and sub-fractionation of PEF, followed by in vitro scratch wound, antimicrobial, and antioxidant activities, yielded a highly active natural antioxidant compound ethyl gallate (EG). In vitro wound healing potentiality of EG was evidenced by a significantly higher percentage of cell migration in L929 fibroblast cells (97.98 ± 0.46% at 3.81μg/ml concentration) compared to a positive control group (98.44 ± 0.36%) at the 48th hour of incubation. A significantly higher rate of wound contraction (98.72 ± 0.41%), an elevated tensile strength of the incised wound (1,154.60 ± 1.42g/mm2), and increased quantity of connective tissue elements were observed in the granulation tissues of the 1% EG ointment treated animal group on the 15th post-wounding day. The accelerated wound healing activity of 1% EG was also exhibited by histopathological examinations through Hematoxylin and Eosin, Masson's trichome, and Toluidine blue-stained sections. Significant up-regulation of enzymatic and non-enzymatic antioxidant contents (reduced glutathione, superoxide dismutase, and catalase) and down-regulation of oxidative stress marker (lipid peroxidation) clearly indicates the effective granular antioxidant activity of 1% EG in preventing oxidative damage to the skin tissues. Further, in vitro antimicrobial and antioxidant activities of EG supports the positive correlation with its enhanced wound-healing activity. Moreover, molecular docking and dynamics for 100ns revealed the stable binding of EG with cyclooxygenase-2 (-6.2kcal/mol) and matrix metalloproteinase-9 (-4.6kcal/mol) and unstable binding with tumor necrosis factor-α (-7.2kcal/mol), suggesting the potential applicability of EG in inflammation and wound treatment.

Chromatography-Spectroscopic Isolated MO11 (Moringa oleifera) and MS06 (Musa sapientum) Positively Immunomodulated ACE2 Levels in Blood, Kidney and Liver of Rats

Moringa oleifera (MO) and Musa sapientum (MS) are ethno-medicinal plants, while cadmium is a carcinogen. SARS-CoV-2 binds to ACE2 for host’s cell invasion and infection. This study evaluated the effects of MO11 (isolated from MO leaves) and MS06 (isolated from MS suckers) on ACE2 levels in cadmium chloride (CdCl)-induced toxicity in rats. Twentyfour adult male Wistar rats were randomly divided into six groups (n = 4). Group 1 was control. Groups 2–4 received single 1.5 mg/kg bodyweight of CdCl (i.p.) (Day 1). Groups 3 and 4 were post-treated with MO11 and MO11 + MS06 doses, respectively (Days 1–17). Groups 5 and 6 received only MO11 and olive oil (vehicle), respectively (Days 1–17). MO leaves and MS suckers were subjected to bioassay-guided fractionation and isolation procedures using chromatography and spectroscopic techniques. ACE2 levels (ELISA) were evaluated in blood, kidney and liver of rats. MO11 and MS06 were the most active antioxidant and antimicrobial compounds isolated from MO leaves and MS suckers, respectively. Liquid chromatography-mass spectrometry showed presence of therapeutic compounds and amino-acids in MO11 and MS06, which are part of 14 shared amino-acids used by SARS-CoV and SARS-CoV-2 for ACE2-binding. Post-treatments of CdCl-exposure with MO11 and MS06 showed decreased ACE2 levels in Group 4 (20.63 ± 4.33 ng/mL and 16.11 ± 0.49 ng/mL in blood and kidney samples, respectively), compared with Group 2 (39.39 ± 3.15 ng/mL and 85.39 ± 3.10 ng/mL in blood and kidney samples, respectively). In conclusion, MO11 and MS06 possess significant ethno-medicinal potentials, may compete with SARS-CoV-2 for ACE2 binding, and are recommended for evaluations as anti-SARS-CoV-2 agents.

Isolation of Bioactive Compounds from Fruit Body of Lentinula edodes (Berk.) Pegler and In Silico Approach using Tyrosinase Target Protein Involved in Melanin Production

Traditionally, Lentinula edodes are used in many Asian countries for the treatment of various chronic diseases. In the current study, ethyl acetate fraction of fruit body of Lentinula edodes was obtained by standard bioassay-guided fractionation procedure. This fractionation resulted in the isolation of three bioactive compounds (compound 1 already reported, compound 2 and 3 first time reported from this mushroom) and their structures were characterized using various spectroscopic techniques. Further, all the compounds were studied using molecular docking analysis. Compounds 1, 2, 3 and standard active ingredient used for the treatment of hyperpigmentation i.e. arbutin was prepared as program database files, was docked with the target receptor (tyrosinase, program database ID: 5M8L) which plays a vital role in melanogenesis pathway. The study mainly focuses on better results of compounds as potent tyrosinase inhibitors to down-regulate the melanogenesis pathway. The present study revealed that due to the presence of compound 1 and 3 in the fruit body of Lentinula edodes, it may be preferred as a cosmetic product in extract form to obtain effective skin lightening properties or treatment of melisma. Compounds 2 and 3 was first isolated from Lentinula edodes. The study further concludes that compound 3 could act as a potential lead molecule for the target gene tyrosinase with slight modification or optimization of chemical structure to obtain more effective, less toxic lead molecule and further exhibits remarkable skin lightening properties via inhibiting melanin production.

The investigation of the potential antidepressant-like activity of Xanthium orientale subsp. italicum (Moretti) Greuter in rodents

Ethnopharmacological relevanceEthnobotanical surveys revealed that Xanthiumorientale subsp. italicum (Moretti) Greuter has been used against central nervous system disorders in Turkish folk medicine. The aim of the present study is to verify the folkloric assertion on this plant. The compounds responsible for the activity were investigated using bioassay-guided fractionation procedures. Materials and methodsThe antidepressant activity of the aqueous, n-hexane, ethyl acetate (EtOAc), methanol (MeOH) extracts; fractions and isolated compounds from active MeOH extract were evaluated by using the in vitro MAO inhibition assay and three different in vivo models namely forced swimming test, tail suspension test, and antagonism of tetrabenazine-induced ptosis, hypothermia, and suppression of locomotor activity. The results were compared with control and reference groups, and active compounds of the plant have been determined. Through the bioassay-guided fractionation procedures, two compounds were isolated from the active fraction and their structures were elucidated by spectroscopic methods. ResultsThe MeOH extract of the plant was found to possess antidepressant-like activity. This extract was then subjected to chromatographic techniques. Isolated sesquiterpene lactones were elucidated as xanthatin (1) and xanthinosin (2), which were responsible for the antidepressant-like activity. ConclusionsThis study discovered the antidepressant potential of X. orientale subsp. italicum. Using bioassay-guided fractionation and isolation techniques, xanthatin (1) and xanthinosin (2) were determined as the main active components of the leaves.

Bioassay-guided fractionation and identification of wound healing active compound from Pistacia vera L. hull extract.

Pistachio hull has traditionally been used to treat peptic ulcer, hemorrhoids, oral and cutaneous wounds. On the basis of its traditional uses and previous pharmacological reports, a bioassay guided fractionation procedures on pistachio (Pistacia vera L.) hulls was performed to define the fractions and bioactive compound that are responsible for wound healing activity of hulls. A bioassay-guided fractionation of the total extract (MeOH 80%) of Pistacia vera L. hulls was carried out to evaluate wound healing activity by scratch assay on NIH/3T3 murine fibroblast cells. A combination of solvent-solvent partitioning, column chromatography, preparative thin layer chromatography and crystallization were used to obtain fractions/sub-fractions and pure compound. The wound healing potential of isolated compound was examined by fibroblasts migration and proliferation using scratch assay and CFSC dilution assay, respectively. In addition, we evaluated the gene expression of some inflammatory markers which are involved in healing process using Real Time PCR. Chemical structure of active compound was elucidated by spectrometric methods. Due to the higher wound healing activity of CHCl3 fraction from P. vera hulls, it was fractionated by successive chromatographic techniques to yield the active compound. 3-Epimasticadienolic acid was isolated and crystallized as a white powder. This active compound (200 μg/ml) significantly increased the fibroblast proliferation and migration, resulting in reduction of the scratch area about 45%. It showed a strong inhibitory effect on gene expression of IL-6 and TNF-α, and a stimulation effect on NF-κB gene expression at the same dose. The present study supported the traditional uses of P. vera hulls for wound-healing and 3-epimasticadienolic acid showed significantly potent on wound repair.

Psiadia punctulata major flavonoids alleviate exaggerated vasoconstriction produced by advanced glycation end products.

Exaggerated vasoconstriction plays important roles in vascular complication in aging and many diseases like diabetes. Here, we investigated the protective effect of Psiadia punctulata (PP) on advanced glycation end products (AGEs)-induced aggravated vasoconstriction. The effect of total methanol extract of PP leaves (PPT) on AGE-induced vascular injury was studied through bioassay-guided fractionation procedures in order to find the bioactive fraction and isolate the bioactive compounds. Vascular reactivity was studied using the isolated artery technique by adding cumulative concentrations of phenylephrine (PE) or acetyl choline (ACh). In addition, the antiglycating effect, as well as the effect on AGEs intermediates dityrosine and N`-formylkynurenine and their radical scavenging activity were measured. The results showed that PPT alleviated the AGEs-induced aggravated vasoconstriction in a concentration-dependent manner. The bioassay guided fractionation procedures suggested the chloroform fraction (Fr I) to be responsible for the activity. Chemical investigation of this fraction resulted in isolation of four major bioactive compounds that were identified as: umuhengerin (1), gardenin (2), luteolin-3`,4`-dimethyl ether (3), and 5,3`-dihydroxy-6,7,4`,5`-tetramethoxyflavone (4). The four compounds alleviated the exaggerated vasoconstriction in a dose dependent manner. In search for their mechanism of action, we observed that PPT, Fr. I and the isolated compounds did not improve the impaired vasodilation associated with AGEs exposure. PPT, Fr. I and the isolated compounds 1–4 inhibited AGEs formation and their protein oxidation intermediates. Furthermore, PPT, Fr. I and the isolated compounds 1–4 showed weak radical scavenging activity with compound 4 as the most potent. In conclusion, PPT appears to protect against AGEs-induced exaggerated vasoconstriction through antiglycation and radical scavenging activities.

Cytochalasans Act as Inhibitors of Biofilm Formation of Staphylococcus Aureus.

During the course of our ongoing work to discover new inhibitors of biofilm formation of Staphylococcus aureus from fungal sources, we observed biofilm inhibition by cytochalasans isolated from cultures of the ascomycete Hypoxylon fragiforme for the first time. Two new compounds were purified by a bioassay-guided fractionation procedure; their structures were elucidated subsequently by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). This unexpected finding prompted us to test further cytochalasans from other fungi and from commercial sources for comparison. Out of 21 cytochalasans, 13 showed significant inhibition of Staphylococcus aureus biofilm formation at subtoxic levels. These findings indicate the potential of cytochalasans as biofilm inhibitors for the first time, also because the minimum inhibitory concentrations (MIC) are independent of the anti-biofilm activities. However, cytochalasans are known to be inhibitors of actin, making some of them very toxic for eukaryotic cells. Since the chemical structures of the tested compounds were rather diverse, the inclusion of additional derivatives, as well as the evaluation of their selectivity against mammalian cells vs. the bacterium, will be necessary as next step in order to develop structure-activity relationships and identify the optimal candidates for development of an anti-biofilm agent.

Bioactivity-Based Molecular Networking for the Discovery of Drug Leads in Natural Product Bioassay-Guided Fractionation.

It is a common problem in natural product therapeutic lead discovery programs that despite good bioassay results in the initial extract, the active compound(s) may not be isolated during subsequent bioassay-guided purification. Herein, we present the concept of bioactive molecular networking to find candidate active molecules directly from fractionated bioactive extracts. By employing tandem mass spectrometry, it is possible to accelerate the dereplication of molecules using molecular networking prior to subsequent isolation of the compounds, and it is also possible to expose potentially bioactive molecules using bioactivity score prediction. Indeed, bioactivity score prediction can be calculated with the relative abundance of a molecule in fractions and the bioactivity level of each fraction. For that reason, we have developed a bioinformatic workflow able to map bioactivity score in molecular networks and applied it for discovery of antiviral compounds from a previously investigated extract of Euphorbia dendroides where the bioactive candidate molecules were not discovered following a classical bioassay-guided fractionation procedure. It can be expected that this approach will be implemented as a systematic strategy, not only in current and future bioactive lead discovery from natural extract collections but also for the reinvestigation of the untapped reservoir of bioactive analogues in previous bioassay-guided fractionation efforts.

Highly Oxidized Guaianolide Sesquiterpenoids with Potential Anti-inflammatory Activity from Chrysanthemum indicum

Ten new highly oxidized monomeric (1-8) and dimeric guaianolides (9 and 10), along with two known guaianolide derivatives (11 and 12), were isolated from the aerial parts of Chrysanthemum indicum using a bioassay-guided fractionation procedure. The new compounds were characterized by the basic analysis of the spectroscopic data obtained, and the absolute configurations were determined by both empirical approaches and ECD calculations. Inhibitory effects of 1-12 on nitric oxide production were investigated in lipopolysaccaride (LPS)-mediated RAW 264.7 cells, and most of them (1-8 and 11) displayed IC50 values in the range 1.4-9.7 μM. Moreover, a mechanistic study revealed that the potential anti-inflammatory activity of compound 1 appears to be mediated via suppression of an LPS-induced NF-κB pathway and down-regulation of MAPK activation.

Anti-hyperglycemic and antioxidant potential of Croton bonplandianus. Bail fractions in correlation with polyphenol content.

Objective(s):Diabetes mellitus, a carbohydrate metabolic disorder, occurs due to absolute or relative deficiency of insulin. Current treatment strategies involve either preventing or delaying the intestinal absorption of glucose to lower the levels of postprandial hyperglycemia (PPHG). Herbal remedies have been since ancient times for treating diabetes mellitus. Therefore, identifying novel phytocompounds with α-amylase and α-glucosidase inhibitory activity that would reduce the glucose absorption as well as the rise in postprandial blood glucose level is vital. Consequently, the present study was aimed to investigate the anti-hyperglycemic activity of Croton bonplandianus against these pancreatic enzymes.Materials and Methods:The methanol extract of C. bonplandianus leaf was prepared and further fractionation was performed with n-hexane, ethyl acetate and chloroform. The antioxidant activity and anti-hyperglycemic activity of the extracts and its fractions were determined. Further, GC-MS analysis was performed for the leaf extract.Results:The chloroform fraction (ChF) was found to contain highest quantity of polyphenols (114.28 µg/ml of GAE), flavonoids (95.68 µg/ml of quercetin) and tannins (63.80 µg/ml of GAE) and also possessed effective inhibitory activity against α amylase (IC5095.78 µg/ml) and α glucosidase (IC50 126.81µg/ml). The antioxidant activity of ChFwas also higher when compared to other fractions. Further, GC-MS analysis of ChF showed the presence of various components that may be responsible for the above mentioned activities.Conclusion:The study findings suggest that the components present in the leaves of C. bonplandianus, may provide a potential therapeutic source in developing treatment forhyperglycemia. Further bioassay guided fractionation procedure is required to identify the active constituents.

In vitro anti-herpes simplex virus-2 activity of Salvia desoleana Atzei & V. Picci essential oil.

Salvia desoleana Atzei & V. Picci is an indigenous species in Sardinia island used in folk medicine to treat menstrual, digestive and central nervous system diseases. Nowadays, it is widely cultivated for the pleasant smell of its essential oil (EO), whose antimicrobial and antifungal activities have already been screened. This study evaluated the in vitro anti-Herpes Simplex Virus-2 (HSV-2) activity of S. desoleana EO, fractions and main components: linalyl acetate, alpha terpinyl acetate, and germacrene D. Phytochemical composition of S. desoleana EO was studied by GC-FID/MS analysis and the active fraction(s) and/or compounds in S. desoleana EO were identified with a bioassay-guided fractionation procedure through in vitro assays on cell viability and HSV-2 and RSV inhibition. S. desoleana EO inhibits both acyclovir sensitive and acyclovir resistant HSV-2 strains with EC50 values of 23.72 μg/ml for the former and 28.57 μg/ml for the latter. Moreover, a significant suppression of HSV-2 replication was observed with an EC50 value of 33.01 μg/ml (95% CI: 26.26 to 41.49) when the EO was added post-infection. Among the fractions resulting from flash column chromatography on silica gel, the one containing 54% of germacrene D showed a similar spectrum of activity of S. desoleana EO with a stronger suppression in post-infection stage. These results indicated that S. desoleana EO can be of interest to develop new and alternative anti-HSV-2 products active also against acyclovir-resistant HSV-2 strains.

Phenolics from Garcinia mangostana alleviate exaggerated vasoconstriction in metabolic syndrome through direct vasodilatation and nitric oxide generation.

BackgroundExaggerated vasoconstriction plays a very important role in the hypertension, a major component of metabolic syndrome (MetS). In the current work, the potential protective effect of methanol extract of fruit hulls of Garcinia mangostana L. on the exaggerated vasoconstriction in MetS has been investigated. In addition, the bioactive fraction and compounds as well as the possible mechanism of action have been illustrated.MethodsThe effect of methanol extract of G. mangostana (GMT) fruit hulls on the vascular reactivity of aorta isolated from animals with MetS was investigated through bioassay-guided fractionation procedures. GMT was partitioned with chloroform (I) and the remaining mother liquor was fractionated on a Diaion HP-20 with H2O, 50 and 100 % methanol to give fractions II, III, and IV, respectively. The effect of total extract (GMT), bioactive fraction and the bioactive compounds on the vasoconstriction were examined in aortae isolated from animals with MetS by incubation for 30 min before exposing aortae to cumulative concentrations of phenylephrine (PE). The direct relaxant effect was also examined by adding cumulative concentrations of the bioactive fraction and its bioactive compounds to PE precontracted vessels. In addition, aortic nitric oxide (NO) and reactive oxygen species (ROS) production was investigated.ResultsBioassay-guided fractionation of GMT revealed isolation of garcimangosone D (1), aromadendrin-8-C-β-D-glucopyranoside (2), 2,4,3′-trihydroxy benzophenone-6-O-β-D-glucopyranoside (3), maclurin-6-O-β-D-glucopyranoside (rhodanthenone) (4), epicatechin (5), and 2,3′,4,5′,6-pentahydroxy benzophenone (6). Only compounds 2, 4, and 5 significantly alleviated the exaggerated vasoconstriction of MetS aortae and in the same time showed significant vasodilation of PE pre-contracted aortae. To further illustrate the mechanism of action, the observed vasodilation was completely blocked by the nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine methyl ester hydrochloride and inhibited by guanylate cyclase inhibitor, methylene blue. However, vasodilation was not affected by the potassium channel blocker, tetraethylammonium or the cyclooxygenase inhibitor, indomethacin. In addition, compounds 2, 4, and 5 stimulated NO generation from isolated aortae to levels comparable with acetylcholine. Furthermore, 4 and 5 inhibited reactive oxygen species generation in MetS aortae.ConclusionThe phenolic compounds 2, 4, and 5 ameliorated the exaggerated vasoconstriction in MetS aortae through vasodilatation-NO generation mechanism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1340-5) contains supplementary material, which is available to authorized users.

Antioxidant and Antiglycating Constituents from Leaves of Ziziphus oxyphylla and Cedrela serrata.

Ziziphus oxyphylla and Cedrela Serrata plants have a folkloric use in Pakistan for treatments of different ailments, i.e., Jaundice, Hepatitis, Diabetes, and antimicrobial. Until now, none of the research studies have reported any phytochemical work on leaves of these two plants. This study aimed to isolate and perform phytochemical analysis in order to search for the constituent having the active role in treatment of the aforementioned ailments. A bioassay-guided fractionation and isolation procedure was used to isolate the concerned phytochemicals present in leaf extracts of Z. oxyphylla and C. serrata. The process involved the hyphenated techniques, i.e., Flash Chromatography, Semi-Preparative HPLC/DAD, UPLC/MS, and NMR in order to isolate and elucidate the structure of the phytochemicals. Seven compounds (1–7) were isolated and identified as flavonoids, more in particular glycosides of quercetin and kaempferol. They showed DPPH scavenging activity, compound 3 (isoquercitrin) being the most active one with an IC50 of 10.8 µg/mL (positive control quercetin; IC50 3.6 µg/mL). The superoxide-radical scavenging and total antioxidant (ABTS) assays indicated IC50 values ranging from 200 to 910 µg/mL and 170 to 320 µg/mL, respectively (positive control quercetin: 374 and 180 µg/mL, respectively). Furthermore, these compounds had low IC50 values for inhibition of protein glycation (AGEs inhibition), ranging from 530 to 818 µg/mL, comparable to aminoguanidine (510 µg/mL) used as a positive control. This study resulted in the identification of seven flavonoid glycosides for the first time from the leaves of Z. oxyphylla and C. serrata with antioxidative and antiglycating activities.

Bioassay-guided isolation of wound healing active compounds from Echium species growing in Turkey

Ethnopharmacological relevanceThe roots and root barks of Echium sp. have been used to treat ulcers, burns and wounds in traditional Turkish medicine. Aim of the studyOn the basis of them traditional use and literature references, four Echium species were selected for evaluation of them wound healing potential. Isolation of active component(s) from the active extracts through the bioassay guided fractionation procedures. Material and methodsIn vivo the wound healing activity of the plants was evaluated by linear incision experimental models. The chloroform extract of Echium italicum L. was fractionated by successive chromatographic techniques. Wound healing activity of each fraction was investigated following the bioassay-guided fractionation procedures. Moreover, the tissue samples of isolated compounds were examined histopathologically. The healing potential was comparatively assessed with a reference ointment Madecassol®, which contains 1% extract of Centella asiatica. ResultsSignificant wound healing activity was observed from the ointment prepared with ethanol extract at 1% concentration. The ethanol root extract treated in groups of animals showed a significant increase (37.38%, 40.97% and 35.29% separately for E. italicum L, Echium vulgare L. and Echium angustifolium Miller) wound tensile strength in the incision wound model. Subfractions showed significant but reduced wound healing activity on in vivo wound models. Shikonin derivatives “Acetylshikonin”, “Deoxyshikonin” and “2-methyl-n-butyrylshikonin+Isovalerylshikonin”, were isolated and determined as active components of active final subfraction from E. italicum L. roots. The results of histopathological examination supported the outcome of linear incision wound models. ConclusionThe experimental study revealed that Echium species display remarkable wound healing activity.

Pseudoverticin B, a novel geldanamycin analog obtained as new cell cycle inhibitor from Streptomyces pseudoverticillus YN17707

Pseudoverticin B (1), a novel naturally occurring geldanamycin analog with cell cycle inhibitory activity, was isolated from the fermentation broth of Streptomyces pseudoverticillus YN17707, together with the known ansamycin antibiotic, hydroquinone geldanamycin (2), through bioassay-guided fractionation procedures. The structure of compound 1 was elucidated by spectroscopic methods, being characterized by an ansa bridge, same as that in geldanamycin and a novel hydroquinone-derived moiety. Compounds 1 and 2 arrested the cell cycle of tsFT210 cells at the G0/G1 phase with the minimum inhibitory concentration values of 10.1 and 20.2 μmolL−1, respectively.

Inhibitory effect of the methanolic extract of Verbascum latisepalum Hub.-Mor. on endothelium-dependent relaxation in rat thoracic aorta.

The leaves and flowers of Verbascum species are used to treat respiratory disorders, haemorrhoids, rheumatic pain, and wounds as well as for the treatment of eczema and other types of inflammatory skin conditions in traditional Turkish medicine. We examined the effect of the methanolic extract of the aerial parts of Verbascum latisepalum Hub.-Mor. on the endothelium-dependent relaxation response in rat aortic rings which is mediated by nitric oxide (NO). Six fractions, A-F, were obtained from the methanolic extract through bioassay-guided fractionation procedures. The phenylethanoid glycoside verbascoside was isolated from fraction D and its structure elucidated by spectral techniques. The inhibitory effects of the extract, its fractions, and verbascoside on the acetylcholine-induced relaxation response in phenylephrine-precontracted aorta was examined in the absence and presence of L-arginine, a precursor in the synthesis of NO. The observation that the effects of the methanolic extract, of fraction D, and of verbascoside were reversed by L-arginine, indicates that verbascoside has an inhibitory effect on the synthesis of NO. This effect should be taken into consideration in view of the wide range of uses of Verbascum species in Turkish folk medicine.

Isolation of an oleanane-type saponin active from Bellis perennis through antitumor bioassay-guided procedures

Context: Bellis perennis L. (Asteraceae) (common daisy) is a herbaceous perennial plant known as a traditional wound herb; it has been used for the treatment of bruises, broken bones, and wounds. Bellis perennis has also been used in the treatment of headache, common cold, stomachache, eye diseases, eczema, skin boils, gastritis, diarrhea, bleeding, rheumatism, inflammation, and infections of the upper respiratory tract in traditional medicine.Objective: Antitumor activities of different fractions of B. perennis flowers at different concentrations were evaluated and through bioassay-guided fractionation and isolation procedures a saponin derivative (1) was isolated from the active fraction obtained from the n-butanol extract of flowers of the title plant by column chromatography.Materials and methods: Antitumor activities of different fractions of B. perennis flowers at different concentrations were evaluated using Potato Disc Tumor Induction Bioassay. Structure elucidation of 1 was accomplished by spectroscopic methods [1D- and 2D-NMR, and LC-ESI(APCI)-TOF-MS(MSn)].Results: The present study showed the antitumor activity of fractions obtained from B. perennis flowers for the first time. The most active fraction showed 99% tumor inhibition at 3000 mg/L. An oleanane-type saponin was isolated through bioassay-guided studies.Discussion and conclusion: Through antitumoral bioassay-guided fractionation and isolation procedures, 1 was isolated from the active fraction of B. perennis. The detailed NMR data of compound 1 is given for the first time.

Natural antifouling compounds produced by a novel fungus Aureobasidium pullulans HN isolated from marine biofilm

A fungus, Aureobasidium pullulans, was isolated from marine biofilm and identified. A bioassay-guided fractionation procedure was developed to isolate and purify antifouling compounds from A. pullulans HN. The procedure was: fermentation broth—aeration and addition of sodium thiosulfate–graduated pH and liquid–liquid extraction—SPE purification—GC–MS analysis. Firstly, the fermentation broth was tested for its toxicity. Then it was treated with aeration and addition of sodium thiosulfate, and its toxicity was almost not changed. Lastly, antifouling compounds were extracted at different pH, the extract had high toxicity at pH 2 but almost no toxicity at pH 10, which suggested the toxicants should be fatty acids. The EC50 of the extract against Skeletonema costatum was 90.9μgml−1, and its LC50 against Balanus amphitrete larvae was 22.2μgml−1. After purified by HLB SPE column, the EC50 of the extract against S. costatum was 49.4μgml−1. The myristic and palmitic acids were found as the main toxicants by GC–MS.

Intestinal antispasmodic effects of Helichrysum italicum (Roth) Don ssp. italicum and chemical identification of the active ingredients

Ethnopharmacological relevanceIn the Mediterranean Area, the flowers of Helichrysum italicum ssp. italicum are a traditional remedy for the treatment of intestinal complaints and are used as herbal tea for curing digestive, stomachic and intestinal diseases. In order to find scientific evidence for the traditional utilization of this plant, the effect of an ethanolic extract of Helichrysum italicum was investigated by using in vivo and in vitro experimental models. Then, through bioassay-guided fractionation procedures, active component(s) were identified. Material and methodsContractility in vitro was evaluated by stimulating the isolated ileum, in an organ bath, with acetylcholine and barium chloride; motility in vivo was evaluated by measuring upper gastrointestinal transit, both in control mice and in mice with experimental intestinal inflammation induced by croton oil. Chromatographic separation techniques such as HPLC and silica gel columns have yielded the active principles of Helichrysum italicum. ResultsWe found that the ethanolic extract of Helichrysum italicum ssp. italicum flowers elicited antispasmodic actions in the isolated mouse ileum and inhibited transit preferentially in the inflamed gut. A bioassay guided fractionation of the extract yielded the known compounds 12-acetoxytremetone (1) and 2,3-dihydro-2-[1-(hydroxymethyl)ethenyl]-5-benzofuranyl]-ethanone (2). ConclusionPresent study supported the traditional use of Helichrysum italicum ssp. italicum flowers for intestinal complaints and through bioassay-guided fractionation procedures from the crude extract we showed that 12-acetoxytremetone (1) and 2,3-dihydro-2-[1-(hydroxymethyl)ethenyl]-5-benzofuranyl]-ethanone (2) acted in a synergistic way to produce an intestinal antispasmodic effect.