Study on the mechanisms of the bronchodilator effects of Folium Eriobotryae and the selected active ingredient on isolated guinea pig tracheal strips

Context: Folium Eriobotryae (FE), the dry leaf of Eriobotrya japonica (Thunb.) Lindl. (Rosaceae), has been widely used to treat respiratory disorders.

Objective: To examine the bronchodilatory activity of FE and the potential mechanisms involved.

Materials and methods: The effects of ethyl acetate fraction of FE (EFE) (0.05–0.3?mg/mL) on the isolated tracheal strips, and ursolic acid (UA) (5–30?μg/mL) that was the main constituent of EFE, were tested in vitro. Meanwhile, acetylcholine (Ach) and histamine (His)-induced bronchospasm were conducted in vivo in guinea pig. Furthermore, mechanisms of relaxant effects of EFE and UA were evaluated in the absence and presence of specific inhibitors.

Results: With in vitro studies, the contractile response evoked by Ach or His (EC_50?=_?0.21 and 0.16?mg/mL) was decreased by EFE, and UA caused a concentration-dependent relaxation precontracted by His (EC_50?=_?23.2?μg/mL). With in vivo studies, EFE strongly prolonged preconvulsive time similar to isoprenalin. The bronchodilator effects of EFE could be blocked by propranolol (1?μM), N^G-nitro-l-arginine methyl ester (l-NAME) (100?μM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) (1?μM). EFE also inhibited the contraction in Ca²⁺-free medium and produced rightward parallel displacement of CaCl₂ curves. In addition, the relaxant effects of UA could only be blocked by l-NAME and ODQ.

Discussion and conclusion: These results suggest that bronchodilator activities of EFE were related to activation of β-adrenoceptor and NO/cGMP pathway. Blockage of Ca^2+?channels and inhibition of IP₃R-mediated internal Ca^2+?release were also involved. Additionally, UA produced relaxant effects by the NO/cGMP pathway.     

Mechanism of action of relaxant effect of Agastache mexicana ssp.mexicana essential oil in guinea-pig trachea smooth muscle

Context: Agastache mexicana ssp. mexicana (Kunth) Lint &; Epling (Lamiaceae), popularly known as ‘toronjil morado’, is used in Mexican traditional medicine for the treatment of several diseases such as hypertension, anxiety and respiratory disorders.

Objective: This study investigates the relaxant action mechanism of A. mexicana ssp. mexicana essential oil (AMEO) in guinea-pig isolated trachea model.

Materials and method: AMEO was analyzed by GC/MS. The relaxant effect of AMEO (5–50?μg/mL) was tested in guinea-pig trachea pre-contracted with carbachol (3?×?10?^??⁶?M) or histamine (3?×?10?^??⁵?M) in the presence or absence of glibenclamide (10?^??⁵?M), propranolol (3?×?10?^??⁶?M) or 2′,5′-dideoxyadenosine (10?^??⁵?M). The antagonist effect of AMEO (10–300?μg/mL) against contractions elicited by carbachol (10?^??¹⁵–10?^??³?M), histamine (10?^??¹⁵–10?^??³?M) or calcium (10–300?μg/mL) was evaluated.

Results: Essential oil composition was estragole, d-limonene and linalyl anthranilate. AMEO relaxed the carbachol (EC_50?=_?18.25?±?1.03?μg/mL) and histamine (EC_50?=_?13.3?±?1.02?μg/mL)-induced contractions. The relaxant effect of AMEO was not modified by the presence of propranolol, glibenclamide or 2′,5′-dideoxyadenosine, suggesting that effect of AMEO is not related to β₂-adrenergic receptors, ATP-sensitive potassium channels or adenylate cyclase activation. AMEO was more potent to antagonize histamine (pA₂′?=??1.507?±?0.122) than carbachol (pA₂′?=??2.180?±?0.357). Also, AMEO antagonized the calcium chloride-induced contractions.

Conclusion: The results suggest that relaxant effect of AMEO might be due to blockade of calcium influx in guinea-pig trachea smooth muscle. It is possible that estragole and d-limonene could contribute majority in the relaxant effect of AMEO.     

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

1.?Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms.

2.?UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms.

3.?UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC₅₀ 0.391?±?0.013?μM; K_i 0.185?±?0.015?μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 2.651?±?0.201?μM; K_i 1.334?±?0.146?μM).

4.?OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC₅₀ 0.336?±?0.013?μM; K_i 0.176?±?0.007?μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 5.468?±?0.697?μM; K_i 6.298?±?0.891?μM).

5.?Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.     

Unlocking the in vitro anti-inflammatory and antidiabetic potential of Polygonum maritimum

Context: Several Polygonum species (Polygonaceae) are used in traditional medicine in Asia, Europe and Africa to treat inflammation and diabetes.

Objective: Evaluate the in vitro antioxidant, anti-inflammatory and antidiabetic potential of methanol and dichloromethane extracts of leaves and roots of the halophyte Polygonum maritimum L.

Material and methods: Antioxidant activity was determined (up to 1?mg/mL) as radical-scavenging activity (RSA) of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), copper (CCA) and iron (ICA) chelating activities and iron reducing power (FRAP). NO production was measured in lipopolysaccharide (LPS)-stimulated macrophages for 24?h at concentrations up to 100?μg/mL and antidiabetic potential was assessed by α-amylase and α-glucosidase inhibition (up to 10?mg/mL) assays. The phytochemical composition of the extracts was determined by gas chromatography-mass spectrometry (GC-MS).

Results: The methanol leaf extract had the highest activity against DPPH? (IC₅₀ =?26?μg/mL) and ABTS⁺? (IC₅₀ =?140?μg/mL), FRAP (IC₅₀ =?48?μg/mL) and CCA (IC₅₀ =?770?μg/mL). Only the dichloromethane leaf extract (LDCM) showed anti-inflammatory activity (IC₅₀ =?48?μg/mL). The methanol root (IC₅₀ =?19?μg/mL) and leaf (IC₅₀ =?29?μg/mL) extracts strongly inhibited baker’s yeast α-glucosidase, but LDCM had higher rat’s α-glucosidase inhibition (IC₅₀ =?2527?μg/mL) than acarbose (IC₅₀ =?4638?μg/mL). GC-MS analysis identified β-sitosterol, stigmasterol, 1-octacosanol and linolenic acid as possible molecules responsible for the observed bioactivities.

Conclusions: Our findings suggest P. maritimum as a source of high-value health promoting commodities for alleviating symptoms associated with oxidative and inflammatory diseases, including diabetes.     

Antioxidant,anticholinesterase and antibacterial activities of Stachys guyoniana and Mentha aquatica

Context: Stachys guyoniana Noë ex. Batt. and Mentha aquatica L. are two Algerian Lamiaceae used in folk medicine.

Objective: To investigate their antioxidant, anticholinesterase and antibacterial activities.

Material and methods: n-Butanol (BESG), ethyl acetate (EESG) and chloroform (CESG) extracts of S. guyoniana and methanol (MEMA) and chloroform (CEMA) aerial part extracts of M. aquatica and methanol (MERMA) and acetone (AERMA) roots extracts of M. aquatica were evaluated for their antioxidant activity by the β-carotene-linoleic acid, DPPH^? and ABTS^?+?scavenging, CUPRAC and metal chelating assays. The anticholinesterase activity was tested against AChE and BChE. The antibacterial activity was assessed by MICs determination against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella heidelberg, Klebsiella pneumoniae, Enterobacter aerogenes and Morganella morganii strains.

Results: In the β-carotene test, the CESG (IC₅₀: 2.3?±?1.27?μg/mL) exhibited the highest activity. The BESG was the best scavenger of DPPH^? (IC₅₀: 2.91?±?0.14?μg/mL). In the ABTS test, AERMA was the most active (IC₅₀: 4.21?±?0.28?μg/mL). However, with the CUPRAC, the BESG exhibited the best activity (A_0.50: 0.15?±?0.05?μg/mL) and was active in metal chelating assay with 48% inhibition at 100?μg/mL. The BESG was the best AChE inhibitor (IC₅₀: 5.78?±?0.01?μg/mL) however, the AERMA showed the highest BChE inhibitory activity (IC₅₀: 19.23?±?1.42?μg/mL). The tested extracts exhibited a good antibacterial activity.

Conclusion: This study demonstrated good antioxidant, anticholinesterase and antibacterial potential of S. guyoniana and M. aquatica, which fits in well with their use in folk medicine.     

Interactions of the active components of Punica granatum (pomegranate) with the essential renal and hepatic human Solute Carrier transporters

Context: Solute carrier transporters (SLCs) are membrane proteins responsible for cellular influx of various substances including many pharmaceutical agents; therefore, they largely impact on drug disposition and elimination in body. Punica granatum Linnaeus (Lythraceae), pomegranate, is a fruit with antidiabetic potential. Oleanolic acid (OA), ursolic acid (UA), and gallic acid (GA) are the major bioactive components of pomegranate. Co-administration of these compounds with other drugs could result in altered drug pharmacokinetics, possibly due to competing for transporter proteins.

Objective: We investigated the interactions of these three compounds with the essential hepatic and renal SLC transporters.

Materials and methods: Uptake of radiolabeled transporter model substrates was assessed in HEK293 cells over-expressing SLC transporters including the organic anion transporters (OATs), organic anion transporting polypeptides (OATPs) and organic cation transporters (OCTs), in the presence or absence of 10.0?µM UA, OA, or GA. Their IC₅₀ values on specific SLC transporters were also evaluated using varying concentrations of the particular compound (ranging from 0.10?nM to 80.0?µM).

Results: Our results demonstrated UA could significantly inhibit OAT3 and OATP2B1 uptake (IC₅₀: 18.9?±?8.20?µM and 11.0?±?5.00?µM, respectively) and GA has a pronounced inhibitory effect on OATP1B3 uptake (IC₅₀: 1.60?±?0.60?μM).

Discussion and conclusion: Our study reports the interactions of OA, UA, and GA with the essential SLC transporters. This information may contribute to elucidating the drug–drug/herb interactions involved with these three compounds and form the basis of therapeutic optimization when drugs are co-administered.     

Anti-cryptococcal activity of ethanol crude extract and hexane fraction from Ocimum basilicum var. Maria bonita: mechanisms of action and synergism with amphotericin B and Ocimum basilicum essential oil

Context: Ocimum basilicum L. (Lamiaceae) has been used in folk medicine to treat headaches, kidney disorders, and intestinal worms.

Objective: This study evaluates the anti-cryptococcal activity of ethanol crude extract and hexane fraction obtained from O. basilicum var. Maria Bonita leaves.

Materials and methods: The MIC values for Cryptococcus sp. were obtained according to Clinical and Laboratory Standards Institute in a range of 0.3–2500?μg/mL. The checkerboard assay evaluated the association of the substances tested (in a range of 0.099–2500?μg/mL) with amphotericin B and O. basilicum essential oil for 48?h. The ethanol extract, hexane fraction and associations in a range of 0.3–2500?μg/mL were tested for pigmentation inhibition after 7?days of treatment. The inhibition of ergosterol synthesis and reduction of capsule size were evaluated after the treatment with ethanol extract (312?μg/mL), hexane fraction (78?μg/mL) and the combinations of essential oil?+?ethanol extract (78?μg/mL?+?19.5?μg/mL, respectively) and essential oil?+?hexane fraction (39.36?μg/mL?+?10?μg/mL, respectively) for 24 and 48?h, respectively.

Results: The hexane fraction presented better results than the ethanol extract, with a low MIC (156?μg/mL against C. neoformans T₄₄₄ and 312?μg/mL against C. neoformans H99 serotype A and C. gattii WM779 serotype C). The combination of the ethanol extract and hexane fraction with amphotericin B and essential oil enhanced their antifungal activity, reducing the concentration of each substance needed to kill 100% of the inoculum. The substances tested were able to reduce the pigmentation, capsule size and ergosterol synthesis, which suggest they have important mechanisms of action.

Conclusions: These results provide further support for the use of ethanol extracts of O. basilicum as a potential source of antifungal agents.     

Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages

Context: Punica granatum L (Punicaceae) flower is an important diabetes treatment in oriental herbal medicine.

Objective: This study investigates the inflammation effects of pomegranate flower (PFE) ethanol extract in LPS-induced RAW264.7 cells.

Materials and methods: PFE (10, 25, 50, 100?μg/mL) was applied to 1?μg/mL LPS-induced RAW 264.7 macrophages in vitro. Levels of nitric oxide (NO), prostaglandin E₂ (PGE₂) and pro-inflammatory cytokines interleukin (IL)-1β (IL-1β), interleukin (IL)-6 (IL-6) and tumor necrosis factor (TNF-α) in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), phosphorylation of mitogen-activated protein kinase (MAPK) subgroups extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38, as well as nuclear factor-κB (NF-κB) activation in extracts were detected via Western blot.

Results: 10–100?μg/mL PFE decreased the production of NO (IC₅₀ value?=?31.8?μg/mL), PGE₂ (IC₅₀ value?=?54.5?μg/mL), IL-6 (IC₅₀ value?=?48.7?μg/mL), IL-1β (IC₅₀ value?=?71.3?μg/mL) and TNF-α (IC₅₀ value?=?62.5?μg/mL) in LPS-stimulated RAW 264.7 cells significantly. A mechanism-based study showed that phosphorylation of ERK1/2, p38, JNK and translocation of the NF-B p65 subunit into nuclei were inhibited by the PFE treatment.

Discussion and conclusion: These results show that PFE produced potential anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process.     

Chemical composition,antioxidant, antitumor,anticancer and cytotoxic effects of Psidium guajava leaf extracts

Context Psidium guajava L. (Myrtaceae) leaves are used in traditional medicines for the treatment of cancer, inflammation and other ailments.

Objective The current study explores scientific validation for this traditional medication.

Materials and methods We used ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picryl hydrazil (DPPH) assays to estimate antioxidant activity of P. guajava leaf extracts (methanol, hexane and chloroform). Antitumour and in vivo cytotoxic activities were determined using potato disc assay (PDA) and brine shrimp lethality assay, respectively. Three human carcinoma cell lines (KBM5, SCC4 and U266) were incubated with different doses (10–100?μg/mL) of extracts and the anticancer activity was estimated by MTT assay. NF-κB suppressing activity was determined using electrophoretic mobility shift assay (EMSA). Chemical composition of the three extracts was identified by GC-MS. Total phenolic and flavonoid contents were measured by colorimetric assays.

Results and discussions The order of antioxidant activity of three extracts was methanol?>?chloroform?>?hexane. The IC₅₀ values ranged from 22.73 to 51.65?μg/mL for KBM5; 22.82 to 70.25?μg/mL for SCC4 and 20.97 to 89.55?μg/mL for U266 cells. The hexane extract exhibited potent antitumour (IC_50? value?=_?65.02?μg/mL) and cytotoxic (LC_50? value?=_?32.18?μg/mL) activities. This extract also completely inhibited the TNF-α induced NF-κB activation in KBM5 cells. GC-MS results showed that pyrogallol, palmitic acid and vitamin E were the major components of methanol, chloroform and hexane extracts. We observed significant (p?<?0.05) difference in total phenolic and flavonoid contents of different solvent extracts.

Conclusion The present study demonstrates that P. guajava leaf extracts play a substantial role against cancer and down-modulate inflammatory nuclear factor kB.     

Evaluation of genotoxic effects of 3-methyl-5-(4-carboxycyclohexylmethyl)-tetrahydro-2H-1,3,5-thiadiazine-2-thione on human peripheral lymphocytes

Context: Tranexamic acid is commonly used for curing abnormal bleeding in a variety of diseases. In a previous study, 12 different tetrahydro-2H-1,3,5-thiadiazine derivatives were synthesized from the amine group of tranexamic acid. Their antifibrinolytic and antimicrobial activities were compared with tranexamic acid. 3-Methyl-5-(4-carboxycyclohexylmethyl)-tetrahydro-2H-1,3,5-thiadiazine-2-thione (3-MTTT) was the most remarkable one, which may be used as a drug.

Objectives: In vitro genotoxicity of 3-MTTT was investigated using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN) and comet assays.

Materials and methods: Various concentrations 0.78, 1.56, 3.13, 6.25, 12.50 and 25.00?μg/mL of 3-MTTT were applied to lymphocytes obtained from two donors for periods of 24 and 48?h. A negative (distilled water), a solvent (2:1 PBS:10% NaOH for cultured lymphocyte, and PBS for isolated lymphocytes) and a positive control (MMC for cultured lymphocytes and H₂O₂ for isolated lymphocytes) were also maintained.

Results: While this compound did not increase the frequency of abnormal cells and CA/cell ratio compared to negative control (except 48?h, 25?μg/mL), it significantly increased the frequency of SCEs at the four highest concentrations at both treatment periods (except 6.25?μg/mL, 48?h). It significantly decreased the MI in all the concentrations at 24?h (except 0.78?μg/mL) and in the highest three concentrations at 48?h. This compound did not significantly increase the frequency of MN and DNA damage compared to negative control. This compound did not affect the replication and nuclear division index.

Discussion and conclusion: Our results demonstrated that this compound does not represent a significant risk at the genetic level in in vitro human lymphocytes.     

Glutathione sulfotransferase inhibition activity of a self-fermented beverage,Kanji

Context: Kanji, a liquid preparation of roots of Daucus carota L. ssp. sativus (Hoffm.) Arcang. var. vavilovii Mazk. (Apiaceae), may inhibit glutathione sulfotransferase (GST) activity due to ferulic acid content.

Objectives: GST inhibition activity and characterization of Kanji and methanol extract of D. carota roots, and oral absorption pattern of ferulic acid from Kanji in rats.

Materials and methods: GST inhibition activity of Kanji and methanol extract of D. carota roots in concentration range 0.001–100.00?mg/mL was determined using Sprague Dawley rat liver cytosolic fraction. Methanol extract upon column chromatography gave ferulic acid, which was used to characterize Kanji and determine its oral absorption pattern in Wistar rats.

Results: The GST inhibition activity of Kanji (100.00?μg/mL), methanol extract of D. carota roots (100.00?μg/mL) and tannic acid (10.00?μg/mL, positive control) was found to be 0.162?±?0.016, 0.106?±?0.013 and 0.073?±?0.004?μM/min/mg, respectively. Different Kanji samples and methanol extract contained ferulic acid (0.222–0.316?mg/g) and 0.77?mg/g, respectively. Ferulic acid did not appear in plasma after oral administration of Kanji.

Discussion: Kanji having solid contents 80.0?μg/mL, equivalent to 0.0025?μg/mL ferulic acid, does not inhibit the activity of GST. The oral administration of Kanji, in human equivalent dose (528?mg/kg, 16.67?μg ferulic acid), to rats indicated poor absorption of ferulic acid.

Conclusion: Kanji having solid contents 14–36?mg/mL does not inhibit GST activity, hence may not interfere with drugs that are the substrates of GST, if taken concomitantly.     

Antimicrobial activity of Buchenavia tetraphylla against Candida albicans strains isolated from vaginal secretions

Context: Buchenavia tetraphylla (Aubl.) RA Howard (Combretaceae: Combretoideae) is an ethnomedicinal plant with reported antifungal action.

Objective: This study evaluates the antimicrobial activity of B. tetraphylla leaf extracts against clinical isolates of Candida albicans. The morphological alterations, combinatory effects with fluconazole and the cytotoxicity of the active extract were analyzed.

Materials and methods: Extracts were obtained using different solvents (hexane: BTHE; chloroform: BTCE; ethyl acetate: BTEE; and methanol: BTME). Antimicrobial activity was determined by the broth microdilution method using nine strains of C. albicans isolated from vaginal secretions and one standard strain (UFPEDA 1007).

Results: All extracts showed anti-C. albicans activity, including against the azole-resistant strains. The MIC values ranged from 156 to 2500?μg/mL for the BTHE; 156 to 1250?μg/mL for the BTCE; 625 to 1250?μg/mL for the BTME and 625?μg/mL to 2500?μg/mL for the BTEE. BTME showed the best anti-C. albicans activity. This extract demonstrated additive/synergistic interactions with fluconazole. Scanning electron microscopy analysis suggested that the BTME interferes with the cell division and development of C. albicans. BTME showed IC₅₀ values of 981 and 3935?μg/mL, against J774 macrophages and human erythrocytes, respectively. This extract also enhanced the production of nitric oxide by J774 macrophages.

Discussion and conclusion: Buchenavia tetraphylla methanolic extract (BTME) is a great source of antimicrobial compounds that are able to enhance the action of fluconazole against different C. albicans strains; this action seems related to inhibition of cell division.     

Evaluation of the antileishmanial potency,toxicity and phytochemical constituents of methanol bark extract of Sterculia villosa

Context: Visceral leishmaniasis is a protozoan disease caused by Leishmania donovani parasite. The genus Sterculia (Malvaceae) possesses ethnobotanical potential against this protozoan infection.

Objective: Determining the potential role of methanol bark extracts from Sterculia villosa Roxb (SVE) and its phytoconstituents against Leishmania donovani promastigotes.

Materials and methods: SVE was analysed by TLC, UV–Vis, IR spectroscopy and biochemical assays. Antileishmanial potential of SVE (0.5–130?μg/mL for 72?h) was characterized by MTT assay. Fluorescent microscopy was performed to validate the IC₅₀ dose. To determine the effect of SVE on promastigotes, reactive oxygen species (ROS) and superoxide generation, lipid peroxidation and DNA fragmentation assays were performed. Molecular aggregation of compounds was determined by atomic force microscopy (AFM). Extent of cytotoxicity of SVE at IC₅₀ dose was determined against RAW 264.7 macrophages, peritoneal macrophages and murine RBCs. In vivo cytotoxicity of SVE was evaluated in BALB/c mice.

Result: SVE exhibited reverse dose dependent antileishmanial activity when 130–0?μg/mL doses were tested against promastigotes. The IC₅₀ and IC₇₀ values were found to be 17.5 and 10?μg/mL, respectively. SVE at IC₅₀ dose demonstrated elevated level of ROS, superoxide, lipid peroxidation and DNA fragmentation against promastigotes with no cytotoxicity. AFM analysis suggested increasing size of molecular aggregation (31.3?nm Discussion and conclusions: The study elucidates the antileishmanial potential of SVE against Leishmania donovani promastigotes by exerting oxidative stress and DNA damage. In sum, SVE can be explored as an immunotherapeutic candidate against leishmaniasis and other infectious diseases.     

Simultaneous screening and analysis of antiplatelet aggregation active alkaloids from Rhizoma Corydalis

Context: The rising problem of atherosclerosis and ischemic heart disease emphasizes the need to look for new antithrombotic components with effective modes of action. Corydalis yanhusuo (Y.H. Chou & Chun C. Hsu) W.T. Wang ex Z.Y. Su & C.Y. Wu (Papaveraceae) (Rhizoma Corydalis) has been used in the traditional medicines for the treatment of cardiovascular disease.

Objective: The antiplatelet aggregation compounds in Rhizoma Corydalis were screened to validate its traditional medicinal use.

Material and methods: Total alkaloid extract (TAE) of Rhizoma Corydalis was obtained by refluxing 100?g Rhizoma Corydalis powder with 600?mL 70% ethanol, and purified by acidification (20% HCl) and alkalization (5 M NaOH) process. Potential antiplatelet aggregation compounds in TAE were screened by a method involving platelet bio-specific extraction and HPLC-DAD/LC–MS analysis. Further in vitro antiplatelet aggregation activity confirmation of TAE and seven main alkaloids were achieved by turbidimetry method within 3?h after blood collection from rabbit carotid artery, and all the test drugs were at the concentration range of 25–350?μg/mL. Finally, HPLC-DAD was employed for the quantitative determination of seven main components in TAE.

Results: Five alkaloids, identified as glaucine, dehydrocorydaline, canadine, tetrahydrocoptisine and corydaline, can be specifically extracted with platelets. The results indicated that all these five alkaloids can inhibit thrombin-induced platelet aggregation in a low dose (IC₅₀ of glaucine, dehydrocorydaline, canadine, tetrahydrocoptisine and corydaline were 49.057, 34.914, 33.547, 84.261 and 54.164?μg/mL, respectively) as compared to TAE (IC_50?=_?175.426?μg/mL) and aspirin (IC_50?=_?300.340?μg/mL), while the unbound compounds (palmatine and tetrahydropalmatine) had a very weak antiplatelet effect (IC_50?>_?200?μg/mL).

Discussion and conclusion: This study is the first reported work for antiplatelet components screening in Rhizoma Corydalis. Seven compounds were detected and identified by HPLC-DAD/LC–MS, of which five platelet-targeted compounds were discovered.     

Inhibitory effects of Pinus massoniana bark extract on hepatitis C virus in vitro

Abstract

Context: Given that Pinus massoniana Lamb (Pinaceae) bark extract (PMBE) is a safe and non-toxic flavonoid found abundantly in nature, it was considered a promising novel candidate agent in the treatment of virus infection.

Object: Experiments were conducted to assay the antiviral character of PMBE against Hepatitis C virus (HCV).

Materials and methods: Assay of PMBE cytotoxicity, HCV replication, infectious HCV production, and its potential influence on the pathways for IFN-ISRE and NS3 protease were conducted.

Results: HCV replication was suppressed when the concentration of PMBE raised greater than 5?μg/mL and its EC₅₀ was 9.58?μg/mL. In the 10?μg/mL group, HCV replication was suppressed for 48?h. When the concentration increased to 40?μg/mL, HCV replication was significantly suppressed (luciferase activity was only 10% at 96?h). PMBE could inhibit HCV virus production efficiently (PMBE group was 5?FFU). Cell viability was affected by 40?μg/mL of PMBE. The F/R ratio ranged from 98% to 101%. The rate of OD₄₅₀ ranged from 96% to 102%. NS3 catalytic activity ranged from 5% (40?μg/mL PMBE) to 45% (5?μg/mL PMBE). Even when used in a low amount (5?μg/mL), NS3 catalytic activity was significantly inhibited (p?<?0.01).

Conclusions: The results suggest that PMBE is effective for use in the stabilization of HCV replication and active liver inflammation.     

Biological activity of terpene compounds produced by biotechnological methods

Context Biotransformation systems are profitable tools for structural modification of bioactive natural compounds into valuable biologically active terpenoids.

Objective This study determines the biological effect of (R)-(+)-limonene and (?)-α-pinene, and their oxygenated derivatives, (a) perillyl alcohol and (S)-(+)- and (R)-(?)-carvone enantiomers and (b) linalool, trans-verbenol and verbenone, respectively, on human colon tumour cells and normal colonic epithelium.

Materials and methods Biotransformation procedures and in vitro cell culture tests were used in this work. Cells were incubated for 24?h with terpenes at concentrations of 5–500?μg/mL for NR, MTT, DPPH, and NO assays. IL-6 was determined by ELISA with/without 2?h pre-activation with 10?μg/mL LPS.

Results trans-Verbenol and perillyl alcohol, obtained via biotransformation, produced in vitro effect against tumour cells at lower concentrations (IC₅₀ value?=?77.8 and 98.8?μg/mL, respectively) than their monoterpene precursors, (R)-(+)-limonene (IC₅₀ value?=?171.4?μg/mL) and (?)-α-pinene (IC₅₀ value?=?206.3?μg/mL). They also showed lower cytotoxicity against normal cells (IC₅₀?>?500 and?>?200?μg/mL, respectively). (S)-(+)-Carvone was 59.4% and 27.1% more toxic to tumour and normal cells, respectively, than the (R)-(?)-enantiomer. (R)-(+)-limonene derivatives decreased IL-6 production from normal cells in media with or without LPS (30.2% and 13.9%, respectively), while (?)-α-pinene derivatives induced IL-6 (verbenone had the strongest effect, 60.2% and 29.1% above control, respectively). None of the terpenes had antioxidative activity below 500?μg/mL.

Discussion and conclusions Bioactivity against tumour cells decreased in the following order: alcohols?>?ketones?>?hydrocarbons. (R)-(+)-limonene, (?)-α-pinene, and their derivatives expressed diverse activity towards normal and tumour cells with noticeable enantiomeric differences.     

Anti-inflammatory and anti-osteoarthritis effect of Mollugo pentaphylla extract

Context: Mollugo pentaphylla L. (Molluginaceae) extract (MPE) has been reported to have anti-inflammatory effect on MSU-induced gouty arthritis in a mouse model.

Objective: This study examined the anti-inflammatory activities of an MPE in vitro and anti-osteoarthritis effects on monosodium iodoacetate (MIA)-induced osteoarthritis (OA) in vivo.

Materials and methods: The dried whole plants of M. pentaphylla were extracted with 70% ethanol under reflux. The anti-inflammatory effect of MPE was evaluated in vitro in lipopolysaccharide (LPS)-treated RAW264.7 cells. The anti-osteoarthritic effect of MPE was investigated in a Sprague–Dawley rat model of MIA-induced OA. Each seven male rats were orally administered MPE (75, 150 or 300?mg/kg) or the positive control drug indomethacin (1?mg/kg) 3?days before MIA injection and once daily for 11?days thereafter. After the treatment with MPE, no evidence of systemic adverse effects was observed in any study group.

Results: MPE exhibited anti-inflammatory activity via inhibition of the production of NO (57.8%), PGE2 (97.1%) and IL-6 (93.2%) in LPS-treated RAW264.7 cells at 200?μg/mL. In addition, MPE suppressed IL-1β (60.9%), TNF-α (37.9%) and IL- 6 (40.9%) production and suppressed the synthesis of MMP-2, MMP-9 and COX-2 in the MIA-induced OA rat model.

Conclusions: These results demonstrate that MPE exerts potent anti-inflammatory activities and protects cartilage in an OA rat model. This might be a potential candidate for therapeutic OA treatment.     

Studies on phytochemical,antioxidant, anti-inflammatory,hypoglycaemic and antiproliferative activities of Echinacea purpurea and Echinacea angustifolia extracts

Context: Echinacea (Asteraceae) is used because of its pharmacological properties. However, there are few studies that integrate phytochemical analyses with pharmacological effects.

Objective: Evaluate the chemical profile and biological activity of hydroalcoholic Echinacea extracts.

Materials and methods: Density, dry matter, phenols (Folin–Ciocalteu method), flavonoids (AlCl₃ method), alkylamides (GC-MS analysis), antioxidant capacity (DPPH and ABTS methods), antiproliferative effect (SRB assay), anti-inflammatory effect (paw oedema assay, 11 days/Wistar rats; 0.4?mL/kg) and hypoglycaemic effect (33 days/Wistar rats; 0.4?mL/kg) were determined in three Echinacea extracts which were labelled as A, B and C (A, roots of Echinacea purpurea L. Moench; B, roots, leaves, flowers and seeds of Echinacea purpurea; C, aerial parts and roots of Echinacea purpurea and roots of Echinacea angustifolia DC).

Results: Extract C showed higher density (0.97?g/mL), dry matter (0.23?g/mL), phenols (137.5?±?2.3 mEAG/mL), flavonoids (0.62?±?0.02 mEQ/mL), and caffeic acid (0.048?mg/L) compared to A and B. A, B presented 11 alkylamides, whereas C presented those 11 and three more. B decreased the oedema (40%) on day 2 similar to indomethacin. A and C showed hypoglycaemic activity similar to glibenclamide. Antiproliferative effect was only detected for C (IC₅₀ 270?μg/mL; 8171?μg/mL; 9338?μg/mL in HeLa, MCF-7, HCT-15, respectively).

Discussion and conclusion: The difference in the chemical and pharmacological properties among extracts highlights the need to consider strategies and policies for standardization of commercial herbal extracts in order to guarantee the safety and identity of this type of products.     

Anti-inflammatory and anti-proliferative activities of the wild edible cruciferous: Diplotaxis simplex

Context The present study deals with new biological properties of the wild edible Diplotaxis simplex (Viv.) Spreng (Brassicaceae).

Objectives The current study evaluates the antioxidant, the anti-inflammatory and the anti-cancer properties of ethyl acetate and ethanol extracts from D. simplex flowers.

Materials and methods The anti-proliferative activity of the extracts (10–70?μg/mL) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) against human colon cancer cell line Caco-2. The anti-inflammatory potential was evaluated by the inhibitory effect of the extracts (1.5–7.5?mg/mL) on phospholipase A2 activity as well as on carrageenan-induced paw oedema in mice. Extracts (200?mg/kg) or indomethacin (50?mg/kg) as positive control were injected intraperitoneally for albino mice prior to the induction of the oedema by carrageenan. Antioxidant activities were investigated using various complementary methods.

Results Flower extracts contained a high level of polyphenolics (17.10–52.70?mg GAE/g) and flavonoids (74.20–100.60?mg QE/g), which correlate with its appreciable antioxidant potential in β-carotene peroxidation (IC₅₀ value: 12.50–27.10?μg/mL), DPPH^? radical-scavenging (IC₅₀ value: 0.20–0.40?mg/mL), Fe^3+?reducing (EC₅₀ value: 0.10–0.14?mg/mL) and Fe^2+?chelating (IC₅₀ value: 0.20–0.60?mg/mL) assays. These extracts were effective in inhibiting cancer cell growth (IC₅₀ value: 62.0–63.25?μg/mL). Besides, the ethyl acetate extract inhibited phospholipase A2 activity (IC₅₀ value: 2.97?mg/mL) and reduced the paw oedema in mice (from 0.38?±?0.01 to 0.24?±?0.01?cm), 4?h post-carrageenan challenge.

Conclusion These data suggest that D. simplex may be useful as a candidate in the treatment of inflammation and the colon cancer.     

Antioxidant and cytotoxic activity of carotenes produced by Dunaliella salina under stress

Context Dunaliella salina Teodoresco (Dunaliellaceae) is one of the promising microalgae consumed as food and medicine for many years.

Objective Dunaliella salina was grown under different stress conditions for enhancing carotene production. The carotene enriched extract was evaluated for antioxidant and cytotoxic activity.

Materials and methods Carotene content was calculated under salinity, nitrogen and temperature stress conditions. Antioxidant activity was determined through DPPH assay by incubating the samples for 45?min with 250?μg/mL of extract and reducing power assay was performed with 50, 100, 150 and 200?μg/mL of extract. Cytotoxicity was determined by incubating ～2?×?10⁴ MCF-7 (breast cancer) cells with 250?μg of extract in each well for 72?h by MTT assay.

Result Carotene content was significantly increased to 9.8 (3.5 M NaCl), 13.9 (37?°C), 8.2 (250?mM KNO₃) and 10.6?μg/mL (nitrogen-depleted medium) as compared with 3.2?μg/mL in normal conditions (1.7 M NaCl, 0.75?mM KNO₃ and 28?°C). Free radical scavenging activity increased at 3.0 and 3.5 M NaCl (27.8 and 57.5%, respectively), 37?°C (31.4%) and in nitrogen-depleted medium (41.9%) compared with normal (15%) conditions. Carotene content and scavenging activity were positively correlated under salinity (r?=?0.97), temperature (r?=?0.85) and nitrogen (r?=?0.7) stress conditions. Cytotoxicity against MCF-7 cell lines increased due to increase in carotene content suggesting that cytotoxicity may be associated with carotene accumulation.

Discussion and conclusions Carotene content enhanced by D. salina under stress conditions increased the antioxidant and cytotoxic activity.