Muscarinic Receptor Activity of Some Malaysian Plant Species

Abstract

Muscarinic receptor binding activity was tested on 224 plant extracts obtained from more than 50 plant families found in Malaysia. The plant extracts were evaluated by a 96-well microplate filtration–based radioligand competitive assay, centered on the ability of the plant extracts to competitively displace the radioligand, [³H]N.-methylscopolamine, from binding to the muscarinic membrane receptors. The screening assay was initially carried out at 50 µg/assay point, and those showing inhibition at and above 61% were retested at 10 µg/assay point. The extracts of Ficus septica. Burm. f. (Moraceae) [65.85±3.75% inhibition; mean (n = 3)±SD], Polyalthia microtus. Miq. (Annonaceae) (32.63±1.38% inhibition), and Popowia odoardoi. Diels (Annonaceae) (35.79±7.11% inhibition) at 10 µg/assay point exhibited muscarinic properties, which are worthy of further investigation.     

Flavonoids in Scutellaria immaculata and S. ramosissima (Lamiaceae) and their biological activity

Objectives The aim of this study was to investigate the flavonoid composition of Scutellaria immaculata and S. ramosissima (Lamiaceae) and the in‐vitro biological activity of their extracts and flavonoids. Methods The flavonoid composition of S. immaculata (Si) and S. ramosissima (Sr) were analysed using LC‐MS. Antimicrobial activity was studied in vitro against a range of bacteria and fungi using diffusion and microdilution methods. Anti‐trypanosomal and cell proliferation inhibitory activity of the extracts and flavonoids was assessed using MTT. The antioxidant activity of the flavonoids and extracts were evaluated using DPPH* test. Key findings LC‐MS investigation of Si and Sr plants allowed the identification, for the first time, of an additional 9 and 16 flavonoids, respectively. The methanol, chloroform and water extracts from these plants and six flavonoids (scutellarin, chrysin, apigenin, apigenin‐7‐O‐glucoside, cynaroside and pinocembrine) exhibited significant inhibition of cell growth against HeLa, HepG‐2 and MCF‐7 cells. The chloroform extract of Sr showed potent cytotoxic effects with IC50 (drug concentration which resulted in a 50% reduction in cell viability) values of 9.25 ± 1.07 µg/ml, 12.83 ± 1.49 µg/ml and 17.29 ± 1.27 µg/ml, respectively. The highest anti‐trypanosomal effect against T. b. brucei was shown by the chloroform extract of Sr with an IC50 (drug concentration which resulted in a 50% inhibition of the biological activity) of 61 µg/ml. The pure flavonoids showed an IC50 range between 3 and 29 µm , with cynaroside as the most active compound with an IC50 value of 3.961 ± 0.133 µm . The chloroform extract of Sr has potent antimicrobial activity against Streptococcus pyogenes (minimum inhibitory concentration, MIC = 0.03 mg/ml). Pinocembrine exhibited a strong activity against the all bacteria except Escherichia coli and yeasts. Water extracts of Sr and Si exhibited potent antioxidant activity with IC50 values of 5.62 ± 0.51 µg/ml and 3.48 ± 0.02 µg/ml, respectively. Scutellarin exerted stronger antioxidant activity than other flavonoids. Conclusions This is the first study reporting an in‐vitro biological investigation for Si and Sr. Especially the chloroform extract of Sr showed potent anticancer and antimicrobial activity. Cynaroside had a highly selective and strong cytotoxicity against T. b. brucei while showing only mild effects against cancer cells.     

Vasorelaxant effect of Valeriana edulis ssp. procera (Valerianaceae) and its mode of action as calcium channel blocker

Objectives The aim was to evaluate the relaxant effect of extracts from Valeriana edulis and determine the possible mechanism of action of the hexanic extract as vasorelaxant agent. Methods Extracts from rhizomes obtained by maceration (hexanic (HEVe), dichloromethanic (DEVe), methanolic (MEVe) and hydroalcoholic (HAEVe) (3.03–500 µg/ml)) were evaluated on aortic rat rings with and without endothelium. Key findings Extracts induced a significant concentration‐dependent and endothelium‐independent relaxation on isolated rat aorta pre‐contracted with noradrenaline (0.1 µm ). HEVe, the most potent extract (0.15–50 µg/ml), induced relaxation in aortic rings pre‐contracted with KCl (80 mm ), with an IC50 value of 34.61 ± 1.41 µg/ml and E_max value of 85.0 ± 4.38%. Pretreatment with HEVe (30 µg/ml) also inhibited contractile responses to noradrenaline and CaCl₂. HEVe (9.98 ± 2.0 µg/ml) reduced noradrenaline‐induced transient contraction in Ca²⁺‐free solution, and inhibited contraction induced by KCl (80 mm ). In endothelium‐denuded rings, the vasorelaxant effect of HEVe was not modified by 1‐H‐[1,2,4]‐oxadiazolo‐[4,3a]‐quinoxalin‐1‐one (1 µm ), tetraethylammonium (5 mm ), glibenclamide (10 µm ) or 2‐aminopyridine (100 µm ). Conclusions Our results suggest that HEVe induces relaxation through an endothelium‐independent pathway, involving blockade of Ca²⁺ channels, and this effect could be related to the presence of valepotriates.     

Ameliorative effect of polyphenols from Padina boergesenii against ferric nitrilotriacetate induced renal oxidative damage: With inhibition of oxidative hemolysis and in vitro free radicals

The aim of this study was to evaluate the antioxidant activities of diethyl ether (DEE) and methanol (M) extracts from brown alga Padina boergesenii using in vitro and in vivo antioxidant assay, which may help to relate the antioxidant properties with the possible outline of its ameliorative effect. M extract showed higher radical scavenging activity through ferric reducing antioxidant power 139.11 µmol tannic acid equivalent/g; DPPH 71.32 ± 0.56%; deoxyribose radical 88.31 ± 0.47%, and total antioxidant activity 0.47 ± 0.02 mg ascorbic acid equivalents/g. Oxidative red blood cell (RBC) hemolysis inhibition rate was significantly higher in M extract (150 mg/kg body weight) in reference to total phenolic content (r = 0.935). Rats administered with DEE and M extracts (150 mg/kg body weight) for seven days before the administration of ferric nitrilotriacetate (9 mg of Fe/mg/kg bodyweight). Rats pretreated with extracts significantly changed the level of renal microsomal lipid peroxidation, glutathione, and antioxidant enzymes in post‐mitochondrial supernatant (P < 0.05). Ameliorative effect of extracts against renal oxidative damage was evident in rat kidney through changes in necrotic and epithelial cells. HPTLC technique has identified the presence of rutin with reference to retardation factor (R_f) in both the extracts. These findings support the source of polyphenols (rutin) from P. boergesenii had potent antioxidant activity; further work on isolation of bioactive compounds can be channeled to develop as a natural antioxidant. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 865–876, 2015.     

Evaluation of selected South African plant species for antioxidant,antiplatelet, and cytotoxic activity

The antioxidant, antiplatelet, and cytoxoxic effects of seven South African plant extracts, namely, Combretum vendae A.E. van Wyk (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae), and Protorhus longifolia (Bernh. Ex C. Krauss) Engl. (Anacardiaceae), were evaluated using established in vitro assays. All the extracts showed comparably low toxicity except for the extract of C. harveyi that showed high hemagluttination assay titer value, which indicates toxicity. The extracts of P. longifolia, K. wilmsii, O. natalitia, L. alata, C. harveyi, and C. vendae exhibited antioxidant properties in the qualitative assay using DPPH. In the quantification of antioxidation using ABTS, only the extracts of P. longifolia, L. alata, and C. vendae showed antioxidant activity with respective TEAC values of 1.39, 1.94, and 2.08. Similarly, in the quantitative DPPH assay, L. alata (EC₅₀, 3.58?±?0.23?µg/mL) and K. wilmsii (EC₅₀, 3.57?±?0.41?µg/mL) did not differ significantly (p?≤?0.05) from the control. K. anthotheca showed a higher EC₅₀ (176.40?±?26.56?µg/mL) value, and differed significantly (p?≤?0.05) from all the other extracts and control. In addition, the extracts of C. vendae and C. harveyi showed significant (p?≤?0.05) antiplatelet activity and did not differ from the control (aspirin) with EC₅₀ of 0.06?±?0.01?µg/mL and 0.19?±?0.00?µg/mL, respectively. Lower EC₅₀ values in the antioxidant and antiplatelet studies are indicative of superior activity of the plant extract against oxidation and platelet aggregation.     

Discovery of novel PDE10 inhibitors by a robust homogeneous screening assay

Aim:

To develop a homogeneous assay for high-throughput screening (HTS) of inhibitors of phosphodiesterase 10 (PDE10).

Methods:

Purified human PDE10 enzyme derived from E coli, [³H]-cAMP and yttrium silicate microbeads were used to develop an HTS assay based on the scintillation proximity assay (SPA) technology. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Preliminary structure-activity relationship (SAR) studies were initiated through limited structural modifications of the hits.

Results:

The IC₅₀ value of the control compound (papaverine) assessed with the SPA approach was comparable and consistent with that reported in the literature. Signal to background (S/B) ratio and Z'' factor of the assay system were evaluated to be 5.24 and 0.71, respectively. In an HTS campaign of 71 360 synthetic and natural compounds, 67 hits displayed reproducible PDE10 inhibition, of which, 8 were chosen as the scaffold for structural modifications and subsequent SAR analysis.

Conclusion:

The homogeneous PDE10 SPA assay is an efficient and robust tool to screen potential PDE10 inhibitors. Preliminary SAR studies suggest that potent PDE10 inhibitors could be identified and developed through this strategy.     

Prospective neurobiological effects of the aerial and root extracts and some pure compounds of randomly selected Scorzonera species

Context: Scorzonera L. species (Asteraceae) are edible and as medicinal plants are used for various purposed in folk medicine.

Objective: The methanol extracts of the aerial parts and roots from 27 Scorzonera taxa were investigated for their possible neurobiological effects.

Materials and methods: Inhibitory potential of the Scorzonera species was tested against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and tyrosinase (TYRO) at 100?µg?mL^?1 using ELISA microtiter assay. Antioxidant activity of the extracts was tested with radical scavenging activity, metal-chelation capacity, ferric- (FRAP), and phosphomolibdenum-reducing antioxidant power (PRAP) assays. Chlorogenic acid, hyperoside, rutin, and scorzotomentosin-4-O-β-glucoside were also screened in the same manner. Total phenol and flavonoid quantification in the extracts were determined spectrophotometrically.

Results: The aerial parts of Scorzonera pisidica (40.25?±?0.74%) and chlorogenic acid (46.97?±?0.82%) displayed the highest TYRO inhibition, while the remaining samples showed only trivial inhibition against cholinesterases (2.08?±?1.35%–25.32?±?1.37%). The same extract of S. pisidica was revealed to be the most potent in scavenging of all three radicals and FRAP assay.

Discussion and conclusion: Out of 27 taxa, S. pisidica, in particular, may deserve further investigation for its neuroprotective potential.     

New chromogenic dipeptide substrate for continuous assay of the d‐alanyl‐d‐alanine dipeptidase VanX required for high‐level vancomycin resistance

Abstract: A direct continuous UV–Vis spectrophotometric assay has been developed for VanX, a d ‐alanyl‐d ‐alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate d ‐alanyl‐α‐(R)‐phenylthio‐glycine d ‐Ala‐d ‐Gly(S‐Ph)‐OH (H‐d Ala‐d Psg‐OH, 5a ). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis–Menten kinetics with a k_cat of 76 ± 5/s and a K_M of 0.83 ± 0.08 mm (k_cat = 46 ± 3/s and K_M = 0.11 ± 0.01 mm for d ‐Ala‐d ‐Ala). Determination of the kinetic parameters of the previously reported mechanism‐based inhibitor d ‐Ala‐d ‐Gly(SΦp‐CHF₂)‐OH (H‐d ‐Ala‐d Pfg‐OH, 5c ) [Araoz, R., Anhalt, E., René, L., Badet‐Denisot, M.‐A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971–15979] using the substrate reported in the present study yielded values of K_irr of 22 ± 1 μm and k_inact of 9.3 ± 0.4/min in good agreement with values previously obtained in our laboratory (K_irr = 30 ± 1 mm ; k_inact = 7.3 ± 0.3/min). In addition, inhibition by the competing substrate d ‐Ala‐d ‐Ala resulted in determination of a K_i = 70 ± 6 μm close to the previously reported K_M value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.     

Antioxidant and anti-cholinesterase activity of Sargassum wightii

Abstract

Context. Sargassum has been used in traditional Chinese medicine since the eighth century AD to treat goiter. Sargassum wightii Greville (Sargassaceae) is a major source of alginic acid used widely in food and drug industries.

Objective: To evaluate the anti-Alzheimer potential of S. wightii through evaluation of antioxidant and cholinesterase inhibitory activities.

Materials and methods: Successive extraction was done using solvents of varying polarity. Solvent extracts (100–500?µg/mL) were employed for all the antioxidant assays. Free radical scavenging activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl, OH?, H₂O₂ radical scavenging assay. The reducing power of the seaweed was evaluated by ferric reducing antioxidant power and reducing power assay. Cholinesterase (ChE) inhibitory activity was evaluated and the Km, Vmax and Ki were calculated. Further, compound characterization was done by GC-MS analysis.

Results: The non-polar extracts were found to possess significant antioxidant activity. At 100?μg/mL, petroleum ether, hexane, benzene and dichloromethane extracts showed significant ChE inhibitory activity with IC₅₀ values of 19.33?±?0.56, 46.81?±?1.62, 27.24?±?0.90, 50.56?±?0.90?µg/mL, respectively, for AChE, and 17.91?±?0.65, 32.75?±?1.00, 12.98?±?0.31, 36.16?±?0.64?µg/mL, respectively, for BuChE. GC-MS reveals that 1,2-benzenedicarboxylic acid, diisooctyl ester is the major compound present in dichloromethane extract of S. wightii. The mode of inhibition exhibited by dichloromethane extract against the cholinesterases was found to be competitive type.

Discussion and conclusion: The presence of high amount of terpenoids could be the possible reason for its potential antioxidant and ChE inhibitory activity.     

Direct analysis of traditional Chinese medicines using surface‐enhanced raman scattering (SERS)

Surface‐Enhanced Raman Scattering (SERS) spectrometry provides an excellent tool to characterize chemical constituents in Traditional Chinese Medicines (TCMs) without requiring separation and extraction procedures. This study involved the use of SERS to analyze two TCMs, namely Coptis chinensis and Phellodendron amurense, and their main active constituent, berberine. Using silver nanospheres as SERS‐active probes, the decoctions of two raw TCMs and their counterfeits were analyzed. Density functional theory (DFT) was used to calculate the expected Raman spectrum of berberine, and liquid chromatography‐ mass spectrometry (LC‐MS) was used as a comparative technique to quantify the amount of berberine in the samples. The results of the SERS measurements were consistent with the results of DFT calculations and LCMS analyses. To our knowledge, this is the first time that the potential of SERS was demonstrated as a sensitive, rapid, and non‐destructive method to qualitatively and quantitatively analyze the active constituents in raw TCM products. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of antihemolytic and antioxidant activities of Geranium tuberosum subsp. tuberosum with in vitro models

Context: There are 33 Geranium species growing in Turkey characterized by the presence of polyphenolic compounds. Some Geranium (Geraniaceae) species are used as antidiabetics, hemostatics, antihemorrhoidals, antidiarrheics and for the treatment of pain, fevers, and gastrointestinal ailments, or are consumed as food.

Objective: The in vitro antioxidant activity and antihemolytic effect of ethyl acetate (EtOAc), n-butanol (BuOH), methanol (MeOH) and water extracts of Geranium tuberosum L. subsp. tuberosum (Geraniaceae), a medicinal food plant, have been evaluated.

Materials and methods: The two antioxidant enzyme activities of human erythrocyte, namely superoxide dismutase (SOD) and catalase (CAT), after in vitro incubation with the extracts, were examined in order to see whether the observed effects are related to altered enzymatic efficiency. Reduced glutathione (GSH) levels were also measured as oxidative stress marker. Antihemolytic activity of extracts was shown by hemolysis assay in erythrocytes. Furthermore, total phenolic content of extracts was measured by Folin–Ciocalteu method.

Results: All extracts enhanced GSH levels, and the activity of SOD and CAT. The EtOAc extracts seems to be the most potent antioxidant at 100 µg/mL (SOD activity 173.736?±?8.33, CAT activity 133.218?±?3.31, GSH level 2.264?±?2.21). However, apart from the MeOH extracts at 100 µg/mL (68.699?±?3.93), they didn’t increase the resistance of erythrocytes to H₂O₂ induced cytotoxicity. Therefore, while a significant antioxidant effect was observed in these samples, antihemolytic effect was not determined.

Discussion and conclusion: The title plant has shown high antioxidant activity without cytotoxicity up to 100 µg/mL, thus could be a potent source as natural antioxidant.     

Preclinical Drug Metabolism in the Age of High-Throughput Screening: An Industrial Perspective

With the advent of genomics, combinatorial paradigms and high-throughput screen (HTS)-based pharmacological testing, the number of compounds flowing through the discovery pipeline is likely to escalate. At the same time, with increased knowledge of the human drug-metabolizing enzymes and the availability of in vitro absorption-metabolism (AM) models, Preclinical Drug Metabolism is poised to meet the challenges of HTS. In order to be successful, however, a rational HTS strategy (vs. serendipitous HTS) has to be employed. Such a strategy is based on automation, validation and integration of in vitroAM models and database management (AVID). A generalized strategy for rational (AVID-based) HTS in Preclinical Drug Metabolism is described briefly.     

Effects of 20-hydroxyecdysone, Leuzea carthamoides extracts, dexamethasone and their combinations on the NF-κB activation in HeLa cells

Objectives The plant steroid 20‐hydroxyecdysterone (20E) and 20E‐containing extracts from Leuzea carthamoides (Willd.) DC are sold with claims of anabolic and immunomodulatory effects. Yet their effect on the activation of nuclear factor kappa B (NF‐κB), a key player in immune response and cell fate, and their influence on the NF‐κB‐inhibiting activity of steroidal anti‐inflammatory drugs is still unknown. Methods The ability of 20E, Leuzea extracts and selected steroidal/non‐steroidal anti‐inflammatory drugs to influence the activation of NF‐κB was explored using, as the experimental model, human cervical cancer HeLa‐IL‐6 cells stably transfected with an IL‐6‐bound reporter gene. Effects on cell viability and proliferation were monitored (MTT assay). HPLC‐DAD was used to establish links between chemical patterns of Leuzea extracts and their bioactivities. Key findings 20E inhibited NF‐κB activation (IC50 31.8 µm ) but was less active than other plant metabolites (xanthohumol 3.8 µm , withaferin A 1.4 µm ). Leuzea extracts with high content in 20E had a fair activating effect, but in contrast, some extracts with low 20E content significantly inhibited NF‐κB activation at IC50s ranging from 3.5 to 6.2 µg/ml. Combination tests confirmed that 20E does not explain the NF‐κB modulation achieved by Leuzea extracts. The extracts but not 20E itself showed a significant modulation of the NF‐κB inhibitory effect of dexamethasone. Conclusions 20E is unlikely a major player in the NF‐κB inhibitory effects displayed by some Leuzea extracts in vitro. If confirmed in vivo, caution should prevail towards marketed Leuzea extracts that are non‐standardised or standardised on 20E only, since different starting materials and extracts may even cause opposite effects. More importantly, our results indicate the interaction potential of Leuzea with steroidal anti‐inflammatory drugs.     

流感病毒神经氨酸酶抑制剂筛选模型的建立和应用

目的建立适用于高通量筛选的流感病毒神经氨酸酶(neuraminidase,NA)抑制剂筛选模型。方法从甲型及乙型流感病毒中制备神经氨酸酶,以2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid(MUNANA)作为底物,建立检测神经氨酸酶活性的荧光测定法及其抑制剂体外筛选方法,用高通量筛选系统对1 200个化合物与提取物进行初筛。结果神经氨酸酶酶促反应以pH 3.5,二价阳离子浓度为2～6 mmol·L^-1及37℃孵育时酶活性最佳;甲、乙型流感病毒不同株神经氨酸酶的米氏常数(Km)的范围为(4.89～5.94) μmol·L^-1;初筛发现12个化合物对流感病毒神经氨酸酶有可重复的抑制活性。结论优化了神经氨酸酶反应体系,建立的体外模型可用于抗甲、乙型流感病毒药物的高通量筛选及酶抑制动力学的研究。     

Antioxidant activity and total phenolic content of methanol extracts of Ixora coccinea

Objective: To investigate the in vitro antioxidant activity of methanol extracts of Ixora coccinea L. (Rubiaceae) flower, leaf and stem.

Materials and methods: The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, total antioxidant capacity (TAC) and xanthine oxidase inhibition assay were carried out to evaluate the antioxidant potential of the extract. The IC₅₀ values were calculated for the DPPH and xanthine oxidase assays in order to evaluate the antioxidant efficiency of each of the I. coccinea extracts. The phenol contents were also determined.

Results: I. coccinea flowers revealed the best antioxidant property, presenting much lower IC₅₀ value (6.6?mg/mL for DPPH assay). The flower extract showed a significantly higher antioxidant capacity compared to the other extracts. Furthermore, the highest phenolic content (polyphenols) was found in the flower extract (210.55?±?6.31 µg GAE/mg extract). Moreover, I. coccinea extracts scavenged the superoxide radical generated by the xanthine/xanthine oxidase system. The xanthine oxidase inhibition activity was in the order of allopurinol > leaf > flower > stem with the percentage of inhibition ranged from 39.7% to 77.3% for the plant parts investigated. The highest phenolic contents (polyphenols) were found in the flower extracts (210.55?±?6.31 µg GAE/mg extract).

Conclusions: I. coccinea could be considered as a potential source of natural antioxidant.     

Novel endomorphin‐2 analogs with μ‐opioid receptor antagonist activity

Abstract: A series of position 4‐substituted endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) analogs containing 3‐(1‐naphthyl)‐alanine (1‐Nal) or 3‐(2‐naphthyl)‐alanine (2‐Nal) in l ‐ or d ‐configuration, was synthesized. The opioid activity profiles of these peptides were determined in the μ‐opioid receptor representative binding assay and in the Guinea‐Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot‐plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the μ‐opioid receptor was reduced compared with endomorphin‐2. The two most potent analogs were [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]endomorphin‐2, with IC₅₀ values 14 ± 1.25 and 19 ± 2.1 nm , respectively, compared with 1.9 ± 0.21 nm for endomorphin‐2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot‐plate test. Antinociception induced by endomorphin‐2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]‐endomorphin‐2, indicating that these analogs were μ‐opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 μg per animal naloxone almost completely inhibited antinociceptive action of endomorphin‐2, while [d ‐1‐Nal⁴]endomorphin‐2 in about 46%.     

Unravelling the cardiovascular effects induced by α‐terpineol: A role for the nitric oxide–cGMP pathway

1. α‐Terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by α‐terpineol in rats. 2. In normotensive rats, administration of α‐terpineol (1, 5, 10, 20 and 30 mg/kg, i.v.) produced a dose‐dependent hypotension (?10 ± 3, ?20 ± 8, ?39 ± 16, ?52 ± 21 and ?57 ± 23 mmHg, respectively; n = 5) followed by tachycardia. The hypotensive responses to 1, 5, 10, 20 and 30 mg/kg, i.v., α‐terpineol were significantly attenuated following the administration of N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME; 20 mg/kg, i.v.; ?2 ± 1, ?5 ± 2, ?7 ± 3, ?22 ± 9 and ?22 ± 10 mmHg, respectively; P < 0.05; n = 5). 3. In 10 μmol/L phenylephrine (PE)‐precontracted mesenteric artery rings, α‐terpineol (10^?12 to 10^?5 mol/L) caused a concentration‐dependent relaxation (maximum relaxation 61 ± 6%; n = 7). After removal of the endothelium, the vasorelaxation elicited by α‐terpineol was attenuated (maximum relaxation 20 ± 1%; P < 0.05; n = 7). In addition, vasorelaxation induced by α‐terpineol in rings pretreated with 100 or 300 μmol/L l ‐NAME, 30 μmol/L hydroxocobalamin or 10 μmol/L 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one was attenuated (maximum relaxation 18 ± 3, 23 ± 3, 24 ± 7 and 21 ± 1%, respectively; n = 6; P < 0.05). 4. Furthermore, in a rabbit aortic endothelial cell line, 10^?6, 10^?5 and 10^?4 mol/L α‐terpineol induced concentration‐dependent increases in nitric oxide (NO) levels (12 ± 6, 18 ± 9 and 34 ± 12%Δ fluorescence, respectively; n = 3). 5. In conclusion, using combined functional and biochemical approaches in the present study, we were able to demonstrate that α‐terpineol‐induced hypotension and vasorelaxation are mediated, at least in part, by the endothelium, most likely via NO release and activation of the NO–cGMP pathway.     

Antioxidant,antimicrobial and wound healing properties of Struthanthus vulgaris

Context: Struthanthus vulgaris (Vell.) Mart. (Loranthaceae) has been widely used in traditional medicine in Brazil to bathe wounds.

Objective: The objective of this study is to investigate the in vitro wound healing effects, together with the antioxidant and antimicrobial activities of S. vulgaris leaf and branch extracts.

Material and methods: Ethanol leaf and branch extracts of S. vulgaris were investigated at 1–100?µg/ml concentrations in the scratch assay after 14?h. Antioxidant activity was investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay, and the antibacterial activity was tested at concentrations up to 1000?µg/ml against Gram-positive and Gram-negative bacteria by the microdilution test after 24?h. The total phenolic and flavonoid contents were determined by colorimetric methods.

Results: Struthanthus vulgaris leaf and branch extracts at 100?µg/ml concentration stimulated migration and proliferation of fibroblasts and enhanced cell numbers by 56.2% and 18.6%, respectively. Antioxidant activity exhibited IC₅₀ values of 24.3 and 18.9?µg/ml for the leaf and branch extracts, respectively. The ethanol leaf extract showed antimicrobial activity against the Gram-positive Staphylococcus mutans and Staphylococcus aureus bacteria, exhibiting minimum inhibitory concentration values of 125 and 500?µg/ml, respectively. An appreciable total phenolic content in the leaves (813.6?±?2.7?mg/g) and branches (462.8?±?9.6?mg/g), and relatively low concentration of flavonoids in the leaves (13.3?±?4.3?mg/g) and branches (1.9?±?0.2?mg/g), was detected.

Discussion and conclusion: The antioxidant and antibacterial activities, together with the strong ability to stimulate proliferation and migration of fibroblasts, provide some support for the traditional use of S. vulgaris.     

Effect of earthworm active protein on fibroblast proliferation and its mechanism

Context: Earthworms have been used as a traditional medicine in China from thousands of years. In recent years, research has demonstrated that earthworm extracts might promote wound healing; however, its mechanism is still unknown.

Objective: The study investigates the mechanism and effects of earthworm active protein (EAP), on mouse embryonic fibroblast (NIH/3T3) proliferation.

Materials and methods: The effects of earthworm active protein (EAP) in different concentrations (0, 25, 50, 100, 150, and 200?μg/mL) on NIH3T3 cell were detected by the MTT and Brdu incorporation assay (50, 100, and 150 μg/mL). The effects of EAP (37.5, 75, and 150?μg/mL) on the cell cycle were detected by flow cytometry. The cell signaling pathways of EAP-promoting NIH3T3 cell proliferation were studied by the MTT and Western blot by using different signaling pathway inhibitors.

Results: The results showed that EAP (50, 100, and 150 μg/mL) could promote NIH3T3 fibroblasts proliferation (36.4 ± 4.4%, 59.1 ± 4.9%, and 71.5 ± 5.7%). The mechanism of EAP promoting NIH3T3 cell proliferation should be as follows: EAP elevated cyclin D1 expression by activating MEK/ERK signaling pathway, and then promoted cell cycle from G1 to S phase, finally caused the proliferation of NIH3T3 cell. PI3K signaling pathway may be the upstream of MEK/ERK signaling pathway.

Discussion and conclusion: The study demonstrates that EAP is effective in promoting effects on proliferation and migration activity of NIH3T3 cell, and the proliferation activity of EAP on NIH3T3 cell may be achieved through the PI3K→Rac→PAK→MEK signaling pathway.     

Isoenzyme‐ and Allozyme‐Specific Inhibitors: 2,2′‐Dihydroxybenzophenones and Their Carbonyl N‐Analogues that Discriminate between Human Glutathione Transferase A1‐1 and P1‐1 Allozymes

The selectivity of certain benzophenones and their carbonyl N‐analogues was investigated towards the human GSTP1‐1 allozymes A, B and C involved in MDR. The allozymes were purified from extracts derived from E. coli harbouring the plasmids pEXP5‐CT/TOPO‐TA‐hGSTP1*A, pOXO4‐hGSTP1*B or pOXO4‐hGSTP1*C. Compound screening with each allozyme activity indicated three compounds with appreciable inhibitory potencies, 12 and 13 with P1‐1A 62% and 67%, 11 and 12 with P1‐1C 51% and 70%, whereas that of 15 fell behind with P1‐1B (41%). These findings were confirmed by IC₅₀ values (74–125 μm ). Enzyme inhibition kinetics, aided by molecular modelling and docking, revealed that there is competition with the substrate CDNB for the same binding site on the allozyme (K_i(13/A) = 63.6 ± 3.0 μm , K_i(15/B) = 198.6 ± 14.3 μm , and K_i(11/C) = 16.5 ± 2.7 μm ). These data were brought into context by an in silico structural comparative analysis of the targeted proteins. Although the screened compounds showed moderate inhibitory potency against hGSTP1‐1, remarkably, some of them demonstrated absolute isoenzyme and/or allozyme selectivity.