In vitro study on α-amylase inhibitory activity of an Indian medicinal plant, Phyllanthus amarus

Objective:

The objective of this study was to evaluate the α-amylase inhibitory activity of different extracts of Phyllanthus amarus against porcine pancreatic amylase in vitro.

Materials and Methods:

The plant extracts were prepared sequentially with ethanol, chloroform, and hexane. Each extract was evaporated using rotary evaporator, under reduced pressure. Different concentrations (10, 20, 40, 60, 80, and 100 μg/mL) of each extract were made by using dimethyl sulfoxide (DMSO) and subjected to α-amylase inhibitory assay using starch azure as a substrate. The absorbance was read at 595 nm using spectrophotometer. Using this method, the percentage of α-amylase inhibitory activity and IC₅₀values of each extract was calculated.

Results:

The chloroform extract failed to inhibit α-amylase activity. However, the ethanol and hexane extracts of P. amarus exhibited appreciable α-amylase inhibitory activity with an IC50 values 36.05 ± 4.01 μg/mL and 48.92 ± 3.43 μg/mL, respectively, when compared with acarbose (IC₅₀value 83.33 ± 0.34 μg/mL).

Conclusion:

This study supports the ayurvedic concept that ethanol and hexane extracts of P. amarus exhibit considerable α-amylase inhibitory activities. Further, this study supports its usage in ethnomedicines for management of diabetes.     

Improved Pharmacokinetics of Aceclofenac Immediate Release Tablets Incorporating its Inclusion Complex with Hydroxypropyl-β-Cyclodextrin

The present investigation reports the various pharmacokinetic parameters of immediate release aceclofenac tablets incorporating its inclusion complex with hydroxypropyl-β-cyclodextrin. The tablets were prepared using aceclofenac: hydroxypropyl-β-cyclodextrin in a 1:1 molar ratio by the direct compression method (TKN). The results were compared with those of the marketed brand (MKT) and pure drug (TAC). The P-values indicated that mean plasma concentrations were significantly different among all three formulations administered (P<0.05, P<0.01). TKN showed significantly higher plasma levels when compared to the pure drug (P<0.01). The C_max and AUC_(0-∞) of TKN were significantly higher (P<0.05) compared to the pure drug and marketed formulation. Furthermore, the first-order overall elimination rate constant (K_el) of TKN was also significantly higher (P<0.05) compared to the pure drug and its marketed formulation. These results suggested that tablets prepared by incorporating the AC-HPβCD inclusion complex (TKN) would provide a more rapid onset of pharmacological effects in comparison to the marketed formulation and pure drug.     

Antileukemic activity of the leaf extract of Bischofia javanica blume on human leukemic cell lines

Objective:

Leaves of Bichofia javanica (BJ) have been traditionally used for many ailments including cancer. In the present study, antileukemic activity of the leaf extract was evaluated on human leukemic cell lines.

Materials and Methods:

Human leukemic cell lines U937, K562, and HL60 were purchased from National Facility for Animal Tissue and Cell Culture, Pune, India. The cells were routinely maintained in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum. Cultures were maintained at 37ºC in a humidified atmosphere containing 5% CO₂ in air. The methanol extract of BJ (MEBJ) was dissolved in PBS and used at the concentrations of 5, 10, and 15 μg/ml for cell viability and cytotoxicity studies (MTT assay). Cell counts were made in quadruplicate samples at the interval of 24, 48, and 72 h and cytarabine (20 μg/ml) served as standard drug. The apoptotic pathway of cytotoxicity was assessed by DNA agarose gel electrophoresis technique and confirmed by fluorescence and confocal microscopic methods at the concentration of 10 μg/ml.

Results:

MEBJ showed significant cytotoxicity (P<0.001) in leukemic cell lines in the in-vitro cell proliferation assay. IC₅₀ of MEBJ was very low (3.5 μg/ml) at 72 h in the HL60 cell line. The apoptotic pathway of cytotoxicity was observed at 10 μg/ml of MEBJ by the fragmented DNA pattern in the apoptosis assay, chromatin condensation, and apoptotic body formation as revealed in the fluorescence and confocal microscopic studies.

Conclusion:

The present findings support the ethno-medicinal use of BJ for cancer by mediating through the apoptosis pathway.     

Antiplasmodial Potential of the African Mistletoe: Agelanthus dodoneifolius Polh and Wiens

Preparations of Agelanthus dodoneifolius have been used in the traditional Nigerian medicine to treat malaria and this practice has remained till date without scientific validation. The antiplasmodial property of the water extract of Agelanthus dodoneifolius was evaluated in vivo and in vitro against Plasmodium berghei and clinical isolates of Plasmodium falciparum, respectively. There was a dose-dependent inhibition of parasitaemia in the in vivo antiplasmodial tests likewise, the in vitro screening demonstrated a strong and concentration-dependent activity (21.54 μg/ml < IC₅₀ < 50 μg/ml) of the extract against the clinical isolates of Plasmodium falciparum. The phytochemical analysis revealed the presence of tannins, saponins, sterols, glycosides, phenols, anthraquinones, terpenes, reducing sugars and resins. It also showed a strong free-radical scavenging activity on 2, 2-diphenyl-2-picrylhydrazyl. The oral median lethal dose (LD₅₀) in mice was estimated to be greater than 5000 mg/kg. Our results evidence that Agelanthus dodoneifolius may contain biologically active principles those are relevant in the treatment of malaria, thus supporting further studies of its active components.     

Vasorelaxant and antihypertensive effects of formononetin through endothelium-dependent and -independent mechanisms

Aim:

To investigate the mechanisms underlying the vasorelaxant effect of formononetin, an O-methylated isoflavone, in isolated arteries, and its antihypertensive activity in vivo.

Methods:

Arterial rings of superior mesenteric arteries, renal arteries, cerebral basilar arteries, coronary arteries and abdominal aortas were prepared from SD rats. Isometric tension of the arterial rings was recorded using a myograph system. Arterial pressure was measured using tail-cuff method in spontaneously hypertensive rats.

Results:

Formononetin (1–300 μmol/L) elicited relaxation in arteries of the five regions that were pre-contracted by KCl (60 mmol/L), U46619 (1 μmol/L) or phenylephrine (10 μmol/L). The formononetin-induced relaxation was reduced by removal of endothelium or by pretreatment with L-NAME (100 μmol/L). Under conditions of endothelium denudation, formononetin (10, 30, and 100 μmol/L) inhibited the contraction induced by KCl and that induced by CaCl₂ in Ca²⁺-free depolarized medium. In the absence of extracellular Ca²⁺, formononetin (10, 30, and 100 μmol/L) depressed the constriction caused by phenylephrine (10 μmol/L), but did not inhibit the tonic contraction in response to the addition of CaCl₂ (2 mmol/L). The contraction caused by caffeine (30 mmol/L) was not inhibited by formononetin (100 μmol/L). Formononetin (10 and 100 μmol/L) reduced the change rate of Ca²⁺-fluorescence intensity in response to KCl (50 mmol/L). In spontaneously hypertensive rats, formononetin (5, 10, and 20 mg/kg) slowly lowered the systolic, diastolic and mean arterial pressure.

Conclusion:

Formononetin causes vasodilatation via two pathways: (1) endothelium-independent pathway, probably due to inhibition of voltage-dependent Ca²⁺ channels and intracellular Ca²⁺ release; and (2) endothelium-dependent pathway by releasing NO. Both the pathways may contribute to its antihypertensive effect.     

Studies of Antiviral Activity and Cytotoxicity of Wrightia tinctoria and Morinda citrifolia

Different extracts of leaf parts of Wrightia tinctoria and fruit powder of Morinda citrifolia have been studied against replication of HIV-1(IIIB) in MT-4 cells and HCV in Huh 5.2 cells. Chloroform extract of Wrightia tinctoria exhibited a maximum protection of 48% against the cytopathic effect of HIV-1(IIIB) in MT-4 cells. Fruit juice of Morinda citrifolia exhibited a displayed marked cytotoxic activity in lymphocyte (MT-4) cells (CC50: 0.19 mg/ml). The 50% effective concentration for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells by Morinda citrifolia was 0.98 μg/ml and by chloroform extract of Wrightia tinctoria was 10 μg/ml. The concentration that reduced the growth of exponentially proliferating Huh 5-2 cells by 50% was greater than 50 μg/ml.     

In vitro and in vivo Evaluation of CYP1A Interaction Potential of Terminalia Arjuna Bark

Terminalia arjuna Wight and Arn. (Combretaceae) is a tree having an extensive medicinal potential in cardiovascular disorders. Triterpenoids are mainly responsible for cardiovascular properties. Aqueous, hydroalcoholic and alcoholic extract of T. arjuna, arjunic acid and arjungenin were examined for their potential to inhibit CYP1A enzyme in rat and human liver microsomes. IC₅₀ values of aqueous, hydroalcoholic and alcoholic extract of T. arjuna was found to be 11.4, 28.9 and 44.6 μg/ml in rat liver microsomes while 30.0, 29.7 and 39.0 μg/ml in human liver microsomes, respectively for CYP1A. However IC₅₀ values of arjunic acid and arjungenin for both rat liver microsomes and human liver microsomes were found to be >50 μM. Arjunic acid and arjungenin did not show inhibition of CYP1A enzyme up to concentrations of 50 μM. These in vitro data indicate that Terminalia arjuna extracts contain constituents that can potently inhibit the activity of CYP1A, which could in turn lead to undesirable pharmacokinetic drug–herb interactions in vivo. Based on the in vitro data, interaction potential of the aqueous extract of Terminalia arjuna orally in rats was investigated. A probe substrate, phenacetin, was used to index the activity of CYP1A. In vivo pharmacokinetic study of coadministration of aqueous extract of Terminalia arjuna and phenacetin, revealed that the aqueous extract did not lead to any significant change in the pharmacokinetic parameters of phenacetin as compared with control group. Though there was no in vivo–in vitro correlation, drug interactions could arise with drugs having a narrow therapeutic range and extensively cleared by CYP1A enzyme, which could lead to undesirable side effects.     

Formulation and Evaluation of Rizatriptan Benzoate Mouth Disintegrating Tablets

The present investigation deals with development of mouth disintegrating tablets of rizatriptan benzoate to produce the intended benefits. Mouth disintegrating tablets of rizatriptan benzoate were prepared using superdisintegrants crospovidone, carboxymethylcellulose calcium, Indion 414 and Indion 234 using the direct compression method. The tablets prepared were evaluated for thickness, uniformity of weight, content uniformity, hardness, friability, wetting time, in vitro and in vivo disintegration time, mouth feel, in vitro drug release and assay by high performance liquid chromatography. The tablets disintegrated in vitro and in vivo within 4 to 7 s and 6 to 19 s, respectively. Almost 90% of drug was released from all formulations within 20 min. The drug release from the formulations followed first order kinetics. Stability studies of the tablets at 40±2°/75%±5% RH for 1 mo showed non significant drug loss. The formulation containing combination of crospovidone and Indion 234 was found to give the best results. Apart from fulfilling all official and other specifications, the tablets exhibited higher rate of release.     

Preparation and In Vitro Characterization of Mucoadhesive Hydroxypropyl Guar Microspheres Containing Amlodipine Besylate for Nasal Administration

Amlodipine besylate microspheres for intranasal administration were prepared with an aim to avoid first-pass metabolism, to achieve controlled blood level profiles and to improve therapeutic efficacy. Hydroxypropyl Guar, a biodegradable polymer, was used in the preparation of microspheres by employing water in oil emulsification solvent evaporation technique. The formulation variables were drug concentration, emulsifier concentration, temperature, agitation speed and polymer concentration. All the formulations were evaluated for particle size, particle shape and surface morphology by scanning electron microscopy, percentage yield, drug entrapment efficiency, in vitro mucoadhesion test, degree of swelling and in vitro drug diffusion through sheep nasal mucosa. The microspheres obtained were free flowing, spherical and the particles ranged in size from 13.4±2.38 μm to 43.4±1.92 μm very much suitable for nasal delivery. Increasing polymer concentration resulted in increased drug entrapment efficiency and increased particle size. Amlodipine besylate was entrapped into the microspheres with an efficiency of 67.2±1.18 % to 81.8±0.64 %. The prepared microspheres showed good mucoadhesion properties, swellability and sustained the release of the drug over a period of 8 h. The data obtained were analysed by fitment into various kinetic models; it was observed that the drug release was matrix diffusion controlled and the release mechanism was found to be non-Fickian. Stability studies were carried out on selected formulations at 5±3°, 25±2°/60±5% RH and 40±2°/75±5% RH for 90 days. The drug content was observed to be within permissible limits and there were no significant deviations in the in vitro mucoadhesion and in vitro drug diffusion characteristics.     

In vitro Antioxidant and Antibacterial Activities of Methanol Extract of Kyllinga nemoralis

The present study was designed to evaluate the antioxidant and antibacterial activity of methanol extract of Kyllinga nemoralis. Six different in vitro antioxidant assays including 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radical, superoxide anion radical, hydrogen peroxide radical, ferric reducing antioxidant power assay and reducing power were carried out to ensure the scavenging effect of the plant on free radicals. In addition, total antioxidant capacity assay, total phenolic contents, tannins, flavonoids and flavonol contents of the plant were also analysed by the standard protocols. Kyllinga nemoralis exhibited high antioxidant activity on 2,2-diphenyl-1-picrylhydrazyl assay (IC₅₀= 90 μg/ml), superoxide radical scavenging assay (IC₅₀= 180 μg/ml) and hydrogen peroxide radical scavenging assay (IC₅₀= 200 μg/ml), compared with standards. These observations provide comprehensible supporting evidence for the antioxidant potential of the plant extract. Reducing power (IC₅₀= 213.16 μg/ml) and hydroxyl radical scavenging activity (IC₅₀= 223 μg/ml) of the plant extract was remarkable. The methanol extract of K. nemoralis exhibited significant antimicrobial activity against Gram-positive human pathogenic bacteria. Standard in vitro antioxidant assays assessed the electron donating ability of the plant extract in scavenging free radicals. The inhibitory effect of the plant extract against bacterial pathogens may be due to the presence of phytochemicals. Thus, the results suggest that Kyllinga nemoralis is a potential source of antioxidants and could serve as the base for drug development.     

Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G₂/M arrest

Aim:

To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms.

Methods:

The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis.

Results:

Genipin reduced the viability of K562 cells with an IC₅₀ value of approximately 250 μmol/L. Genipin 200–400 μmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 μmol/L significantly increased the caspase 3 activity from 8–24 h and arrested the cells in the G₂/M phase. After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200–500 μmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 μmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L.

Conclusion:

These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.     

Design and Evaluation of Niacin Microspheres

Present study aims to prepare and evaluate niacin microspheres. Niacin-ethyl cellulose microspheres were prepared by water-in-oil-in-oil double emulsion solvent diffusion method. Spherical, free flowing microspheres having an entrapment efficiency of 72% were obtained. The effect of polymer-drug ratio, surfactant concentration for secondary emulsion process and stirring speed of emulsification process were evaluated with respect to entrapment efficiency, in vitro drug release behavior and particle size. FT-IR and DSC analyses confirmed the absence of drug-polymer interaction. The in vitro release profile could be altered significantly by changing various processing and formulation parameters to give a controlled release of drug from the microspheres. The percentage yield was 85%, particle size range was 405 to 560 μm. The drug release was controlled for 10 h. The in vitro release profiles from optimized formulations were applied on various kinetic models. The best fit with the highest correlation coefficient was observed in Higuchi model, indicating diffusion controlled principle. The in vitro release profiles of optimized formulation was studied and compared with commercially available niacin extended release formulation.     

In Vitro Antileishmanial,Trypanocidal, and Mammalian Cell Activities of Diverse N,N′ -Dihetaryl Substituted Diamines and Related Compounds

The leishmaniasis and Chagas diseases constitute a serious public health problem worldwide with few and ineffective treatment options. The search for new antiparasitic candidates at the initial steps of drug discovery and development is still necessary. The synthesis of 22 de novo synthetized N,N′-dihetaryl-alkyldiamine derivatives and in vitro antiparasitic activity were evaluated for the first time against intracellular and extracellular forms of Leishmania (Leishmania) infantum, L. (Viannia) panamensis, L. (Leishmania) amazonensis, and Trypanosoma cruzi. Additionally, the toxicity on mammalian cells was determined. Some of these substituted N,N′-diamines (25–35 % of the tested compounds) showed interesting results against free-living forms of parasites with activities at the inhibitory concentration (IC₅₀) level of 1.96 to 28.83 μM for L. (L.) infantum promastigotes and IC₅₀ of 0.02 to 5.31 μM for T. cruzi epimastigotes. No activity at the IC₅₀ level on intracellular amastigotes of T. cruzi was observed. However, N¹,N²-dibenzylethane-1,2-diamine 5a revealed an important activity against the intracellular amastigotes of L. infantum (IC₅₀ 25.42 μM ±0.33) and L. panamensis (IC₅₀ 58.20 μM ±3.23), while their analogue N¹,N⁴-dibenzylbutane-1,4-diamine 5c resulted in activity only against L. panamensis (IC₅₀ 11.19 μM ±0.20) without toxicity on Vero and THP-1 mammalian cells. The active compounds against intracellular parasites with low toxicity in mammalian cells may be considered for future studies in experimental models.     

Effects of excitatory and inhibitory neurotransmission on motor patterns of human sigmoid colon in vitro

Background and purpose:

To characterize the in vitro motor patterns and the neurotransmitters released by enteric motor neurons (EMNs) in the human sigmoid colon.

Experimental approach:

Sigmoid circular strips were studied in organ baths. EMNs were stimulated by electrical field stimulation (EFS) and through nicotinic ACh receptors.

Key results:

Strips developed weak spontaneous rhythmic contractions (3.67±0.49 g, 2.54±0.15 min) unaffected by the neurotoxin tetrodotoxin (TTX; 1 μM). EFS induced strong contractions during (on, 56%) or after electrical stimulus (off, 44%), both abolished by TTX. Nicotine (1–100 μM) inhibited spontaneous contractions. Latency of off-contractions and nicotine responses were reduced by N^G-nitro-L-arginine (1 mM) and blocked after further addition of apamin (1 μM) or the P2Y₁ receptor antagonist MRS 2179 (10 μM) and were unaffected by the P2X antagonist NF279 (10 μM) or α-chymotrypsin (10 U mL⁻¹). Amplitude of on- and off-contractions was reduced by atropine (1 μM) and the selective NK₂ receptor antagonist Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH₂ (1 μM). MRS 2179 reduced the amplitude of EFS on- and off-contractions without altering direct muscular contractions induced by ACh (1 nM–1 mM) or substance P (1 nM–10 μM).

Conclusions and implications:

Latency of EFS-induced off-contractions and inhibition of spontaneous motility by nicotine are caused by stimulation of inhibitory EMNs coreleasing NO and a purine acting at muscular P2Y₁ receptors through apamin-sensitive K⁺ channels. EFS-induced on- and off-contractions are caused by stimulation of excitatory EMNs coreleasing ACh and tachykinins acting on muscular muscarinic and NK₂ receptors. Prejunctional P2Y₁ receptors might modulate the activity of excitatory EMNs. P2Y₁ and NK₂ receptors might be therapeutic targets for colonic motor disorders.     

Sensitive assay for measurement of volatile borneol,isoborneol, and the metabolite camphor in rat pharmacokinetic study of Borneolum (Bingpian) and Borneolum syntheticum (synthetic Bingpian)

Aim:

Both Borneolum (Chinese name Bingpian; dextrorotatory borneol) and Borneolum syntheticum (synthetic Bingpian; a mixture of optically inactive borneol and isoborneol) have been used for medicinal purposes in Chinese traditional medicine. The aim of this study was to develop a sensitive assay for measuring volatile ingredients borneol, isoborneol, and their metabolite camphor in pharmacokinetic study of Bingpian.

Methods:

Rat plasma samples were prepared using liquid-liquid microextraction: 70 μL of plasma sample (containing 125 nmol/L naphthalene as the internal standard) was extracted with 35 μL of n-hexane. The resulting n-hexane extract (20 μL) was introduced into a gas chromatography/mass spectrometry system using programmable temperature vaporizing-based large-volume injection. The assay was validated to demonstrate its reliability for the intended use. Using this assay, pharmacokinetic studies of Bingpian, synthetic Bingpian, and Fufang-Danshen tablets (containing synthetic Bingpian) were conducted in rats.

Results:

The extraction efficiency for the analytes and the internal standard from plasma was almost constant with decrease in n-hexane-to-plasma volume ratio, thus enabling a small volume of extracting solvent to be used for sample preparation, and enhancing the assay sensitivity. The lower quantification limit for measuring borneol, isoborneol, and camphor in plasma was 0.98 nmol/L, which was 33–330 times more sensitive than those reported earlier for Bingpian and synthetic Bingpian. The applicability of the miniaturized liquid-liquid extraction technique could be extended to measure other volatile and nonvolatile medicinal compounds in biomatrices, which can be predicted according to the analytes'' octanol/water distribution coefficient (logD) and acid dissociation constant (pK_a).

Conclusion:

This assay is sensitive, accurate and free of matrix effects, and can be applied to pharmacokinetic studies of Bingpian, synthetic Bingpian, and Bingpian-containing herbal products.     

Formulation and bioequivalence of two valsartan tablets after a single oral administration

The aim of this study is to assess the quality of Valzan^® tablet (160 mg, valsartan immediate release test formulation) by comparing its pharmacokinetic parameters with Diovan^® tablet (160 mg, valsartan reference formulation). Valzan^® tablets were prepared according to a dry granulation method (roll compaction). To assess the bioequivalence of Valzan^® tablets a randomized, two-way, crossover, bioequivalence study was performed in 24 healthy male volunteers. The selected volunteers were divided into two groups of 12 subjects. One group was treated with the reference formulation (Diovan^®) and the other one with the generic Valzan^®, with a cross-over after the drug washout period of 14 days. Blood samples were collected at fixed time intervals and valsartan concentrations were determined by a validated HPLC assay method. The pharmacokinetic parameters AUC_0–48, AUC_0–∞, C_max, T_max, K_e and T_1/2 were determined for both the tablets and were compared statistically to evaluate the bioequivalence between the two brands of valsartan, using the statistical model recommended by the FDA. The analysis of variance (ANOVA) did not show any significant difference between the two formulations and 90% confidence intervals (CI) fell within the acceptable range for bioequivalence. Based on this statistical evaluation it was concluded that the test tablets (Valzan^®) is well formulated, since it exhibits pharmacokinetic profile comparable to the reference brand Diovan^®.     

The Rhizomes of Acorus gramineus and the Constituents Inhibit Allergic Response In vitro and In vivo

The rhizomes of Acorus gramineus have frequently been used in traditional medicine mainly for sedation as well as enhancing brain function. In this study, the anti-allergic activity of A. gramineus was investigated. The 70% ethanol extract of the rhizomes of A. gramineus was found to inhibit the allergic response against 5-lipoxygenase (5-LOX)-catalyzed leukotriene (LT) production from rat basophilic leukemia (RBL)-1 cells and β-hexosaminidase release from RBL-2H3 cells with IC₅₀’s of 48.9 and >200 μg/ml, respectively. Among the 9 major constituents isolated, β-asarone, (2R,3R,4S,5S)-2,4-dimethyl-1,3-bis (2'',4'',5''-trimethoxyphenyl)tetrahydrofuran (AF) and 2,3-dihydro-4,5,7-trimethoxy-1-ethyl-2-methyl-3-(2,4,5-trimethoxyphenyl)indene (AI) strongly inhibited 5-LOX-catalyzed LT production in {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-treated RBL-1 cells, AI being the most potent (IC₅₀=6.7 μM). Against β-hexosaminidase release by antigen-stimulated RBL-2H3 cells, only AI exhibited strong inhibition (IC₅₀=7.3 μM) while β-asarone and AF showed 26.0% and 39.9% inhibition at 50 μM, respectively. In addition, the ethanol extract of A. gramineus showed significant inhibitory action against the hapten-induced delayed hypersensitivity reaction in mice by oral administration at 200 mg/kg. Therefore, it is suggested that A. gramineus possesses anti-allergic activity and the constituents including β-asarone and AI certainly contribute to the anti-allergic activity of the rhizomes of A. gramineus.     

Ginsenoside Rbl protects cardiomyocytes against CoCl2-induced apoptosis in neonatal rats by inhibiting mitochondria permeability transition pore opening

Aim:

To investigate whether mitochondria permeability transition pore (mPTP) opening was involved in ginsenoside Rb1 (Gs-Rb1) induced anti-hypoxia effects in neonatal rat cardiomyocytes ex vivo.

Methods:

Cardiomyocytes were randomly divided into 7 groups: control group, hypoxia group (500 μmol/L CoCl₂), Gs-Rb1 200 μmol/L group (CoCl₂ intervention+Gs-Rb1), wortmannin (PI3K inhibitor) 0.5 μmol/L group, wortmannin+Gs-Rb1 group, adenine 9-β-D-arabinofuranoside (Ara A, AMPK inhibitor) 500 μmol/L group, and Ara A and Gs-Rb1 group. Apoptosis rate was determined by using flow cytometry. The opening of the transient mPTP was assessed by using co-loading with calcein AM and CoCl₂ in high conductance mode. Expression of GSK-3β, cytochrome c, caspase-3 and poly (ADP-ribose) polymerase (PARP) was measured by using Western blotting. ΔGSK-3β was defined as the ratio of p-Ser9-GSK-3β to total GSK-3β.

Results:

CoCl₂ significantly stimulated mPTP opening and up-regulated the level of ΔGSK-3β. There was a statistically significant positive correlation between apoptosis rate and mPTP opening, between apoptosis rate and ΔGSK-3β, and between mPTP opening and ΔGSK-3β. Gs-Rb1 significantly inhibited mPTP opening induced by hypoxia (41.3%±2.0%, P<0.001) . Gs-Rb1 caused a 77.3%±3.2% reduction in the expression of GSK-3β protein (P<0.001) and a significant increase of 1.182±0.007–fold (P=0.0001) in p-Ser9-GSK-3β compared with control group. Wortmannin and Ara A significantly inhibited the effect of Gs-Rb1 on mPTP opening and ΔGSK-3β. Gs-Rb1 significantly decreased expression of cytochrome c (66.1%±1.7%, P=0.001), caspase-3 (56.5%±2.7%, P=0.001) and cleaved poly ADP-ribose polymerase (PARP) (57.9%±1.4%, P=0.001).

Conclusion:

Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3β-mediated mPTP opening.     

Plantago ovata Mucilage in the Design of Fast Disintegrating Tablets

In the present work, fast disintegrating tablets of prochlorperazine maleate were designed with a view to enhance patient compliance by direct compression method. In this method mucilage of Plantago ovata and crospovidone were used as superdisintegrants (2-8% w/w) along with microcrystalline cellulose (20-60% w/w) and directly compressible mannitol (Pearlitol SD 200) to enhance mouth feel. The prepared batches of tablets were evaluated for hardness, friability, drug content uniformity, wetting time, water absorption ratio and in vitro dispersion time. Based on in vitro dispersion time (approximately 8 s), the two formulations were tested for the in vitro drug release pattern (in pH 6.8 phosphate buffer), short-term stability (at 40°/75% relative humidity for 3 mo) and drug-excipient interaction (IR spectroscopy). Among the two promising formulations, the formulation prepared by using 8% w/w of Plantago ovata mucilage and 60% w/w of microcrystalline cellulose emerged as the overall best formulation (t_50% 3.3 min) based on the in vitro drug release characteristics compared to conventional commercial tablets formulation (t_50% 17.4 min). Short-term stability studies on the formulations indicated that there are no significant changes in drug content and in vitro dispersion time (p<0.05).     

Antioxidant Activities of Some Cameroonian Plants Extracts Used in the Treatment of Intestinal and Infectious Diseases

Antioxidant activity test using two different methods namely 2,2-diphenyl-1-picrylhydrazyl and 2,2''-azinobis(3-ethylbenzothialozinesulfonate) diammonium salt free radical scavenging test has been carried out on three Cameroonian plant extracts used in the treatment of intestinal and infectious diseases: Pittosporum mannii Hook f. (Pittosporaceae), Vepris heterophylla R. Letouzey (Rutaceae) and Ricinodendron heudelotii (Baill) Pierre ex Pax (Euphorbiaceae). Results of this study in the 2,2-diphenyl-1-picrylhydrazyl scavenging test show that the ethyl acetate extract of P. mannii and the methanol extract of V. heterophylla exhibit high free radical scavenging activities with IC₅₀ values of 177.74 and 204.69 μg/ml, respectively while the methanol/dichloromethane (1+1) extract of R. heudelotii showed weak free radical scavenging activities as compared to Trolox (939.19 μg/ml) used as standard. In the same manner, 2,2''-azinobis(3-ethylbenzothialozinesulfonate) diammonium salt radical scavenging test of these extracts was in accordance of the result of 2,2-diphenyl-1-picrylhydrazyl test. The antioxidant properties of these extracts probably explain partly, the use of these plants in traditional medicine for the treatment of infectious diseases and inflammations.