Chloral Hydrate Solution Research Articles

MORPHOLOGICAL CHANGES IN THE HIPPOCAMPUS OF THE RAT BRAIN IN ISCHEMIA AND IN CONDITIONS OF COMBINED PRECONDITIONING

BACKGROUND: Preconditioning is an effective method of increasing the body's resistance to hypoxia/ischemia.
 AIM: The aim is to evaluate morphological changes in the most hypoxia-sensitive fields of the hippocampus CA1 and CA3 in cerebral ischemia in rats and under conditions of combined preconditioning.
 MATERIALS AND METHODS. Cerebral ischemia was simulated in rats under anesthesia (8% chloral hydrate solution 400 mg/kg) by bilateral ligation of the common carotid arteries. The combined preconditioning method (CPreC) included the alternate use of two preconditional factors – pharmacological (amtisol at a dose of 25 mg /kg) and hypoxic (hypobaric hypoxia, 410 mmHg, exposure time 60 min). Morphometric assessment of brain damage was performed a day after modeling ischemia in the CA1 and CA3 fields of the hippocampus.
 RESULTS. KPreK has a positive effect on the morphometric parameters of the brain during its ischemia, increasing the survival of neurons in the early and late periods of ischemia modeling, preventing the formation of necrotically and apoptotically altered neurons, hyperactivation of microglial cells and contributing to the preservation of endotheliocytes. 
 CONCLUSION. KPreK (amtisol + hypobaric hypoxia) has a neuroprotective effect in cerebral ischemia.

Controlling chloroform content to a safe level in a pharmaceutical oral solution of chloral hydrate

Abstract Chloral hydrate is a halogenated hydrocarbon with sedative and hypnotic properties and has been used for its medicinal properties for many years. Its solubility and permeability determine it as a Biopharmaceutical Classification System class 1 compound, and as such is ideal to be pharmaceutically presented as an aqueous oral liquid presentation. Its primary route of degradation is by hydrolysis to form formic acid and chloroform and hence licensed pharmaceutical presentations require validated methods to analyse these degradants. The content of the toxic chloroform degradation product was determined via a head-space gas chromatograph with a flame ionisation detector, and its content was controlled to a safe pharmaceutically acceptable level. This can be achieved by solution pH optimisation in the oral solution, incorporating a buffering system to control the rate of this degradation mechanism. Where other behavioural and pharmacological therapies have failed for the treatment of short-term severe insomnia, a chloral hydrate 500 mg/5 ml oral solution presentation with an optimised safety profile has been developed as a medication for prescription. Extensive analytical and formulation development work has arrived at a room temperature stable oral solution, with a multiyear expiry date. Key findings: The use of Chloral hydrate in medicinal practice is often associated with high levels of the carcinogen chloroform as a degradation product. The research described herein has shown that the rate of formation of chloroform can be reduced by controlling the impact that the pH has on the stability of the oral solution. The Ph Eur monograph for Chloral hydrate oral solution (unlicensed medicines) does not include a criterion for chloroform quantification, hence Specials manufacturers of this oral solution are potentially risking the safety of patients without appropriate control of chloroform. This research has used validated analytical techniques to support the detection of chloroform, and other possible impurities, thus ensuring a safe product can be provided to the patient.

ВЛИЯНИЕ ВИДА АНЕСТЕЗИИ НА ПОКАЗАТЕЛИ КРОВООБРАЩЕНИЯ У КРЫС

The study aims to detect the effects of non-inhaled anesthetics of two different types on the parameters of the central hemodynamics and microcirculation in an occlusive reactive hyperemia test in two groups of male Wistar rats weighting 400–500 g. The “ChH” group received 6 % chloral hydrate solution at a dose of 300 mg/kg intraperitoneally, while the “zol + xyl” group received 20 mg/kg of tiletamine/zolazepam (Zoletil 100, France) mixed with 5 mg/kg of xylazine (Xylanit, Russia) intraperitoneally. The following parameters were measured: blood pressure, average heart rate, duration of PQ, QRS, and QTc intervals according to the electrocardiography, cutaneous blood flow with laser Doppler flowmetry, arterial blood gas composition and acid-base state. The mean value of perfusion was recorded simultaneously with electrocardiography and blood pressure. The parameters of a post-occlusive reactive hyperemia were analyzed to conduct an occlusive reactive hyperemia test. Blood pressure was statistically significantly lower in “ChH” group than in “zol + xyl” group when analyzing the central hemodynamics parameters. The “ChH” group demonstrated higher maximum asodilation in reactive hyperemia (CVCmax) and time of its achievement (Tmax). Both types of anesthesia demonstrated compensated and subcompensated mixed acidosis and moderate arterial hypoxemia when comparing parameters of arterial blood gas composition and acid-base state in rats.

ИССЛЕДОВАНИЕ АНАТОМИЧЕСКОГО СТРОЕНИЯ КОРНЕЙ МАЛИНЫ ОБЫКНОВЕННОЙ (RUBUS IDAEUS L.)

Abstract. Raspberry (Rubus idaeus L.) Rosaceae is a perennial semi-shrub. Raspberry fruits are used in medicine as a diaphoretic. Underground organs are also of interest for medical use, thanks to the history of use in folk medicine and scientific research data. It is important not only to study the chemical composition, pharmacological action, but also to determine the authenticity of raw materials. The aim of the work was to determine the diagnostic signs of raspberry roots. Samples of raspberry roots were harvested on the territory of the Ilyinsky district of Perm Krai in the blueberry spruce forest. The material for the study was previously fixed in a mixture of glycerin - water - ethyl alcohol for 3 days. Cross-sections were made from the prepared materials. The micro-preparations were examined in a solution of chloral hydrate, and they were also stained with phloroglucin and Lugol's solution. When studying the cross-section of the raspberry root, it was found that the root has a secondary structure. The covering tissue is the periderm. Parenchymal cells of the phelloderm contain starch grains. Conducting system of non-ductile structure. The phloem has a typical structure for angiosperms and is located in a solid ring. The secondary xylem is represented by large vessels that surround the sclerenchyme. During the study, the following anatomical diagnostic signs of raspberry roots were established: the root has a secondary non–bulky structure, stony cells and idioblast - druze cells were found in the phelloderm. The established anatomical diagnostic features can be used in the development of normative documentation for raw materials, as well as to establish the authenticity of raspberry roots.

LEAF ANATOMY OF INULA PEACOCKIANA (AITCH. & HEMSL.) KOROVIN

Objective: Inula peacockiana, which is called "uzunandızotu" in Turkey, is a stout perennial herbaceous plant with yellow flowers, grow up to 2 m. It is a plant belonging to the Asteraceae family, which has medicinal value. The safe use of medicinal plants depends on their correct diagnosis. The anatomical features of the leaves are valuable in diagnosing plants correctly. In this study, the anatomical features of the basal leaf, petiole and cauline leaf of I. peacockiana were investigated. Material and Method: Plant materials were collected from Van (Turkey). A voucher specimen was deposited in the VANF Herbarium. The samples for anatomical studies were preserved in 70% alcohol. The transverse and surface sections were cutted by hand with razor blade in microscopic preparation form. The chloral hydrate solution was used in microscopic examinations. Result and Discussion: The basal and cauline leaves are dorsiventral and contain cover and glandular hairs. The main vein protrudes outward in the midrib of the both leaves. Different the basal leaf, the cauline leaf contains more glandular hairs on the upper surface and cover hairs on the lower surface. In both leaves, ranunculaceae type stomata were observed in the upper and lower epidermis. The simple arc-shaped petiole includes of numerous separate bundles embedded in the parenchymatous cells. The epidermal layer consist of numerous cover and glandular hairs.

Comparison of Chloral Hydrate Solution, Hydroxyzine Syrup, and Lidocaine/Prilocaine Cream as Premedication for Lumbar Puncture in Children: A Double-Blind Study.

Objectives This study aimed to compare the clinical effectiveness of oral hydroxyzine and chloral hydrate to topical lidocaine/prilocaine 2.5% cream as premedication in pediatric leukemia patients.Materials & MethodsThis double-blind clinical trial study was conducted on 70 leukemic and nonleukemic patients aged 3-11 years. The patients were divided into four groups. In group A, chloral hydrate solution was given to 18 patients. In group B, hydroxyzine syrup was used for 18 patients. In group C, chloral hydrate solution and lidocaine/prilocaine cream were used for 17 patients. In group D, hydroxyzine syrup and lidocaine/prilocaine cream were used for 17 patients. These groups were assessed and judged based on the visual analog scale (VAS). The side effects of the drugs were also recorded. Results In this study, 54.3% (n=38) and 45.7% (n=32) of the cases were female and male, respectively. Furthermore, 77%, 7.2%, and 15.8% of the cases were reported with acute lymphocytic leukemia, acute myeloid leukemia, and infectious disease, respectively. The VAS results showed no difference in these four groups. Nontraumatic lumbar puncture (LP) (red blood cell<50) was observed in 97.1% of the cases.Conclusion Although premedications for LP with hydroxyzine syrup and chloral hydrate solution were not statistically effective in pain relief, they increased patient and parent satisfaction. Moreover, adding lidocaine/prilocaine cream does not improve the effectiveness of the drugs.

The microscopic diagnostic signs of some Fabaceae L. genus representatives. Message ІІ. Plant conducting system

The world plant products market is expanded rapidly and trade in them tends to grow by 15–25 % annually. The number of reports is increased proportionally about accidental contamination or deliberate, economically motivated falsification of plant raw materials. 27 % of the nearly 6.000 herbal preparations that are sold in 37 countries have contained undeclared contaminants, substitutes, or other components, according to the literature. We have conducted a plant conduction system microscopic analysis of the individual members’ genus Clover (Trifoliae L.), Fabaceae L. to identify morphoanatomical characteristics. Clover has anti-inflammatory, antiseptic, choleretic, diaphoretic, diuretic, hemostatic, expectorant, astringent properties and is used in many diseases. Aim. The finding common features and those that differ and can be used as diagnostic during studying the conducting system structure of genus Trifolium L. leaves and stems. Materials and methods. Plant material (herb) from Trifolium pratense L., T. incarnatum L., T. repens L. and T. fragiferum L. was harvested during the active flowering period – (May – June) and was dried in a well-ventilated place. Leaves and stems preparations were pre-boiled in 5 % sodium hydroxide water solution and fixed in chloral hydrate solution. Cross-sections were made with a microtome. The BIOLAM LOMO light microscope (Russia) and OLYMPUS SH-21 digital camera were used to record the data about identify the conducting apparatus of the plant’s leaf, petiole, and stem. Results. It has been examined the central vein structure of T. pratense L. and T. fragiferum L. leaves, it was determined that the conductive system is covered with a crystalline coating and there is one closed collateral bundle in the center, which is not typical for dicotyledonous plants. The petioles of T. incarnatum L., T. fragiferum L., and T. repens L. in cross-section are several different shapes. There are kidney-shaped and round. The conducting apparatus T. incarnatum L. and T. repens L. have arranged in a circle, closed and collateral. The leafstalk structure type of T. fragiferum L. is bunchles. It contradicts too the information about the structure conducting system of dicotyledonous plants. The stem’s conducting bundles are collateral and open. Conclusions. We have paid attention to the structural peculiarities of the conductive system of the central vein and petiole of objects for study: Trifolium pratense L., T. incarnatum L., T. repens L., and T. fragiferum L. when searching for differentiating features in some members of the genus Trifoliae L. in pharmacognostic analysis. The Dicotyledonae representatives are not characteristic of the closed type of conductive bundles, which we observed during microscopic examination.

Comparative Morphological and Anatomical Studies on Morus Species (Moraceae) in Turkey

Morus alba L., Morus nigra L., and Morus rubra L. are widely cultivated in many countries due to their nutritive, economic, and medicinal value. In this study, comparative morphological and anatomical studies on three common Morus L. species found in Turkey were carried out. According to the results, differences regarding the morphological and anatomical features of these species were described, and the data were displayed in detailed photographs. Specimens collected from different provinces of Turkey were studied. In the anatomical studies, investigations were performed on transversal and superficial sections of the leaves. All sections were stained with chloral hydrate and Sartur solution and were then examined using an Olympus BH2 light microscope. Significant diagnostic characteristics were found, such as trichome types, stomatal measurements, the stomatal index, and the density ratio of the parenchyma and collenchyma layers. Some morphological features of the leaves also showed prominent differences. Our results may contribute to the taxonomy of Morus species for future work and be helpful in species diagnosis.

Влияние хронического ионизирующего излучения на вариабельность митотической активности тканей Pinus sylvestris L.

The development of nuclear power engineering will increase in the future, due to the continued likelihood of radionuclides entering the environment. The relevance of studying the genetic material of conifers as a reliable bioindicator was revealed. It is known that under the influence of ionizing radiation there is an accumulation of genetic abnormalities in pine (Pinus sylvestris L.) progeny. The stages of gametogenesis, zygotes and young seedlings of plant organisms are most sensitive to ionizing radiation. It is important to determine the variability of the mitotic index (MI) of tissues of Scots pine (Pinus sylvestris L.) progeny in areas with different levels of ionizing radiation, including in the exclusion zone. The exposure dose rate was measured at the soil level and 1 m from the ground surface. Germination of seeds was carried out in a thermostat on wet filter paper. Roots of seedlings 0.5–1 cm long were fixed in a mixture of 96 % ethanol and glacial acetic acid. Root staining was carried out in a solution of acetocarmine. Tissue softening was carried out with a strong chloral hydrate solution. The total number of dividing cells, as well as pathological mitoses (PM) were counted on squash preparations using a microscope. The mitotic index and the duration of the phases of mitosis were determined. An increase in the level of radioactive contamination increases the cell division rate in prophase, anaphase, and telophase, but decreases the duration of metaphase, and also changes the relative duration of mitosis phases. With an increase in the exposure dose rate, the number of pathological mitoses naturally increases. The spectrum of mitosis disorders is represented by various abnormalities of the chromosome apparatus in anaphase: exit and lagging of chromosomes, bridges. Herewith, the number of anaphases with bridges increases significantly with simultaneous exit and lagging of chromosomes.

Stability-of-extemporaneously-compounded-chloral-hydrate-oral-solution

The objective of this study is to look into the stability of Chloral Hydrate 40 mg per ml formulated as oral solution in X-temp Oral Suspension System in order to select proper storage conditions and establish beyond-use date. X-temp is a novel oral, flavoured sugar-free extemporaneous compounding vehicle to assist in the preparation of extemporaneous dosage forms. The compounded solution of 40 mg/ml was prepared by dissolving chloral hydrate powder in X-temp vehicle. The solution was then packed in amber HDPE containers, and were stored at refrigeration 5 ± 3°C and room temperature 30°C. Physical, chemical and microbiological parameters were evaluated at predetermined time-points up to 180 days. Samples were tested using a validated stability–indicating assay. Chloral hydrate concentration was assayed by high-performance liquid chromatography (HPLC). The stability results indicated that the solution remained unchanged in visual appearance or pH at both refrigeration and room temperature for up to 180 days. The HPLC results showed that all the stability studies maintained 90 – 100% of initial drug concentration. There was no substantial changes in the microbiological stability. Chloral hydrate solution prepared in X-temp Oral Suspension System was stable for up to 180 days when stored at room temperature and refrigeration conditions. These results demonstrated that X-temp is a suitable vehicle for extemporaneous compounding for chloral hydrate.

Metodología de detección de Gliclazida en muestras biológicas

Objetivo: Desarrollar un método de detección de gliclazida en muestras biológicas para el diagnóstico analítico de los fallecimientos por sobredosis del fármaco. Materiales y métodos: Método de CCF: placas para cromatografía Merck silica gel 60 F254 de 10×10 cm de tamaño, fase móvil - acetato de etilo, revelador - solución al 1% de vainillina o solución al 5% de hidrato de cloral. Método de HPLC: cromatógrafo líquido «Milihrom-A-02» con detección de UV (Rusia); columna de fase inversa Prontosil-120-5-C18-AQ de Ø2 x 75 mm, con graneado de 5 mm («Bischoff Analysetechnik und Geräte GmbH», Alemania); elución gradiente con una mezcla de dos eluyentes (eluyente A - [0,2 M solución de LiClO4 - solución de HClO 0,005 M4] y eluyente B - acetonitrilo); la longitud de onda es de 230 nm. Resultados y discusión: Se desarrolló una metodología de determinación de la gliclazida en muestras biológicos, que incluye un método eficaz de aislar la gliclazida de los objetos de estudio, unos métodos cromatográficos específicos y selectivos de detección y cuantificación de la sustancia tóxica en los extractos obtenidos. Se constató que al aplicarse el método de CCF la gliclazida ha sido identificada de acuerdo con el valor Rf 0,48±0,01; el límite de detección es de 3,0 µg. Al aplicarse el método de HPLC se constató lo siguiente: el tiempo de retención de la gliclazida es de 7,80 min (RSD=0,13%), el límite de detección es de 0,050 µg/ml, el límite de cuantificación es de 0,157 µg/ml, la linealidad del método está en el intervalo de 0,1-20,0 µg/ml. En condiciones diseñadas la gliclazida se detectó a un nivel de 17,48±1,32µg/ml (RSD=6,06%). Conclusiones. Los resultados obtenidos confirman que la metodología desarrollada de detección de la gliclazida en muestras biológicos es adecuada para el diagnóstico analítico de los fallecimientos por sobredosis del fármaco.

PHARMACOGNOSY SIGNSOF AERIAL PARTS MEDICINAL PLANT ECHINOPS ALBICAULIS KAR.ET KIR

Objective: This work is intended to provide you with information about pharmacognosy, herbs and medicinal plants Echinops L., structured from the main issues, such as macro-morphological features of organs leaves, to the deeper and more challenging questions, pharmacognosy as the analysis of active ingredients and other substances or compounds use and healing properties. Methods: Pieces of leaves were boiled on chloral hydrate solution - water (1:1) for 5-10 minutes to enlightenment. Then, the study objects were placed on a slide in a drop of glycerol and separated into two parts with dissecting needle. The object was covered with a cover glass. The object was viewed from both sides with the microscope. First, the object was viewed with a small (×180), then at high (×720), an increase by MS-300 microscope (MICROS, Austria) on the technique of microscopic and microchemical studies of medicinal plants. Results: This manuscript is intended to provide you with information about pharmacognosy, herbs and medicinal plants Echinops L., structured from the main issues, such as macro-morphological features of organs leaves, to the deeper and more challenging questions, pharmacognosy as the analysis of active ingredients and other substances or compounds use and healing properties. Conclusion: This work is offered as a study on the biological characteristics of medicinal plants to identify diagnostic markers and signs for the standardization of herbal medicines.

ДОСЛІДЖЕННЯ АНАТОМІЧНОЇ БУДОВИ НАДЗЕМНИХ ОРГАНІВ PRIMULA JULIAE KUSN

Вперше здійснено дослідження особливостей анатомічної будови надземних органів первоцвіту (примули) Юлії родини первоцвіті, в результаті якого виявлено їх характерні мікроскопічні діагностичні ознаки. Встановлені мікродіагностичні ознаки дають можливість ідентифікувати лікарську рослинну сировину і можуть бути використані для розробки проекту АНД.

Quantitative standarization and CPC-based recovery of pharmacologically active components from Polygonum tinctorium Ait. leaf extracts

Polygonum tinctorium Ait. (dyer’s knotweed) is a commonly known Asian perennial containing indole alkaloids – indigo and indirubin. Its leaves constitute a pharmacopoeial material (Chinese Pharmacopoeia) known for its antineoplastic, antipsoriatic, antiviral and antibacterial actions. This article presents the results of a detailed study into the optimization of the extraction and separation (CPC-based purification) process, and also the quantitative analysis of the extracts. The applied extraction parameters considered the impact of the extraction temperature, its duration and the solvent selection on the quantitative analysis of both indigo and indirubin. The study concludes that a 30min long extraction of the leaves, in a 2% solution of chloral hydrate in dichloromethane, was found to be most beneficial and reproductive. The compounds’ poor solubility, low stability and high toxicity of the so far used solvents, were some of the challenges encountered. CPC purification by heptane:ethyl acetate:methanol:water (6:1:6:1) in ascending mode led to the most satisfactory results in terms of purity, solvent consumption and duration. The elaborated method can be used as a suitable tool for the detailed quantitative indigo and indirubin standardization in the leaves of the P. tinctorium. The indirubin recovery model may serve for further scale-up for industrial applications.

Video Case 1: Extraction of Impacted Coin in Upper Esophagus of a 5- Years Old Boy

A 5-years old Egyptian boy presented by mild dysphagia for solids for one year with repeated chest infections and fever. On plain chest X ray impacted coin was found in upper esophagus. After sedation by 2.5 mg midazolam I.M. and oral chloral hydrate solution ; the coin was extracted endoscopically by shark tooth forceps.

Effects of aspirin on the expression of nuclear factor-κB in a rat model of acute pulmonary embolism.

Acute pulmonary embolism (APE) is a disorder involving the pulmonary circulation resulting from a blockage of the pulmonary artery. The present study aimed to investigate the effects of aspirin on the nuclear factor-κB (NF-κB) activity in a rat model of APE. A total of 108 healthy male Sprague-Dawley rats were randomly assigned into six groups (n=18 rats per group): control group, sham operation group, APE model group, and low-, medium- and high-dose aspirin groups. Six, 24, and 72 hours after the induction of APE, rats in the low-, medium- and high-dose aspirin groups were given aspirin at a respective daily dose of 150, 300, and 600 mg/kg by gavage for three consecutive days. Rats in the other groups were treated with equal volumes of normal saline. Six rats in each group were anesthetized with 10% chloral hydrate solution at each time point, and then the lung tissues were collected and analyzed using immunohistochemical staining. Positive immunohistochemical staining was present in the bronchial epithelial cells, alveolar cells, macrophages, and surrounding bronchial smooth muscle cells. When compared with the APE model group, the number of positive cells was significantly lower in the other groups at each time point (P<0.001). Statistically significant differences were also observed among the aspirin-treated groups at 6 hours (P<0.05, P<0.001). Compared with the APE model group, NF-κB protein expression was reduced in the other groups at each time point (P<0.05, P<0.001). Rats from the APE model group had thrombosis, damaged alveolar walls, and pulmonary hemorrhage, along with different degrees of inflammatory cellular infiltration at each time point. However, pathological changes such as pulmonary hemorrhage and infiltration of inflammatory cells were attenuated after the aspirin treatment. Aspirin can significantly inhibit NF-κB activity in the lung of rats with APE in a dose-dependent manner, and can alleviate lung injury after APE.

The Dung- and Carrion-Feeding Scarabs (Coleoptera: Scarabaeoidea) of an Amazonian Blackwater Rainforest: Results of a Continuous, 56-Week, Baited-PitfallTrap Study

Abstract A guild of Neotropical rainforest dung- and carrion-feeding beetles (Scarabaeinae, Aphodiinae, Trogidae) at Reserva Florestal Ducke in Amazonian Brazil was inventoried using baited pitfall traps continuously for 56 weeks during September 1977 to October 1978. The 20 traps were baited with either dung or carrion, and the preservatives in the traps were solutions of either chloral hydrate or picric acid so that all combinations of bait and preservatives were present. A total of 19,416 specimens, 66 species, 20 genera, two subfamilies of Scarabaeidae (Scarabaeinae and Aphodiinae), and two families (Scarabaeidae and Trogidae) were collected during the 56-week study. The by-catch of approximately 300,000 other insects was retained at the Instituto Nacional de Pesquisas da Amazonia (INPA) for further studies. In the dung traps, 14,413 specimens were collected (74% of all scarabaeoids collected). In the carrion traps, 5,003 specimens were collected (26% of all scarabaeoids collected). The mean number of...

An improved clearing and mounting solution to replace chloral hydrate in microscopic applications.

• Premise of the study: This study presents Visikol™, a new proprietary formulation that can be used as an efficient replacement for chloral hydrate as a clearing agent for microscopic examination. In the United States, chloral hydrate is regulated and therefore difficult to acquire.• Methods and Results: Fresh and dry samples of the following plants: ginger (Zingiber officinale), maté (Ilex paraguariensis), lime basil (Ocimum americanum), oregano (Origanum vulgare), and mouse-ear cress (Arabidopsis thaliana), were cleared using Visikol or chloral hydrate solution and compared using a light microscope.• Conclusions: This new method can be used successfully to clear specimens, allowing identification of diagnostic characteristics for the identification of plant materials. Visikol is as effective as chloral hydrate in providing clarity and resolution of all tissues examined. Tissues become transparent, allowing observation of deeper layers of cells and making it effective in research, botanical and quality control, and for educational applications.

RECOVERY AND COUNTER-CURRENT SEPARATION OF INDIGO AND INDIRUBIN FROM POLYGONUM TINCTORIUM AIT. LEAVES

s. PHYTOPHARM 2012 M 74 Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] ТОМ 10/2012/2 RECOVERY AND COUNTER-CURRENT SEPARATION OF INDIGO AND INDIRUBIN FROM POLYGONUM TINCTORIUM AIT. LEAVES © Kukula-Koch Wirginia, Glowniak Kazimierz Chair and Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1, Chodzki str., 20– 093 Lublin, Poland, virginia.kukula@gmail.com Indigo naturalis (“qing dai”) has been used in traditional Chinese medicine (TCM) as a heat remover. Polygonum tinctorium Ait. belongs to the indigoferous plants together with Indigofera tinctoria l., Isatis indigotica fort., Isatis tinctoria l., and Baphicacanthus cusia brem. its leaves are rich sources of indigo dye, indirubin (a well-known antipsoriatic and antibacterial agent), isatan b, and tryptanthrine. interest in the drug was fostered when activity against myelocitic leukemia of indirubine was discovered. Currently indirubin is known to inhibit cyclin-dependent kinases (Cdk1/cyclin b and Cdk5/p25) as well. Current report is to show the recent advances in the isolation of the precious constituents of P. tinctorium leaf extracts. Suggested by Chinese Pharmacopoeia extraction protocols remain unfavourable, as they employ the usage of highly toxic solvents (chloroform). herewith we present the optimization of extraction process, introducing the 2 % solution of chloral hydrate in dichloromethane as the best from the investigated solvent systems characterized by lower toxicity. Separation of P. tinctorium Ait. metabolites was performed using environmentally friendly separatory technique: fast Centrifugal Partition Chromatography. Among several tested solvent systems, the mixture of n-heptane: ethyl acetate: methanol: water (6:1:6:1) was perceived as the best one. The one led to obtaining of indigo and indirubin of high purity already after the first injection of crude extract. indigo content in P. tinctorium Ait. samples from Taiwan were found to be similar to its quantity determined in the leaves grown in Poland and collected in the late summer. it may prove, that the cultivation of the investigated species could be spread to Europe. A STUDY ON PERIPHERAL UTILIZATION OF GLUCOSE AND ALDOSE REDUCTASE INHIBITORY ACTIVITY OF HOUTTUYNIA CORDATA FRACTIONS © Kumar Manish, Laloo Damiki, Prasad Satyendra K., Hemalatha S Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi-221005, India, Email: mkumar. rs.phe@itbhu.ac.in The plant Hottuynia cordata, used as a medicinal salad for lowering the blood sugar level in the North Eastern parts of india. Our earlier investigation showed significant antidiabetic effect of H.cordata in streptozotocin induced diabetic rats (1). Therefore, the aim of present study was to evaluate the glucose uptake by isolated rat hemi-diaphragm in an in vitro model. We have also evaluated in vitro aldose reductase inhibitory activity (ARi) and their kinetic profile on different fractions of H. cordata Thunb. (Saururaceae) for their potential use in diabetic cataract. Total phenolic and flavonoids content of different fraction of H.cordata was determined by folin-Ciocalteu and aluminium chloride method respectively. Glucose uptake by isolated rat hemi-diaphragm was evaluated in different fractions of H. cordata using insulin as a reference standard. Rats lens were enucleated through posterior approach and their homogenate was prepared and centrifuged to obtain clear supernatant for the determination of AR activity and protein content. The kinetic studies of inhibitory activity against aldose reductase of different fractions were analyzed using the lineweaver-burk plot with varying concentrations of glyceraldehydes. k m and V max were also estimated. k i value in presence of different fractions was determined by applying Cheng-Prusoff equation (2). The result showed significant level of phenol and flavonoids in ethyl acetate and aqueous fraction compared to hexane and chloroform fraction. Ethyl acetate fraction showed maximum glucose utilization by rat hemidiaphragm and was also found to have the most potent ARi activity which was significant when compared with all other fractions. Their iC 50 values with descending order of activity was reported to be: ethyl acetate [64.62 ± 3.9 μg/ml] > aqueous [90.69 ± 7.5 μg/ml] > hexane [134.59 ± 4.9 μg/ml] > chloroform [151.58 ± 3.3 μg/ml] respectively. from the kinetic studies, it was found that the ethyl acetate fraction showed a non-competitive inhibition while all other fractions showed competitive inhibition. The present study concluded that ethyl acetate fraction of H.cordata exhibited maximum glucose utilization and ARi activity which may be due to the presence of high phenols and flavonoids content.

Light and Oxygen Are Not Required for Harpin-induced Cell Death

Nicotiana sylvestris leaves challenged by the bacterial elicitor harpin N(Ea) were used as a model system in which to determine the respective roles of light, oxygen, photosynthesis, and respiration in the programmed cell death response in plants. The appearance of cell death markers, such as membrane damage, nuclear fragmentation, and induction of the stress-responsive element Tnt1, was observed in all conditions. However, the cell death process was delayed in the dark compared with the light, despite a similar accumulation of superoxide and hydrogen peroxide in the chloroplasts. In contrast, harpin-induced cell death was accelerated under very low oxygen (<0.1% O(2)) compared with air. Oxygen deprivation impaired accumulation of chloroplastic reactive oxygen species (ROS) and the induction of cytosolic antioxidant genes in both the light and the dark. It also attenuates the collapse of photosynthetic capacity and the respiratory burst driven by mitochondrial alternative oxidase activity observed in air. Since alternative oxidase is known to limit overreduction of the respiratory chain, these results strongly suggest that mitochondrial ROS accumulate in leaves elicited under low oxygen. We conclude that the harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts but that mitochondrial ROS could be important in the orchestration of the cell suicide program.