Effect of head covering on phototherapy induced hypocalcemia in term neonates with hyperbilirubinemia: A randomised controlled study.

BACKGROUND: Phototherapy is the primary treatment for hyperbilirubinemia in neonates. Hypocalcemia is a lesser known but potential detrimental effect of phototherapy. It has been hypothesized that phototherapy inhibits pineal secretion of melatonin, which blocks the effect of cortisol on bone calcium. Therefore, unchecked cortisol increases bone uptake of calcium and induces hypocalcemia. Covering head during phototherapy in order to prevent light reaching to the pineal gland which eventually leads to the prevention of hypocalcemia is hypothesized to prevent hypocalcemia but it lacks sufficient evidence worldwide. METHOD: It is a prospective, randomized controlled study. 112 neonates were randomized into two groups of 56 neonates. Group A underwent phototherapy without head cover and group B with head covered by a cap. RESULT: The mean decline in serum ionic calcium after 48 hours of phototherapy in group A and group B was 0.57±0.37 mg/dl and 0.34±0.24 mg/dl respectively. This decline in serum ionic calcium was significantly higher in group A. (p < 0.001). 26.8% newborns from group A developed hypocalcemia while in group B only 14.3% developed hypocalcemia however it was not found to be statistically significant. Incidence of symptomatic hypocalcemia between the two groups was also not significant. CONCLUSION: There was significant reduction in serum calcium in neonates undergoing phototherapy without head cover as compared to neonates with head cover but risk of hypocalcemia was not significant. Further studies with larger sample size including preterm are recommended.

Alpha lipoic acid supplementation ameliorates the wrath of simulated tropical heat and humidity stress in male Murrah buffaloes.

A supplement which ameliorates temperature-humidity menace in food producing livestock is a prerequisite to develop climate smart agricultural packages. A study was conducted to investigate the heat stress ameliorative efficacy of alpha lipoic acid (ALA) in male Murrah water buffaloes (Bubalus bubalis). Eighteen animals (293.61 ± 4.66Kg Bwt) were randomly allocated into three groups (n = 6); NHSC (non-heat-stressed control), HS (heat-stressed) and HSLA (heat-stressed-supplemented with ALA@32 mg/kg Bwt orally) based on the temperature humidity index (THI) and ALA supplementation. HS and HSLA were exposed to simulated heat challenge in a climatically controlled chamber (40 °C) for 21 consecutive days, 6 h daily. Physiological responses viz. Respiration rate (RR), Pulse rate (PR) and Rectal temperature (RT) were recorded daily before and after heat exposure. Blood samples were collected at the end of heat exposure on days 1, 6, 11, 16, and 21 and on day 28 (7th day post exposure which is considered as recovery) for peripheral blood mononuclear cells (PBMCs) separation, followed by RNA and Protein extraction for Real time quantitative PCR and Western blot analysis respectively, of heat shock proteins (HSPs). Two-way repeated measure ANOVA was performed between groups at different experimental periods. RR (post exposure) in HS and HSLA was significantly higher (P < 0.05) than NHSC from day 1 onwards but HSLA varied significantly from the HS 8th day onwards. Post exposure RT and PR in both HS and HSLA varied (P < 0.05) from NHSC throughout the study; but between HS and HSLA, RT significantly varied on initial 2 days and last 6 days (from days 16 to 21). HSP70 mRNA expression significantly up regulated in high THI groups with respect to the low THI group throughout the experimental period. During chronic stress (days 16 and 21) HSP70 significantly (P < 0.05) increased in HS but not in HSLA (P > 0.05) with respect to NHSC. ALA supplementation up-regulates and sustains (P < 0.05) the expression of HSP90 in HSLA in comparison to the HS and NHSC. HSP105 expression was significantly up-regulated (P < 0.05) in HS on days 16 and 21 (during long-term exposure) but only on day 21 (P < 0.05) in HSLA. HSP70, HSP90, and HSP105 protein expression dynamics were akin to the mRNA transcript data between the study groups. In conclusion, supplementing ALA ameliorates the deleterious effect of heat stress as reflected by improved physiological and cellular responses. ALA supplementation improved cellular antioxidant status and sustained otherwise easily decaying heat shock responses which concertedly hasten the baton change from a limited window of thermo tolerance to long run acclimatization.

Cryopreservation of Axillary Buds of Cannabis sativa L. by V-Cryoplate Droplet-Vitrification: The Critical Role of Sucrose Preculture.

BACKGROUND: Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of important germplasm. Recent use of the cryo-plate system has proven beneficial in further simplifying the cryopreservation protocols. OBJECTIVE: Developing an efficient protocol for the cryopreservation of axillary buds of Cannabis sativa elite cultivars (MX and V1-20) by the V-cryoplate droplet-vitrification technique. MATERIALS AND METHODS: Stem segments (~5 cm in length) with mature axillary buds collected from indoor-grown plants were surface sterilized and then either precultured on MS basal medium with 0.1 M sucrose (1st step preculture) for 72 h or non-precultured. All mature axillary buds (~1 mm) were aseptically excised from stem segments and precultured for an additional 48 h on MS basal medium with sucrose (0.3 M) and 5% DMSO prior to cryopreservation (2nd step preculture). Biomass samples of fully mature mother plants and regrown cryopreserved plants were analyzed for Δ9-THC and CBD content using gas chromatography-flame ionization detector (GC/FID). RESULTS: The survival and regrowth rates of cryopreserved axillary buds of cultivar MX following this two-step preculture were 45% and 42% respectively, while those of cultivar V1-20 were 47% and 44% respectively. A direct preculture of axillary buds (2nd step preculture) on high sucrose (0.3M sucrose) significantly decreased both the survival and regrowth levels of axillary buds of cultivar MX (5% and 3% respectively) as well as those of cultivar V1-20 (20% and 17% respectively). Δ9-THC and CBD content of mother plants and regrown cryopreserved plants were found to be highly comparable to each other. CONCLUSION: The resulting plants after cryopreservation appeared normal without any callus formation or morphogenetic variation. On maturity, mother plants and re-grown cryopreserved plants were comparable in terms of Δ9-THC and CBD content. This report provides an efficient protocol for cryopreservation of axillary buds of Cannabis sativa cultivars which may be applicable to other important cultivars, plant parts and other related medicinal plants.

Chemical composition and biological activities of Artemisia judaica essential oil from southern desert of Jordan.

ETHNOPHARMACOLOGIC RELEVANCE: Artemisia judaica L. (Arabic name: Beithran), is a medicinal and aromatic plant growing in the valley bottoms of desert areas, particularly in the southern desert of Jordan nearest to the Jordan-Saudi Arabia borders and in Wadi Araba in the Southern Badia. In Jordan, A. judaica is widely used in traditional medicine being recommended by aboriginal Bedouins in the North Badia region of Jordan as calmative. Furthermore, it is used for the treatment of stomach ache, heart diseases, sexual weakness, diabetes, gastro-intestinal disorders and external wounding. Additionally, other folk medicines of the Arabic region commonly use this aromatic plant for the treatment of inflammatory-related diseases, for instance fungal infections, diabetes, atherosclerosis, cancer and arthritis. AIM OF THE STUDY: Considering the traditional medicinal uses and the lack of scientific studies addressing the cellular and molecular mechanisms behind A. judaica claimed activities, the present study was designed to validate some of the traditional uses ascribed to this species, specifically the antifungal and anti-inflammatory activities of A. judaica essential oil at doses devoid of cytotoxicity to mammalian cells. MATERIALS AND METHODS: Chemical analysis of A. judaica essential oil isolated by hydrodistillation from aerial parts was carried out by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The antifungal activity (minimal inhibitory concentrations and minimal lethal concentrations) was evaluated against yeasts, dermatophyte and Aspergillus strains. In order to deeply explore the mechanisms behind the anti-fungal effect of the essential oil, the germ tube inhibition assay and the biofilms formation assay were evaluated using Candida albicans. The assessment of cell viability was accomplished using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in both hepatocytes and macrophages. Furthermore, the in vitro anti-inflammatory potential of A. judaica oil was evaluated by measuring nitric oxide (NO) production using lipopolysaccharide (LPS)-stimulated mouse macrophages. RESULTS: Oxygen containing monoterpenes are a representative group of constituents (68.7%) with piperitone (30.4%), camphor (16.1%) and ethyl cinnamate (11.0%) as main compounds. The highest antifungal activity of the oil was observed against Cryptococcus neoformans, with a MIC value of 0.16µL/mL. The oil revealed an important inhibitory effect on germ tube formation in C. albicans with 80% inhibition of filamentation at a concentration of 0.16µL/mL. Importantly, the oil also interfered with pre-formed biofilms by reducing the amount of the attached biomass. Furthermore, the essential oil significantly inhibited NO production evoked by LPS on macrophages at concentrations with very low toxicity (0.32µL/mL) or without toxicity (0.16µL/mL) to both macrophages and hepatocytes. CONCLUSIONS: The present study revealed that A. judaica essential oil from Jordan significantly inhibited germ tube formation and disrupted preformed biofilms of C. albicans, emphasizing the therapeutic potential for the treatment of disseminated candidiasis. Additionally, safe concentrations of this essential oil significantly inhibited NO production elicited by LPS in macrophages, highlighting its potential anti-inflammatory activity. Overall, A. judaica bears promising therapeutic potential for further drug development. Importantly, this work also validates some of the traditional uses of A. judaica.

Modulation of ethanol toxicity by Asian ginseng (Panax ginseng) in Japanese ricefish (Oryzias latipes) embryogenesis.

Alcohol consumption by women during pregnancy often induces fetal alcohol spectrum disorder (FASD) in children who have serious central nervous system (CNS), cardiovascular, and craniofacial defects. Prevention of FASD, other than women abstaining from alcohol drinking during pregnancy, is not known. A limitation of the use of synthetic anti-alcoholic drugs during pregnancy led us to investigate herbal products. In particular, many plants including Asian ginseng (Panax ginseng) have therapeutic potential for the treatment of alcoholism. We used Japanese ricefish (medaka) (Oryzias latipes), an animal model of FASD, for identifying herbal medicines that can attenuate ethanol toxicity. Fertilized eggs in standard laboratory conditions were exposed to ginseng (PG) root extract (0-2 mg/mL) either 0-2 (group A) or 1-3 (group B) day post fertilization (dpf) followed by maintenance in a clean hatching solution. The calculated IC50 as determined 10 dpf in A and B groups were 355.3±1.12 and 679.7±1.6 µg/mL, respectively. Simultaneous exposure of embryos in sub-lethal concentrations of PG (50-200 µg/mL) and ethanol (300 mM) for 48 h disrupted vessel circulation and enhanced mortality. However, PG (100 µg/mL) may partially protect trabecular cartilage (TC) deformities in the neurocranium in B group embryos induced by ethanol (300 mM). To understand the mechanism, embryonic ethanol concentration was measured at 2 dpf and adh5, adh8, aldh2, aldh9a, catalase, GST, and GR mRNAs were analyzed at 6 dpf. It was observed that although ethanol is able to reduce adh8 and GST mRNA contents, the simultaneous addition of PG was unable to alter ethanol level as well as mRNA contents in these embryos. Therefore, antagonistic effects of PG on ethanol toxicity are mediated by a mechanism which is different from those regulating ethanol metabolism and oxidative stress.

4-epi-Pimaric acid: a phytomolecule as a potent antibacterial and anti-biofilm agent for oral cavity pathogens.

The present study focused on the antibacterial and biofilm inhibitory potential of 4-epi-pimaric acid isolated from aerial parts (stem and leaves) of Aralia cachemirica L. (Araliaceae) against oral cavity pathogens. 4-epi-Pimaric acid exhibited minimum inhibitory concentration (MIC) in the range of 4-16 µg/ml and minimum bactericidal concentration (MBC) two- to four-folds higher than MIC. There was significant inhibition in the biofilm formation by Streptococcus mutans on the saliva coated surface (P < 0.05), and confocal microscopy revealed that 4-epi-pimaric acid inhibited the clumping and attachment of S. mutans. At 8 × MIC concentration, it significantly prevented the pH drop and reduced S. mutans biofilms (P < 0.05). Increased propidium iodide staining and leakage of 260- and 280-nm absorbing material by 4-epi-pimaric acid treated cells of S. mutans suggested that it probably causes disruption of the cytoplasmic membrane structure. It also exhibited significant suppression of TNF-α expression in human neutrophils, suggestive of its anti-inflammatory activity. Furthermore, the compound was found to be significantly safe (IC(50) >100 µg/ml) in the MTT assay on AML-12 cell lines. In conclusion, 4-epi-pimaric acid showed promising antibacterial, anti-biofilm and anti-inflammatory potency and this compound can be exploited for therapeutic application in oral microbial infections.

A comprehensive approach to identifying and authenticating botanical products.

Whether they are being taken as dietary supplements by the general public or being evaluated in a clinical study, the authenticity of botanical products is a matter of paramount concern. Botanical specimens and the dietary supplements derived from them can vary in quality and in chemical constituent profiles because of a number of factors. Subtle variations in botanical specimens are known to have profound effects on the quality, efficacy, and safety of botanical dietary supplements and can potentially alter the results of clinical studies that rely on these materials. A complete array of authentication and evaluation tools can be utilized to provide a well-rounded scientific approach to the authentication of botanical products. It is vital that the authenticity of botanical supplements be established using appropriate analysis tools regardless of whether the end products are being considered for evaluation in clinical studies or are being developed for the consumer market.

Quantitative determination of vasicine and vasicinone in Adhatoda vasica by high performance capillary electrophoresis.

A new method of capillary electrophoresis was developed for the quantitative determination of vasicine and vasicinone from Adhatoda vasica (L.) Nees. The electrophoretic separation was performed using a 47 cm x 50 microm ID (38.5 cm effective length) fused silica capillary. The samples were injected by pressure for 3 s at 50 mbar and the running voltage was 19 kV at the injector end of the capillary. The capillary temperature was maintained at 40 degrees C. The separation of the two alkaloids has been achieved within 11 min with good repeatability. The method was validated in terms of reproducibility, linearity, accuracy and applied for the quantitative determination of vasicine and vasicinone in A. vasica plant samples/extracts. Parameters affecting the resolution such as pH, temperature, organic modifier, buffer concentration and capillary dimensions were reported.

Transport of sennosides and sennidines from Cassia angustifolia and Cassia senna across Caco-2 monolayers--an in vitro model for intestinal absorption.

Laxative effects of Senna preparations are mainly mediated by rheinanthrone, a metabolite formed in the intestinal flora from dianthrones. Nevertheless, it was not clear whether dianthrones are bioavailable at all and contribute to the overall effects of this important medicinal plant. Using the Caco-2 human colonic cell line as an in vitro model of the human intestinal mucosal barrier, the bioavailability of dianthrones was studied in apical to basolateral (absorptive) and basolateral to apical (secretive) direction. Permeability coefficients (P(c)) and percent transport were calculated based on quantitations by HPLC. From the data obtained it was concluded that sennosides A and B, as well as their aglycones sennidine A and B are transported through the Caco-2 monolayers in a concentration-dependent manner and their transport was linear with time. The absorption in apical to basolateral direction was poor and P(c) values were comparable to mannitol. The transport was higher in the secretory direction, indicating a significant efflux (e.g. by efflux pumps) of the (poorly) absorbed compounds in the intestinal lumen again. Our findings support the general understanding that the laxative effects of Senna are explainable mainly by metabolites and not by the natively present dianthrones.

Simultaneous identification and quantification by liquid chromatography of benzethonium chloride, methyl paraben and triclosan in commercial products labeled as grapefruit seed extract.

A HPLC method has been developed which permits the quantification of methyl paraben, benzethonium chloride and triclosan in various samples of grapefruit seed extract (GSE). The best results were obtained with a Phenomenex Gemini C18 column using gradient mobile phase of water (0.1% acetic acid) and acetonitrile (0.1% acetic acid) with a flow rate of 1.0 mL per minute. The detection wavelength was 254 nm for methyl paraben, and 275 nm for benzethonium chloride and triclosan. The main synthetic antimicrobial agent identified in commercial GSE samples was benzethonium chloride in concentrations from 0.29-21.84%. Positive ion electrospray MS of a commercial GSE sample showed a molecular ion at m/z 412 [M+], which matched that of a standard of benzethonium chloride. Triclosan was detected in two samples at 0.009 and 1.13%concentrations; while methyl paraben was not detected in the samples analyzed.

Quantitative and qualitative HPLC analysis of thermogenic weight loss products.

An HPLC qualitative and quantitative method of seven analytes (caffeine, ephedrine, forskolin, icariin, pseudoephedrine, synephrine, and yohimbine) in thermogenic weight loss preparations available on the market is described in this paper. After 45 min the seven analytes were separated and detected in the acetonitrile: water (80:20) extract. The method uses a Waters XTerra RP18 (5 microm particle size) column as the stationary phase, a gradient mobile phase of water (5.0 mM SDS) and acetonitrile, and a UV detection of 210 nm. The correlation coefficients for the calibration curves and the recovery rates ranged from 0.994 to 0.999 and from 97.45% to 101.05%, respectively. The qualitative and quantitative results are discussed.

The predatory mite Typhlodromus pyri (Acari: Phytoseiidae) causes feeding scars on leaves and fruits of apple.

Typhlodromus pyri Scheuten (Acari: Phytoseiidae) is the most important predator of Panonychus ulmi (Koch) (Acari: Tetranychidae) in orchards and vineyards. It was recently found that adult T. pyri females cause microscopic scars on apple leaves. The present laboratory experiments were carried out to confirm the production of scars on apple leaves and to assess if females cause scars on fruits as well. Scar production on apple leaves and/or fruits was investigated under various nutritional conditions: no food, pollen of Scots pine (Pinus sylvsestris L.) only, nymphs of P. ulmi only, and pollen + prey. Both on leaves and fruits, either offered alone or in combination, feeding scars were produced under all nutritional conditions, but mostly in the 'no food' treatment. The predators consumed significantly more P. ulmi nymphs when offered alone than when offered in combination with pollen. T. pyri laid eggs under all nutritional conditions, but mostly in the 'pollen + prey' treatment and least when no food was offered. T. pyri females caused scars on both leaves and fruits when offered simultaneously, but more on leaves than on fruits. The scars were also bigger on leaves than on fruits in all experiments. T. pyri survived and reproduced on plant material in the absence of other food sources. Whether the scars produced on leaves and fruits harm the quality of fruits or the yield of apple cannot be concluded from the present experiments.

Analysis of products suspected of containing Aristolochia or Asarum species.

Aristolochia species have been administered by those trained in traditional Chinese medicine (TCM) for centuries. After determining Aristolochia fangchi was an adulterant that caused death due to renal failure in a number of patients at a Belgian weight loss clinic, many countries took steps to regulate products containing Aristolochia fangchi as well as other Aristolochia species. The US FDA issued a Consumer Advisory 'advising consumers to stop using any products that may likely contain aristolochic acid'. The Aristolochia and Asarum genera both have been found to contain aristolochic acids. A number of websites have been found from which individuals can order products containing either Aristolochia or Asarum as an ingredient through US merchants. We purchased 25 products from such sites and analyzed them for the presence of aristolochic acid I and II by HPLC with PDA. Six of the products contained detectable amounts of I and II.

Authentication of medicinal plants using molecular biology techniques to compliment conventional methods.

Medicinal plants have become extremely popular in the United States as botanical supplements, herbal medicines and sources of lead compounds for pharmaceutical development. It is estimated that in 1997 Americans used or consumed 5.1 billion US dollars worth of herbal medicines. For the protection of consumers, authentication of medicinal plants is a critical issue. Ideally, authentication should occur from the harvesting of the plant material to the final product. Unfortunately there is no single or superior method to assure 100 percent authentication during the entire process, but the goal can be achieved through the application of a variety of different methodologies. The whole process starts with good voucher specimens that act as reference material and to prove chain of custody. Macroscopic and microscopic examinations can be used as rapid and inexpensive identification techniques. Chemical analysis is by far the best method for the detection of contaminants and can be an excellent method for plant identification. Each of these methodologies has limitations and more analytical methods are needed to assist in the authentication process. Molecular biology offers an assortment of techniques that can be very useful for authentication of medicinal plants. This review covers various aspects of authentication methods, with special emphasis on molecular biology techniques.

Constituents of Peucedanum zenkeri seeds and their antimicrobial effects.

The methanol extract of Peucedanum zenkeri L. seeds showed antimicrobial activity which is concentrated in the n-hexane fraction. Bioactivity-guided chromatographic fractionation of the seeds of P. zenkeri led to the isolation and characterization of five major coumarins, umbelliprenin, imperatorin, bergapten, isopimpinellin and byakangelicin, as well as two minor coumarins, 7-methoxy coumarin and 5-hydroxy-8-methoxy psoralen. Amongst the isolated compounds only imperatorin, bergapten and isopimpinellin were found to possess anti-microbial activity.

HPLC analysis of neo-clerodane diterpenoids from Teucrium chamaedrys.

A simple, rapid analytical method for the quantitative determination of nine neo-clerodane diterpenoids was developed. The neo-clerodane diterpenoids present in the plant material and extracts were separated with an acetonitrile-water gradient at a flow rate of 1 mL per minute. The HPLC separation was performed on a Phenomenex Luna C18(2) (150 x 4.6 mm I.D., particle size 5 microm) reversed phase column with detection at 220 nm. The limit of detection was 0.24-0.90 microg/mL. The relative standard deviation (RSD) values for the determination of neo-clerodane diterpenoids in plant extracts were less than 3.20%. This is the first analytical method developed for qualitative and quantitative analysis of nine neo-clerodane diterpenoids by HPLC with PDA detection.

An improved HPLC method for quantitative determination of six triterpenes in Centella asiatica extracts and commercial products.

An improved HPLC qualitative and quantitative method of six triterpenes (asiaticoside, madecassoside, asiatic acid, madecassic acid, terminolic acid, and asiaticoside-B) in Centella asiatica (raw plant material and preparations) is described in this paper. After 50 minutes the six active triterpenes were separated and detected in the methanolic extract at a limit of 0.01 microg/ml. The method uses a Phenomenex Aqua 5mu C18 (200 A) column as the stationary phase, a gradient mobile phase of water (0.1% TFA), acetonitrile (0.1% TFA), and methyl tert-butyl ether (0.1% TFA), and UV detection at 206 nm. The correlation coefficients for the calibration curves and the recovery rates ranged from 0.995 to 0.999 and from 98.39% to 100.02%, respectively. The qualitative and quantitative results are discussed.

Microbial transformation of the phthalideisoquinoline alkaloid, (-)-beta-hydrastine.

The phthalideisoquinoline alkaloid (-)-beta-hydrastine is one of the main active constituents of the medicinal plant, Hydrastis canadensis, which is used in many dietary supplements intended to enhance the immune system. Treatment of hydrastine with the fermentation broth of Polyporous brumalis (ATCC 34487) as a model for mammalian metabolism, gave a new alkaloid, (1S)-hydroxyhydrastine. Structure elucidation was based primarily on NMR and chiroptical studies.

Quantitative HPLC analysis of withanolides in Withania somnifera.

One of the most widely used herbs in Ayurvedic medicine is Ashwaghanda, Withania somnifera, a shrub commonly found on the Indian subcontinent. As this plant is increasingly becoming a popular adaptogenic in the western world, analytical methods for its identification and quality control are in demand. Thus, a HPLC method for the determination of withaferin A and withanolide D was developed. The system was successfully used to investigate the presence of the markers in different W. somnifera plant parts as well as to analyze their content in market products.

Determination of aristolochic acid I and II in North American species of Asarum and Aristolochia.

Wild ginger, Asarum canadense, which has folk uses as a medicinal and food plant, has been reported to contain aristolochic acid I. Rhizomes of North American species of Aristolochiaceae were surveyed for the presence of aristolochic acids by HPLC. Aristolochic acid I (1) and aristolochic acid II (2) were present in Aristolochia species and Hexastylis; 1 alone was detected in multiple accessions of A. canadense and Asarum caudatum, though not in Asarum wagneri. Concentrations in A. canadense were highly variable, reaching as much as 0.037 percent of dry weight.