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Anticancer potential of rosmarinic acid and its improved production through biotechnological interventions and functional genomics

Swamy MK, Sinniah UR, Ghasemzadeh A

Appl Microbiol Biotechnol, 2018 Sep;102(18):7775-7793.
PMID: 30022261 DOI: 10.1007/s00253-018-9223-y

Rosmarinic acid (RA) is a highly valued natural phenolic compound that is very commonly found in plants of the families Lamiaceae and Boraginaceae, including Coleus blumei, Heliotropium foertherianum, Rosmarinus officinalis, Perilla frutescens, and Salvia officinalis. RA is also found in other members of higher plant families and in some fern and horned liverwort species. The biosynthesis of RA is catalyzed by the enzymes phenylalanine ammonia lyase and cytochrome P450-dependent hydroxylase using the amino acids tyrosine and phenylalanine. Chemically, RA can be produced via methods involving the esterification of 3,4-dihydroxyphenyllactic acid and caffeic acid. Some of the derivatives of RA include melitric acid, salvianolic acid, lithospermic acid, and yunnaneic acid. In plants, RA is known to have growth-promoting and defensive roles. Studies have elucidated the varied pharmacological potential of RA and its derived molecules, including anticancer, antiangiogenic, anti-inflammatory, antioxidant, and antimicrobial activities. The demand for RA is therefore, very high in the pharmaceutical industry, but this demand cannot be met by plants alone because RA content in plant organs is very low. Further, many plants that synthesize RA are under threat and near extinction owing to biodiversity loss caused by unscientific harvesting, over-collection, environmental changes, and other inherent features. Moreover, the chemical synthesis of RA is complicated and expensive. Alternative approaches using biotechnological methodologies could overcome these problems. This review provides the state of the art information on the chemistry, sources, and biosynthetic pathways of RA, as well as its anticancer properties against different cancer types. Biotechnological methods are also discussed for producing RA using plant cell, tissue, and organ cultures and hairy-root cultures using flasks and bioreactors. The recent developments and applications of the functional genomics approach and heterologous production of RA in microbes are also highlighted. This chapter will be of benefit to readers aiming to design studies on RA and its applicability as an anticancer agent.

Matched MeSH terms: Organ Culture Techniques
Isolation and characterization of α-elapitoxin-Bf1b, a postsynaptic neurotoxin from Malaysian Bungarus fasciatus venom

Rusmili MR, Tee TY, Mustafa MR, Othman I, Hodgson WC

Biochem Pharmacol, 2014 Mar 15;88(2):229-36.
PMID: 24440452 DOI: 10.1016/j.bcp.2014.01.004

Bungarus fasciatus is one of three species of krait found in Malaysia. Envenoming by B. fasciatus results in neurotoxicity due to the presence of presynaptic and postsynaptic neurotoxins. Antivenom, either monovalent or polyvalent, is the treatment of choice in systemically envenomed patients. In this study, we have isolated a postsynaptic neurotoxin which we named α-elapitoxin-Bf1b. This toxin has an approximate molecular weight of 6.9 kDa, with LCMS/MS data showing that it is highly homologous with Neurotoxin 3FTx-RI, a toxin identified in the Bungarus fasciatus venom gland transcriptome. α-Elapitoxin-Bf1b also shared similarity with short-chain neurotoxins from Laticauda colubrina and Pseudechis australis. α-Elapitoxin-Bf1b produced concentration- and time-dependent neurotoxicity in the indirectly-stimulated chick biventer cervicis muscle preparation, an effect partially reversible by repetitive washing of the preparation. The pA2 value for α-elapitoxin-Bf1b of 9.17 ± 0.64, determined by examining the effects of the toxin on cumulative carbacol concentration-response curves, indicated that the toxin is more potent than tubocurarine and α-bungarotoxin. Pre-incubation of Bungarus fasciatus monovalent and neuro polyvalent antivenom failed to prevent the neurotoxic effects of α-elapitoxin-Bf1b in the chick biventer cervicis muscle preparation. In conclusion, the isolation of a postsynaptic neurotoxin that cannot be neutralized by either monovalent and polyvalent antivenoms may indicate the presence of isoforms of postsynaptic neurotoxins in Malaysian B. fasciatus venom.

Matched MeSH terms: Organ Culture Techniques
Treatment with salvianolic acid B restores endothelial function in angiotensin II-induced hypertensive mice

Ling WC, Liu J, Lau CW, Murugan DD, Mustafa MR, Huang Y

Biochem Pharmacol, 2017 Jul 15;136:76-85.
PMID: 28396195 DOI: 10.1016/j.bcp.2017.04.007

Salvianolic acid B (Sal B) is one of the most abundant phenolic acids derived from the root of Danshen with potent anti-oxidative properties. The present study examined the vasoprotective effect of Sal B in hypertensive mice induced by angiotensin II (Ang II). Sal B (25mg/kg/day) was administered via oral gavage for 11days to Ang II (1.2mg/kg/day)-infused C57BL/6J mice (8-10weeks old). The vascular reactivity (both endothelium-dependent relaxations and contractions) in mouse arteries was examined by wire myography. The production of reactive oxygen species (ROS), protein level and localization of angiotensin AT1 receptors and the proteins involved in ROS formation were evaluated using dihydroethidium (DHE) fluorescence, lucigenin-enhanced chemiluminescence, immunohistochemistry and Western blotting, respectively. The changes of ROS generating proteins were also assessed in vitro in human umbilical vein endothelial cells (HUVECs) exposed to Ang II with and without co-treatment with Sal B (0.1-10nM). Oral administration of Sal B reversed the Ang II-induced elevation of arterial systolic blood pressure in mice, augmented the impaired endothelium-dependent relaxations and attenuated the exaggerated endothelium-dependent contractions in both aortas and renal arteries of Ang II-infused mice. In addition, Sal B treatment normalized the elevated levels of AT1 receptors, NADPH oxidase subunits (NOx-2 and NOx-4) and nitrotyrosine in arteries of Ang II-infused mice or in Ang II-treated HUVECs. In summary, the present study provided additional evidence demonstrating that Sal B treatment for 11days reverses the impaired endothelial function and with a marked inhibition of AT1 receptor-dependent vascular oxidative stress. This vasoprotective and anti-oxidative action of Sal B most likely contributes to the anti-hypertensive action of the plant-derived compound.

Matched MeSH terms: Organ Culture Techniques
Cardiovascular autonomic effects of transcutaneous auricular nerve stimulation via the tragus in the rat involve spinal cervical sensory afferent pathways

Mahadi KM, Lall VK, Deuchars SA, Deuchars J

Brain Stimul, 2019 05 06;12(5):1151-1158.
PMID: 31129152 DOI: 10.1016/j.brs.2019.05.002

BACKGROUND: Electrical stimulation on select areas of the external auricular dermatome influences the autonomic nervous system. It has been postulated that activation of the Auricular Branch of the Vagus Nerve (ABVN) mediates such autonomic changes. However, the underlying neural pathways mediating these effects are unknown and, further, our understanding of the anatomical distribution of the ABVN in the auricle has now been questioned.
OBJECTIVE: To investigate the effects of electrical stimulation of the tragus on autonomic outputs in the rat and probe the underlying neural pathways.
METHODS: Central neuronal projections from nerves innervating the external auricle were investigated by injections of the transganglionic tracer cholera toxin B chain (CTB) into the right tragus of Wistar rats. Physiological recordings of heart rate, perfusion pressure, respiratory rate and sympathetic nerve activity were made in an anaesthetic free Working Heart Brainstem Preparation (WHBP) of the rat and changes in response to electrical stimulation of the tragus analysed.
RESULTS: Neuronal tracing from the tragus revealed that the densest CTB labelling was within laminae III-IV of the dorsal horn of the upper cervical spinal cord, ipsilateral to the injection sites. In the medulla oblongata, CTB labelled afferents were observed in the paratrigeminal nucleus, spinal trigeminal tract and cuneate nucleus. Surprisingly, only sparse labelling was observed in the vagal afferent termination site, the nucleus tractus solitarius. Recordings made from rats at night time revealed more robust sympathetic activity in comparison to day time rats, thus subsequent experiments were conducted in rats at night time. Electrical stimulation was delivered across the tragus for 5 min. Direct recording from the sympathetic chain revealed a central sympathoinhibition by up to 36% following tragus stimulation. Sympathoinhibition remained following sectioning of the cervical vagus nerve ipsilateral to the stimulation site, but was attenuated by sectioning of the upper cervical afferent nerve roots.
CONCLUSIONS: Inhibition of the sympathetic nervous system activity upon electrical stimulation of the tragus in the rat is mediated at least in part through sensory afferent projections to the upper cervical spinal cord. This challenges the notion that tragal stimulation is mediated by the auricular branch of the vagus nerve and suggests that alternative mechanisms may be involved.

Matched MeSH terms: Organ Culture Techniques
Organotypic culture of human amnion cells in air-liquid interface as a potential substitute for skin regeneration

Fatimah SS, Chua K, Tan GC, Azmi TI, Tan AE, Abdul Rahman H

Cytotherapy, 2013 Aug;15(8):1030-41.
PMID: 23830235 DOI: 10.1016/j.jcyt.2013.05.003

The aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture.

Matched MeSH terms: Organ Culture Techniques
Transdermal anti-inflammatory activity of bilayer film containing olive compound hydroxytyrosol: physical assessment, in vivo dermal safety and efficacy study in Freund's adjuvant-induced arthritic rat model

Ng SF, Tan LS, Buang F

Drug Dev Ind Pharm, 2017 Jan;43(1):108-119.
PMID: 27588411 DOI: 10.1080/03639045.2016.1224893

Previous studies have shown that hydroxytyrosol (HT) can be a potential alternative therapeutic agent for the treatment of rheumatoid arthritis (RA). However, HT is extensively metabolized following oral administration, which leads to formulating HT in a topical vehicle to prolong drug action as well as to provide a localized effect. Hidrox-6 is a freeze-dried powder derived from fresh olives and contains a high amount of HT (∼3%) and other polyphenols. Alginate bilayer films containing 5% and 10% Hidrox-6 were formulated. The films were characterized with respect to their physical, morphology, rheological properties; drug content uniformity; and in vitro drug release. Acute dermal irritancy tests and a skin sensitization study were carried out in rats. An efficacy study of the bilayer films for RA was conducted using Freund's adjuvant-induced polyarthritis rats. Animal data showed that the bilayer film formulations did not cause skin irritancy. The efficacy in vivo results showed that the Hidrox-6 bilayer films lowered the arthritic scores, paw and ankle circumference, serum IL-6 level and cumulative histological scores compared with those measured for controls. The topical Hidrox-6 bilayer films improve synovitis and inflammatory symptoms in RA and can be a potential alternative to oral RA therapy.

Matched MeSH terms: Organ Culture Techniques
Preparation and characterization of dutasteride-loaded nanostructured lipid carriers coated with stearic acid-chitosan oligomer for topical delivery

Noor NM, Sheikh K, Somavarapu S, Taylor KMG

Eur J Pharm Biopharm, 2017 Aug;117:372-384.
PMID: 28412472 DOI: 10.1016/j.ejpb.2017.04.012

Dutasteride, used for treating benign prostate hyperplasia (BPH), promotes hair growth. To enhance delivery to the hair follicles and reduce systemic effects, in this study dutasteride has been formulated for topical application, in a nanostructured lipid carrier (NLC) coated with chitosan oligomer-stearic acid (CSO-SA). CSO-SA has been successfully synthesized, as confirmed using1H NMR and FTIR. Formulation of dutasteride-loaded nanostructured lipid carriers (DST-NLCs) was optimized using a 23full factorial design. This formulation was coated with different concentrations of stearic acid-chitosan solution. Coating DST-NLCs with 5% SA-CSO increased mean size from 187.6±7.0nm to 220.1±11.9nm, and modified surface charge, with zeta potentials being -18.3±0.9mV and +25.8±1.1mV for uncoated and coated DST-NLCs respectively. Transmission electron microscopy showed all formulations comprised approximately spherical particles. DST-NLCs, coated and uncoated with CSO-SA, exhibited particle size stability over 60days, when stored at 4-8°C. However, NLCs coated with CSO (without conjugation) showed aggregation when stored at 4-8°C after 30days. The measured particle size for all formulations stored at 25°C suggested aggregation, which was greatest for DST-NLCs coated with 10% CSO-SA and 5% CSO. All nanoparticle formulations exhibited rapid release in an in vitro release study, with uncoated NLCs exhibiting the fastest release rate. Using a Franz diffusion cell, no dutasteride permeated through pig ear skin after 48h, such that it was not detected in the receptor chamber for all samples. The amount of dutasteride in the skin was significantly different (p<0.05) for DST-NLCs (6.09±1.09μg/cm2) without coating and those coated with 5% CSO-SA (2.82±0.40μg/cm2), 10% CSO-SA (2.70±0.35μg/cm2) and CSO (2.11±0.64μg/cm2). There was a significant difference (p<0.05) in the cytotoxicity (IC50) between dutasteride alone and in the nanoparticles. DST-NLCs coated and uncoated with CSO-SA increased the maximum non-toxic concentration by 20-fold compared to dutasteride alone. These studies indicate that a stearic acid-chitosan conjugate was successfully prepared, and modified the surface charge of DST-NLCs from negative to positive. These stable, less cytotoxic, positively-charged dutasteride-loaded nanostructured lipid carriers, with stearic acid-chitosan oligomer conjugate, are appropriate for topical delivery and have potential for promotion of hair growth.

Matched MeSH terms: Organ Culture Techniques
Fulltext Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system

Yang C, Li X, Li S, Chai X, Guan L, Qiao L, et al.

J Cell Mol Med, 2019 03;23(3):1813-1826.
PMID: 30565384 DOI: 10.1111/jcmm.14080

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)-positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30-mm culture dish and 1 ml of slice culture media was added. We show that during serum-free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured-tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum-free medium cultured-tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.

Matched MeSH terms: Organ Culture Techniques/methods*
An ex vivo culture model for orthodontically induced root resorption

Wan Hassan WN, Stephenson PA, Waddington RJ, Sloan AJ

J Dent, 2012 May;40(5):406-15.
PMID: 22342686 DOI: 10.1016/j.jdent.2012.02.002

Root resorption is a ubiquitous although undesirable sequela to orthodontic treatment. Current methods to investigate the pathophysiology have certain limitations. In pursuit to understand and develop treatment modalities for orthodontically induced root resorption, the ability to manipulate cells within their natural extracellular matrix in a three dimensional organotypic model is invaluable. The study aimed to develop a laboratory-based organotypic model to investigate the effect of orthodontic forces on the periodontium.

Matched MeSH terms: Organ Culture Techniques
Vasorelaxation effect of Glycyrrhizae uralensis through the endothelium-dependent Pathway

Tan CS, Ch'ng YS, Loh YC, Zaini Asmawi M, Ahmad M, Yam MF

J Ethnopharmacol, 2017 Mar 06;199:149-160.
PMID: 28161542 DOI: 10.1016/j.jep.2017.02.001

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza uralensis (G. uralensis) is one of the herbs used in traditional Chinese medicine (TCM) and serves as an envoy medicinal. Since G. uralensis plays a major role in the anti-hypertensive TCM formulae, we believe that G. uralensis might possess vasorelaxation activity.
AIM OF THE STUDY: This study is designed to investigate the vasorelaxation effect of G. uralensis from various extracts and to study its pharmacology effect.
MATERIALS AND METHODS: The vasorelaxation effect of G. uralensis extracts were evaluated on thoracic aortic rings isolated from Sprague Dawley rats.
RESULTS: Among these three extracts of G. uralensis, 50% ethanolic extract (EFG) showed the strongest vasorelaxation activity. EFG caused the relaxation of the aortic rings pre-contracted with phenylephrine either in the presence or absence of endothelium and pre-contracted with potassium chloride in endothelium-intact aortic ring. Nω-nitro-L-arginine methyl ester, methylene blue, or 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one inhibit the vasorelaxation effect of EFG in the presence of endothelium. On the other hand, in the presence of the potassium channel blockers (tetraethylammonium and barium chloride), the vasorelaxation effect of EFG was not affected, but glibenclamide and 4-aminopyridine did inhibit the vasorelaxation effect of EFG. With indomethacin, atropine and propranolol, the vasorelaxation effect by EFG was significantly reduced. EFG was also found to be effective in reducing Ca(2+) release from sarcoplasmic reticulum and the blocking of calcium channels.
CONCLUSIONS: The results obtained suggest that EFG is involved in the NO/sGC/cGMP pathway.

Matched MeSH terms: Organ Culture Techniques
Fulltext Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus

Greenwood M, Greenwood MP, Mecawi AS, Loh SY, Rodrigues JA, Paton JF, et al.

Mol Brain, 2015 Oct 26;8(1):68.
PMID: 26503226 DOI: 10.1186/s13041-015-0159-1

BACKGROUND: Arginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression.
RESULTS: The effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1. The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress.
CONCLUSION: Here we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP expression is mediated through CREB3L1. This data provides the connection between CREB3L1, a newly identified transcription factor of AVP expression, with the previously proposed mechanism of Avp transcription which extends our understanding in transcription regulation of Avp in the hypothalamus.

Matched MeSH terms: Organ Culture Techniques
Quercetin, a flavonoid antioxidant, modulates endothelium-derived nitric oxide bioavailability in diabetic rat aortas

Machha A, Achike FI, Mustafa AM, Mustafa MR

Nitric Oxide, 2007 Jun;16(4):442-7.
PMID: 17513143 DOI: 10.1016/j.niox.2007.04.001

The present work examined the effect of chronic oral administration of quercetin, a flavonoid antioxidant, on blood glucose, vascular function and oxidative stress in STZ-induced diabetic rats. Male Wistar-Kyoto (WKY) rats were randomized into euglycemic, untreated diabetic, vehicle (1% w/v methylcellulose)-treated diabetic, which served as control, or quercetin (10mgkg(-1) body weight)-treated diabetic groups and treated orally for 6 weeks. Quercetin treatment reduced blood glucose level in diabetic rats. Impaired relaxations to endothelium-dependent vasodilator acetylcholine (ACh) and enhanced vasoconstriction responses to alpha(1)-adrenoceptor agonist phenylephrine (PE) in diabetic rat aortic rings were restored to euglycemic levels by quercetin treatment. Pretreatment with N(omega)-nitro-l-arginine methyl ester (l-NAME, 10microM) or methylene blue (10microM) completely blocked but indomethacin (10microM) did not affect relaxations to ACh in aortic rings from vehicle- or quercetin-treated diabetic rats. PE-induced vasoconstriction with an essentially similar magnitude in vehicle- or quercetin-treated diabetic rat aortic rings pretreated with l-NAME (10microM) plus indomethacin (10microM). Quercetin treatment reduced plasma malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HNE) content as well as increased superoxide dismutase activity and total antioxidant capacity in diabetic rats. From the present study, it can be concluded that quercetin administration to diabetic rats restores vascular function, probably through enhancement in the bioavailability of endothelium-derived nitric oxide coupled to reduced blood glucose level and oxidative stress.

Matched MeSH terms: Organ Culture Techniques
Fulltext Vasorelaxant properties of Vernonia amygdalina ethanol extract and its possible mechanism

Ch'ng YS, Loh YC, Tan CS, Ahmad M, Asmawi MZ, Wan Omar WM, et al.

Pharm Biol, 2017 Dec;55(1):2083-2094.
PMID: 28832263 DOI: 10.1080/13880209.2017.1357735

CONTEXT: Vernonia amygdalina Del. (VA) (Asteraceae) is commonly used to treat hypertension in Malaysia.
OBJECTIVE: This study investigates the vasorelaxant mechanism of VA ethanol extract (VAE) and analyzes its tri-step FTIR spectroscopy fingerprint.
MATERIALS AND METHODS: Dried VA leaves were extracted with ethanol through maceration and concentrated using rotary evaporator before freeze-dried. The vasorelaxant activity and the underlying mechanisms of VAE using the cumulative concentration (0.01-2.55 mg/mL at 20-min intervals) were evaluated on aortic rings isolated from Sprague Dawley rats in the presence of antagonists.
RESULTS: The tri-step FTIR spectroscopy showed that VAE contains alkaloids, flavonoids, and saponins. VAE caused the relaxation of pre-contracted aortic rings in the presence and absence of endothelium with EC50 of 0.057 ± 0.006 and 0.430 ± 0.196 mg/mL, respectively. In the presence of Nω-nitro-l-arginine methyl ester (EC50 0.971 ± 0.459 mg/mL), methylene blue (EC50 1.203 ± 0.426 mg/mL), indomethacin (EC50 2.128 ± 1.218 mg/mL), atropine (EC50 0.470 ± 0.325 mg/mL), and propranolol (EC50 0.314 ± 0.032 mg/mL), relaxation stimulated by VAE was significantly reduced. VAE acted on potassium channels, with its vasorelaxation effects significantly reduced by tetraethylammonium, 4-aminopyridine, barium chloride, and glibenclamide (EC50 0.548 ± 0.184, 0.158 ± 0.012, 0.847 ± 0.342, and 0.304 ± 0.075 mg/mL, respectively). VAE was also found to be active in reducing Ca2+ released from the sarcoplasmic reticulum and blocking calcium channels.
CONCLUSIONS: The vasorelaxation effect of VAE involves upregulation of NO/cGMP and PGI2 signalling pathways, and modulation of calcium/potassium channels, and muscarinic and β2-adrenergic receptor levels.

Matched MeSH terms: Organ Culture Techniques
An evaluation of crude palm oil (CPO) and tocotrienol rich fraction (TRF) of palm oil as percutaneous permeation enhancers using full-thickness human skin

Singh I, Nair RS, Gan S, Cheong V, Morris A

Pharm Dev Technol, 2019 Apr;24(4):448-454.
PMID: 30084268 DOI: 10.1080/10837450.2018.1509347

The drawbacks associated with chemical skin permeation enhancers such as skin irritation and toxicity necessitated the research to focus on potential permeation enhancers with a perceived lower toxicity. Crude palm oil (CPO) is obtained by direct compression of the mesocarp of the fruit of the oil palm belonging to the genus Elaeis. In this research, CPO and tocotrienol-rich fraction (TRF) of palm oil were evaluated for the first time as skin permeation enhancers using full-thickness human skin. The in vitro permeation experiments were conducted using excised human skin mounted in static upright 'Franz-type' diffusion cells. The drugs selected to evaluate the enhancing effects of these palm oil derivatives were 5-fluorouracil, lidocaine and ibuprofen: compounds covering a wide range of Log p values. It was demonstrated that CPO and TRF were capable of enhancing the percutaneous permeation of drugs across full-thickness human skin in vitro. Both TRF and CPO were shown to significantly enhance the permeation of ibuprofen with flux values of 30.6 µg/cm2 h and 23.0 µg/cm2 h respectively, compared to the control with a flux of 16.2 µg/cm2 h. The outcome of this research opens further scope for investigation on the transdermal penetration enhancement activity of pure compounds derived from palm oil.

Matched MeSH terms: Organ Culture Techniques
Fulltext "A Step and a Ceiling": mechanical properties of Ca2+ spark vasoregulation in resistance arteries by pressure-induced oxidative activation of PKG

Csato V, Kadir SZSA, Khavandi K, Bennett H, Sugden S, Gurney AM, et al.

Physiol Rep, 2019 Nov;7(22):e14260.
PMID: 31782255 DOI: 10.14814/phy2.14260

We investigated the biomechanical relationship between intraluminal pressure within small mesenteric resistance arteries, oxidant activation of PKG, Ca2+ sparks, and BK channel vasoregulation. Mesenteric resistance arteries from wild type (WT) and genetically modified mice with PKG resistance to oxidative activation were studied using wire and pressure myography. Ca2+ sparks and Ca2+ transients within vascular smooth muscle cells of intact arteries were characterized using high-speed confocal microscopy of intact arteries. Arteries were studied under conditions of varying intraluminal pressure and oxidation. Intraluminal pressure specifically, rather than the generic stretch of the artery, was necessary to activate the oxidative pathway. We demonstrated a graded step activation profile for the generation of Ca2+ sparks and also a functional "ceiling" for this pressure --sensitive oxidative pathway. During steady state pressure - induced constriction, any additional Ca2+ sensitive-K+ channel functional availability was independent of oxidant activated PKG. There was an increase in the amplitude, but not the Area under the Curve (AUC) of the caffeine-induced Ca2+ transient in pressurized arteries from mice with oxidant-resistant PKG compared with wild type. Overall, we surmise that intraluminal pressure within resistance arteries controls Ca2+ spark vasoregulation through a tightly controlled pathway with a graded onset switch. The pathway, underpinned by oxidant activation of PKG, cannot be further boosted by additional pressure or oxidation once active. We propose that these restrictive characteristics of pressure-induced Ca2+ spark vasoregulation confer stability for the artery in order to provide a constant flow independent of additional pressure fluctuations or exogenous oxidants.

Matched MeSH terms: Organ Culture Techniques
Bronchodilator effects of Lignosus rhinocerotis extract on rat isolated airways is linked to the blockage of calcium entry

Lee MK, Lim KH, Millns P, Mohankumar SK, Ng ST, Tan CS, et al.

Phytomedicine, 2018 Mar 15;42:172-179.
PMID: 29655683 DOI: 10.1016/j.phymed.2018.03.025

BACKGROUND: Lignosus rhinocerotis (Cooke) Ryvarden is a popular medicinal mushroom used for centuries in Southeast Asia to treat asthma and chronic cough. The present study aimed to investigate the effect of this mushroom on airways patency.
MATERIALS AND METHODS: The composition of L. rhinocerotis TM02 cultivar was analyzed. Organ bath experiment was employed to study the bronchodilator effect of Lignosus rhinocerotis cold water extract (CWE) on rat isolated airways. Trachea and bronchus were removed from male Sprague-Dawley rats, cut into rings of 2 mm, pre-contracted with carbachol before adding CWE into the bath in increasing concentrations. To investigate the influence of incubation time, tissues were exposed to intervals of 5, 15 and 30 min between CWE concentrations after pre-contraction with carbachol in subsequent protocol. Next, tissues were pre-incubated with CWE before the addition of different contractile agents, carbachol and 5-hydroxytrptamine (5-HT). The bronchodilator effect of CWE was compared with salmeterol and ipratropium. In order to uncover the mechanism of action of CWE, the role of beta-adrenoceptor, potassium and calcium channels was investigated.
RESULTS: Composition analysis of TM02 cultivar revealed the presence of β-glucans and derivatives of adenosine. The extract fully relaxed the trachea at 3.75 mg/ml (p 

Matched MeSH terms: Organ Culture Techniques
Fulltext Squamous epitheliotropism of Enterovirus A71 in human epidermis and oral mucosa

Phyu WK, Ong KC, Kong CK, Alizan AK, Ramanujam TM, Wong KT

Sci Rep, 2017 03 21;7:45069.
PMID: 28322333 DOI: 10.1038/srep45069

Hand-foot-and-mouth disease is a self-limiting paediatric infectious disease commonly caused by Enterovirus A71 (Genus: Enterovirus, Family: Picornaviridae). Typical lesions in and around the hands, feet, oral cavity and other places may rarely be complicated by acute flaccid paralysis and acute encephalomyelitis. Although virus is readily cultured from skin vesicles and oral secretions, the cellular target/s of Enterovirus A71 in human skin and oral mucosa are unknown. In Enterovirus A71-infected human skin and oral mucosa organotypic cultures derived from the prepuce and lip biopsies, focal viral antigens and viral RNA were localized to cytoplasm of epidermal and mucosal squamous cells as early as 2 days post-infection. Viral antigens/RNA were associated with cytoplasmic vacuolation and cellular necrosis. Infected primary prepuce epidermal keratinocyte cultures showed cytopathic effects with concomitant detection of viral antigens from 2 days post-infection. Supernatant and/or tissue homogenates from prepuce skin organotypic cultures and primary prepuce keratinocyte cultures showed viral titres consistent with active viral replication. Our data strongly support Enterovirus A71 squamous epitheliotropism in the human epidermis and oral mucosa, and suggest that these organs are important primary and/or secondary viral replication sites that contribute significantly to oral and cutaneous viral shedding resulting in person-to-person transmission, and viraemia, which could lead to neuroinvasion.

Matched MeSH terms: Organ Culture Techniques
Fulltext Transcription factor CREB3L1 regulates vasopressin gene expression in the rat hypothalamus

Greenwood M, Bordieri L, Greenwood MP, Rosso Melo M, Colombari DS, Colombari E, et al.

J Neurosci, 2014 Mar 12;34(11):3810-20.
PMID: 24623760 DOI: 10.1523/JNEUROSCI.4343-13.2014

Arginine vasopressin (AVP) is a neurohypophysial hormone regulating hydromineral homeostasis. Here we show that the mRNA encoding cAMP responsive element-binding protein-3 like-1 (CREB3L1), a transcription factor of the CREB/activating transcription factor (ATF) family, increases in expression in parallel with AVP expression in supraoptic nuclei (SONs) and paraventicular nuclei (PVNs) of dehydrated (DH) and salt-loaded (SL) rats, compared with euhydrated (EH) controls. In EH animals, CREB3L1 protein is expressed in glial cells, but only at a low level in SON and PVN neurons, whereas robust upregulation in AVP neurons accompanied DH and SL rats. Concomitantly, CREB3L1 is activated by cleavage, with the N-terminal domain translocating from the Golgi, via the cytosol, to the nucleus. We also show that CREB3L1 mRNA levels correlate with AVP transcription level in SONs and PVNs following sodium depletion, and as a consequence of diurnal rhythm in the suprachiasmatic nucleus. We tested the hypothesis that CREB3L1 activates AVP gene transcription. Both full-length and constitutively active forms of CREB3L1 (CREB3L1CA) induce the expression of rat AVP promoter-luciferase reporter constructs, whereas a dominant-negative mutant reduces expression. Rat AVP promoter deletion constructs revealed that CRE-like and G-box sequences in the region between -170 and -120 bp are important for CREB3L1 actions. Direct binding of CREB3L1 to the AVP promoter was shown by chromatin immunoprecipitation both in vitro and in the SON itself. Injection of a lentiviral vector expressing CREB3L1CA into rat SONs and PVNs resulted in increased AVP biosynthesis. We thus identify CREB3L1 as a regulator of AVP transcription in the rat hypothalamus.

Matched MeSH terms: Organ Culture Techniques
Bisnicalaterines B and C, atropisomeric bisindole alkaloids from Hunteria zeylanica, showing vasorelaxant activity

Hirasawa Y, Hara M, Nugroho AE, Sugai M, Zaima K, Kawahara N, et al.

J Org Chem, 2010 Jun 18;75(12):4218-23.
PMID: 20469917 DOI: 10.1021/jo1006762

Two new bisindole alkaloids, bisnicalaterines B and C (1 and 2) consisting of an eburnane and a corynanthe type of skeletons, were isolated from the bark of Hunteria zeylanica. Their absolute structures were determined by combination of NMR, CD, and computational methods, and each of them was shown to be in an atropisomeric relationship. Bisnicalaterines B and C (1 and 2) showed potent vasorelaxant activity on isolated rat aorta.

Matched MeSH terms: Organ Culture Techniques
Comparison of chitosan scaffold and chitosan-collagen scaffold: a preliminary study

Norazril SA, Aminuddin BS, Norhayati MM, Mazlyzam AL, Fauziah O, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:186-7.
PMID: 15468880

Chitosan has similar structure to glycosaminoglycans in the tissue, thus may be a good candidates as tissue engineering scaffold. However, to improve their cell attachment ability, we try to incorporate this natural polymer with collagen by combining it via cross-linking process. In this preliminary study we evaluate the cell attachment ability of chitosan-collagen scaffold versus chitosan scaffold alone. Chitosan and collagen were dissolved in 1% acetic acid and then were frozen for 24 hours before the lyophilizing process. Human skin fibroblasts were seeded into both scaffold and were cultured in F12: DMEM (1:1). Metabolic activity assay were used to evaluate cell attachment ability of scaffold for a period of 1, 3, 7 and 14 days. Scanning electron micrographs shows good cell morphology on chitosan-collagen hybrid scaffold. In conclusion, the incorporation of collagen to chitosan will enhance its cell attachment ability and will be a potential scaffold in tissue engineering.

Matched MeSH terms: Organ Culture Techniques/methods*

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