Characterization of an anti-hyperglycemic coumarin from the fruits of Averrhoa

Objectives: The present study was carried out to isolate and characterize the anti-hyperglycemic principles of the fruits of Averrhoa carambola.Methods: The fresh ripe fruits of A. carambola were collected from Mathugama area (Southern Province) in July 2014. The fruits were cut into small pieces and dried at 400 C. The dried and powdered fruits of A. carambola were successively extracted with n-hexane, CH2Cl2 and CH3OH using soxhlet apparatus. The CH2Cl2 extract was subjected to silica gel column chromatography (CC), eluting in a stepwise gradient with hexane and ethylacetate (EtOAc) mixtures. The fractions eluted with hexane: EtOAC (60:40) yielded a mixture which with preparative thin layer chromalography (CH2Cl2 : EtOAC – 9:1) afforded JW-AC-3 (20 mg). The structure of JW-AC-3 was elucidated on the basis of its UV, IR, MS, NMR including DEPT,COSY, NOESY, HMQC, HMBC experiments and direct comparison with reported data.Results: The JW-AC-3 was identified as scopoletin by direct comparison of its spectral data with reported data of scopoletin. Although this compound has previously been isolated from various plant species, this is the first report of this compound from A. carambola.Conclusions: Scopoletin has been shown to possess significant anti-hyperglycemic activity by previous workers. The results clearly indicated that the majority of the anti-hyperglycemic activity of the fruits of A. carambola could be attributed to scopoletin. As the fruits are used in traditional medicine for anti-hyperglycemic effects, the results obtained in this study could be used in the rationalization of ethnomedical use of the fruits of A. carambola

Quality and stability studies of metformin hydrochloride tablets marketed in Sri Lanka

Objective: An attempt was made to assess the quality and stability of generic and branded products of metformin hydrochloride tablets B.P.500mg marketed in Sri Lanka. Method: two generic and three branded products including the brand leader were used in this study. Three batches were selected randomly from each of the five products. Three samples of tablets from each batch were collected from different market places in this study. Each sample was tested according to British Pharmacopoeial specifications. The real time stability test was carried out for a period of six months. Results: Our results showed that all the batches of five products passed the disintegration, identification and dissolution. However one batch of brand leader did not comply with the uniformity of weight test and one batch of another branded product was found to be substandard due to failure in assay test. Hence this study showed that out of fifteen batches tested only thirteen batches are of acceptable quality at present. All the batches except the one which failed to meet the pharmacopoeial specifications for assay have passed the assay and stability test. However, considering the rate of loss of potency during stability test period it can be concluded that two of the five products will not retain the stability throughout the shelf life of the product. Conclusion: All three branded products of metformin hydrochloride tablets manufactured by foreign manufactures were found to be substandard. Hence through this study it is possible to contribute towards the concept of rational use of generic medicines in Sri Lanka.

Isolation of cytotoxic triterpenes from the mangrove plant, Scyphiphora hydrophyllacea C.F.Gaertn (Rubiaceae)

Purpose: To isolate active cytotoxic compounds from the hexane and chloroform  extracts of the leaves of the mangrove plant, Scyphiphora hydrophyllacea C.F. Gaertn (Rubiaceae), grown in Sri Lanka.Methods: Dried pulverized leaves of S. hydrophyllacea were extracted with hexane and chloroform. Vacuum liquid chromatography (VLC), column chromatography (size exclusion chromatography, Sephadex LH-20) and reversed phase preparative recycling high performance liquid chromatography (HPLC) techniques were used to isolate three compounds (compounds 1, 2 and 3). The structures of the isolated compounds were established with the aid of 1H, 13C and two-dimensional nuclear magnetic resonance (2D-NMR) and electron ionization-mass spectrometry (EI-MS) techniques. 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effects of the compounds on oestrogen receptor positive breast (MCF-7) and non-small cell lung (NCIH- 292) cancer cells.Results: The isolated compounds were identified as oleanolic acid (1), ursolic acid (2) and eichlerianic acid (3). Ursolic acid and eichlerianic acid showed strong  cytotoxic effects {IC50- ursolic acid: 8.47 μg/mL (24 h, MCF-7), 7.78 μg/mL (24 h, NCI-H292) and eichlerianic acid: 8.86 μg/mL (24 h, MCF-7), 10.15 μg/mL (24 h, NCI-H292)} in MCF-7 and NCI-H292 cancer cells at 24, 48 and 72 h  post-incubation periods.Conclusion: Hexane and chloroform extracts of the leaves of S. hydrophyllacea yielded three compounds namely oleanolic acid, eichlerianic acid and ursolic acid. Ursolic acid and eichlerianic acid have been isolated for the first time from the leaves of S. hydrophyllacea grown in Sri Lanka and demonstrate in-vitro cytotoxic effects in oestrogen receptor positive (MCF-7) and non-small lung cancer (NCI-H-292) cells.Keywords: Scyphiphora hydrophyllacea, eichlerianic acid, ursolic acid, oleanolic acid, MCF-7, NCI-H-29

A survey on the knowledge, perceptions and practices regarding unwanted medicine disposal among pharmacists in Sri Lanka

Background: Unwanted medicines are defined as expired, unused, damaged or contaminated pharmaceutical products. Improper disposal of unwanted medicines leads to many health and environmental hazards. The World Health Organisation recommends that unwanted medicines should always be disposed properly. The main objective of this study was to assess the knowledge, practices and perceptions on the disposal of unwanted medicines among pharmacists in Sri Lanka.Methods: A cross-sectional study was carried out among pharmacists in 40 private retail pharmacies in the Northern, Eastern and the Western provinces within a period of three months. The pharmacies were selected via stratified randomised sampling in each district. The most experienced pharmacist in each pharmacy was recruited for data collection. A pre-tested, self-administered questionnaire was used. The ethics approval was obtained (Ref: EC-12-190). The data was represented using simple descriptive statistics.Results: The data was collected from 40 pharmacies. Among the pharmacists, 65% were males. The majority answered that burning and landfill as the most appropriate methods of disposal for most of the types of medicinal waste. A significant number of pharmacists were not aware about the method of disposal for anti-infective agents and anti-neoplastic agents. The majority perceived the seriousness of environmental damage caused by disposal via trash or sink. A majority was not agreeing to have pharmacies as collecting centers for unwanted medicines. A discrepancy between the pharmacists’ perceptions and the practices was observed.Conclusions: The level of knowledge, practices and perceptions among pharmacists on unwanted medicines disposal was substandard and needs attention

Seed dormancy and germination of five tropical montane species belonging to the family Fabaceae in Sri Lanka

The  family  Fabaceae  consists  of  economically  and  ecologically  important  plant  species   (as crops,  cropwild  relatives,  cover  crops,  shade  crops,  weeds,  and  invasive  species  and  as  members  in   m a n yi m p or t a n t   p l a n t   c o m m u n i t i e s ).    Information  on   s e e d   d or m a n cy   a n d   germination  is  essentialin  propagation  of  useful  species  and  in  eradication  of  unwanted species.  However,  information  onseed  dormancy  and  germination  is  rare  on the  species  belongs  to  Fabaceae  family  occurring  in tropical  montane  forests.  Thus,  the  objective  of  the  study  was  to  investigate   the  seed  dormancy  type  offive species  of   Fabaceae  family  ie.    Tadehagi  triquetrum,  Calpurnea  aurea,  Clotelaria scabrella,  Erithriasubumbrans  and  Cassia  didymobotrya.  Available  information  suggested  that  seeds  of these  species  havePhysical  Dormancy  (PY),  Physiological  Dormancy (PD),  combinational   dormancy  and  non  dormancy. Seeds  were  collected  from  various  montane  forests  in  Sri  Lanka.  Seed  moisture  content  (SMC),imbibition  and  germination  of non  scarified  (NS)  and  manually  scarified  (MS)  seeds  were determinedfor  each  species separately.SMC  of  T. triquetrum,  C.  aurea, C. scabrella  and  C.  didymobotrya  was  ≤15%  indicating  that seeds  areOrthodox.  However,  SMC  of  E.  subumbrans   was  34%,  suggesting  that  they  are recalcitrant.  Significantlyhigher  mass  increment  was  observed  in  MS  seeds  than  that  of  NS seeds  of  all  the  species  except  for  E.subumbrans.  Thus,  it  reveals  that  seeds  of  these  four species  have  PY.  The  germinating  test  inlight/dark  condition  confirmed  the  PY  of  these  four species.  MS  seeds  of  C.  aurea  C.  scabrella  and  C.didymobotrya  germinated  >50  %  while,  NS  seeds  only  germinated  to  <  15  %.  Although,  within  first  14days  only  <  50  %  of  the  NS seeds  of  T.  triquetrum  germinated,  within  30  days  it  germinated  >  90%indicating  that  they  have  a  slight   dormancy.  Interestingly,  both  NS  and  MS  seeds  of  E.  subumbransimbibed  water   and  >  90%  of  them  germinated  during  the  30  day  germinated  within  14  days.  Thus, seeds of E. subumbrans have no dormancy. Key words: orthodox seeds, physical dormancy, recalcitrant seed

Very-Long-Chain Acyl-Co-Enzyme A Dehydrogenase Deficiency Presenting as Rhabdomyolysis: First Case Report from Sri Lanka

Background. Rhabdomyolysis can be either inherited or acquired such as in metabolic myopathies. Very-long-chain acyl-CoA dehydrogenase deficiency is a rare fatty acid oxidation disorder which presents with different phenotypes, and the mild adult form can present as intermittent rhabdomyolysis. Here, we present the first adult case of very-long-chain acyl-CoA dehydrogenase deficiency presenting as rhabdomyolysis in a Sri Lankan patient. Case Presentation. A 36-year-old Sri Lankan man who was born to consanguineous parents presented with severe generalized muscle pain, stiffness, and dark-coloured urine for three days following prolonged low-intensity activity. Since fourteen years of age, he has had multiple similar episodes, where one episode was complicated with acute kidney injury. His eldest brother also suffered from the similar episode. Examination revealed only generalized muscle tenderness without any weakness. His creatine phosphokinase level was above 50,000 IU/L, and he had myoglobinuria. Molecular genetic tests confirmed the diagnosis of very-long-chain acyl-CoA dehydrogenase deficiency. Following a successful recovery devoid of complications, he remained asymptomatic with lifestyle adjustments. Conclusion. Very-long-chain acyl-CoA dehydrogenase deficiency is a rare inherited cause of metabolic myopathy that gives rise to intermittent rhabdomyolysis in adults. Prompt diagnosis is essential to prevent complications and prevent its recurrence

Role of hypothalamic prostaglandin production and signalling in regulating energy balance during sickness

Under normal physiological conditions, hypothalamus maintains energy homeostasis, through a neuronal network that produces orexigenic and anorexigenic neuropeptides such as NPY and POMC respectively. During sickness, inflammatory mediators released by the host immune system alter the interplay of these neuronal systems, leading to a state of negative energy balance. Prostaglandins (PG) primarily of the E2 type play a significant role in altering these changes in energy balance within the hypothalamus. Production of PG depends on the expression of cyclooxygenase (COX) enzymes, of which there are 2 types. PGE2 binds to 4 receptor subtypes termed EP1-4, expressed throughout the brain. The following thesis project aims to characterize the role of COX-2 dependent PG production and signalling during sickness induced states of negative energy balance, with a focus on hypothalamic neuropeptide systems as key mediators. We hypothesized that during sickness induced-inflammatory state, energy balance is regulated by the modulation of PGE2 production and signaling through hypothalamic neuropeptide systems. The present project also aims to elucidate the distribution of EP receptors and its region-specific role in regulating energy balance with an emphasis on EP3 and EP4 distribution within hypothalamic regions known to regulate energy balance. Furthermore we will also attempt to understand the immunosuppressive effects of PGE2 on hypothalamic microglial activity. C57/bl6 mice were pretreated with a diet inclusive of the COX-2 specific inhibitor, celecoxib (50mg/day/kg of b.w) and injected with either saline or LPS (100µg/kg of b.w) and perfused 4 hrs after for gene expression analysis. The indices of energy balance (food intake, locomotor activity, 02 consumption, C02 Expiration) were measured 24hrs before and after the LPS/Veh injections. Similarly, Mice implanted with intracerebroventricular cannula were injected with PGE2 (100nml/animal) or saline, and perfused 4hrs post injections for gene expression analysis. Indices of energy balance were also monitored 24hrs before and after injections. Pretreatment with celecoxib attenuated LPS induced fever, hypophagia, and delayed decreases in RER and activity, suggesting COX-2 is involved in regulating delayed inflammatory signaling in response to LPS. Similar decrease in indices of energy balance were observed in mice given central infusions of PGE2, confirming the critical role of PGE2 in regulating energy balance during sickness. In situ hybridizations and immunohistochemistry revealed that COX-2 dependent PGE2 is capable of regulating key hypothalamic neural systems (POMC/NPY) in order to drive the changes in energy balance. In addition COX-2 inhibition attenuated LPS-induced increase in EP4 receptor within Arc and PVN, suggesting that COX-2 derived PGE2 is the primary regulator of this receptor. Furthermore our results suggest that in acute inflammation, PGE2 is likely to induce microglial activity, while at rest endogenous PGE2 may inhibit glial activity, suggesting a dose dependent immunosuppressive function of PGE2. In conclusion, the studies described in this thesis, highlights the critical role of COX-2 derived PGE2 in energy balance regulation during acute sickness along with the possible neural circuitry, and receptors involved. Further investigations are required to elucidate the specific targets of PGE2 and identify the receptors and neuronal populations involved in regulating energy balance

Development of standards for the quality control of Cyclea peltata and its marketed formulations

Objective: Cyclea peltata (Family Menispermaceae) commonly known as “ Kehi pittan” is a medicinal plant widely used for dyspepsia, infertility, urinary  tract   infections and wound healing in the traditional system of medicine in Sri Lanka. However the scientific parameters are not yet available to authenticate the true plant material. The present study was aimed at development of various pharmacognostic  standards for Cyclea peltata. Method: Morphological, anatomical, powder microscopical, quantitative microscopical and phytochemical investigations were carried out to identity the diagnostic features of leaves, stem and root of C. peltata. Results: Some of the diagnostic features of the leaf drug noted from the powder microscopic study are hexagonal oil cells and calcium oxalate crystals in the upper epidermis. Characteristic fingerprinting thin layer chromatography profiles, which were developed for methanolic and dichloromethane extracts of roots, stems and leaves  have been found to be valuable parameters for the identification of  C. peltata species and for the authentication of marketed raw drugs. Conclusion: The results of this study could be used as precise, reliable and reproducible standards for the quality control of the drug in the crude form and to distinguish the drug from its adulterants. The results will also be useful for the standardization and quality control of  marketed  formulations that include C. peltata.

Role of hypothalamic prostaglandin production and signalling in regulating energy balance during sickness

Under normal physiological conditions, hypothalamus maintains energy homeostasis, through a neuronal network that produces orexigenic and anorexigenic neuropeptides such as NPY and POMC respectively. During sickness, inflammatory mediators released by the host immune system alter the interplay of these neuronal systems, leading to a state of negative energy balance. Prostaglandins (PG) primarily of the E2 type play a significant role in altering these changes in energy balance within the hypothalamus. Production of PG depends on the expression of cyclooxygenase (COX) enzymes, of which there are 2 types. PGE2 binds to 4 receptor subtypes termed EP1-4, expressed throughout the brain. The following thesis project aims to characterize the role of COX-2 dependent PG production and signalling during sickness induced states of negative energy balance, with a focus on hypothalamic neuropeptide systems as key mediators. We hypothesized that during sickness induced-inflammatory state, energy balance is regulated by the modulation of PGE2 production and signaling through hypothalamic neuropeptide systems. The present project also aims to elucidate the distribution of EP receptors and its region-specific role in regulating energy balance with an emphasis on EP3 and EP4 distribution within hypothalamic regions known to regulate energy balance. Furthermore we will also attempt to understand the immunosuppressive effects of PGE2 on hypothalamic microglial activity. C57/bl6 mice were pretreated with a diet inclusive of the COX-2 specific inhibitor, celecoxib (50mg/day/kg of b.w) and injected with either saline or LPS (100µg/kg of b.w) and perfused 4 hrs after for gene expression analysis. The indices of energy balance (food intake, locomotor activity, 02 consumption, C02 Expiration) were measured 24hrs before and after the LPS/Veh injections. Similarly, Mice implanted with intracerebroventricular cannula were injected with PGE2 (100nml/animal) or saline, and perfused 4hrs post injections for gene expression analysis. Indices of energy balance were also monitored 24hrs before and after injections. Pretreatment with celecoxib attenuated LPS induced fever, hypophagia, and delayed decreases in RER and activity, suggesting COX-2 is involved in regulating delayed inflammatory signaling in response to LPS. Similar decrease in indices of energy balance were observed in mice given central infusions of PGE2, confirming the critical role of PGE2 in regulating energy balance during sickness. In situ hybridizations and immunohistochemistry revealed that COX-2 dependent PGE2 is capable of regulating key hypothalamic neural systems (POMC/NPY) in order to drive the changes in energy balance. In addition COX-2 inhibition attenuated LPS-induced increase in EP4 receptor within Arc and PVN, suggesting that COX-2 derived PGE2 is the primary regulator of this receptor. Furthermore our results suggest that in acute inflammation, PGE2 is likely to induce microglial activity, while at rest endogenous PGE2 may inhibit glial activity, suggesting a dose dependent immunosuppressive function of PGE2. In conclusion, the studies described in this thesis, highlights the critical role of COX-2 derived PGE2 in energy balance regulation during acute sickness along with the possible neural circuitry, and receptors involved. Further investigations are required to elucidate the specific targets of PGE2 and identify the receptors and neuronal populations involved in regulating energy balance