Browsing by Author "Thammitiyagodage, M.G."

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Acute and subacute toxicity studies of the Ethyl Acetate soluble proanthocyanidins of the immature inflorescence of Cocos nucifera L. in female Wistar Rats.
(Hindawi Pub. Co., 2019) Ekanayake, C.P.; Thammitiyagodage, M.G.; Padumadasa, S.; Seneviratne, B.; Padumadasa, C.; Abeysekera, A.M.
Ayurvedic and traditional medical practitioners of Sri Lanka use the decoction of the immature inflorescence of Cocos nucifera L. (IC) variety aurantiaca for the treatment of menorrhagia. The progestogenic effect of the ethyl acetate soluble proanthocyanidins (EASPA) of the IC in female rats at a dose of 3.5 mg/kg body weight has been reported. Acute and subacute toxicity studies of EASPA of the IC carried out using female Wistar rats according to Organization for Economic Co-operation and Development (OECD) guidelines 423 and 407, respectively, are reported herein. In the acute toxicity study, a single dose of EASPA (2000 mg/kg body weight) was orally administered to rats, which were monitored for 14 days. In the subacute toxicity study, rats were orally administered with EASPA daily for 28 days at doses of 1.75, 3.5, 7, and 14 mg/kg body weight. No rat in either the acute or subacute toxicity study exhibited mortality or clinical signs of toxicity. Further, these rats did not show any significant change in their mean body weight, food, and water intake, haematological and biochemical parameters as well as in the results of their histopathological examinations compared to those of control group rats. According to results of the acute toxicity, the LD50 of EASPA is estimated to be greater than 2000 mg/kg body weight. Considering the results of the subacute toxicity study, the oral administration of EASPA daily for 28 days was well tolerated up to the dose, 14 mg/kg by rats. These results will be useful in the development of a novel therapeutic agent from EASPA of the IC for the treatment of menorrhagia, which incapacitates a considerable proportion of women worldwide.
Anti-Inflammatory activity is a possible mechanism by which the polyherbal formulation comprised of Nigella sativa (Seeds), Hemidesmus indicus (Root), and Smilax glabra (Rhizome) mediates its antihepatocarcinogenic effects
(Hindawi Pub, 2012) Galhena, P.B.; Samarakoon, S.R.; Thabrew, M.I.; Weerasinghe, G.A.; Thammitiyagodage, M.G.; Ratnasooriya, W.; Tennekoon, K.H.
The present study investigated the anti-inflammatory effects of a polyherbal decoction comprised of Nigella sativa, Hemidesmus indicus, and Smilax glabra in order to justify its claimed antihepatocarcinogenic activity. Activation of hepatic nuclear factor-kappa B (NF-κB), IκB kinase (IKK α/β) proteins, and TNFα and IL-6 expression was investigated in diethylnitrosamine- (DEN-) induced C3H mice-bearing early hepatocarcinogenic changes. Acute phase inflammatory response was evaluated by carrageenan-induced rat paw edema formation. Anti-inflammatory mechanisms were also assessed by determining effect on (a) membrane stabilization, (b) nitric oxide (NO) inhibitory activity, and (c) inhibition of leukocyte migration. A significant inhibition of the paw edema formation was observed in healthy rats as well as in rats bearing early hepatocarcinogenic changes with oral administration of the decoction. As with the positive control, indomethacin (10 mg/kg b.w.) the inhibitory effect was pronounced at 3rd and 4th h after carrageenan injection. A notable IKK α/β mediated hepatic NF-κB inactivation was associated with a significant hepatic TNFα down regulation among mice-bearing hepatocarcinogenic changes subjected to decoction treatment. Inhibition of NO production, leukocyte migration, and membrane stabilization are possible mechanisms by which anti-inflammatory effect is mediated by the decoction. Overall findings imply that anti-inflammatory activity could
Biochemical and histopathological changes in Wistar Rats after consumption of boiled and un-boiled water from high and low disease prevalent areas for Chronic Kidney Disease of Unknown Etiology (CKDu) in North Central Province (NCP) and Its comparison with Low disease prevalent Colombo, Sri Lanka
(BioMed Central,, 2020) Thammitiyagodage, M.G.; de Silva, N.R.; Rathnayake, C.; Karunakaran, R.; Wgss, K.; Gunatillka, M.M.; Ekanayaka, N.; Galhena, B.P.; Thabrew, M.I.
BACKGROUND: Chronic Kidney Disease of unknown etiology (CKDu) is prevalent in North Central Province (NCP) of Sri Lanka. Consumption of un-boiled dug well water has been identified as one of the causative factors. This in-vivo study was performed to investigate some of the suspected factors associated with the pathogenesis of CKDu mediated via ground water. METHOD: Rats were given water, collected from high and low disease prevalent areas from the NCP of Sri Lanka and the results compared with those obtained from previously identified low disease prevalent area; Colombo. Blood Urea Nitrogen, creatinine, urinary microalbumin:creatinine ratio together with ALT and AST levels were analyzed and results were compared using one-way ANOVA and paired t-Test. Histopathology was analyzed using non-parametric method. RESULTS: Rats that ingested water from New Town Medirigiriya (NTM) from high disease prevalent NCP reported significantly elevated microalbumin:creatinine ratios compared to other water sources after 8 months, whilst boiled water from NTM had been able to significantly reduce it. Histopathological findings after the 14 months experimental period revealed significantly high tubular lesion index in rats that ingested water from NCP compared to Colombo. Rats that ingested water from high disease prevalent Divuldamana (DD) from NCP showed the highest kidney lesion index though the fluoride content was relatively low in this area compared to other water sources from high disease prevalent NCP. Rats that ingested boiled and un-boiled water from NTM also developed severe lesions whilst the group from Colombo reported the lowest. Low disease prevalent area from NCP, Huruluwewa (HW) also reported elevated liver enzymes and altered renal histopathology. Association of Na+:Ca2+ ratio in the disease progression was not reflected by the current study. Compared to Colombo, high fluoride, calcium and sodium contents were observed in water from high disease prevalent areas. All the water samples were negative for heavy metals. CONCLUSIONS: Though Fluoride is a known kidney toxic agent it cannot be the sole reason for CKDu in NCP, Sri Lanka. Various toxic elements present in NCP water may contribute to different grade of kidney and liver lesions in Wistar rats. KEYWORDS: BUN; CKDu; Microalbumin:creatinine; NCP.
Biochemical,inflammatory, and histopathological changes in wistar rats given dug well water from high and low disease prevalent villages for chronic kidney disease of unknown etiology (CKDu) in north central province of Sri Lanka
(University of Kelaniya, Sri Lanka, 2021) Thammitiyagodage, M.G.
Chronic Kidney Disease of unknown etiology (CKDu) is prevalent in the North Central Province (NCP) of Sri Lanka. Ingestion of groundwater is identified as one of the causative factors. The einfonley of the population consumes un-boiled dug well water. The objectives of the study were to find out the biochemical, haematological, and immunological changes in Wistar rats that ingested dug well water from high and low disease prevalent areas from the NCP and low disease prevalent Colombo and correlate the findings with their histopathological changes. Sex balance 3 months old Wistar rats (n=60) were used for the study. Their WBC/ DC, cpt CD** gerum cytokines, creatinine, ALT, AST, BUN levels and Microalbumin: creatinine ratios were measured. Rats were randomly divided into 6 groups by assigning 10 rats per group. Groups 1, 2, and 3 were given water from high disease prevalent New Town Medirigiriya (NTM), Bisobandaragama (BB), and Divuldamana (DD) respectively. Group 4 was given boiled water from NTM (NTMB). Groups 5 and 6 were given water from low disease prevalent Huruluwewa (HW) from NCP and tap water from low disease prevalent Colombo (CO) adlibitum quantity. Serum cytokines (IL1β; IL6, TNFα) were measured after 8 months and CD 4+ and CD 8+ counts were measured after 14 months. Histopathology was performed in kidney and liver tissues. Serum TNFα levels were significantly elevated in rats from DD and BB (p<0.05) but hepatic TNFα levels were significantly elevated only in rats from DD (p<0.05)). Rats from high disease prevalent NTM had significantly high CD4+ and CD8+ cells than those from low disease prevalent HW and CO. Immunological findings. were correlated with the histopathological changes. There was a correlation between the kidney Tubular Interstitial (TI) Lesion index and liver lesions. Serum TNFα and CD4+ and CD8+ lymphocytes were closely correlated with renal damage. Rats with severe TI lesions reported a high percentage of portal tracts and parenchymal lesions in the liver and this expression was minimum in CO. It was evident that boiling water was effective against liver damage but not renal damage. The present study is supportive of an immune therapy targeting the CD4+ and CD8+, and TNFα in reducing renal damage in the early stages of the disease. In conclusion, peritubular nonsuppurative interstitial cell infiltrations in kidney tissues were common in all the experimental groups irrespective of the toxicant sicient in water bodies. Observed kidney damage may mediate via cytokine triggered immune response by high sodium, fluoride, calcium and some other unmeasured toxic agents present in NCP water. anid toxicant present in DD from NCP can directly damage the kidney tubules leading to fibrosis.
Investigation of the antioxidant activity of a herbal decoction with anti-carcinogenic properties
(Sri Lanka Association for the Advancement of Science, 2007) Galhena, P.B.; Thabrew, M.I.; Thammitiyagodage, M.G.
A Long-term investigation of the anti-hepatocarcinogenic potential of an indigenous medicine comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra
(BioMed Central, 2006) Iddamaldeniya, S.S.; Thabrew, M.I.; Wickramasinghe, S.M.D.N.; Ratnatunga, N.; Thammitiyagodage, M.G.
BACKGROUND: A decoction comprised of Nigella sativa seeds, Hemidesmus indicus root bark and Smilax glabra rhizome is being recommended for cancer patients by a family of traditional medical practitioners of Sri Lanka. Previous investigations have demonstrated that a short term (10 weeks) treatment with the decoction can significantly inhibit diethylnitrosamine (DEN) mediated expression of Glutathione S-transferase P form (GST-P) in rat liver. The objective of the present investigation was to determine whether long term (16 months) treatment with the decoction would be successful in inhibiting in rat livers, not only DEN- mediated expression of GST-P, but also the carcinogen mediated development of overt tumours (OT) or histopathological changes leading to tumour development (HT). METHODS: Thirty-six male Wistar rats were divided into 3 groups of 12 each. Groups 1 and 2 were injected intraperitoneally (i.p) with DEN (200 mg/kg) while group 3 was injected normal saline (NS). Twenty-four hours later, decoction (DC; 6 g/kg body weight/day) was orally administered to group 1 rats, while groups 2 and 3 (DEN-control and normal control) were given distilled water (DW). Treatment with DC or DW continued for 16 months. At the end of the 9th month and 16th months (study 1 and study 2 respectively), six rats from each group were sacrificed, and livers observed for OT or HT, both visually and by subjecting liver sections to staining with Haemotoxylin and Eosin (H & E), Sweet's Silver stain (for reticulin fibers), Periodic Acid Schiff (PAS) staining (for glycogen), and immunohistochemical staining (for GST-P). RESULTS: At the end of 9 months (study 1) a hepatocellular adenoma (HA) developed in one of the rats in the DEN + DW treated group (group 2). At the end of 16 months (study 2), livers of all rats of group 2 developed OT and HT. Large areas of GST-P positive foci were also observed. No OT, HT or GST-P positive foci were detected in any of the other groups. CONCLUSION: Protection against DEN-mediated carcinogenic changes in rat liver can be achieved by long term treatment with the DC comprised of N. sativa seeds, S. glabra rhizome and H. indicus root bark.
Optimization of reverse transcriptase PCR for selected hepatic cytokines in Wistar Rats
(Faculty of Graduate Studies, University of Kelaniya, 2015) Samaranayake, H.A.E.; Thammitiyagodage, M.G.; Galhena, B.P.; Chakrewarthy, S.; Wickremasinghe, A.R.
Expression patterns of hepatic cytokines elucidates the immune and pathological pathways involved in inflammatory responses. Cytokine mRNA quantification is widely used approach in this regard that involves RNA extraction, cDNA synthesis and real-time polymerase chain reaction of selected targets. In the present study, we optimized the reverse transcriptase PCR conditions for selected hepatic cytokines; TNF alpha and IL 6 in Wistar Rats. Liver tissues obtained from Wistar rats were washed with diethylpyrocarbonate (DEPC) treated water and frozen immediately in liquid nitrogen. Samples were stored at -800C. Total RNA was extracted from 0.1 g of liver tissue using Trizol@ according to the manufacturer‘s instructions. Subsequently, cDNA was synthesized from 2000ng of RNA using random primers and M-MLV reverse transcriptase enzyme. PCR for target cytokines was carried out using newly synthesized cDNA based on following PCR conditions. For TNF alpha, 5'-TTC TGT CTA CTG AAC TTG GGG GTG ATC GGT CC-3' and 5'-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG -3' were used as primers. PCR was optimized with initial denaturation at 940C for 1 and 30 sec followed by 35 cycles of 30 sec denaturation at 940C, 1 min annealing and 1 min extension at 720C. A temperature gradient of 530C, 550C and 570C was used for annealing step. Final extension was done at 720C for 3 min. For IL 6, 5‘-TCC TAC CCC AAC TTC CAA TGC TC-3‘ and 5‘-TTG GAT GGT CTT GGT CCT TAG CC-3‘were used as primers. PCR was optimized with initial denaturation at 940C for 1 and 30 sec followed by 35 cycles of 30 sec denaturation at 940C, 1 min annealing and 1 min extension at 720C. A temperature gradient of 570C, 590C, and 610C was used for annealing step. Final extension was done at 720C 3 min. Based on PCR products of TNF alpha and IL-6 separated by agarose gel electrophoresis, annealing temperatures for both genes were decided as 550C and 590C respectively.
Traditional Remedy with possible antl-hepatocarclnogenlc potential
(Sri Lanka Medical Association, 2004) Iddamaldeniya, S.S.; Thabrew, M.l.; Wickramasinghe, S.M.D.N.; Ratnatunga, N.; Thammitiyagodage, M.G.
OBJECTIVES: To determine whether a decoction (DC) comprised of Nigella sativa seeds, Hemidesmus indicus root, and Srnilax glabra rhizome can protect against diethylnitrosamine (DEN) induced hepatocarcinogenesis (HC) as assessed by (i). Glutathione S-transferase P form (GST-P) expression in rat hepatocytes, and (ii). tumour development in rat livers. DESIGN, SETTING, AND METHODS: (i). Six groups of 10 rats each were used. Groups 1-4 were administered DEN (200mg/kg, i.p). Groups 1 and 2 received orally, DC at 4g/kg /day, and 6g/kg /day, respectively. Groups 3 and 4 received respectively, distilled water (DW), and garlic (20 g/kg /day). Groups 5 and 6 served as the Normal controls and DC controls, respectively. The number, area and staining intensity of GST-P + foci and the number of cells/cm2 of the + foci were recorded. (ii). Two groups of 6 rats each were injected with DEN (200mg/kg). Group 1 was fed with DC (6g/kg/day) for 16 months while group 2 received only DW. Livers were examined for overt tumours (OT) / tumour like lesions. Results; (i). The number and area of DEN- mediated GST-P positive foci, number of cells/cm of foci and staining intensity of the foci were significantly (P>0.001) reduced by the decoction and garlic in the order dose 2 = garlic >dose 1. (ii). OTs / tumour like lesions were observed in all DW fed rats but not in DC treated rats. CONCLUSION: The DC comprised of N. sativa, S. glabra and H. indicus has the potential to protect rat liver against DEN induced hepatocarcinogenesis.