Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent Chlorogenic acid in vitro

Objective:To examine the acanthamoebicidal effects of ethyl acetate,aqueous and butanol fractions of dried flower buds of Lonicera japonica(L.japonica) Thunb.(Flos Lonicerae) in vitro.Methods:Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR.They were exposed to ethyl acetate,water and butanol fractions of L.japonica Thunb.at concentrations ranging from 0.5 mg/m L to 1.5 mg/m L.The extracts were evaluated for growth inhibition at 24,48 and 72 h,respectively.Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment.Results:Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h,49.2% after 48 h and 33.7% after 72 h Chlorogenic acid,the major active constituent of L.japonica Thunb.at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h,84.0% after 48 h and 72.3% after 72 h.This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h.Conclusions:Results obtained from this study show that ethyl acetate fraction at 1.5 mg/m L is the most potent fraction of L.japonica Thunb.and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

Presence of Cryptosporidium parvum and Giardia lamblia in water samples from Southeast Asia: towards an integrated water detection system

Background:Access to clean and safe drinking water that is free from pathogenic protozoan parasites,especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans,is still an issue in Southeast Asia(SEA).This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA,using real-time polymerase chain reaction(qPCR)assays.Methods:A total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia(53),Thailand(120),the Philippines(33),and Vietnam(15).A physicochemical analysis was conducted.The water samples were processed in accordance with the US Environmental Protection Agency’s methods 1622/1623.1,microscopically observed and subsequently screened using qPCR assays.Results:Cryptosporidium oocysts were detected in treated water samples from the Philippines(1/10),with a concentration of 0.06±0.19 oocyst/L,and untreated water samples from Thailand(25/93),Malaysia(17/44),and the Philippines(11/23),with concentrations ranging from 0.13±0.18 to 0.57±1.41 oocyst/L.Giardia cysts were found in treated water samples from the Philippines(1/10),with a concentration of 0.02±0.06 cyst/L,and in untreated water samples from Thailand(20/93),Vietnam(5/10),Malaysia(22/44),and the Philippines(16/23),with concentrations ranging from 0.12±0.3 to 8.90±19.65 cyst/L.The pathogens C.parvum and G.lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene,respectively.C.parvum was detected in untreated water samples from the Philippines(1/23)and Malaysia(2/44),whilst,G.lamblia detected was detected in treated water samples from the Philippines(1/10)and in untreated water samples from Thailand(21/93),Malaysia(12/44),and the Philippines(17/23).Nitrate concentration was found to have a high positive correlation with(oo)cyst(0.993).Conclusion:The presence of(oo)cysts in the water samples means that there is potential risk for zoonotic disease transmission in the studied countries.Detection using qPCR is feasible for quantifying both pathogenic C.parvum and G.lamblia in large water samples.