Microbiological quality of different tea grades produced in diverse agro-climatic regions in Sri Lanka.

In Sri Lanka, several tea grades are produced in factories located in different agro-climatic regions within three geographical elevations. The study aimed to determine the microbial quality of different tea grades and composite tea samples obtained from factories situated at diverse locations. The average APC, yeast & mould counts and coliforms in different tea grades ranged from 3.4 × 103 to 2.0 × 104 cfu/g, 4.8 × 102 to 2.5 × 103 cfu/g and 0.005 to 3.9 × 101 Most Probable Number (MPN)/g respectively. The tea samples collected from different factories had mean values of APC and yeast & mould as 5.3 × 103±1.3 × 103 cfu/g and 9.7 × 102±1.9 × 102 cfu/g. Escherichia coli (E.coli) and Salmonella were not detected either in tea grades or in composite samples. The identified microorganisms in tea samples belong to phyla Firmicutes, Proteobacteria, Ascomycota, Basidiomycota and Zygomycota. The samples collected from the mid country elevation had the highest counts of APC and yeast & mould counts were high in the low country elevation. More than 70 % of the tested samples comply with the SLTB guidelines given for the microbiological quality of black tea. The distribution of bacterial, yeast and mould and coliform densities of tea were significantly variable with respect to geographic areas.

Diagnosis of Apis dorsata venom allergy: use of recombinant allergens of Apis mellifera and a passive basophil activation test.

BACKGROUND: Allergy to Apis dorsata (Giant Asian Honeybee) venom is the commonest insect allergy in Sri Lanka and South East Asia. However, laboratory diagnosis is difficult as the pure venom and diagnostic reagents are not commercially available. OBJECTIVE: This study assessed the use of four recombinant allergens of A. mellifera venom and the passive basophil activation test in the diagnosis of A. dorsata venom anaphylaxis. METHODS: Serum IgE levels to four recombinant allergens of A. mellifera, rApi m 1, 2, 5 and 10 were assessed and compared with serum IgE to the crude venom of A. mellifera or V. vulgaris by Phadia ImmunoCAP, in patients who developed anaphylaxis to A. dorsata stings. Basophil activation in response to venom of A. dorsata or V. affinis was assessed using a passive basophil activation test. Association of the severity of the reaction with basophil activation was compared. RESULTS: rApi m 1 and 10 combinedly had significant correlation (r = 0.722; p < 0.001) with the crude venom of A. mellifera (Western honeybee) and a higher positivity rate of 90% (27/30). Whereas, IgE reactivity to rApi m 2 or 5 had significant correlation (p = 0.02 and p = 0.005 respectively) with V. vulgaris crude venom. All 30 (100%) were positive to A. dorsata venom in passive BAT; 70% (21/30) had over 80% activation, 96.7% (29/30) had over 60% activation and 100% had over 50% activation. Percentage activation of basophils in patients who had mild or moderate reactions (n = 20) was significantly low (p = 0.02) from that of patients who had severe reactions (n = 10). CONCLUSIONS: rApi m 1 and 10 when combined was sensitive for the diagnosis of A. dorsata allergy. This combination had the lowest cross-reactivity rate with Vespula vulgaris. The passive BAT is highly sensitive in A. dorsata allergy. The basophil reactivity was significantly higher in severe anaphylaxis compared to mild/moderate anaphylaxis. This finding should be further explored in further studies.

In Vivo and In Vitro Toxicity Profiles of Hexane Extract of Alpinia malaccensis Rhizome in Rat and Cell Line Models.

The objective of the study was to evaluate the potential toxicity of crude n-hexane extract of Alpinia malaccensis rhizome. The in vivo acute oral toxicity was evaluated by administering a single oral dose of the extract at 0, 300, or 2000 mg/kg body weight to female Wistar rats according to modified OECD Test Guideline 423. For the in vitro cytotoxicity study, A549, HepG2, 3T3, and COS-7 cell lines were exposed to different doses of A. malaccensis extract and cell viability was assessed adopting MTT assay followed by AO/EB staining, Hoechst staining, and comet assay with a view to compare the cellular and molecular mechanisms underlying the toxicity, if any. It was found that administration of 2000 mg/kg bw dose in in vivo oral acute toxicity study did not produce significant toxicity or mortality. No significant (p < 0.05) differences were observed for body weight and hematological and biochemical parameters compared to control after 14 days of treatment. No changes in behavior, body weight, hematological and biochemical parameters, and aspects of histopathology were observed when compared to the control. Thus, the possible oral lethal dose for A. malaccensis extract is above 2000 mg/kg body weight. The in vitro cytotoxicity analysis showed nontoxicity concentrations of the extract to be 2, 1.4, 30, and 1.4 µg/mL for A549, HepG2, 3T3, and COS-7 cells, respectively, where no apoptotic/necrotic cell death and DNA damage were observed. In conclusion, the extract of rhizome of A. malaccensis did not produce apparent cytotoxicity or acute oral toxicity, confirming the scope to use A. malaccensis as a safe food preservative and a natural therapeutic product after further subacute and chronic toxicity studies.

Diagnosis of Vespa affinis venom allergy: use of immunochemical methods and a passive basophil activation test.

BACKGROUND: Allergy to Vespa affinis venom is common in the Asia Pacific region. Venom preparations for diagnosis are not commercially available for this species. METHODS: The prominent allergens in V. affinis venom were identifiedusing immunochemical methods. Use of ImmunoCAP of Vespula vulgaris crude venom/its components and a passive basophil activation test (BAT) in the diagnosis of patients who had anaphylaxis to V. affinis venom (n = 30) were also accessed. The IgE double-positivity rates (positive to both hornet and honeybee) in ImmunoCAP and the passive BAT were determined. RESULTS: High IgE reactivity was seen with the five allergens in V. affinis venom; 96% (29/30) for 34 and 24 kDa, 93% (28/30) for 45 kDa and 90% (27/30) reactivity for the 100 and 80 kDa respectively. IgE cross-reactivity was low with ImmunoCAP using V. vulgaris venom (43%; 13/30) and Ves v1 (3%; 1/30), but relatively high with Ves v5 (73%; 22/30). All patients (100%) were positive to V. affinis venom in passive BAT. In ImmunoCAP, a high double-positivity rate (76%; 23/30) was detected while no double-positivity was detected in passive BAT. CONCLUSIONS: High IgE reactivity for five allergens of V. affinis points to the potential of using these allergens in component resolved diagnosis (CRD). The passive BAT has shown its importance as a promising diagnostic tool with high accuracy. It would be particularly useful in cases with doubtful double-positive results of other diagnostic tests.

Antimicrobial activity and phytochemical screening of Alpinia malaccensis (Ran-kiriya) against food-borne bacteria.

AIMS: Investigation of antimicrobial activity and phytochemicals of Alpinia malaccensis (Ran-kiriya) against foodborne bacteria Staphyloccocus aureus, Listeria monocytogenes, Escherichia coli and Salmonella Typhimurium. METHODS AND RESULTS: Antibacterial activity was tested on the above four foodborne bacteria using agar disc diffusion and broth dilution assay. Alpinia malaccensis rhizome extract chemical composition was determined using gas chromatography-mass spectrometry (GCMS). Active compound was identified using thin-layer chromatography (TLC) and confirmed by nuclear magnetic resonance spectroscopy (NMR). The A. malaccensis rhizome hexane crude extract showed significantly (P < 0·05) higher diameter of inhibition (DIZ) 40 ± 0·52, 38 ± 0·96 and 36 ± 1·45 mm for S. aureusSA113, MSSASS25D and methicillin-resistant S. aureus compared with other tested bacteria. The minimum inhibition concentration and minimum bactericidal concentration were 0·625 and 5 mg ml-1 for S. aureus 113. TLC showed DIZ 39 ± 0·12 mm only for one fraction. The crude extract showed 82·87% a major compound by GCMS which is the active fraction. This purified active fraction was confirmed as 1'acetoxychavicol acetate (1'ACA) by NMR. No significantly different inhibition was observed for crude extract and purified compound. CONCLUSIONS: Bioactive 1'ACA of A. malaccensis showed strong antibacterial activity against S. aureus strains including MRSA strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to identify 1'ACA from A. malaccensis. The crude or purified compound could potentially be developed as antimicrobials.

IgE cross-reactivity of phospholipase A2 and hyaluronidase of Apis dorsata (Giant Asian Honeybee) and Apis mellifera (Western Honeybee) venom: Possible use of A. mellifera venom for diagnosis of patients allergic to A. dorsata venom.

Diagnostic and therapeutic reagents are unavailable for anaphylaxis arising from stings by Apis dorsata. Venom profiles and cross-reactivity of A. dorsata and Apis mellifera were compared, to ascertain whether venom of A. mellifera can be used for diagnosis in A. dorsata allergy. Both venom profiles were similar by High Performance Liquid Chromatography and SDS-PAGE. Sera of 29 of 30 (96.7%) patients with anaphylaxis to A. dorsata stings had IgE to the phospholipase-2 (PLA2) doublet (15 and 16 kDa) of A. dorsata venom by immunoblot, compared to 26 of 30 (86.7%) with the PLA2 of A. mellifera and a purified preparation of PLA2. Twelve patients (40%) with severe anaphylaxis had IgE reactivity to a 39 kDa protein band of venom of both species, a third band, identified in immunoblot as hyaluronidase. The cross-reactivity of PLA2 and hyaluronidase of A. dorsata and A. mellifera were further confirmed by immunoblot inhibition results. Twenty five of 30 (83.3%) of our patients had positive venom specific IgE (>0.35 KUA/L) reactivity to Phadia ImmunoCAPs of A. mellifera venom. The observed IgE cross reactivity suggests the possibility of using A. mellifera venom as a diagnostic test for A. dorsata venom allergy.

Detection of common vetch (Vicia sativa L.) in Lentil (Lens culinaris L.) using unique chemical fingerprint markers.

Detection of adulteration of split red lentil (Lens culinaris L.) seeds with low level addition of split common vetch (Vicia sativa L.) is hampered by a lack of reliable detection methods. An analytical method was developed using high performance liquid chromatography with diode array detection (HPLC-DAD) based on two unique chemical markers found in common vetch: ß-cyanoalanine (BCA) and γ-glutamyl-ß-cyanoalanine (GCA). These two markers were present in samples of common vetch seed grown in Canada and Serbia. Authentic lentil samples grown in Canada, Australia, USA, Turkey, Syria, and Morocco had no detectable levels of these chemical markers. Commercial lentil samples for export from lentil processing plants in Saskatchewan, Canada, also had no detectable levels of GCA and BCA. The presence of vetch in intentionally adulterated lentil samples could be determined via chemical markers with a detection limit of 5% (w/w). The proposed method is a simple sample extraction and rapid HPLC analysis that could be widely used to detect intentional adulteration of lentils with common vetch.

Antioxidant activity of water extract of Scoparia dulcis.

An aqueous extract of Scoparia dulcis showed marked antioxidative activity in vitro, which supports the therapeutic effects claimed by traditional practitioners.

Adverse pregnancy outcome in rats following exposure to a Salacia reticulata (Celastraceae) root extract.

The root extract of Salacia reticulata Wight (family: Celastraceae) is used in Sri Lanka by traditional practitioners as a herbal therapy for glycemic control even during pregnancy. It is recognized that some clinically used antidiabetic drugs have harmful effects on pregnancy but the effects of the S. reticulata root extract on reproductive outcome is unknown and deserves examination. We determined the effects of the S. reticulata root extract on the reproductive outcome of Wistar rats (250-260 g) when administered orally (10 g/kg) during early (days 1-7) and mid- (days 7-14) pregnancy. The root extract significantly (P<0.05) enhanced post-implantation losses (control vs treatment: early pregnancy, 4.7 2.4 vs 49.3 13%; mid-pregnancy, 4.7 2.4 vs 41.7 16.1%). Gestational length was unaltered but the pups born had a low birth weight (P<0.05) (early pregnancy, 6.8 0.1 vs 5.3 0.1 g; mid-pregnancy, 6.8 0.1 vs 5.0 0.1 g) and low birth index (P<0.05) (early pregnancy, 95.2 2.4 vs 50.7 12.9%; mid-pregnancy, 95.2 2.4 vs 58.3 16.1%), fetal survival ratio (P<0.05) (early pregnancy, 95.2 2.4 vs 50.7 12.9; mid-pregnancy, 95.2 2.4 vs 58.3 16.1), and viability index (P<0.05) (early pregnancy, 94.9 2.6 vs 49.5 12.5%; mid-pregnancy, 94.9 2.6 vs 57.1 16.1%). However, the root extract was non-teratogenic. We conclude that the S. reticulata root extract can be hazardous to successful pregnancy in women and should not be used in pregnancy complicated by diabetes.

Adverse pregnancy outcome in rats following exposure to a Salacia reticulata (Celastraceae) root extract

The root extract of Salacia reticulata Wight (family: Celastraceae) is used in Sri Lanka by traditional practitioners as a herbal therapy for glycemic control even during pregnancy. It is recognized that some clinically used antidiabetic drugs have harmful effects on pregnancy but the effects of the S. reticulata root extract on reproductive outcome is unknown and deserves examination. We determined the effects of the S. reticulata root extract on the reproductive outcome of Wistar rats (250-260 g) when administered orally (10 g/kg) during early (days 1-7) and mid- (days 7-14) pregnancy. The root extract significantly (P<0.05) enhanced post-implantation losses (control vs treatment: early pregnancy, 4.7 ± 2.4 vs 49.3 ± 13 percent; mid-pregnancy, 4.7 ± 2.4 vs 41.7 ± 16.1 percent). Gestational length was unaltered but the pups born had a low birth weight (P<0.05) (early pregnancy, 6.8 ± 0.1 vs 5.3 ± 0.1 g; mid-pregnancy, 6.8 ± 0.1 vs 5.0 ± 0.1 g) and low birth index (P<0.05) (early pregnancy, 95.2 ± 2.4 vs 50.7 ± 12.9 percent; mid-pregnancy, 95.2 ± 2.4 vs 58.3 ± 16.1 percent), fetal survival ratio (P<0.05) (early pregnancy, 95.2 ± 2.4 vs 50.7 ± 12.9; mid-pregnancy, 95.2 ± 2.4 vs 58.3 ± 16.1), and viability index (P<0.05) (early pregnancy, 94.9 ± 2.6 vs 49.5 ± 12.5 percent; mid-pregnancy, 94.9 ± 2.6 vs 57.1 ± 16.1 percent). However, the root extract was non-teratogenic. We conclude that the S. reticulata root extract can be hazardous to successful pregnancy in women and should not be used in pregnancy complicated by diabetes