2.1.1 Anthraquinones One gram of ea

2.1.1 Anthraquinones One gram of each tea sample was shaken with 10ml of ferric chloride solution with 5ml of hydrochloric acid (HCl). Each mixture was heated in a water bath for 10-15min, filtered and allowed to cool. The filtrate was extracted with chloroform and shaken gently. The clear layer at the base was pipette into test tubes and 2ml each of ammonia sulphate added. An observation of a delicate pink rose indicated the presence of anthraquinones.
2.1.2 Cardenolides Four grams of each sample was extracted in the test tube with 80% ethanol, and appropriately labeled. They were divided into two portions for Kedde’s test and Keler-Killian’s test. For Kedde’s test, few drops of 10% lead acetate were added to each of the tubes, followed by few drops of distilled water and chloroform. The contents were evaporated to dryness in a water bath. 5% sodium hydroxide was added to each residue and then 2% of 3-5 dinitrobenzene acid. For Keller-Killian’s test, few drops of 10% lead acetate, water and chloroform were added to each test sample. The mixture was evaporated to dryness in the water bath and subsequently a few drops of concentrated sulphulic acid were added. For Keller-Keillan’s test, a brown ring indicated the presence of cardenolides, while for Keddi’s test a brown to purple colour was indicative of cardenolides.
2.1.3 Phenolics To 2ml of alcoholic or aqueous tea extract, 1ml of 1% ferric chloride solution was added. Blue or green colour formation was an indication of phenols.
2.1.4 Flavinoids 2g tea was extracted in 10ml alcohol or water. To 2ml filtrate few drops of concentrated hydrochloric acid (HCl) followed by 0.5g zinc or magnesium turnings was added. After 3min magenta or pink colour formation indicated the presence of flavonoids.
2.1.5 Terpenes To 2ml of aqueous extract, 5mg chloroform, 2ml acetic anhydride, concentrated HCL were added carefully to form a layer. Redish-brown colour of interface was an indication of terpenes.
2.1.6 Cardiac Glycosides To 2ml alcoholic filtrate, 1ml glacial acetic acid and 1-2 drops of ferric chloride were added followed by 1ml of concentrated sulphulic acid. A presence of brown ring at the interface indicated the presence of cardiac glycosides. A violet ring might also appear below the brown ring.
2.1.7 Saponin 5ml of each plant extract was placed into a test tube and diluted with 5ml of distilled water. The mixture was shaken vigorously for 2min.Persistent appearance of foam lasting for 5min or the forming of emulsion when olive oil was added confirmed the presence of sponins.
2.1.8 Alkaloids The tea extracted (2g) was hydrolyzed with 2ml hydrochloric acid (HCl) solution by heating in water bath for 10 min, allowed to cool and 5ml of filtrate was reacted with a few drops of Dragendoff’s Mayer’s Wagner’s reagents, alkaloids were recorded as present in the sample if turbidity or brownish precipitate was observed.
2.2 Antimicrobial Screening Four human pathogenic bacteria made of two gram-positive Staphylococcus aureus and Streptococcus faecalis and two gram-negative Salmonella typhimurium and Escherichia coli were used for antibacterial assay. One yeast Candida albicans was used for antifungal assay. All the organisms were local isolates from the laboratory bacterial
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stock of the Medical Microbiology Department, JKUAT, Kenya. Three to five identical colonies from stored slopes of microorganisms were lifted with a sterile wire loop and transferred into a single strength nutrient broth(sigma) contained in a well labeled screw cap bottles for each bacterium and fungus respectively. The bottles were well shaken and incubated at room temperature for 18-24 hrs for bacteria and 72hrs for fungi. The agar well diffusion method was used to test the tea extract for antimicrobial activity. Briefly 15ml of melted and cooled nutrient agar (Sigma Laboratories, USA) and potato dextrose agar (Sigma Laboratories, USA) were added to 0.2ml in 100 dilutions of bacteria and fungal cultures respectively in sterile petri dishes. The contents were mixed after the gar in each plate solidified, 6 wells of 5mm each were bunched in each plate using aseptic pipette tip. 0.1ml of tea extracts at varying concentrations (50mgml-1, 100mgml-1, 200mgml-1,and 400mgml-1) as well as the standard antibiotic solution was loaded into the wells. Control experiments were set up using streptomycin and cefadroxil (4mgml-1) for the bacteria and fungal assays. The plates were incubated at 37oC for 24hrs for bacteria and 48hr for fungi.
All inoculation procedures were carried out under aseptic conditions. The antimicrobial studies were done in triplicate. With the aid of a transparent ruler the diameter of zones of inhibition around the wells were measured in mm for all the three replicates and the average of the three measurements were calculated as an indication of activity. The results were interpreted according to the modified Kirby-Baur technique. The minimum inhibitory concentration (MIC) of tea extracts was determined using the broth dilution method as described by Salon and Washington (1990). Briefly 1ml of the extract solution at the concentration of 400mgml-1 was added to1 ml of nutrient broth and subsequently transferred to make solution of varying concentration (400mgml-1 200mgml, 100mgml-150mgml-1) in different test tubes. The 1ml of bacterial and fungal suspension and 1ml of extracts at different concentrations was added to each test tube and incubated at 370C for 24 hrs for bacteria and 48hr for fungi. The test tube with the concentration of tea extract at which no detectable growth was observed was considered as the MIC.
2.3 Statistical Analysis Data obtained from diameter zones of inhibition were subjected to ANOVA. Further analysis of the tea extracts to assess variation on disc diameter showed that the crude tea extracts had an activity had great variation on inhibition of the microorganisms (Table 3). There was significant difference (P

Kết quả (Việt) 1:

2.1.1 Anthraquinones One gram of each tea sample was shaken with 10ml of ferric chloride solution with 5ml of hydrochloric acid (HCl). Each mixture was heated in a water bath for 10-15min, filtered and allowed to cool. The filtrate was extracted with chloroform and shaken gently. The clear layer at the base was pipette into test tubes and 2ml each of ammonia sulphate added. An observation of a delicate pink rose indicated the presence of anthraquinones. 2.1.2 Cardenolides Four grams of each sample was extracted in the test tube with 80% ethanol, and appropriately labeled. They were divided into two portions for Kedde’s test and Keler-Killian’s test. For Kedde’s test, few drops of 10% lead acetate were added to each of the tubes, followed by few drops of distilled water and chloroform. The contents were evaporated to dryness in a water bath. 5% sodium hydroxide was added to each residue and then 2% of 3-5 dinitrobenzene acid. For Keller-Killian’s test, few drops of 10% lead acetate, water and chloroform were added to each test sample. The mixture was evaporated to dryness in the water bath and subsequently a few drops of concentrated sulphulic acid were added. For Keller-Keillan’s test, a brown ring indicated the presence of cardenolides, while for Keddi’s test a brown to purple colour was indicative of cardenolides. 2.1.3 Phenolics To 2ml of alcoholic or aqueous tea extract, 1ml of 1% ferric chloride solution was added. Blue or green colour formation was an indication of phenols. 2.1.4 Flavinoids 2g tea was extracted in 10ml alcohol or water. To 2ml filtrate few drops of concentrated hydrochloric acid (HCl) followed by 0.5g zinc or magnesium turnings was added. After 3min magenta or pink colour formation indicated the presence of flavonoids. 2.1.5 Terpenes To 2ml of aqueous extract, 5mg chloroform, 2ml acetic anhydride, concentrated HCL were added carefully to form a layer. Redish-brown colour of interface was an indication of terpenes. 2.1.6 Cardiac Glycosides To 2ml alcoholic filtrate, 1ml glacial acetic acid and 1-2 drops of ferric chloride were added followed by 1ml of concentrated sulphulic acid. A presence of brown ring at the interface indicated the presence of cardiac glycosides. A violet ring might also appear below the brown ring. 2.1.7 Saponin 5ml of each plant extract was placed into a test tube and diluted with 5ml of distilled water. The mixture was shaken vigorously for 2min.Persistent appearance of foam lasting for 5min or the forming of emulsion when olive oil was added confirmed the presence of sponins. 2.1.8 Alkaloids The tea extracted (2g) was hydrolyzed with 2ml hydrochloric acid (HCl) solution by heating in water bath for 10 min, allowed to cool and 5ml of filtrate was reacted with a few drops of Dragendoff’s Mayer’s Wagner’s reagents, alkaloids were recorded as present in the sample if turbidity or brownish precipitate was observed. 2.2 Antimicrobial Screening Four human pathogenic bacteria made of two gram-positive Staphylococcus aureus and Streptococcus faecalis and two gram-negative Salmonella typhimurium and Escherichia coli were used for antibacterial assay. One yeast Candida albicans was used for antifungal assay. All the organisms were local isolates from the laboratory bacterial
202
stock of the Medical Microbiology Department, JKUAT, Kenya. Three to five identical colonies from stored slopes of microorganisms were lifted with a sterile wire loop and transferred into a single strength nutrient broth(sigma) contained in a well labeled screw cap bottles for each bacterium and fungus respectively. The bottles were well shaken and incubated at room temperature for 18-24 hrs for bacteria and 72hrs for fungi. The agar well diffusion method was used to test the tea extract for antimicrobial activity. Briefly 15ml of melted and cooled nutrient agar (Sigma Laboratories, USA) and potato dextrose agar (Sigma Laboratories, USA) were added to 0.2ml in 100 dilutions of bacteria and fungal cultures respectively in sterile petri dishes. The contents were mixed after the gar in each plate solidified, 6 wells of 5mm each were bunched in each plate using aseptic pipette tip. 0.1ml of tea extracts at varying concentrations (50mgml-1, 100mgml-1, 200mgml-1,and 400mgml-1) as well as the standard antibiotic solution was loaded into the wells. Control experiments were set up using streptomycin and cefadroxil (4mgml-1) for the bacteria and fungal assays. The plates were incubated at 37oC for 24hrs for bacteria and 48hr for fungi.
All inoculation procedures were carried out under aseptic conditions. The antimicrobial studies were done in triplicate. With the aid of a transparent ruler the diameter of zones of inhibition around the wells were measured in mm for all the three replicates and the average of the three measurements were calculated as an indication of activity. The results were interpreted according to the modified Kirby-Baur technique. The minimum inhibitory concentration (MIC) of tea extracts was determined using the broth dilution method as described by Salon and Washington (1990). Briefly 1ml of the extract solution at the concentration of 400mgml-1 was added to1 ml of nutrient broth and subsequently transferred to make solution of varying concentration (400mgml-1 200mgml, 100mgml-150mgml-1) in different test tubes. The 1ml of bacterial and fungal suspension and 1ml of extracts at different concentrations was added to each test tube and incubated at 370C for 24 hrs for bacteria and 48hr for fungi. The test tube with the concentration of tea extract at which no detectable growth was observed was considered as the MIC.
2.3 Statistical Analysis Data obtained from diameter zones of inhibition were subjected to ANOVA. Further analysis of the tea extracts to assess variation on disc diameter showed that the crude tea extracts had an activity had great variation on inhibition of the microorganisms (Table 3). There was significant difference (P<0.05). Those that did not show activity had a disc diameter of less than 3mm. Thus in inhibition activity, table 3 gives the variation inhibitory activity on the microorganisms since they show susceptibility to the tea extracts except for Salmonella typhimurium and Candida albicans which were not susceptible to orthodox tea.
3.0 Results The results of the phytochemical screening of the tea samples is presented in Table 2. The secondary metabolites tested wereAlkaloids, Saponin, Phenolics, Tannins, Anthraquinones, Cardenolides, Terpenes, Flavinoids and Cardiac glycosides. The results shows that Alkaloids, Saponin, Phenolics, Tannins, Anthraquinones, Cardenolides, Terpenes ,Flavinoids and Cardiac glycosides are present in all extracts except in green tea. Cardenolides are present in green and black tea but absent in orthodox tea. Phenolics are absent in orthodox tea but present in green and black tea. The results of the antimicrobial screening of the aqueous tea extracts are presented in Table 3, while the minimum inhibitory concentration (MIC) of each extract are shown in table 4. The tea extracts were found to be more effective in the tested bacteria than they were on fungi. Green and orthodox tea extracts showed important inhibition of Salmonella typhimurium and Escherichia coli gram-positive bacteria at the concentrations of 200 and 400mgml-1. All the tea extracts had activity against Staphylococcus aureus and Streptococcus faecalisgram-negative bacteria and only the extract of black tea was active against Candida albicans (a fungus) with the diameter of the zone of inhibition of 4.02mm and the MIC of 100mgml-1.

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Table 1: Sample information of tea studied
Name Locality of Collection Fermentation Status Green tea Kangaita Non-fermented Orthodox tea Kangaita Semi- fermented Black tea Murang’a Fermented
Table 2: Phytochemical constituents of crude extract of tea samples
Green tea Orthodox tea Black tea Phenolics + - + Flavonoids + + + Terpenes + + + Cardia glycosides + + + Cardenolides + - + Anthraquinones - + + Alkaloids + + + Saponins + + + + Presence of secondary metabolite - Absence of secondary metabolite
Table 3: Antimicrobial activity of crude tea extracts of Green tea, Orthodox tea and Black tea
Tea extract Conc. mgml-1
Staph aureus
Escherichia Coli
Salmonella typhim.
Streptococcus faecolis
Cadida albicans
Green tea 50 100 200 400
- 10± 0.0 15± 0.0 20± 0.0
- - 14± 0.0 18 ±0.0
- - 5± 0.0 18± 0.0
- 12.0±00 14.0±00 15.0±00
- - 1±0.0 1±0.4
Orthodox tea 50 100 200 400
- - 4 ±0.2 8 ± 0.0
- - 6± 0.0 14± 0.0
- - - -
-
10± 0.0 12 ±0.0
- - - -
Black tea (Murang’a)
50 100 200 400
- - 6.5± 0.0 7.4± 0.2
- - 3.5 ±0.0 14 ±0.0
- 1± 0.0 3± 0.0 4 ±0.0
- 2±0.0 5±0.0 6±0.2
- 4±.002 6±0.01 9±0.01
Streptomycin 4.0mg/ ml
20±0.0 10±0.0 15±0.0 10±0.0 25±0.0
Cefadoxil 4.0mg/ ml
20±0.0 20±0.0 20±0.0 20±0.0 10±0.0
- Absence of antimicrobial activity Arabic numerals – inhibition diameters in mm
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Table 4: Minimum inhibitory concentrations of aqueous extracts of tea on selected micro-organisms
Tea extract Conc. mgml-1 Staph aureus Escherichia Coli
Salmonella typhimurium
Streptococcus faecolis
Cadida albicans
Green tea 400 200 150 100 50 25
- - - - + +
- - - + + +
- - + + + +
- - - - + +
- - - + + +
Orthodox tea 400 200 150 100 50 25
- - - - + +
- - - + + +
- - + + + +
- - - - + +
+ + + + + +
Black tea (Murang’a)
400 200 150 100 50 25
- - - - + +
- - - + + +
+ + + + + +
- - - - + +
- - - - + +
- No growth observed + Growth observed
4.0 Discussion and Conclusion The presence of the secondary metabolites (alkaloids, terpenes, saponins, tannins, flavonoids, cardiac glycosides, cardenolides anthroquinones and phenols) in tea partly enhances the antimicrobial and anti-parasitic activity of the green, black and orthodox tea. Although the presence of similar secondary metabolites ma