Redissolving Extracts for Quantitative Data
To enable valid comparisons, all data have to be expressed on a quantitative base
(e.g. activity per mg extract). Extracts therefore have to be dried and subsequently
redissolved to make up a known concentration of the extract. Frequently, dried ex-
tracts are not freely soluble even if the same solvent is used and this causes com-
plications. To avoid this problem we do not dry extracts. To determine the concen-
tration of the extract for quantification purposes we take a small aliquot, dry it, and
use the values obtained to calculate the original concentration [37].
5.7.5
Storage of Extracts
Extracts are kept at 3–7 °C, not in a deep freeze where precipitation may take place.
We had difficulties in storing aqueous extracts because in our experience fungal
growth invariably occurs after some time, even at low temperatures. This is prob-
ably because good carbon and nitrogen resources for fungal growth such as sugars
and amino acids may be present in the aqueous extracts.
Selection of containers used to store acetone extracts is important. Acetone plant
extracts lose up to 87% of the acetone if stored in glass containers with polyethy-
lene stoppers at 40 °C for a month. Overall, Teflon film is the best, followed by rub-
ber, aluminum film, and polyethylene stoppers [38].
One would expect that dried extracts would be very stable, but we were surprised
that acetone extracts of members of the Combretaceae retained antibacterial and
anti-inflammatory activity over prolonged periods even when stored in a dissolved
state at room temperature [23]. This may be due to the antibacterial and antifungal
activity of these compounds [21, 27].
Evaluating Quantitative Antimicrobial Activity
Agar diffusion techniques are used widely to assay plant extracts for antimicrobial
activity, but there are problems associated with this technique. A microdilution
technique was developed using 96-well microplates and tetrazolium salts to indi-
cate bacterial growth [39]. p-Iodonitrotetrazolium violet (0.2 mg mL–1) gave better
results than tetrazolium red or thiazolyl blue. The method is quick, worked well
with Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia
coli and with nonaqueous extracts from many different plants. These bac-
5.8 Evaluating Quantitative Antimicrobial Activity 105
terial species were selected because they are responsible for most nosocomial dis-
eases in hospitals [40]. The method gives reproducible results, requires only
10–25 μL of extract to determine minimal inhibitory concentrations (MIC), distin-
guishes between microcidal and microstatic effects and provides a permanent
record of the results. Using S. aureus, and a Combretum molle extract, the technique
was 32 times more sensitive than agar diffusion techniques and was not sensitive
to culture age of the test organism up to 24 h [39]. The S. aureus culture could be
stored up to 10 days in a cold room with little effect on the assay results. This meth-
od is useful in screening plants for antimicrobial activity and for the bioassay-guid-
ed isolation of antimicrobial compounds from plants. MIC values determined for
sulfisoxazole, norfloxacin, gentamicin, and nitrofurantoin were similar to values
indicated in the literature but values obtained with trimethroprim and ampicillin
were higher with some bacteria [39]. This method also works well with fungi [27].
To enable valid comparisons, all data have to be expressed on a quantitative base
(e.g. activity per mg extract). Extracts therefore have to be dried and subsequently
redissolved to make up a known concentration of the extract. Frequently, dried ex-
tracts are not freely soluble even if the same solvent is used and this causes com-
plications. To avoid this problem we do not dry extracts. To determine the concen-
tration of the extract for quantification purposes we take a small aliquot, dry it, and
use the values obtained to calculate the original concentration [37].
5.7.5
Storage of Extracts
Extracts are kept at 3–7 °C, not in a deep freeze where precipitation may take place.
We had difficulties in storing aqueous extracts because in our experience fungal
growth invariably occurs after some time, even at low temperatures. This is prob-
ably because good carbon and nitrogen resources for fungal growth such as sugars
and amino acids may be present in the aqueous extracts.
Selection of containers used to store acetone extracts is important. Acetone plant
extracts lose up to 87% of the acetone if stored in glass containers with polyethy-
lene stoppers at 40 °C for a month. Overall, Teflon film is the best, followed by rub-
ber, aluminum film, and polyethylene stoppers [38].
One would expect that dried extracts would be very stable, but we were surprised
that acetone extracts of members of the Combretaceae retained antibacterial and
anti-inflammatory activity over prolonged periods even when stored in a dissolved
state at room temperature [23]. This may be due to the antibacterial and antifungal
activity of these compounds [21, 27].
Evaluating Quantitative Antimicrobial Activity
Agar diffusion techniques are used widely to assay plant extracts for antimicrobial
activity, but there are problems associated with this technique. A microdilution
technique was developed using 96-well microplates and tetrazolium salts to indi-
cate bacterial growth [39]. p-Iodonitrotetrazolium violet (0.2 mg mL–1) gave better
results than tetrazolium red or thiazolyl blue. The method is quick, worked well
with Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia
coli and with nonaqueous extracts from many different plants. These bac-
5.8 Evaluating Quantitative Antimicrobial Activity 105
terial species were selected because they are responsible for most nosocomial dis-
eases in hospitals [40]. The method gives reproducible results, requires only
10–25 μL of extract to determine minimal inhibitory concentrations (MIC), distin-
guishes between microcidal and microstatic effects and provides a permanent
record of the results. Using S. aureus, and a Combretum molle extract, the technique
was 32 times more sensitive than agar diffusion techniques and was not sensitive
to culture age of the test organism up to 24 h [39]. The S. aureus culture could be
stored up to 10 days in a cold room with little effect on the assay results. This meth-
od is useful in screening plants for antimicrobial activity and for the bioassay-guid-
ed isolation of antimicrobial compounds from plants. MIC values determined for
sulfisoxazole, norfloxacin, gentamicin, and nitrofurantoin were similar to values
indicated in the literature but values obtained with trimethroprim and ampicillin
were higher with some bacteria [39]. This method also works well with fungi [27].
Post a Comment