Medicinal Plants Classification Biosynthesis and ... - Index of

Medicinal Plants: A Tool to Overcome Antibiotic Resistance?
index ≤ 0.5), additive (0.5 < FIC index ≤1), indifference (1 < FIC index ≤ 2) and antagonism
(FIC index > 2) (Mackay et al., 2000).
2.5. Bioautography on Thin-Layer Chromatoplates
TLC-bioautography, a convenient and simple way of testing plant extracts and pure
substances for their effects on pathogenic microorganisms, allows an easy detection of active
fractions (Hostettmann et al., 1997). Three variants of the technique are described: (i) direct
bioautography, in which a broth of the micro-organism is directly applied on the TLC plate;
(ii) contact bio-autography, where the antimicrobial compounds are transferred from the TLC
plate to an inoculated agar plate through direct contact; and (iii) agar-overlay bioautography,
where a seeded agar medium is applied directly onto the TLC plate (Botz et al., 2001). In the
1960's, contact and immersion bioautography in various versions were routinely used, but
nowadays direct bioautography largely prevails (Hamburger et Cordell, 1987).
TLC is usually performed in duplicate with suitable mobile phases and the plates are
dried to remove the eluents, a critical step. One set of plates is used as the "reference"
chromatogram, spots being visualized under UV light or by spraying a specific or a general
reagent (Wagner et Bladt, 1996). The second plate is placed in a Petri dish, aseptically
overlaid by the inoculated medium and incubated overnight. The bioautogram is
subsequently sprayed with an aqueous solution of MTT 0.8 mg/ml and incubated for a few
hours (Botz et al., 2001). The MTT is converted to a formazan dye by the microorganisms
and active compounds are indicated by inhibition zones observed as clear spots against a
purple background (Begue et Kline, 1972). The applicability of TLC-bioautography is
however limited to microorganisms that easily grow on TLC plates and requires complete
removal of residual low volatility solvents, such as n-BuOH, trifluoroacetic acid or ammonia,
which can inhibit the growth of several microorganisms (Nagy et al., 2002).
2.6. Antiparasitic Test
In contast to antibacterial, antifungal and antiviral assays that are based on common test
conditions and endpoints, antiparasitic assays are more complicated and more exclusive since
they tend to be highly species-specific (Plasmodium, Entamoeba, helminthes...). Particular
interest is devoted to malaria, one of the protozoal diseases that have been defined by the
WHO as major health risks. Extracts possibly effective against Plasmodium can be tested on
microorganisms in an erythrocyte infection assay on microtiter plates. Parasitized
erythrocytes are incubated with and without test substances, and the numbers of Plasmodium
organisms after the incubation period are quantitated either by measurements of radioactivity
(radiolabeleld parasites) (Cos et al., 2006) or of the parasite lactate deshydrogenase (pLDH)
activity, using 3-acetyl pyridine NAD (APAD) as a coenzyme; as human red blood cell LDH
carries out this reaction at a very slow rate in the presence of APAD, the measurements
correlate with the levels of parasitemia (Makler et al., 1993).
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