Analysis of phenolic compounds in Carica papaya, Zingiber officinale, Ipomoea batatas and Myrothamnus flabellifolius using RP-HPLC-UV/VIS-DAD, and in vitro evaluation of antifungal activity o

ABSTRACT

Agricultural pesticides use is rising in crop protection against plant invaders of which fungi are

chief in reducing both yield quantity and quality. Botrytis cinerea, Rhizoctonia solani,

Macrophomina phaseolina and Alternaria alternata are polyphagous fungi of economic

importance with severe yield and quality losses worldwide. Environmental concerns on pesticide

use and their resistance now on a worldwide scale. Extensive studies of plant secondary

metabolites have not exhausted their potential use as natural crop proctetants. The antifungal

activity of plant phenolic extracts: Carica papaya leaves and bark; Zingiber officinale rhizomes;

Myrothamnus flabellifolius above ground parts; and Ipomoea batatas leaves were investigated on

the plant pathogenic fungi. Methanol (80%) was used to extract phenolic compounds from dried

samples. Qualitative and quantitative analyses of extracts were done through Reversed-Phase

High Performance Liquid Chromatograph, Ultraviolet-Visible light Diode Array Detector (RPHPLC-

UV/VIS-DAD) analyses. In antifungal in vitro assays, Potato Glucose Agar (PGA) in

petri dishes was incorporated with three concentrations 250 ppm,500 ppm and1000 ppm) of

sample plant phenolic compounds and active ingredients of fungicides (as positive controls); the

negative controls were incorporated with diluent used to reconstitute plant phenolic compounds.

The study was laid in a 7 x 3 factorial treatment in a completely randomized design replicated

three times with the two factors being plant phenolic extracts and extract concentration. A 6mm

diameter disc of the 7-day old pure fungi culture cultivated on PGA was aseptically inoculated at

the centre of the petri dish for each treatment and petri dishes incubated at 20±2°C (B. cinerea)

and 26±2°C (R. solani, M. phaseolina and A. alternata). Mycelial growth (diameter)

measurements (in mm) were taken at 24hour exposure and stopped upon full colony diameter

(85mm) observation in the negative control plates. Quantitative RP-HPLC-UV/VIS-DAD

analyses yielded a strong peak at a Retention Time (RT) =1.01min for Ipomoea batatas however

this could not be elucidated. Other extracts produced nondescript chromatograms that were not

similar to standard phenolic compounds RTs. There were significant differences (ρ ˂ 0.05) in

Total Phenolic Content of plant phenolic extracts yields (mgGAE/g), 55.7±2.2, 7.0±3.3,

106.1±12.0, 33.5±3.4, and 41.9±1.5 for C. papaya leaves, bark, Z. officinale, M. flabellifolius

and I. batatas respectively. Phytochemical test results were varied amongst plant phenolic

extracts, mostly being positive. Antifungal in vitro assays, plant phenolic extract and extract

concentraction interacted significantly (all ρ ˂ 0.05) at all the time intervals for all the species

except for B. cinerea 96 hours interval where only the plant phenolic extract were significant (ρ

˂ 0.05) whilst there was no significant interaction of the two factors. The interaction of the two

factors showed varied results in their inhibition of mycelial growth with differences amongst

plant phenolic extracts and fungi species noted. Percentage Mycelial Reduction (PMR) varied

amongst phenolic extracts and phytopathogenic fungi. Generally, the phenolic extracts showed

less antifungal activity compared to the positive control treatments (fungicides). Carica papaya

bark and Z. officinale rhizomes showed the highest PMR against M. phaseolina

(30%≤PMR≤40%) at the 1000 ppm concentration. Z. officinale showed the highest PMR

(45.3%≤PMR≤ 54.7%) for all the concentrations on A. alternaria. M. flabellifolius showed the

highest PMR (26.5%≤PMR≤53.9%) for all the three concentrations on R. solani. Only M.

flabellifolius had limited PMR of 0-5.9%, whilst other plant phenolic extracts showed a 0% PMR

agaist B. cinerea. The result of PMRs shows that plant phenolic extracts have potential use in

vivo as crop protectants however further research may consider reduced fungicide dosages by

using both synthetic and plant phenolic extract as mixtures.