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International Journal of Pharmacognosy and Phytochemical Research 2013; 5(3); 223-226

ISSN: 0975-4873
Research Article

Antimicrobial Activity and Phytochemical Screening of Extracts from

Leaves of Croton polyandrus Spreng

*1
Fernandes HMB, 1Leão AD, 1Oliveira-Filho AA, 1Sousa JP, 2Oliveira TL, 1Lima EO, 1Silva
MS , 1Tavares JF

1
Graduate Program in Natural Products and Synthetic Bioactive, Federal University of Paraiba, João Pessoa-Paraiba-
Brazil
2
Department of Pharmacy, Federal University of Paraiba, João Pessoa-Paraiba-Brazil

ABSTRACT
Aromatic plants have been used in folk medicine as antimicrobial agents since ancient times. The extracts were subjected
to phytochemical tests for plant secondary metabolites, tannins, saponins, steroid, alkaloids and flavonoids. The
antimicrobial activity of extracts was studied using the microdilution method and determination of minimal inhibitory
concentration (MIC) value. Investigations on the phytochemical screening of Croton polyandrous leaves extracts
revealed the presence of saponins, steroids and tannins. The results obtained with Croton polyandrus ethyl acetate and
dichloromethane extracts showed promising antifungal activity against Candida albicans (ATCC 90028) with MIC of 64
µg/mL and 128 µg/mL, respectively.

Key words: Croton polyandrus, extracts, antimicrobial activity.

INTRODUCTION Based on the medicinal properties of plants of Croton

Historically, plants have provided a source of inspiration genus, the present study aimed to investigate the
for novel drug compounds, as plant derived medicines antimicrobial activity of the extracts isolated from Croton
have made large contributions to human health and well polyandrus belongs to the family Euphorbiaceae.
being1.
Aromatic plants have been used in folk medicine as MATERIALS AND METHODS
antimicrobial agents since ancient times2,3. Although Preparation of plant extract: The leaves of Croton
approximately 20% of the world plants have been polyandrus were collected in the town of Santa Rita,
submitted to pharmacological or biological test, it could Paraiba, Brazil. The botanical material was identified by
be concluded that natural products from plant origin are Drª. Maria de Fatima Agra of the Botany Section of the
an important source to discover new leads with Laboratory of Pharmaceutical Technology "Prof. Delby
economical and pharmaceutical importance and great Fernandes de Medeiros". Voucher specimens of these
possibilities to be developed as drugs, dyes, fragrances plants are deposited in the Herbarium Prof. Lauro Pires
and pesticides, among others4. To obtain novel and Xavier (JPB), exsicata Agra & Gois 1446 (JPB), Federal
promissory substances many plant extracts have to be University of Paraiba.
assayed. For example, Mendes et al. (2011)5 assayed 2 The powder aerial parts - leaves (3000 g) was sprayed
plant extracts and found extracts with strong dried and subjected to maceration with hexane,
antimicrobial activity. dichloromethane, ethyl acetate and ethanol in a stainless
Furthermore, the screening of plant extracts as steel container for 72 hours each extraction. After
antimicrobial agents is necessary to go insight into extraction, the extraction solutions were concentrated in a
medicinal flora and get the molecules responsible for this rotary evaporator under reduced pressure at a temperature
activity and add value to natural resources from tropical of 45 ° C to yield extracts: hexane (40.2 g),
areas6. dichloromethane (46.5 g), ethyl acetate (59.8 g) and
Many Euphorbiaceae are well known in different parts of ethanol (194.4 g).
the world as toxic and/or medicinal plants. The high Phytochemical analysis of the plant extracts: The extracts
diversity of the described effects is a reflex of the high were subjected to phytochemical tests for plant
chemical diversity of this plant group. Croton is a large seconddary metabolites, tannins, saponins, steroid,
genus of Euphorbiaceae, comprising around 1,300 species alkaloids and flavonoids in accordance with Trease et al.
of trees, shrubs and herbs distributed in tropical and (1989)8 and Harborne (1998)9 with little modification.
subtropical regions of both hemispheres7.

*Author for correspondence:E-mail: [email protected]

 Fernandes HMB et al. / Antimicrobial Activity and…

Table 1 - Phytochemical analysis of the extracts from C. polyandrus.

Extracts/Seconddary Saponins Steroids Tannins Alkaloids Flavonoids
metabolites
EtOH extract + + + - -
Acoet extract - + - - -
Hexane extract - + - - -
Dichloro extract - + - - -
(+) presence and (-) absence

Table 2 – Antibacterial activity of the extracts from C. polyandrus.

Substance/Bacteria EtOH extract Acoet Hexane Dichloro Negative Positive control
strain (MIC) extract extract extract control (Chloramphenicol)
(MIC) (MIC) (MIC)
S.aureus ATCC 13150 1024 µg/mL 1024 µg/mL - - - +
S. aureus ATCC 25923 - - - - - +
P. aeruginosa P 03 - - - 1024 µg/mL - +
P. aeruginosa ATCC 1024 µg/mL 1024 µg/mL - 1024 µg/mL - +
25853
E.coli ATCC 25922 - - - - - +
E.coli 5 - - - - - +
(-) no growth inhibition (+) growth inhibition

Table 3: Antifungal activity of the extracts from C. polyandrus.

Substance/Bacteria strain EtOH Acoet Hexane Dichloro extract Negative Positive
extract extract extract (MIC) control control
(MIC) (MIC) (MIC) (Nistatin)
C. albicans ATCC 90028 1024 µg/mL 64 µg/mL - 128 µg/mL - +
C. albicans LM-109 - - - - - +
C. tropicalis ATCC - - - - - +
13803
C. tropicalis LM-P20 - - - - - +
C. krusei LM-13 - - - - - +
C. krusei LM-08 1024 µg/mL - - 1024 µg/mL - +
(-) no growth inhibition (+) growth inhibition
of Croton polyandrus extracts. The 96-well plates were
Bacterial and fungal strains: For antibacterial activity prepared by dispensing 100 µL of double strength
assays, were selected six strains of bacteria Nutrient Broth (NB) inoculated with the bacterium
(Staphylococcus aureus - ATCC 13150, Staphylococcus inoculum into each well prior to the assay. An aliquot
aureus - ATCC 25923, Pseudomonas aeruginosa - P03, (100 µL) of the extracts solutions at their respective
Pseudomonas aeruginosa - ATCC 25853, Escherichia concentrations was transferred into seven consecutive
coli - ATCC 25922 and Escherichia coli - 5) and for wells. The final volume in each well was 200 µL. The
antifungal activity assays were selected 6 strains of fungi solution having the highest extracts concentration was
( Candida added into the first well and the one having the smallest
albicans – ATCC 90028, Candida albicans – LM 109, concentration was added into the antepenultimate well.
Candida tropicalis - ATCC 13803, Candida tropicalis – The penultimate and the last well, containing 200 µL of
LMP 20, Candida krusei – LM 13 and Candida krusei – the NB inoculated with the microorganism suspension
LM 08). All the microorganism strains tested belong to and Chloramphenicol (100 µg/mL) or Nistatin (100
the collection of the Mycology Laboratory, Federal UI/mL), were used as the negative control and positive
University of Paraíba. Bacteria and fungi were kept on control, respectively. The microplate was asseptically
Nutrient Agar sealed, followed by mixing on a plate shaker (300 rpm)
(NA) slants at 4 °C. Inocula were obtained from for 30 seconds and incubated at 37 °C for 24 hours10,11.
overnight cultures grown on NA slants at 37 °C and The antibacterial and antifungal activities were detected
diluted in sterile saline solution (NaCl 0.85% w/v) to using the colorimetric method by adding 200 µL of
resauzurin staining (0.1 g.100 mL-1) aqueous solution in
224

provide a final concentration of approximately 106 Count

-1
Forming Unit per mL (CFU.mL ) adjusted according to each well at the end of the incubation period. MIC was
the turbidity of 0.5 McFarland scale tube. defined as the lowest essential oil concentration able to
Antimicrobial assay: The microplate bioassay was used to inhibit the bacterial growth as indicated by resauzurin
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determine the minimum inhibitory concentration (MIC) staining (dead cells were not able to change the staining

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 Fernandes HMB et al. / Antimicrobial Activity and…

color by visual observation – blue to red)12. All acetate (Acoet) and dichloromethane (Dichloro) extracts
experiments were carried out at least twice with show promising antifungal activity against Candida
consistent results (Table 2 and Table 3). albicans (ATCC 90028) with MIC of 64 µg/mL and 128
µg/mL, respectively. Furthermore, these extracts showed
RESULTS low antifungal activity against Candida krusei (LM 08)
Phytochemical screening of Croton polyandrous leaves with MIC of 1024 µg/mL. The different behavior
ethanolic extracts revealed the presence of saponins, observed between strains of the same species could be
steroids and tannins. Yet the phytochemical screening of justified by the existence of genetic variability among
Croton polyandrous leaves hexane, dichloromethane and different strains20.
ethyl acetate extracts revealed the presence of steroids This antifungal activity against Candida albicans of
(Table 1). extracts from Croton polyandrus has been observed in
The results for antimicrobial activity of the extracts with other studies with extracts of plant species of the family
MIC value are show in Table 2 and Table 3. The activity Euphorbiaceae21.
was measured in terms of presence of microorganism
growth. the extracts from Croton polyandrus show no CONCLUSION
antibacterial activity against either gram (+) or gram (-) Based on these results it can be stated that the Cronton
bacteria. However, results obtained from the in vitro polyandrous extracts have an important antifungal
antifungal assay showed that the ethyl acetate (Acoet) and activity against Candida species, which highlights the
dichloromethane (Dichloro) extracts show antifungal need for further studies with other fungal species to
activity against Candida albicans (ATCC 90028) with investigate the immense therapeutic potential of this plant
MIC of 64 µg/mL and 128 µg/mL, respectively. species and with his isolated secondary metabolites.
Furthermore, these extracts showed low antifungal
activity against Candida krusei (LM 08) with MIC of ACKNOWLEDGEMENTS
1024 µg/mL. The authors thank all the technical staff of the Mycology
Laboratory and the Laboratory of Phytochemistry
DISCUSSION CCS/UFPB.
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