Antimicrobial testing of selected fijian plants :: The University of the South Pacific Library

INSTITUTE OF APPLIED SCIENCES

THE UNIVERSITY OF THE SOUTH PACIFIC

Antimicrobial Testing of

Selected Fijian Plants

IAS Technical Report No. 2000/04

by

Bill Aalbersberg, Paul Ralifo,

Arun Pande, Girish Lakhan,

Semisi Meo, Ritesh Raju,

and Mukesh Sharma

December, 2000

ANTIMICROBIAL TESTING OF SELECTED FIJIAN PLANTS

by

Bill Aalbersberg, Paul Ralifo, Arun Pande, Girish Lakhan,

Semisi Meo, Ritesh Raju, Mukesh Sharma.

Institute of Applied Science,

University of the South Pacific,

Suva, Fiji.

Abstract

Sixteen plants were extracted and tested for antimicrobial activity. These plants

were collected in Viti Levu and some of them have been used traditionally as

herbal remedies for certain illnesses. Seven microbes were used in the tests and

include Salinovibrio costicolla, Escherichia coli, Staphylococcus aureus, Bacillus

subtilis, Candida albicans Trychophyton mentagrophytes and Mirosporum

gypseum. The tests showed a variety of activities against these microorganisms.

1. Introduction

Every year rainforests succumb to deforestation and plant species

become extinct. Hence, it is essential to know and appreciate the potential uses

and values of local plants, in order to facilitate conservation of rain forests and

their invaluable plant species. Many plants have a long history of traditional

medicinal use. Lately, modern scientists have borrowed ideas from traditional

medicinai practices resulting in the development of new drugs.

In this study, the antimicrobial activities of sixteen plant (Table 1) extracts

were investigated . These plants were collected in Viti Levu and various pa1is of

these plants were dried, ground and then extracted with methanol. The extracts

were then exposed to seven microorganisms and their activities recorded. Some

of the plants collected were used traditionally as herbal remedies and their uses

are listed in Table 2.

Four bacteria and three fungi were used in the assays. The four test

bacteria included a marine Gram-negative bacterium, Salinovibrio costicolla, and

three terrestrial bacteria, Escherichia coli, Staphylococcus aureus and Bacillus

subtilis. E. coli is Gram negative and is known to be part of the flora of

microoganisms that inhabit the guts of humans and animals . This bacterium can

produce enterotoxins causing diarrhea in animals and man. S. aureus is a Gram

positive bacterium and is a common inhabitant of skin and nasal passages and

can cause staphylococcal food poisoning, boils, meningitis, impetigo and

pneumonia. B. subtilis is a Gram positive bacterium and is sometimes an

Table 1. List of plants tested in the assays.

Sample code

174D

173

153

157T

162

159

Dilo seed

169D

153T

155T

46D

151

167

170

169

174T

Scientific name

Crossostylis harvei

Flame ginger *

Oolicholobium macgregori

Crossostylis seemanii

Costus speciousus

Heliconia paka

Calophyllum inophyllum

Neuburgia alata

Oolicholobium macgregori

Pittosporum pickeringii

Mikania micrantha

Pittosporum rhytidocarpum

Calanthe sp.

Spathoglottis pacifica

Neuburgia corynocarpa

Agathis macrophyllum

Part of plant used

Leaves

Whole

Leaves and flowers

Trunk

Stem and leaves

Leaves and stalk

Seeds

Leaves

Trunk

Whole

Trunk and leaves

Whole

Trunk

* The scientific name of this plant is not known however has been identified by its

common name of flame ginger.

Table 2. Traditional medicinal uses of plants in Fiji.

Sample code Traditional medicinal use

157T

Used to treat headaches, occurrence of blood in the urine,

unconsciousness and dysentery. A combination of the bark of

this plant and other plants is used to treat fractures.

Dilo seed

Oil from the seed is used to treat rheumatism, wounds and coral

sores and bruises. The leaf is used to treat eye irritations and

conjunctivitis. The plant is also used to treat toothache and

tuberculosis.

155T

The plant is used to treat constipation, cuts, wounds and

stomach trouble.

46D

The leaves are used to treat skin irritations, bees and wasp

stings, stop bleeding and boils of the armpit. It is also used for

stomach aches, high blood pressure and diabetes, cancer and

hastens birth for expectant mothers.

151

The plant is used by mothers after childbirth.

170

Used to treat pain in the joints.

169

The roots and leaves are used together with Mikania micrantha

to treat backache, headache and vaginal bleeding.

opportunistic pathogen causing food poisoning. The three fungi include the yeast

Candida albicans and two moulds Trychophyton mentagrophytes and

Microsporum gypseum. C. albicans causes infections of the mucosa! membranes

of the body known as candidiasis. T. mentagrophytes (var. mentagrophytesi) and

M. gypseum are common human dermatophytes causing cutaneous mycoses

such as tinea pedis (atheletes fo ot), tinea corporis (ringworm of the smooth or

bare parts of the skin), tinea cruris (ringworm of the groin), and tinea unguium

(infection of the nail bed). Due to the pathogenic nature of these microoganisms

these tests were done in a biosafety cabinet (class II) using sterile techniques.

2. Methodology

1. Sample preparations:

All the samples were dried, ground and soaked in methanol overnight.

This extraction process was repeated twice. The combined methanol extracts

of the samples were then filtered and reduced under vacuum.

The dried crude extracts (100mg) were then dissolved in 80% dimethyl

sulfoxide (DMSO) in water to make concentrations of 250mg.mr1. The

dissolved samples (1 0~tl) were then transferred to sterile paper disks (6mm

diameter) and dried and then used in the assays.

2. Antibacterial assay:

This assay employed four bacteria, S. costicol/a, E. coli, B. subtilis and S.

aureus. Seed cultures (3ml) were prepared for each bacterium in the

following way. Nutrient broth (NB) was used to culture E. coli, B. subtilis and

S. aureus, however, marine broth (MB) was used for S. costicolla . The

prepared media (3ml) for each bacterium was autoclaved, cooled ;:md

inoculated with each bacterium using sterile 10µL loops and the culture"

incubated at 30°C overnight while shaking.

For the assay plates, nutrient agar (NA) was used to culture E. coli, B.

subtilis and S. aureus, however, marine agar (MA) was used for S. costicolla.

The media (50ml) for each bacterium was autoclaved and then cooled to

45°C and inoculated with overnight seed cultures. MA (50ml) was inoculated

with 1.5ml of overnight broth culture of S. costicol/a, while for the other test

bacteria, 50ml of NA was inoculated with 50µL of overnight broth culture. The

inoculated media were then poured into tissue culture plates, allowed to

solidify, and then used in the bioassay.

Prepared sample disks were then placed on the assay plates together with

a known standard for that particular bacterium used as a positive control. The

plates were incubated as follows. E. coli and B. subtilis plates were incubated

at 37°C while S. aureus and S. costicol/a were incubated at 30°C. The results

were checked and recorded after 24 hours of incubation by measuring the

diameter of the zone of inhibited microbial growth (mm). Corrected results

were then taken by subtracting the diameter of the sample disks from the

diameter of the zone of inhibition.

3. Anti-Candida assay:

Freshly prepared and autoclaved tryptic soy broth (TSB) was inoculated

with C. albicans and incubated at 35 to 38°C, stationary overnight. After

incubation, the seed culture was used to prepare a 10-1dilution (10ml)

overnight culture with an optical density between 0.05 and 0.5 using a

spectrophotometer (A500). The diluted seed culture (500µL) was then used to

inoculate 125ml of molten Sabourad dextrose agar (SDA) at ~45°C. The

inoculated media was then gently mixed and poured into sterile petri dishes.

The plates were then allowed to dry and the sample disks were placed on

the surface of the inoculated agar. A disk containing nystatin was assayed as

a standard and a disk was also assayed which contained only the solvent.

4. Anti-fungal assay:

Potato dextrose agar (PDA) was autoclaved and poured into sterile petri

dishes and allowed to set and dry at room temperature. The plates were

inoculated with thawed glycerolised fungal colonies. The inoculated plates

were then incubated at 36-38°C for 7 days.

After incubation, a sterile inoculating needle was used to remove spores

from the 7-day fungal colonies and on newly prepared PDA plates, the spore-

loaded needle was dipped at three points. The inoculated plates were then

incubated at 36-38°C for 7 days.

To inoculate the assay plates, mycelium plugs from the 7-day old 3-point

inoculum plates were used . A sterile core bore (size 2) was used to punch

into the agar at points covered with mycelium on the 3-point inoculum plates.

The mycelium plugs were then carefully lifted using sterile forceps and placed

upside down (the mycelium side of the plugs were placed face down) on new

PDA plates. The plates were then sealed and incubated (right side up) at 36-

380C for 4 days.

In the assay, a sample disk was loaded onto the 4-day assay plate at

1.5cm from the mycelium plug. A blank disk loaded with the solvent alone

was placed (1.5cm from the mycelium plug) on the opposite side of the

mycelium plug on the same plate. The assay plates were then incubated

(right side up) at 36 to 38°C for another 4 days. A standard prepared from

9mg of griseofulvin in 1ml of DMSO was also assayed for comparison

· purposes. Positive results we n~ interpreted as having an inhibition zone

between the mycelium growth and the sample disk as compared to the blank.

All positive results were replicated and the average of the diameters of the

zones of inhibited microbial growth was taken.

3. Results and discussion

Table 3. Results of the bioactivity tests for the plant extracts:

Antimicrobial activit, (Ave. zone of inhibition (mm

Plant

(part of plant used)

Cl)

::(:I:J:

0

ls

Cl)

0

(.)

uj

·0 -

(.)

LJ..i

Cl)

::J

@

::J

(IJ

uj

-:.-.~.:.:.:,:

(IJ

~

.Q

::J

Cl)

sJ

C:

(IJ

aj

0

J!l

..~ c::

E:

::J

e0..

i-:

Di

Q)

Cl)

~

..(..I.J..

Di

C:

Q)

~

E:

Crossosty/is harvei

(leaves)

-

Flame ginger

(whole)

2.0

-

Dolicho/obium macgregori

(leaves and flowers)

-

Crossosty/is seemanii

(trunk)

-

Costus speciosus

(stem and leaves)

-

Heliconia paka

(leaves and stalk)

-

Ca/ophy/lum inophyllum

(seeds)

3.0

-

Neuburgia a/ata

(leaves)

-

Do/icho/obium macgregori

(trunk)

-

Pittosporurn pickeringii

(trunk)

-

Mikania micrantha

(whole)

12 .5

-

Pittosporum

rhytidocarpum

-

(trunk and leaves)

Ca/anthe sp.

(whole)

-

Spathoglottis pacifica

(whole)

-

Neuburgia corynocarpa

(whole)

-

Agathis macrophyllum

(trunk)

-

Ampicillin

9.0

-

3.0

-

3.0

4.0

-

3.0

-

8.0

5.0

-

NA

-

3.5

-

1.5

-

1.0

2.0

1.0

-

2.0

-

2.0

-

1.0

1.5

-

NA

Penicillin-G

NA

13.0 30.0

19.0

NA

Nystatin

NA

-

NA

3.0

NA

Griseofulvin

NA

-

NA

6 .5

9.0

All inhibition zones of less than 1mm were recorded as negative. "NA" in

the above table means not applicable. In each of the assays, a standard was

included to ensure that the test system was functioning properly and to compare

the activity of the sample of interests with the standard. In addition, a control,

which includes the solvent alone, was included to measure the effect, if any, of

the solvent in which the samples were dissolved.

The results showed that some of these plant extracts showed variable

antimicrobial activity. None of the extracts however showed any activity against

C. albicans. The strongest antimicrobial activity was recorded for Mikania

micrantha against S. costicolla (even more active than ampicillin) and it showed

activity against S. aureus, B. subti/is and M. gypseum. This plant is used

traditionally in Fiji to treat skin irritations, insect stings, boils of the armpits and is

known to exhibit antimicrobial activity. Calophyllum inophyllum is known to exhibit

antibacterial activity and the leaves of Pittosporum rhytidocarpum are known to

produce antimicrobial compounds. However in the assay, Calophyllum

inophyl/um showed only low activity (3.0mm) against S. costicolla and no activity

against the other microbes for that concentration. Pittosporum rhytidocarpum

also exhibited moderate activity against M. gypseum and no activity against the

other microbes for that concentration.

Not all the plants studied are used traditionally as herbal remedies.

However, these types of studies can identify potential uses of various plant

species which can be of commercial value.

4. References

• Bergey's manual of systematic bacteriology, Vol 1, Kreig, N. R. and Hol, J. G.

(eds), Williams and Wilkins, Baltimore, 1984.

• Bergey's manual of systematic bacteriology, Vol 2, Sneath, P. H. A.; Mair, N.

S.; Sharpe, M. E. and Holt, J. G. (eds.), Williams and Wilkins, Baltimore,

1986.

• Cambie, R. C. and Ash, J. (1994) Fijian medicinal plants, CSIRO, Australia,

365pp.

• Medicinal plants in the South Pacific, WHO Regional Publications Western

Pacific Series No. 19, 1998, 254pp.