Antimicrobial testing of selected fijian plants part 2 :: The University of the South Pacific Library

INSTITUTE OF APPLIED SCIENCES

THE UNIVERSITY OF THE SOUTH PACIFIC

ANTIMICROBIAL TESTING OF

SELECTED FIJIAN PLANTS -

PART2

IAS TECHNICAL REPORT NO. 2002/01

by

Bill Aalbersberg

Paul Ralifo

Marika Tuiwawa

January, 2002

Antimicrobial Testing of Selected Fijian Plants, Part 2

by

Bill Aalbersberg, Paul Ralifo and Marika Tuiwawa

Institute of Applied Sciences,

The University of the South Pacific,

Suva, Fiji.

Abstract

Forty-six plants were extracted and tested for antimicrobial act1v1ty. 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 four

bacteria, Salinovibrio costicolla, Escherichia coli, Staphylococcus aureus, Bacillus

subtilis, and three fungi, Candida albicans, Trychophyton mentagrophytes and

mM1i.rcorospoorgraumm.smgsy.pseum. The tests showed a variety of activities against these

The traditional use of these plants was also compared to the test results. Quite

interestingly, all plants that were used for antibacterial or antifungal purposes showed

this kind of activity in the laboratory tests.

·

1. Introduction

Every year rainforests are succumbing to deforestation and many plant species now

face the danger of being overexploited. 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 and have been used extensively by traditional healers.

Lately, modern scientists have borrowed ideas from traditional medicinal practices

resulting in the development of new drugs.

In this study, the antimicrobial activities of forty six plant (Appendix 1) extracts were

evaluated. These plants were collected in Viti Levu, Fiji and various parts of these

plants were dried, ground and then extracted with methanol (MeOH). The extracts

were then exposed to seven microorganisms, some of which are dermatophytes, and

their activities recorded.

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

Gramnegative and is known to be part of the flora of microorganisms that inhabit the

guts of humans and animals. This bacterium can produce enterotoxins causing

diaiThea in animals and man. S aureus is a Gram-positive bacterium and is a

1

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 opportunistic pathogen causing food

poisoning. The three fungi include the yeast Candida albicans, Trychophyton

mentagrophytes and Microsporum gypseum. C. albicans is a yeast that causes

infections of the mucosal membranes of the body known as candidiasis. This fungus

can grow in many forms such as unicellular, filamentous and as chlamydospores

depending on the medium it is grown in. T mentagrophytes (var. mentagrophytesi)

and M gypseum are common human dermatophytes causing cutaneous mycoses such

as tinea pedis (atheletes foot), 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 microorganisms 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. The

methanol (MeOH) extracts of the samples were then filtered and reduced under

vacuum. This extraction process was repeated twice.

·

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

(DMSO)

in

water

to

make

concentrations

of

250

m

g.mr

1

•

The

dissolved

samples

(10µL) 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. costicolla, E. coli, B. subtilis and S. aureus.

Seed cultures (3rnL) 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 medium

(3mL) for each bacterium was autoclaved, cooled and inoculated with each

bacterium using sterile lOµL loops and the cultures 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. costicolla, 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.

2

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

standard for that particular bacterium and 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. costicolla 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 (l0mL) overnight culture with an

optical density between 0.05 and 0.5 using a spectrophotometer (A6oo). 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 then 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 positive control was also assayed which contains only the solvent.

4. Antifungal 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 faced down) on new PDA

plates. The plates were then sealed and incubated (right side up) at 36-38°C 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

3

plate as a positive control. 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

lmL of DMSO was also assayed for comparison purposes. Positive results were

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

All inhibition zones of less than 1mm were recorded as negative. 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 positive 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 from the microbial assays are listed in Table I. The results of the controls

are listed in Table 2.

Approximately half of the extracts showed activity against one of the bacteria and

about a third of them against one of the fungi. Only the Endospermum macrophyllum

wood extract was active against of yeast Candida albicans. This extract was also

active against with three of the bacteria with large zones of inhibition and _both fungi.

Interestingly, the most extensive compilation of medicinal activity of Fijian plants

(Cambie and Ash, 1994) which summarises all written reports of such activity, makes

no mention of this plant. Activity is repo1ied for 450 of the roughly 2,500 plants

found in Fiji. Of course, many traditional treatments have not been documented in

writing and some of the most important ones may be family secrets.

The use of plants can also depend on availability. Many rare plants in Fiji are not

used and some not even named in the local language due to this rareness.

Of the forty-six plants tested eighteen are not listed in Cambie and Ash. Seventeen

are listed but the conditions for which they are reportedly used are not related to anti-

bacterial or antifungal activity. Nine plants were used for anti-bacterial purposes,

Barringtonia asiatica, Vitex trifolia var. subtrisecta, Epipremnum pinnatum, Premna

taitensis, Syzygium malaccense, Solanum torvum, Mussaenda raiateensis, Cordyline

terminalis and Dillenia bi.flora. All of these tested positive for antimicrobial activity,

and all but Barringtonia asiatica for antibacteria activity. The leaves of two plants,

Heritiera littoralis and Xylocarpus granatum, are used to treat thrush, a yeast

intection of the tongue and both displayed strong antifungal activity.

Overall only 44% of plants that did not appear in Cambie and Ash showed any

activity. For plants that were listed as used medicinally but not for antimicrobial uses

53% showed activity. As mentioned earlier, 100% of the 11 plants that were reported

4

to be used extensively for antimicrobial purposes were also active in the laboratory.

These results indicate a good correlation between traditional use by Fijians and

activity tests in the laboratory.

One plant tested, Homalanthus nutans, has received considerable interest overseas as

it contains prostratin, a potential anti-AIDS medicine. The extract from this plant

also showed antimicrobial activity.

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, WH~ Regional Publications Western

Pacific Series No. 19, 1998, 254pp.

5

TABLE 1: RESULTS OF THE BIOACTIVITY TESTS FOR THE PLANT EXTRACTS

Plant Code Antimicrobial Activity (Zone of inhibition measured in mm).

~

:-:c-::;:

;:;

"'

Cl::i

3D

6D

13D&T

JO

14D

15 D&T

9

16T

9

17D&T

18D

24D

24T

26D

28D

29D

32D

33D

34D

34T

36T

38T

41 D&T

42D

44D

45

45T

47D

SOD

12

51D

8

36D

8

53D

57D

54D

57T

8

58T

9

58D

61T

62D

9

62T

63D

II

73T

76 D

79D

9

SOT

17

84D

87D

92D

95T

9

96T

102D

105D

106D

9

108D

109D

9

110D

9

·--a..

;~ "._:;'

;:;

'-'

<::!

kl

C,:i

7.5

8

8.5

8

10

JO

· 12

II

10

II

9

II

--~-a<.:.:!

C,:i 8"'

s"::'

<::!

u ~-'0-'

§.

!::

5°-0

-~s:: "()'.)

-E' ().)

h E:

~

2

3

2

3 .5

2

4

2

3

2

3

4

3.5

2

-

9

2

1.5

8

2

6

TABLE 2: AVERAGE ZONES OF INHIBITION FOR THE RESPECTIVE CONTROLS

Controls Measured in mm

~

- ~ ~

~

·..~... -

..c

·~

0

(.;)

~

<:I)

:,.._

~

i::::s

c::i

~

tl'j

Penicillin 30

-

39

G

Polymixci -

22

-

nB

Nystatin -

-

Griseoful -

-

vm

~

0

·.~.\\...).

~

tl'j

0

\\.)

-

25

-

~

i::::s

u .·.~\\c.)

~

-

10

-

So ~

-2 ~i::::s ~

h ~ §<

-

~

<:I)

~~

-

6

8

7

Appendix 1. List of Plants Tested in the Assays

Sample Code

Scientific Name

Plant Part

3

Barringtonia asiatica

leaves

6

Heritiera littoralis

leaves

13

Vitex trifolia var subtrisecta

bark

14

Aleurites moluccana

leaves

15

Epipremnum pinnatum

bark

16

Homalanthus nutans

wood

17

Bambusa vulgaris

leaves

18

Cerbera manghas

leaves

24

Premna taitensis

leaves, wood

26

Pandanus caricosus (Whitmeeanus)

leaves

28

Syzygium malaccense

leaves

29

Parasponia andersonii

leaves

32

Xylocarpus granatum

leaves

33

Myristica grandifolia

leaves

34

Lumnitzera littorea

leaves, wood

36

Syzygium richii

wood

38

Maesa tabacifolia

wood

41

Passiflorafoetida var. hispida

wood

42

Physalis peruviana

leaves

44

Hernandia peltata

leaves

45

Tarenna sambucina

wood

47

Coccinea grandis

leaves

50

Alphitonia zizyphoides

leaves

51

Solanum torvum

leaves

53

Ervatamia orientalis

leaves

54

Gironniera celtidifolia

leaves

57

Mussaenda raiateensis

leaves, wood

58

Arytera brackenridgei

leaves, wood

61

Fagraea berteroana

wood

62

Cordyline terminalis

leaves, wood

63

Podocarpus afjinis

leaves

73

Dillenia biflora

wood

76

Ficus theophrastoides

leaves

79

Pittosporum rhytidoca,pum

leaves

80

Endospermum macrophyllum

wood

84

Palaquium vitilevuense

leaves

87

Palaquium porphyreum

leaves

92

Codiaeum variegatum

leaves

95

Adenanthera pavonina

wood

96

Trichospermum richii

wood

102

Merremia peltata

leaves

105

Geissois ternata

leaves

8

106

Garcinia myrtifolia

108

Endiandra gillespiei

109

Turrillia vitiensis

110

Smilax vitiensis

leaves

9