Phytochemical Screening of Solvent Extracts from Hyptis suaveolens LAM for Fungal Growth Inhibition

Abstract

Hyptis suaveolens was targeted on the basis of folkloric uses which suggest its toxicity to microbes, coupled with its importance as food to humans. The pulverized plant material was extracted with 96% ethanol and further partitioned using chloroform, distilled water, petroleum ether and methanol. Soluble solvent extracts of the plant were tested for phytochemicals which revealed the existence of alkaloids, flavonols, flavones, flavonones, terpenoids, tannins, aldehydes and ketones and the absence of steroids, saponins and anthraquinones. Antifungal screening exhibited growth inhibition in some instances which exceeded that of griseofulvin antibiotics. The presence of phytochemicals and activity against Aspergillus niger , Candida albicans , Cryptococcus and Fursarium species support ethno-medicinal uses of plant.

Full text

Pakistan Journal of Nutrition 9 (4): 358-361, 2010
ISSN 1680-5194
© Asian Network for Scientific Information, 2010
358
Phytochemical Screening of Solvent Extracts from
Hyptis suaveolens LAM for Fungal Growth Inhibition
V.C. Mbatchou , S. Abdullatif and R. Glover
1 1 2
Department of Applied Chemistry and Biochemistry, Department of Applied Biology,
1 2
University for Development Studies, Ghana
Abstract: Hyptis suaveolens was targeted on the basis of folkloric uses which suggest its toxicity to
microbes, coupled with its importance as food to humans. The pulverized plant material was extracted with
96% ethanol and further partitioned using chloroform, distilled water, petroleum ether and methanol. Soluble
solvent extracts of the plant were tested for phytochemicals which revealed the existence of alkaloids,
flavonols, flavones, flavonones, terpenoids, tannins, aldehydes and ketones and the absence of steroids,
saponins and anthraquinones. Antifungal screening exhibited growth inhibition in some instances which
exceeded that of griseofulvin antibiotics. The presence of phytochemicals and activity against Aspergillus
niger, Candida albicans, Cryptococcus and Fursarium species support ethno-medicinal uses of plant.
Key words: Hyptis suaveolens, soluble solvent extracts, phytochemicals, Aspergillus niger, Candida
albican, Cryptococcus and Fursarium species, griseofulvin, fungal growth inhibition, zones of
inhibition and toxic components
INTRODUCTION
A wide range of our recently used medicines had their
roots directly or indirectly from plants. Some of these
medicines are no longer synthesized in large
quantities by competitors because they have shown
toxicity to humans and other animals. This has made
it possible for more investigations to be carried out on
plants so as to enable us know the therapeutic status
of newly discovered drugs of plant origin. In this
respect, plant based research has made promising
results in the fields of anticancer and anti-malarial
therapies (De Smet, 1997).
Of the 250,000-500,000 species of existing plants on
earth (Borris, 1996), only about 300 species are being
used worldwide in the pharmaceutical, food,
cosmetics and perfume industries (Robber and
Speedie, 1996; Lee et al., 1997). A relatively small
percentage (1-10 % ) of these plants is consumed as
food by both humans and other animal species, while
more are used for medicinal purposes (Moerman,
1996).
Plant foods contain constituents such as flavonoids,
saponins, tannins, phenolics, etc, which have been
assessed for their anti-oxidant, anti-mutagenic, anti-
carcinogenic and other biological effects
(Krishnaswamy and Raghuramulu, 1998).
Interestingly, natural product research guided by
ethno-pharmacological knowledge has made
substantial contributions to drug innovation by
providing novel chemical structures and or
mechanisms of action (De Smet, 1997).
According to a report by Walker, the medicinal
properties of plants could be seen in their response to
attacks from insect predators and disease organisms.
This is achieved by the accumulation of
phytochemicals at the sites of infection of plants,
several of which are insecticidal, anti-bacterial, anti-
fungal, etc (Walker, 1975; Ameen et al., 2005).
MATERIALS AND METHODS
The plant, Hyptis suaveolens LAM was randomly
collected from the farmland on Navrongo Campus at
University for Development Studies, Ghana. It was
identified by Dr. Walters M. Kpikpi and Dr. Sarkey, both
from Department of Applied Biology, Faculty of Applied
Sciences. The entire plant was air-dried at room
temperature for three weeks and later ground. The
pathogenic fungi: Aspergillus niger, Candida albican,
Cryptococcus and Fursarium species were collected
from the Medical School of University of Legon, Ghana
after characterization and identification using
microbiological procedures of Cowan and Steel
(1965). The solvents and other materials used in this
study were of analytical grades bought from Timster
laboratory Supplies Limited, Accra, Ghana. These
include: 96% ethanol, chloroform, methanol,
petroleum ether, distilled water, Dimethylsulphoxide
(D.M.S.O), sabouaud dextrose agar, Petri dishes,
Whatmann number 1. filter papers, etc.
Extraction procedure: Air-dried, ground plant sample
(600 g) was percolated with 2,200 ml of 96% ethanol
at room temperature for two weeks with intermittent

Pak. J. Nutr., 9 (4): 358-361, 2010
359
shaking. The percolate was evaporated to dryness at 5% ferric chloride. A blue-black or blue-green
room temperature and a crude extract (ethanol
soluble) was obtained. 0.1 g of the crude extract was
then transferred in to a vial and kept at the lower
compartment of a refrigerator until required for use.
The remainder of the crude extract was used in a
fractionation process.
Fractionation of crude extract: The crude extract
prepared as mentioned above was partitioned
between chloroform and distilled water (120, 1:1),
using a separating funnel. The chloroform and
distilled water soluble fractions were separately
evaporated at room temperature to residues. 0.1 g of
the chloroform soluble residue and all the distilled
water soluble residue were transferred in to two
distinct vials and kept at the lower compartment of a
refrigerator until required for use. The remainder of the
chloroform soluble residue was also partitioned
between methanol and petroleum ether (120, 1:1).
These solvent soluble fractions were separated and
concentrated as the chloroform and distilled water
soluble fractions to their respective residues which
were transferred in to distinct vials and kept as other
residues until required for use.
Qualitative phytochemical evaluation: Phytochemical
screening was conducted to determine the presence
of natural products in the extracts obtained from the
entire parts of Hyptis suaveolens LAM.
Alkaloids (Wakama test): 0.2 g of each extract was re-
extracted with 1% HCl for 24 h. 2 ml portion of the
filtrate was taken and tested for alkaloids by adding
drops of Meyer’s reagent. Alkaloids formed a picric
yellow precipitate with the reagent.
Flavonoids (Willistatter test): To methanol solution of
each extract, a piece of magnesium ribbon was added
followed by drop-wise addition of concentrated HCl.
Colors ranging from orange to red indicated flanones,
red to crimson indicated flavonols and crimson to
magenta indicated flavonones.
Terpenoids and steroids (Liebermann buchart test):
A small quantity of each extract was dissolved in
trichloromethane and a minimum amount of
concentrated sulphuric acid was then added to its
content. A blue or green color or a mixture of these two
shades was taken as positive test for steroidal
compounds, while red, pink or violet color indicates
the presence of terpenoids.
Tannins: 0.2 g of each extract was re-extracted with
ethanol. The solution obtained was later treated with
appearance was taken as positive test for tannins.
Saponins: A small portion of each extract was added
to 2 ml of distilled water and boiled for 3-5 min. The
resultant mixture was filtered, allowed to cool with the
filtrate shaken vigorously. Honey comb froth higher
than the aqueous layer was taken as strongly positive
for saponins. Froth as high as the aqueous layer was
taken as moderate and lower than this as negative for
the presence of saponins.
Anthraquinones (Bornstrager-test Kraus modified):
About 0.5 g of each extract was boiled for a few
minutes with 12 ml of 0.5 M potassium hydroxide and
2 ml of hydrogen peroxide (10 %). The mixture
obtained was then cooled, filtered, acidified and
extracted with a small quantity of ammonium
hydroxide solution. A red color formed in the alkaline
layer indicated the presence of anthraquinones.
Aldehydes: To 1 ml of freshly prepared Tollen’s
reagent in a tilted test tube rinsed with 3 Molar sodium
hydroxide solution, one drop of aqueous solution of
each extract was slowly added. A silver mirror on the
surface of the test tube is a positive test for aldehydes.
Ketones: To 1 ml of aqueous solution of each extract
in a test tube, a few drops of 3 molar sodium
hydroxide solution was added, followed by a slow
addition of 3 drops of iodine solution. The test tube
was stopped and shaken vigorously. A positive test
resulted from a brown color of the mixture
disappearing and a yellow iodo-form solid
precipitating out of solution.
Antifungal bioassay: The spreading method of
Cruickshanks et al. (1980) and dose (agar) diffusion
method were used.
Five days old cultures of Aspergillus niger, Candida
albicans, Cryptococcus and Fursarium species to be
tested were used. 0.1 ml solution of cultures were
uniformly spread over the surface of sabouraud
dextrose agar with the aid of a sterile inoculating loop.
The solvent soluble extracts and griseofulvin
antibiotics employed in the test were diluted to obtain
different concentrations of 1,500, 1,000 and 500 µg /ml
using Dimethylsluphoxide (D.M.S.O). 0.1 ml of various
concentrations of the prepared extracts and
griseofulvin antibiotics were used to fill holes bored by
5mm improvised cork borer in the inoculated agar.
Three plates were made for each extract and
organism-griseofulvin, standard drug. The plates were
then incubated at 37 C for 24 h. Diameters of zones of
o
inhibition were measured manually in millimeters for

Pak. J. Nutr., 9 (4): 358-361, 2010
360
the created holes from which sample means were
calculated.
RESULTS AND DISCUSSION
From Table 2 results, the crude ethanol extract of
Hyptis suaveolens LAM did not reveal the presence of
alkaloids, flavones, flavonols and flavonones, while
subsequent fractions did. This can be explained by
masking effect which often occurs when different
phytochemicals form a mixture. In this effect, the
presence of a particular phytochemical is not noticed
because it is being masked or inhibited by other
phytochemicals that make up the mixture. In search for
phytochemicals from the plant, qualitative analyses of
solvent soluble extracts/fractions revealed the
presence of alkaloids, flavonoids, terpenoids, tannins,
aldehydes and ketones, whereas steroids, saponins
and anthraquinones were absent.
Results in Table 1 illustrate the growth inhibitory effect
of Hyptis suaveolens LAM extracts/fractions and
griseofulvin, standard antifungal drug on Aspergillus
niger. At concentrations of 500, 1,000 and 1,500 µg/ml
of the plant extracts/fractions and griseofulvin there
was a uniform trend of increase in zones of inhibition.
Of all the tested at 500, 1,000 and 1,500 µg/ml
concentrations, the distilled water soluble fraction of
the plant presented the highest growth inhibitory effect
on the isolate. It recorded mean zones of inhibition of
7.5±0.10, 12.0±0.10 and 16.5±0.10. This was closely
followed by the chloroform soluble fraction with mean
zones of inhibition of 6.0±0.20, 11.0±0.27 and
15.0±0.10. Both the distilled water and chloroform
soluble fractions of Hyptis suaveolens LAM showed
higher growth inhibitory effects on Aspergillus niger
than the antifungal drug. It is an indication that these
soluble fractions contained more toxic components
which inhibited the growth of the isolate.
Similarly, from Table 4 results it is observed that the
plant extracts/fractions and griseofulvin antibiotics
inhibited the growth of Candida albicans in an
increasing trend at the concentrations of 500, 1,000
and 1,500 µg/ml. The most toxic soluble fraction of
Hyptis suaveolens LAM to the isolate is the chloroform
soluble fraction with mean zones of inhibition of
5.0±0.10, 10.50±0.10 and 12.50±0.10 in an increasing
order at concentrations of 500, 1,000 and 1,500 µg/ml.
The methanol soluble fraction of the plant showed the
least growth inhibitory effect on Candida albicans just
as it did on Aspergillus niger with mean zones of
inhibition of 1.5±0.10, 3.5±0.20 and 5.50±0.15.
The results in Table 3 clearly revealed the resistance
to Hyptis suaveolens LAM extracts/fractions and
griseofulvin antibiotics by Cryptococcus species.
There were no growth inhibitory effects for griseofulvin
Table 1: Texture, color and weight of solvent soluble
extracts/fractions obtained from Hyptis suaveolens
LAM
Extract/fraction Texture Color Weight (g)
EtOH Sticky Dark brown 2.40
CHCl Sticky Dark brown 0.60
3
Distilled H O Sticky Dark brown 0.90
2
MeOH Sticky Dark brown 0.20
Petroleum ether Sticky Dark brown 0.60
Table 2: Phytochemical screening results of solvent soluble
extracts/fractions from Hyptis suaveolens LAM
Distilled Petroleum
Phytochemicals EtOH H O CHCl MeOH ether
2 3
Alkaloids -ve +ve -ve +ve +ve
Flavones -ve +ve +ve +ve +ve
Flavonols -ve +ve +ve +ve -ve
Flavonones -ve +ve +ve -ve -ve
Terpenoids +ve +ve +ve +ve +ve
Steroids -ve -ve -ve -ve -ve
Tannins +ve +ve +ve +ve +ve
Saponins -ve -ve -ve -ve -ve
Anthraquinones -ve -ve -ve -ve -ve
Aldehydes +ve +ve +ve +ve +ve
Ketones +ve +ve +ve +ve +ve
+ve means phytochemical is present; -ve means phytochemical
is absent
antibiotics, methanol and petroleum ether soluble
fractions at the concentrations of 500, 1,000 and 1,500
µg/ml. It was only at 1,500 µg/ml concentration that the
ethanol soluble extract, the distilled water and
chloroform soluble fractions of the plant presented
mean zones of inhibition of 5.5±0.10, 6.0±0.20 and
9.0±0.10 respectively. This is an indication that these
three soluble fractions of Hyptis suaveolens LAM
contain toxic components that inhibit the growth of
Cryptococcus species.
Contrary to results in Table 5 and 6 results showed
griseofulvin antibiotics and extracts/fractions of Hyptis
suaveolens LAM to be toxic to Fursarium species with
the methanol soluble fraction presenting the least
growth inhibitory effect. At concentrations of 500, 1,000
and 1,500 µg/ml, the distilled water soluble fraction
recorded the highest mean zones of inhibition of
12.50±0.10, 19±0.27 and 22.50±0.10 respectively.
This is closely followed by the chloroform soluble
fraction with mean zones of inhibition of 9.0±0.21,
11.05±0.10 and 18.0±0.10.
A comparison of results from Table 3, 4, 5 and 6
revealed that the soluble fraction of Hyptis suaveolens
LAM with the highest mean zones of inhibition is
alternating between the distilled water and chloroform
soluble fractions. To some extend, these two soluble
fractions are more active against the isolates than the
antifungal drug and therefore contain ingredients
which could serve as drugs.

Pak. J. Nutr., 9 (4): 358-361, 2010
361
Table 3: Growth inhibitory effect of Hyptis suaveolens LAM extracts/fractions and griseofulvin antibiotics on Aspergillus niger
Zone of inhibition (mm)
Concentration -----------------------------------------------------------------------------------------------------------------------------------------------------------
(µg/ml) Griseofulvin EtOH CHCl Distilled H O MeOH Petroleum ether
3 2
500 4.0±0.10 5.5 ±0.20 6.0±0.20 7.5±0.10 0.0±0.0 5.0±0.10
1,000 6.0±0.10 6.0 ±0.10 11±0.27 12.0 ±0.10 0.50±0.01 7.0±0.30
1,500 12.0±0.17 8.0±0.17 15±0.10 16.5 ±0.10 2.0 ±0.20 12.5±0.01
Table 4: Growth inhibitory effects of Hyptis suaveolens LAM extracts/fractions and griseofulvin antibiotics on Candida albicans
Zones of inhibition (mm)
Concentration -----------------------------------------------------------------------------------------------------------------------------------------------------------
(µg/ml) Griseofulvin EtOH CHCl Distilled H O MeOH Petroleum ether
3 2
500 4.5±0.10 4.0±0.20 5.0±0.10 4.0±0.10 1.5±0.10 5.0±0.20
1,000 10.0±0.20 10±0.10 10.5±0.10 8.0±0.10 3.5±0.20 8.5±0.10
1,500 12.0±0.10 10.5±0.29 12.5±0.10 11.5±0.10 5.5±0.15 11.0±0.10
Table 5: Growth inhibitory effects of Hyptis suaveolens LAM extracts/fractions and griseofulvin antibiotics on Cryptococcus species
Zones of inhibition (mm)
Concentration -----------------------------------------------------------------------------------------------------------------------------------------------------------
(µg/ml) Griseofulvin EtOH CHCl Distilled H O MeOH Petroleum ether
3 2
500 0 0 0 0 0 0
1,000 0 0 0 0 0 0
1,500 0 5.5±0.10 9.0±0.10 6.0±0.20 0 0
Table 6: Growth inhibitory effects of Hyptis suaveolens LAM extracts/fractions and griseofulvin antibiotics on Fursarium species
Zones of inhibition (mm)
Concentration -----------------------------------------------------------------------------------------------------------------------------------------------------------
(µg/ml) Griseofulvin EtOH CHCl Distilled H O MeOH Petroleum ether
3 2
500 5.5±0.10 8.0±0.10 9.0±0.21 12.50±0.10 0 9.0±0.20
1,000 7.0±0.17 12±0.10 11.05±0.1 19.0±0.27 3.0±0.10 12.0±0.10
1,500 10.0±0.10 13±0.17 18±0.10 22.5±0.10 6.0±0.10 14.0±0.10
Conclusion: The investigation was able to prove that Cruickshanks, R., J.P. Oluguid, B.P. Mormoin and
Hyptis suaveolens LAM contained phytochemicals or
agents which were effective against Aspergillus niger,
Candida albicans, Cryptococcus and Fursarium
species. It also went further to explain that bioactive
agents of the plant were more effective in inhibiting the
growth of isolates than griseofulvin, antifungal drug.
These findings justify the ethno-medicinal uses of the
plant and could be of interest to pharmaceutical
companies.
ACKNOWLEDGEMENTS
Authors are grateful to the Medical School of University
of Ghana, Legon, Ghana, for the fungal isolates
provided in the investigation.
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