These are general bioassays that are
inexpensive, rapid, technologically simple, animal sparring and require little
technical training. They are capable of detecting a board spectrum of
bioactivity present in crude extracts; BSLT is a general bioassay; the
inhibition of crown gall tumors on discs of potato tubers is an antitumor bioassay,
while antimicrobial activity and antioxidant activity helps to ascertain the
presence of cytotoxicity and any significant
biological activity16-22.
MATERIALS
AND METHODS:
Plant material
The flowering whole plants were collected from the
fields around the campus of Indira Gandhi Krishi Vishvavidyalaya
(Agriculture University), Raipur (Chhattisgarh), India, during the months of
December and January 2004-05, and the identification of plant was done by Prof.
P. Jayaraman, Director Plant Anatomy Research Centre;
Chennai- India. A specimen was vouchered, and
deposited at Institute of Pharmacy, Pt. Ravishankar Shukla Vishvdyalaya, Raipur; Chhattisgarh, India.
Test
microorganisms: The microorganisms were procured
from IMTECH, Chandigarh, India. Agarobacterium tumefaciens (MTCC 3329), Staphylococcus aureus (MTCC 96), Bacillus subtilis
(MTCC 619), Candida albicans (MTCC 227), Escherichia
coli (MTCC 1687) and Shigella sonnei (MTCC 2957).
Brine shrimps: The brine shrimp eggs were obtained from
Southern Aquaculture, Chennai, India.
Preparation of the extract
One kilogram of powdered drug was packed in soxhlet apparatus and extracted with petroleum ether (60-80âC) to defat the drug. Defatted powdered drug was then extracted
with ethanol (95%). The alcoholic extract thus obtained was further fractioned
with chloroform into chloroform soluble and insoluble fractions. The solvents
were removed by distillation and the last traces of solvent being removed under
reduced pressure. The extracts and fractions were weighed and thereafter, were
stored in refrigerator for further experimental work.
Preparations of dilutions of
crude extract for antibacterial assay
The methods of Akujobi et
al., (2004)23 and Esimone et al., (1998)24 were adopted with minor
modifications. The crude extracts were dissolved in 30% dimethylsulphoxide
(DMSO) and further diluted to obtain 5 mg/ml, 25 mg/ml, 50 mg/ml, 100 mg/ml and
200 mg/ml concentrations. These were stored at 15âC until required.
Disc diffusion antimicrobial
bioassay
The main aim of this assay was to determine if the
plant extract under investigation possess any antibiotic activity. The
antimicrobial assays were performed by minimum inhibitory concentration (MIC)
determination and diffusion disc methods as described earlier24- 27
with modifications.
In-vitro Antioxidant activity
In
our work we have performed some more popular methods that were easy to perform
in our laboratory conditions.
DPPH assay: The method followed here was introduced by Marsden
Blois, more than 50 years ago28. The free radical scavenging
activity of the extracts and butylated hydroxy toluene (BHT) was measured in terms of hydrogen
donating or radical-scavenging ability using the stable radical DPPH29.
H2O2 radical scavenging activity:
The ability of the extracts
to scavenge hydrogen peroxide was determined according to Sundararajan
et al., 200629. A solution
of H2O2 was prepared in phosphate buffer (pH 7.4). H2O2
concentration was determined spectroscopically
measuring absorption with extinct coefficient for H2O2.
Different concentrations of extracts in distilled water were added to a H2O2
solution (0.6 ml, 40 mM). Absorbance of H2O2
at 230 nm was determined 10 min later against a blank solution containing the
phosphate buffer without H2O2. a-tocopherol was
used as the standard. The % of H2O2 scavenging of both
the extracts and standard compound was calculated by the equation:
Where, Acont is the absorbance of the control reaction
and Atest is the absorbance in the
presence of the sample of the extracts.
Determination of reducing power: The reducing power of extracts was
determined according to the method of Rajeshwar, et al., 200530. Various
concentrations of the extracts in 1.0 ml of deionized
water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 1% potassium
ferricyanide (2.5 ml). The mixture was incubated at
50°C for 20 min. A portion (2.5 ml) of trichloroacetic
acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for
10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water
(2.5 ml) and FeCl3 (0.5 ml. 0.1%) and the absorbance was measured at 700 nm.
Increased absorbance of the reaction mixture indicated increased reducing
power. Butylated hydroxy
toluene (BHT) was used as a standard31.
Brine shrimp lethality test: A general bioassay that is very useful to
assess the bioactivity of the plant extracts. The procedure described here has
been adapted from previous studies32, 33. Brine shrimps (Artemia salina)
were hatched using brine shrimp eggs in a glass vessel (1L), filled with
sterile artificial seawater (prepared using sea salt 38 g/L and adjusted to pH
8.5 using 1N NaOH) under constant aeration and
artificial light provided for 48 h. After hatching, active nauplii
free from egg shells were collected from brighter portion of the hatching
chamber and used for the assay. Nauplii were drawn
through a glass capillary and placed in each vial containing 4.5 ml of brine
solution and their numbers counted. In each experiment, 0.5 ml. of the plant
extract was added to 4.5 ml of brine solution and maintained at room
temperature for 24 h under the light and surviving larvae were counted.
Experiments were conducted along with control (vehicle treated) and different
concentrations of the test substances in a set of six tubes per dose. The
percentage lethality was determined by comparing the mean surviving larvae of
the test and control tubes and LC50 values were obtained.
Crown gall tumor assay: Goal of scientists over the past years had
been the development and application of crown gall tumor assay as a bench-top
bioassay that is simple enough for phytochemists to
use34. Modification
of this assay was done by McLaughlin to perform a procedure that is rapid,
inexpensive, safe, animal sparing, and a statistically reliable prescreen for in vivo murine
leukemia antitumor activity34,35.
For crown gall tumor assay, the procedure described by Oran47 and Hussain et al. 49 was followed with slight
modification. Using sterile technique, 2 ml of bacterial growth, 0.5 ml of the
sample at different concentrations (replaced by distilled water in the case of
the standard), and 5 ml of distilled water were used to prepare the inoculums. Agrobacterium tumefaciens
were grown on a broth medium that contained 0.5 g sucrose, 0.8 g nutrient
broth, and 0.1 g yeast extract and completed to 100 ml distilled water. After
sterilization in an autoclave for 12 min at 121°C and cooling, a loop
containing Agrobacterium tumefaciens was
added and the mixture was placed in dark for 48 h below 30°C. One and a half
grams of agar were dissolved in 100 ml distilled water. After autoclaving for
15 min, 20 ml of the media were poured in each petri
dish under sterile conditions and cooled. Potatoes were washed with tap water
and soaked in bleach. After taking out of bleach they were cut into cylinders
using a potato borer and put in a tray. The cylinders then were cut into discs,
and 5 discs were placed in each Petri dish. For each sample 2–3 Petri dishes
were used, in addition to those used for control. One drop of the inoculums was
then added on each disc, the dishes then were wrapped with parafilm
strips and kept in the dark at 27°C for 21 days. After 21 days, the discs were
stained with Lugol’s solution (10% KI and 5% I2) for
30 minutes and then observed under dissecting microscope. Numbers of tumors per
disc were counted and percent inhibition for each concentration was determined
by the formula given below.
Statistical analysis
All the data were subjected to statistical analysis
using SPSS 14.0 for Windows. The statistical analysis was performed by using
one-way analysis-of-variance (ANOVA) followed by Dunnet’s
test for individual comparison of groups with control. p-values <0.05 were
considered as significant. The values are represented as the mean ± SEM for six
sets of observation.
RESULTS AND DISCUSSION:
Disc diffusion antimicrobial
bioassay
The disc
diffusion method was used to determine the inhibition zones of Hyptis suaveolens extract. Results of the disc
diffusion antimicrobial bioassay of the different concentrations of the
extract on the test isolates and the minimum inhibitory concentrations of the
extract on the test isolates are shown in table 1. The results are indicative
of the fact that an increase in concentration of the extracts as well as the
standard drugs results in augmented zone of inhibitions. The extract had
pronounced effect on E. coli with a
ZOI of 48 mm at a concentration of 200 mg/ disc and it was least effective on Shigella sonnei. The
extract had moderate effect on rest of the microorganisms tested. The minimum
inhibitory concentrations were 2.48, 5.61 8.52, 8.34 and 6.13 mg/ml for E.coli, B.subtilis, S.aureus, Shigella sonnei and C.albicans
respectively.
In-vitro Antioxidant activity
DPPH scavenging activity, H2O2
radical scavenging activity and reducing power of the extract at different
concentrations was evaluated to determine its antioxidant potential. The
results of antioxidant activities are shown in table 2, 3 and 4, which
illustrates that the tested extract has a very moderate, but definitive
antioxidant capability in each of the models tested. It has an IC50 value
of 30.06 µg/ ml in DPPH scavenging model compared to 18.92 µg/ml of the
standard, while in H2O2 scavenging model the IC50
was 178.47mg/ml, compared to
129.84mg/ml of α-tocopherol that was used as standard. The extract had a
comparable reducing power to the standard drug BHT (butylated
hydroxy toluene)
Brine shrimp lethality test
The results of BSLT are shown
in table 5. The chloroform
soluble and insoluble fractions at concentration of 10, 25, 50, 100 and 150
µg/ml was tested and showed LD50 values of 43.08 µg/ml and 66.93
µg/ml..
Crown gall tumor assay
Result shows that chloroform
soluble fraction and chloroform insoluble fraction exhibit inhibition of tumors
in potato discs in dose dependent manner and gave LD50 values of 29.71 µg/ml and 74.23 µg/ml respectively (Table 6).
The results of
antimicrobial and antioxidant activities have shown that H. suaveolens extract exhibited both
antibacterial as well as anti fungal activities. It also successfully
illustrated free radical scavenging and reducing abilities.
