Evaluation of Antibacterial, Analgesic and Antipyretic Activity of Leucas aspera Spreng
P. Padmakumari, Mary Reshma,
Mercy Sulochana, N. Vijaya,
V. Preethi, K. Abbulu
Malla Reddy Institute of Pharmaceutical
Sciences, Secunderabad, Andhra Pradesh, India.
Aim
of the present study is to investigate preliminary phyto-chemical
screening and to evaluate plant extracts for analgesic, antipyretic and
antimicrobial activities of different parts of Leucas aspera spreng by standard experimental
methods which include anti-writhing method for analgesic activity, cup-plate
diffusion method for antimicrobial activity and baker’s yeast induced pyrexia
method for antipyretic activity. Different parts of the plant like bud, root,
leaf, and stem were selected and extracted by cold maceration for 72 hrs. by using ethyl acetate and methanol as solvents. All crude
extracts were used for preliminary phyto chemical
screening. Ethyl acetate extracts were found be positive for alkaloids,
glycosides, steroids, carbohydrates and terpenes. Methonolic extracts were found to be positive for
alkaloids, glycosides, carbohydrates and terpenes and
negative for steroids, flavonoids. In antimicrobial
study all crude extract were found to be active against tested organisms. Ethyl
acetate extracts were active against Gram-negative bacteria. Methonolic extracts were active against Gram-positive
bacteria .Among all the parts bud has maximum anti-bacterial activity followed
by root, leaf, stem .In analgesic activity ethyl acetate extracts of bud
produce significant inhibition in acetic acid induced writhing’s
in mice at the dose of 100mg/kg and 200mg/kg. The same ethyl acetate extracts
also showed significant antipyretic activity than methonolic
extracts. These results suggest conformation of its traditional uses
(antimicrobial, analgesic as well as antipyretic activities) of a plant and
among different parts of plant bud has shown maximum activity
KEYWORDS: Leucas aspera, cup-plate method,
writhing method, Brewer’s induced pyrexia method.
Leucas aspera spreng
(Labiatae)commonly known as tummi. It is an annual ,branched
,herb erecting to a height of 15-60cm. Traditionally whole plant is used as
insecticide, hepatoprotective, febrifuge and to
relieve painful swellings. Leaves are used in psoriasis, chronic skin
eruptions, in chronic rheumatism. Bruised leaves are applied locally in skin
bites. Mrs. Moni Saha et al 1 performed In
Vitro Nitric Oxide Scavenging Activity of Leucas aspera, K. Mangathayaru et al 2 studied effect of
Leucas aspera spreng on hepatotoxicity in
rats, Selvaraj et al 3evaluated
toxicity of herbal smoke and synthetic mosquito mat on Culexquinquefasciatus.,
Rao B, Narasimha et
al4 studied antimicrobial action of some
essential oils of the plant. Rahman et al5 performed preliminary
antioxidant and cytotoxic activities of Leucas aspera root.
S. Satish et al6studied Anti-fungal
evaluation of the plant.
MATERIAL AND METHODS:
Plant material:
The
whole plant of Leucas aspera
was collected from forest academy near Medchal, Secunderabad. The plant was identified and authenticated by
taxonomist from department of botany, Government Degree College, Midthur, Kurnool District, Andhra
Pradesh.
Preparation of extract:
The
whole plant was collected and washed to remove debris and earthy material .Then
root, leaves, buds, stems are separated individually dried in shade and reduced
to coarse powder ,subjected to cold maceration with methanol, ethyl acetate for
72hrs respectively. Then the extracts are concentrated by using rotary
evaporator and used for phytochemical screening and
pharmacological evaluation.
Phytochemical screening7, 8
Phytochemical
screening of extracts was done for the presence of different chemical constituents
by using chemical tests. Results are shown in table: 1
Table. 1- Phytochemical screening of extracts
|
Constituents
|
E.A.E
|
M.E
|
|
Alkaloids
|
+
|
+
|
|
Glycosides
|
+
|
-
|
|
Steroids
|
+
|
-
|
|
Carbohydrates
|
+
|
+
|
|
Terpenes
|
+
|
+
|
|
Tannins
|
+
|
-
|
|
Flavanoids
|
-
|
-
|
E.A.E=
Ethyl Acetate Extract, M.E=Methanolic Extract (+) =
Present, (-) = Absent
Antibacterial activity9
All
Ethyl acetate extracts and methanolic extracts of
different parts of Leucas aspera were studied for its Antibacterial activity
using E. coli and S. aureus at
50,100,200ug/disc by cup-plate method. Ciprofloxacin is used as standard at
10µg/disc. Plates were incubated at 370/48 hrs. Zone of inhibition
are measured for each concentration of the drug in comparison to the standard
antibiotic. For each value average of 3 determinations were recorded. Results
are shown in fig.-1 and table- 2.
Peripheral analgesic Activity10:
Writhing tests
Male
Swiss albino mice weighing in the range 25-30g were used for the study. All the
mice were kept for fasting (8 hrs.) and provided water and divided in to 3
groups (n=5) as follows. An irritating agent such as acetic acid was injected intraperitoneally to mice and the stretching reaction is
evaluated. In this test both central and peripheral analgesics can be detected.
Results are shown in table: 3
Antipyretic Activity11
Animals:
Male
Wister rats (weighing 180 + 10g) were used for the study. The animals
were fed with mouse cubes and water was provided ad libitum.
Animals were divided into 3 groups. Each group comprising of five rats
each.
Method:
Animals
presenting initial rectal temperature between 360 and 370c
were selected for antipyretic tests. The animals were administered Brewer’s
yeast in the back and 17hrs later rectal temperature of the hyperpyrexia rats
was measured. After one hour, the fractions saline and extract were orally
administered to the animals. Paracetamol is used as standard drug. Basal rectal
temperature and changes in rectal temperature were expressed as Means ± SEM.
Results are shown in table: 4
Fig.-1. Antibacterial activity of
extract
Table 2: Evaluation of Antibacterial Activity of Plant
Extracts by cup-plate method
|
Compound
|
Concentration
(µg/ml)
|
Zone of
inhibition E. coli (mm)
|
Zone of
inhibition S. aureus (mm)
|
|
Ciprofloxacin(std)
|
10
|
28.4
|
21.4
|
|
Control
|
-
|
-
|
-
|
|
Leaves(E.A.E)
|
100
|
13.75
|
12.45
|
|
200
|
18.2
|
15.25
|
|
Leaves
(M.E)
|
100
|
9.65
|
10.65
|
|
200
|
13.95
|
12.95
|
|
Buds
(E.A.E)
|
100
|
17.65
|
-
|
|
200
|
27.95
|
-
|
|
Buds(M.E)
|
100
|
11.25
|
8.65
|
|
200
|
12.75
|
12.65
|
|
Stem
(E.A.E)
|
100
|
9.85
|
-
|
|
200
|
12.35
|
-
|
|
Stem(M.E)
|
100
|
11.95
|
8.75
|
|
200
|
14.65
|
12.35
|
|
Root
(E.A.E)
|
100
|
11.65
|
8.7
|
|
200
|
12.25
|
11.2
|
|
Root(M.E)
|
100
|
7.95
|
9.95
|
|
200
|
11.25
|
12.15
|
E.A.E =
Ethyl Acetate Extract M.E = Methanolic Extr
Table 3: Evaluation of analgesic activity of Leucas
aspera spreng by acetic
acid induced writhing method.
|
Group
|
Treatment
(n=4)
|
Dose (mg/kg)
|
No. of.
Writhes mean + SEM
|
Mean % inhibition
|
|
I
|
Acetic
Acid
|
-
|
60.5+
0.16
|
-
|
|
II
|
Ibuprofen
(std)
|
200mg/kg
|
4 +
0.5
|
93.4
|
|
III
|
Buds
(E.A.E)
|
200mg/kg
|
14.5 +
0.16
|
76.4
|
|
IV
|
Buds
(M.E)
|
200mg/kg
|
21.25 +
0.55
|
64.8
|
|
V
|
Root(E.A.E)
|
200mg/kg
|
16+
0.8
|
73.5
|
|
VI
|
Root
(M.E)
|
200mg/kg
|
23+
0.6
|
61.9
|
|
VII
|
Leaf(E.A.E)
|
200mg/kg
|
16.1+
0.1
|
73.3
|
|
VIII
|
Leaf(M.E)
|
200mg/kg
|
23.3+
0.3
|
61.4
|
Table.4:
Effect of Leucasasperasprengextracts (bud and
root) on brewer’s yeast induced pyrexia in rats
|
Treatment
|
Dose(mg/kg)
|
0h
|
1h
|
2h
|
3h
|
4h
|
|
Control
|
-
|
37.02+0.11
|
37.10+0.10
|
37.07+0.08
|
37.03+0.11
|
37.07+0.16
|
|
Standard
|
200mg/kg
|
37.10+0.10
|
35.82+0.08
|
35.72+0.13*
|
35.8+0.09*
|
35.75+0.15*
|
|
Bud(E.A.E)
|
200mg/kg
|
37.13+0.12
|
36.37+0.10
|
36.15+0.06*
|
36.45+0.09*
|
36.43+0.11*
|
|
Bud(M.E)
|
200mg/kg
|
37.08±0.12
|
36.72± 0.10*
|
36.57± 0.10*
|
36.68± 0.11*
|
36.72± 0.13*
|
|
Root(E.A.E)
|
200mg/kg
|
37.13+0.12
|
37.11+0.12
|
37.08+0.10
|
37.06+0.09
|
37.02+0.04
|
|
Root(M.E)
|
200mg/kg
|
37.13+0.14
|
37.12+0.12
|
37.10+0.11
|
37.09+0.10
|
37.06+0.08
|
Values are expressed as mean ± S.E.M. (n = 5);
* p < 0.05, compared to control E.A.E, Ethyl acetate extract; M.E, Methanol
extract
Statistical analysis:
Data
was expressed as mean ± standard error of mean. . The results were analyzed
statistically by ANOVA followed by Dunnet’s test.
DISCUSSIONS:
1. The extracts were obtained by cold maceration process.
A preliminary investigation was done by different chemical tests. Ethyl acetate
extracts of stem, leaf, bud, root was found to be positive for alkaloids,
glycosides, steroids, carbohydrates, terpenes and are
negative for flavonoids
2. Methanolic extracts of stem,
leaf, bud, and root were found to be positive for alkaloids, glycosides, carbohydrates, terpenes and are negative for steroids, tannins, flavonoids.
3. All the extracts obtained were evaluated for antibacterial
activity by using Cup-plate method. Ciprofloxacin was used as standard drug at
a concentration of 10µg/ml. E.coli and S. aureus are the two different strains used for the
activity. Plant extracts were used at a concentration 100µg/ml and 200µg/ml.
Zone of inhibition was measured in mm.
4. All extracts showed antibacterial activity at 100µg/ml and
200µg/ml. Ethyl acetate extracts of different parts are active against gram
negative bacteria. Methanolic extracts are active
against gram positive bacteria. Ethyl acetate extract of bud showed maximum
zone of inhibition (27.95mm) against E. coli.
Methanolic extract of leaf showed maximum zone of
inhibition (12.95) against S. aureus.
5. Analgesic activity is evaluated by writhing test. Rats
are used for the study. Bud, Root, Leaf extracts were used for the study. The effects of crude
extracts on acetic acid-induced abdominal contractions were observed. Ibuprofen
was taken as standard (200mg/kg (i.p). The test drugs
at a dose of 200mg/kg (i.p) showed significant
activity compared to control at p < 0.001, whereas there was no significance
difference between the standard and bud and root ethyl acetate extracts at p
< 0.001. Bud has shown maximum inhibition compared with other extracts.
6. Antipyretic activity was done by brewer’s yeast method. Among all paracetamol and bud ethyl extract showed significant
antipyretic activity throughout the test period of 6 h.
CONCLUSION:
Leucas aspera plant parts are evaluated for
antibacterial and analgesic activity. Among all crude extracts ethyl acetate
extracts are active against gram
negative bacteria (E. coli) and methanol extracts are active against
gram positive bacteria (S. aureus).All the
crude extracts exhibited significant antibacterial activity analgesic and antipyretic activity among them bud has shown maximum activity
followed by root, leaf and stem. However, further investigation is required to
isolate the active Constituents responsible for these activities and to elucidate
the exact mechanisms of action.
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