Evaluation of Antinociceptive and Antioxidant Activities of Whole Plant Extract of Bacopa monniera
Subrata Kumar Biswas,
Utpal Kumar Karmakar
Bacopa monniera commonly known as Brahmi grows in Bangladesh. The objective
of the present study was to investigate the antinociceptive and antioxidant
activities of ethanol extract of the whole plant of (B. monniera). The
antinociceptive potential of the plant was determined using acetic acid induced
writhing method. The results showed 39.79% (p<0.05) inhibition of writhings
in the experimental animals when the plant extract was given intraperitoneally
(i.p.) at 250 mg kg-1 b.wt. However, the extract produced maximum
56.14% (p<0.05) acetic acid induced writhing inhibition in mice at the dose
of 500 mg kg-1 b.wt. This was also found to be comparable to the
standard drug, Diclofenac-Na at 25 mg kg-1 b.wt. which inhibited
76.52% (p<0.05) of writhing reflex. The plant extract showed a dose dependent
antinociceptive activity in acetic induced writhing model in mice. It was also
found that the obtained p-values calculated by students
t-test were statistically significant. In order to determine the antioxidant
activity of B. monniera, the extract was also assessed by 1,1-diphenyl-2-picrylhydrazyl
(DPPH) free radical scavenging method. The plant extract showed significant
DPPH free radical scavenging effect compared to the standard antioxidants such
as ascorbic acid and Butylated Hydroxy Anisole (BHA). The IC50 values
of the extract, ascorbic acid and BHA were 79.84, 9.45 and 14.15 μg mL-1,
respectively. Thus, the present study confirmed the antinociceptive and antioxidant
activities of the ethanol extract of the whole plant of B. monniera. Finally,
it is suggested to do further research for isolation and identification of the
chemical structures of the phytochemical compounds responsible for antinociceptive
and antioxidant activities of B. monniera.
to cite this article:
Subrata Kumar Biswas, Joysree Das, Anusua Chowdhury, Utpal Kumar Karmakar and Hemayet Hossain, 2012. Evaluation of Antinociceptive and Antioxidant Activities of Whole Plant Extract of Bacopa monniera. Research Journal of Medicinal Plants, 6: 607-614.
Received: June 27, 2012;
Accepted: July 05, 2012;
Published: December 05, 2012
Antioxidants are chemical compounds that can slow or prevent the oxidation
of other compounds through a chain of reactions (Jospeh
et al., 2010). These chain reactions can be terminated by antioxidants
through the elimination of reactive free radical intermediates (Sies,
1997). It is reported that oxidative stress are developed due to low quantity
of antioxidant in the cells which damages or kills the cells resulting in cancer
and coronary diseases (Jospeh et al., 2010).
The human body having endogenous antioxidant defense mechanism is not able to
stop completely the oxidative stress in the body (Willcox
et al., 2004). The phytochemical compounds are one of the most important
and huge sources of natural antioxidants (Al-Mustafa and
Al-Thunibat, 2008). However, medicinal plants, foods or antioxidant supplements
may be utilized to reduce oxidative stress in the body (Malekirad
et al., 2011; Adesegun et al., 2007).
The plants might show their antioxidant activities against oxidative damage
due to the presence of phenolic, flavonoid and tannins constituents but there
is still a demand to find more information on the antioxidant potential of plant
species (Frankle and Meyer, 2000; Rahman
et al., 2011). Moreover, several free radicals play crucial roles
to induce short-term algesia (Chung, 2004). It is reported
that the plants are also used traditionally as analgesics (Calixto
et al., 2000; Almeida et al., 2001).
Naturally occurring pain killers are still required to substitute conventional
steroid or non steroidal anti-inflammatory drugs having their severe adverse
effects (Alam et al., 2012).
B. monniera Linn. belonging to the family of Scrophulariaceae is commonly
known as Brahmi and has important applications in Ayurveda system. The plant
is also referred to as Bacopa monnieri, Herpestis monniera and
water hyssop (Nandave et al., 2007; Mukherjee
and Dey, 1996). Several phytochemical compounds such as brahmine and herpestine,
saponins d-mannitol, hersaponin, betulinic acid, stigmasterol, beta-sitosterol,
bacosides A and B were isolated from the plant (Kapoor, 1990).
The plant possesses significant antiulcerogenic (Sairam
et al., 2001) and cardioprotective activities (Nandave
et al., 2007) and has wide applications in the treatment of anxiety,
depression, mental fatigue (Bhattacharya and Ghosal, 1998;
Singh and Singh, 1981), epilepsy (Ganguly
and Malhotra, 1967), bronchitis and asthma (Channa
et al., 2003; Dar and Channa, 1997). Moreover,
in vitro research has revealed the anticancer activity of Bacopa saponin
fractions against sarcoma-180 cells. This might be due to inhibition of DNA
replication in the cancerous cell line (Elangovan et
al., 1995). Literature review indicated that no studies have so far
been done for antinociceptive and antioxidant potentials of the ethanol extract
of the whole plant of B. monniera. Thus, the present study was aimed
to evaluate the antinociceptive and antioxidant potentials of the ethanol extract
of the whole plant of B. monniera.
MATERIALS AND METHODS
Collection and identification of plant material: The whole plant of
B. monniera was collected in the month of September, 2009 from the hilly
areas adjacent to the University of Chittagong, Bangladesh. The plant was taxonomically
classified and identified scientifically by Department of Botany, University
of Chittagong, Bangladesh.
Preparation of plant extract: The fresh plants were washed with water
immediately after collection. The collected plants were chopped into small pieces,
air dried at room temperature for about 10 days and ground into powder form
which was stored in an airtight container. The 900 g powder was macerated in
2.5 L pure ethanol for 5 days at room temperature with occasional stirring and
then mixture was filtered with Whatman No. 1 filter paper. The obtained filtrate
was concentrated under reduced pressure below 50°C through rotatory vacuum
evaporator (Bibby RE200, Sterlin Ltd., England). The concentrated filtrate was
collected and allowed to air dry for complete evaporation of ethanol. The whole
process was repeated three times and finally, 50 g of blackish-green colored,
concentrated extract was obtained (yield approx. 5.55% w/w) which was kept in
refrigerator at 4°C before use.
Test animals and drugs: Young Swiss-albino mice, either sex of 3-4 weeks
of age weighing 20 g, were used for in vivo pharmacological screening.
Mice were purchased from the Animal Research Branch of the International Centre
for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B). They were kept in
standard environmental conditions at animal house of Pharmacy Department of
BGC Trust University Bangladesh, Chittagong and fed with rodent diet and water
ad libitum. The standard drug Diclofenac-Na was used for this study and obtained
from Square Pharmaceuticals Ltd., Bangladesh as a gift. 1,1-Diphenyl-2-picrylhydrazyl
(DPPH), ascorbic acid and Butylated Hydroxy Anisole (BHA) were obtained from
Sigma Chemical Co., USA. Ethanol and Tween 80 were of analytical grade and purchased
from Merck, Germany.
In vitro assay for antioxidant activity of plant extract: The
antioxidant activity of B. monniera extract was assessed in comparison
to standard antioxidant ascorbic acid depending on the scavenging effect of
DPPH free radical. The DPPH free radical scavenging activity of the ethanol
extract of the plant was performed according to the method described by Chang
et al. (2001). The radical scavenging activity was expressed as the
percentage inhibition (% inhibition) and calculated as per following equation:
where, A(blank) is the absorbance of the control (containing all
reagents except the test compound), and A(sample) is the absorbance
of the experimental sample with all reagents. IC50 value (the concentration
of sample required to scavenge 50% of DPPH free radical) was calculated from
the plot of inhibition (%) against the log concentration of the extract. Here,
ascorbic acid and BHA were used as standards.
Antinociceptive activity of the plant extract: The antinociceptive activity
of the crude ethanol extract of B. monniera was studied using acetic
acid induced writhing model in mice (Whittle, 1964).
The percentage inhibition of writhing was calculated according to the following
Statistical analysis: The results of the experiment were expressed as
Mean±Standard Deviation (SD). All determinations were carried out in
triplicate and average of the results was noted. Students t-test (GraphPad
Software) was used to determine a significant difference between the control
and experimental groups where p values of less than 5% (p<0.05) was chosen
as the level of significance.% inhibition was plotted against log concentration
and IC50 (Inhibition Concentration 50) value was calculated by linear
DPPH free radical scavenging activity: It was found from the study results
that the plant extract exhibited 86.62% inhibition of DPPH free radical scavenging
activity at 100 μg mL-1 whereas, ascorbic acid and BHA showed
99.34 and 90.38% inhibition, respectively at the same concentration. DPPH free
radical scavenging activity of the crude ethanol extract of B. monniera was increased with the increase of concentration of the extract (Fig.
1). Moreover, IC50 value of the extract, ascorbic acid and BHA
was calculated by linear regression analysis. It was found that IC50
value of the extract was 79.84 μg mL-1 with regression coefficient
value (R2) of 0.988 as shown in Fig. 2. On the
other hand, Fig. 3 showed the IC50 value of ascorbic
acid (9.45 μg mL-1) and the regression coefficient value (R2)
was 0.963 whereas, Fig. 4 revealed the IC50 value
of BHA (IC50: 14.15 μg mL-1) with R2 value
of 0.946. From the above results, it was found that all of the results showed
linearity and IC50 value of the standards and the extracts increased
in the following order: Ascorbic acid <BHA <ethanol extract of B. monniera.
The IC50 value of the extract was found to be significant and
to be comparable to that of ascorbic acid and BHA.
Antinociceptive activity of the plant extract: Antinociceptive activity
of ethanol extract of the whole plant of B. monniera was done using acetic
acid induced writhing model in mice. The extract produced 39.79 and 56.14% (p<0.05)
writhing inhibition in mice at the dose of 250 and 500 mg kg-1 body
weight, respectively which were comparable to Diclofenac-Na (76.52% writhing
inhibition, p<0.05) at the dose of 25 mg kg-1 body weight as shown
in Table 1.
The above findings may play an important role in the field of phytochemistry
and pharmacology and this may be helpful for the future scientists to identify
novel analgesics and antioxidants.
||DPPH free radical scavenging activity (% inhibition) of the
ethanol extract of B. monniera, ascorbic acid and BHA. The values
are the average of triplicate experiments and are represented as Mean±SD
||IC50 value of the ethanol extract of B. monniera.
The values are the average of triplicate experiments and are represented
||IC50 value of ascorbic acid. The values are the
average of triplicate experiments and are represented as Mean±SD
||IC50 value of BHA. The values are the average of
triplicate experiments and are represented as Mean±SD
|| Effect of ethanol extract of B. monniera on acetic
acid-induced writhing in mice
|Values are expressed as Mean±SD, SD: Standard deviation,
n: No. of mice, *Significant at 5% significance level, *p <0.05 vs. control
The antinociceptive activity of the plant extract was tested by acetic acid
induced writhing method in mice which is usually used for investigation of antinociceptive
activities (Koster et al., 1959). Acetic acid
produces analgesia and liberates endogenous compounds which in turn stimulate
the pain nerve endings (Taesotikul et al., 2003).
Moreover, acetic acid increases the levels of prostaglandins such as PGE2
and PGF2α in the peritoneal fluid which are responsible for
pain sensation (Deraedt et al., 1980) and the
analgesic activity can be achieved by the inhibition of prostaglandin productions
(Gupta et al., 2007). Few sympathetic nervous
system mediators are also liberated by acetic acid (Hokanson,
1978). The ethanol extract of B. monniera showed significant inhibition
of writhing reflex which was comparable to the standard drug, Diclofenac-Na.
Bhaskar and Jagtap (2011) also reported the antinociceptive
activity of aqueous extract of B. monniera at the dose of 160 mg kg-1
b.wt. They also suggested that the aqueous extract of B. monniera worked
on the endogenous adrenergic, serotonergic and opioidergic systems for analgesic
activity. The present study results also provide evidence of antinociceptive
activity of the ethanol extract of B. monniera at the dose of 500 mg
On the other hand, antioxidant activity of the plant extract was tested by
DPPH free radical scavenging method. Yamaguchi et al.
(1998) and Mokbel and Hashinaga (2006) reported
that the antioxidants showed DPPH scavenging activities due to their proton
donating abilities. The study results revealed that the scavenging effect (%
inhibition of DPPH activity) of the extract and standards (ascorbic acid and
BHA) was dependent on concentration. The scavenging power of the extract and
standards increased with increasing of concentration. The DPPH free radical
scavenging power of the extract and standards decreased in the following order:
Ascorbic acid (99.34%)>BHA (90.38%)>ethanol extract of B. monniera
(86.62%) with IC50 of 79.84, 14.15 and 79.84 μg mL-1,
respectively. However, oxidative free radical scavenging activity of B. monniera
was reported by Bhattacharya et al. (2000). The
plant extract has also possessed abilities to scavenge superoxide anion and
hydroxyl radical as antioxidant activities (Tripathi et
al., 1996). The results of the present study showed again its significant
antioxidant potentials of the ethanol extract of the whole plant of B. monniera
and the present study supported the results of the previous studies done on
the same plant by the other researchers.
Finally, it is concluded that the ethanol extract of the whole plant of B.
monniera has significant antinociceptive and antioxidant activities. Although,
the antinociceptive and antioxidant activities are relatively lower than those
of ascorbic acid and BHA, the plant is supposed to be an important source of
active phytochemical compounds for pharmacological efficacy. Thus, it is recommended
for the future scientists to isolate, purify and elucidate structures of the
active phytochemical compounds responsible for antinociceptive and antioxidant
activities of B. monniera. Additionally, in vivo experiments for
the pharmacological activities of the plant must be carried out to confirm the
appropriateness of the results obtained from in vitro experiments.
The authors are grateful to International Centre for Diarrhoeal Disease and
Research, Bangladesh (ICDDR, B) for providing the experimental animals. The
authors are also grateful to the authority of Square Pharmaceuticals Ltd., for
the standard drug, Diclofenac-Na.
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