International Journal of Pharmacy
International Journal of Pharmacy
International Journal of Pharmacy
Utpal Kumar Karmakar1*, Sharmin Sultana1, Sonya Nishi1, Nripendra Nath Biswas1, Lokman
Hossain2, Sharmin Sheikh3
1
Pharmacy Discipline, Khulna University, Khulna-9208, Bangladesh
2
Department of Pharmacy, Stamford University, Dhaka-1209, Bangladesh
3
Department of Pharmacy, East West University, Dhaka-1212, Bangladesh
*Corresponding author e-mail: ukk146@gmail.com
Received on: 19-02-2018; Revised on: 22-03-2018; Accepted on: 30-03-2018
ABSTRACT
The present study was performed to evaluate phytochemical groups, antioxidant, antibacterial, analgesic and anthelmintic activity of
ethanol extract of seeds of Bixa orellana (Bixaceae). Preliminary phytochemical screening of ethanol extract exhibited the presence of
reducing sugar, carbohydrate, saponin, terpenoids, tannins, alkaloids, acidic compound, proteins, steroids, and flavonoids. In the TLC-
based qualitative antioxidant assay using DPPH, B. orellana extract showed the free radical scavenging properties that was indicated
by the presence of yellow spot on a purple background on the TLC plate. In the quantitative free radical scavenging assay by DPPH,
IC50 value was approximately 47 µg/mL, where IC50 value of the ascorbic acid was approximately 16 µg/mL. B. orellana extract
displayed hydroxyl radical scavenging activity (SC50 = 157 mg/L) which was comparable to that of standard ascorbic acid (SC 50 = 81
mg/L). The Total Phenolic Content (TPC) and Total Tannin Content (TTC) was found to be ~ 52 and ~ 25 mg GAE/gm of dried
extract respectively using Gallic acid calibration curve. Total Flavonoid Content (TFC) was ~ 41 mg QE/gm of dried extract using
Quercetin calibration curve. The extract showed antibacterial activity against S. aureus, S. dysenteriae, P. aeruginosa, S. typhi, and E.
coli at the doses of 250 and 500 µg/disc in comparison with standard drug Fucloxacillin (30 µg/disc) and Ciprofloxacin (30 µg/disc).
The extract showed significant (p<0.01) acute peripheral analgesic activity at the doses of 250 (40.34% writhing inhibition) and 500
mg/kg body weight (67.05% writhing inhibition) determined by acetic acid induced writhing method in mice as compared to
Diclofenac sodium (79.55%). In anthelmintic activity test, time taken for paralysis and death at the concentration of 25 and 50 mg/mL
was approximately 33 minutes and 39 minutes and 23 minutes & 30 minutes respectively whereas standard showed approximately 7
and 16 minutes at the dose of 15 mg/mL respectively against the parasite. The phytochemical groups present in this plant may be
responsible for the aforementioned pharmacological effects.
Keywords: Bixa orellana, Phytochemical study, Antioxidant activity, Antibacterial activity, Analgesic activity, Anthelmintic activity.
INTRODUTION
Modern synthetic drugs are very effective in curing disease Utkana, Lip stick tree, Achiolte, has been investigated because
but cause a number of side effects. Therefore, researchers of their numerous traditional uses for the treatment of many
in recent years, especially due to the constant emergence of B. orellana is native to tropical American area and cultivated in
resistant to conventional drugs. Bixa orellana L. (Family: warm regions of the world. In Bangladesh it is cultivated in
Bixaceae), commonly known as Annatta, Latkan, Belatihaldi, Chittagong Hill Tracts, sporadically in other districts [1]. It is a
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tall shrub to small evergreen tree and 6–10 m (20–33 ft) high, Animals
trunk is up to 10 cm in diameter. Moreover it has evergreen Young Swiss albino mice aged 4-5 weeks, average weight (28-
ovate leaves with reddish veins; pink, white, or some 35) gm were used for analgesic experiment. The mice were
combination flowers; two-valved, woody capsule covered collected from the animal house of Jahangirnagar University,
either with dense soft bristles or a smooth surface fruit Savar, Dhaka-1342. They were kept in the animal house of the
approximately 4 cm wide, appear in a variety of colors [2]. Pharmacy Discipline, Khulna University, Khulna under standard
Traditionally the root bark of the plant is antiperiodic and Laboratory condition (relative humidity (55-60)%, room
antipyretic and also used in intermittent, remittent and temperature (25 ± 2)0C and 12 hours (light: dark cycle) for
continued fevers. Root is used in jaundice, and fever. Seeds period of 14 days prior to performing the experiment. The
are cordial, astringent, febrifuge, diuretic, laxative, digestive; animals were provided with standard laboratory food and tap
anti-dysenteric, and also used in epilepsy, skin diseases and water.
gonorrhoea. Fresh pulp (the colouring matter, surrounding Microorganisms
the seeds) is astringent; applied to burns to prevent blisters For antibacterial test both gram positive and gram negative
and scars [3-5]. American Indians use the pulp to paint their bacterial strains were taken. These bacteria were collected from
body, which they believe, prevents mosquito bites. Seeds the Microbiology Lab. of Pharmacy Discipline, Khulna
fatty oil is used in leprosy. Leaves are febrifuge, useful in University, Khulna. The live parasites, Nematodes and
jaundice, dysentery; decoction of the leaves is used as a Trematodes were collected from freshly slaughtered cattle at
gargle for sore throat. In developing countries the plant is local abattoirs for anthelmintic test and stored in 0.9 %
also used as folk medicine for the treatment of common Phosphate buffered saline (PBS) of pH 7.4.
infections and as an anti-parasitic agent [6]. The seeds of this Drugs
plant produce dyes which are most frequently used The standard drugs Flucloxacillin, Ciprofloxacin disc,
worldwide, not only in food products but also in the textile, Diclofenac sodium, and Albendazole were collected from local
paint, and cosmetic industries. So it is also known as “Lip pharmacy in Khulna.
stick tree” [7]. Identification of phytochemical constituents
MATERIALS & METHODS The crude extract was subjected to preliminary phytochemical
Collection and extraction screening for the detection of major functional groups [8]. Then,
The seeds of Bixa orellana (L.) were collected from the extract was used for pharmacological screening.
Komolgonj, Moulobibazar, Sylhet, Bangladesh in May 25, Estimation of antioxidant components
2016 at the daytime. The sample was identified by the DPPH free radical scavenging assay
experts of Bangladesh National Herbarium, Mirpur, and DPPH free radical produces a violet solution in ethanol and
Dhaka (Accession No.: 43217 DACB). The collected seeds shows a characteristic spectrum with a maximum absorbance
were shade-dried for about 15 days. The dried seeds were close to 517 nm. Based on electron transfer reaction, free radical
then powdered with the help of a suitable grinder. The seeds which is stable at room temperature is reduced to a light yellow
were extracted by cold extraction method. About 350 gm or colorless ethanol solution in the presence of an antioxidant
powder was soaked in 1500 mL ethanol in a glass container molecule by electron transfer [9].
for fifteen days accompanying regular shaking and stirring. DPPH based qualitative analysis was determined on the basis of
Then the extract was separated from the plant debris by their scavenging activity of DPPH free radical by TLC technique
filtration with a piece of clean, white cotton cloth. The filtrate using commercially prepared TLC plate [10]. The sample and
obtained was evaporated using rotary evaporator. After a few the standard ascorbic acid were spotted on TLC plate. The
days, the concentrated extract (approx. yield value 1.29%) chromatogram was developed by ascending technique using
was transferred into a small beaker and the opening of the three types of solvent systems i.e. non-polar (n-hexane: Ethyl
beaker was wrapped by a sheet of aluminum foil on which acetate = 2: 1), medium polar (CHCl3: CH3OH = 5: 1) and polar
perforation was done. The extract thus obtained was stored in (CHCl3: CH3OH: H2O = 40:10:1) solvent system. The solvent
a cool, dark and dry place. system was allowed to move up to a previously marked line.
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After drying the plates, they were viewed under UV detector absorbance was measured at 765 nm against blank for each
both in short wavelength (254 nm) and long (366 nm) concentration.
wavelength. The plates were then sprayed with 0.02 % In Total Tannin Content determination, 0.1 mL of different
ethanol solution of DPPH by spray gun. Finally the plates concentrations (0.5, 0.4, 0.3, 0.2, 0.1 mg/mL) of standard and
were dried with a current of air suitably. extract sample solution was taken separately in different test
DPPH based quantitative analysis of seeds of B. orellana was tube. Then 7.5 mL of distilled water and 0.5 mL of FC reagent
performed by microplate technique [11]. At first plant extract was added to the test tube. After that 1mL of 35% Na2CO3 was
and ascorbic acid as positive control was dissolved in DMSO added to the test tube and the solution was diluted Q.S. to 10 mL
to make the required concentrations by dilution method. Then with distilled water. Then all test tubes vortex for 15 second and
10 µL of different concentrations (1, 2, 4, 8, 16, 32, 64, 128, kept at room temperature for 30 minutes. Absorbance of the
256, and 512 μg/mL) of plant extract and ascorbic acid were solution was measured at 725 nm against blank for each
taken in each well of the microplate and 190 µL DPPH in concentration.
methanol was added to each well. Here, 190 µL of DPPH Determination of total flavonoid content
was applied in two well of the plate as blank. After mixing The total flavonoid content was determined by aluminum
for 5 minutes, the plate was kept standing at room chloride method [16]. In such case, quercetin is usually used as
temperature in light for 30 minutes, and finally the reference standard and the result is usually expressed as
absorbance was measured at 517 nm using micro plate quercetin equivalence.
reader. In this test, 1 mL of different concentrations (1, 0.75, 0.5, 0.25,
Hydroxyl radical scavenging assay and 0 mg/mL) of standard and sample extract solution were
Hydroxyl radical scavenging activity of the extract was taken separately into different test tubes. Then 4 mL of distilled
measured based on the method of Halliwell, Gutteridge et al., water was added and 0.3 mL of 5% w/v NaNO2 was added to
[12] with a slight modification according to Jiang, Bank et every test tube and kept for 5 minutes. Then 0.3 mL of 10% w/v
al., [13] In this test 0.5 mL 2-deoxy-D-ribose solution was AlCl3 solution and 2 mL of 1M NaOH were also added to every
mixed with 12.5 µL of different concentrations (6.25, 12.5, test tube and added distilled water Q.S. to10 ml of the solution.
25, 50, 100, 200, 400, and 800 mg/L) of sample or standard. They were kept for 15 minutes at room temperature and the
Then 1 mL of 200 µM FeCl3, 1 mL of 1.04 mM EDTA, 0.5 absorbance was measured at 510 nm against blank for each
mL of 1 mM H2O2 was added to prepare the reaction mixture. concentration.
0
After incubation at 37 C for 1 hour, 3.75 mL of 2.8% TCA Antibacterial activity test
and 3.75 mL of 1% TBA were added and kept at 1000C for The evaluation of antibacterial potential of ethanol extract of
20 minutes. Finally the absorbance was measured at 530 nm seeds of Bixa orellana was carried out by Disc Diffusion method
against blank for each concentration. using both gram positive and gram negative bacterial strains
Polyphenolic compound determination [17,18]. In these test two types of standard antibiotic disc was
Determination of total phenolic content and total used as positive control such as Flucloxacillin antibiotic disc and
tannin content the commercial Ciprofloxacin antibiotic disc. For preparing the
Total phenolic and tannin content was determined by Folin- Flucloxacillin antibiotic disc, a 250 mg flucloxacillin capsule
Ciocalteau Colometry method [14]. In such case, Gallic acid was used and 3 mg equivalent weight of the powder was then
is usually used as reference standard and the result is usually dissolved in 1 mL 50% ethanol to prepare standard solution [19].
expressed as gallic acid equivalence [15]. Sample impregnated discs (250 and 500 μg/disc), standard
For the determination of total phenolic content, 0.5 mL of antibiotic discs (30 µg/disc) and negative control discs (10 µL
different concentrations (0.15, 0.1, 0.08, 0.06, 0.04, 0.02 50% ethanol/disc) were placed gently on the seeded agar plates
mg/mL) of standard and extract solution were taken separately with the help of sterile forceps to assure complete contact with
into different test tubes. Then 5 mL FC reagent (1/10) and 7% medium surface. The plates were then incubated at 37°C for (16-
Na2CO3 were added to every test tube. Then they were kept 18) hours for observation. After proper incubation, the
for 30 minutes at 40ºC temperature. After 30 minutes the UV antibacterial activity of the test agent was determined by
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measuring the diameter of zone of inhibition in terms of writhing, so two half-writhing were taken as one full writhing.
millimeter with a slide calipers. Anthelmintic activity test
Analgesic activity test Anthelmintic activity of the plant extract was determined
The analgesic activity of the sample was investigated using according to Hossain et al. [21]. For this test four petridishes
acetic acid induced writhing method in mice [20]. were taken denoted as Control group, Positive control group and
Experimental animals were randomly selected and divided Test group I and Test group II consisting of six parasites in each
into four groups denoted as Control group, Positive control petridish. 10 mL of 0.1 % Tween-80 in PBS as negative control,
group and Test group I and Test group II consisting of five Albendazole at the dose of 150 mg/10 mL as positive control
(05) mice in each group. and suspension of the ethanol extract at the dose of 250 and 500
Test samples, positive and negative control solution were mg/10 mL were taken in different petridishes. Time taken for
given orally by using feeding needle. Control group received paralysis for each parasite was recorded when no movement was
1% Tween-80 at the dose of 10 mg/kg body weight and observed unless shaken vigorously. Time taken for death for
Positive control group received Diclofenac sodium at the each parasite was recorded after evaluating that the parasites did
dose of 25 mg/kg body weight. Test group I and Test group II not move when shaken vigorously, dipped in warm water (50°C)
were treated with test sample at the dose of 250 and 500 or subjected to external stimuli.
gm/kg body weight. A thirty minutes interval was given to STATISTICAL ANALYSIS
ensure proper absorption of the administered substances. Student’s t-test was used to determine significant differences
Then the writhing inducing chemical, acetic acid solution between the control group and test group.
(0.7%) was administered intra-peritoneally to each of the RESULTS
animal of a group. After an interval of 5 minutes, which was In the preliminary phytochemical analysis of seeds of B.
given for absorption of acetic acid, number of writhing was orellana extract exhibited the presence of reducing sugar,
counted for 15 minutes. The animals do not always perform carbohydrate, saponin, terpenoids, tannins, alkaloids, acidic
full writhing. The incomplete writhing was taken as half- compound, proteins, steroids, and flavonoids (Table 1).
Table 1: Results of preliminary phytochemical analysis.
Acidic compound
Carbohydra
Reducing sugar
Plant Extract
Flavonoids
Glycosides
Alkaloids
Saponins
Proteins
Steroids
Tannins
te
Ethanol
extract of
+ - + + + + + + + +
Bixa
orellana
+ = Presence - = Absence
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Figure 1: Comparison of TLC plate for B. orellana with the standard after applying DPPH.
128 μg/mL
256 μg/mL
512 μg/mL
16 μg/mL
32 μg/mL
64 μg/mL
1 μg/mL
2 μg/mL
4 μg/mL
8 μg/mL
Standard 5.67 13.1 21.38 33.37 51.92 68.36 81.71 90.48 95.18 96.54
Sample 6.18 11.87 18.67 24.47 30.28 37.45 45.98 54.01 64.28 75.77
Figure 2: Comparison of % inhibition vs. log concentration graph for standard (ascorbic acid) vs. B. orellana in DPPH scavenging assay.
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6.25 mg/mL
12.5 mg/mL
100 mg/mL
200 mg/mL
400 mg/mL
800 mg/mL
25 mg/mL
mg/mL
50
Standard 8.69 15.95 27.64 40.31 52.28 65.95 79.34 92.45
In polyphenolic compound determination, the Total Phenolic Flucloxacillin (30 μg/disc) and Ciprofloxacin (30 μg/disc). The
Content (TPC) & Total Tannin Content (TTC) was found to maximum zone of inhibition was found against Staphylococcus
be ~ 52 mg GAE/gm (Table 4 and Figure 4) and ~ 25 mg aureus that was 20.5 and 26.5 mm when Flucloxacillin was used
GAE/gm (Table 4 and Figure 5) of dried extract respectively as positive control (Table 5 and Figure 7) and 21 and 27 mm
using Gallic acid calibration curve and Total when Ciprofloxacin was used as positive control (Table 6 and
Figure 8) at the dose of 250 and 500 μg/disc respectively.
(mg GAE/gm)
(mg GAE/gm)
(mg QE/gm)
Total tannin
flavonoid
content
content
content
Sample
Total
B. orellana extract ~ 52
~ 25 ~ 41
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1.8
y = 9.0907x + 0.1724
1.6
R² = 0.9812
1.4
Absorbance
1.2
Absorbance
0.8
Linear (Absorbance)
0.6
0.4
0.2
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
Concentration
Figure 4: Quercetin calibration curve for the determination of total flavonoids content.
0.7
0.6
absorbance
0.5
0.4
0.3
0.2
0.1
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Concentration
Figure 5: Gallic acid calibration curve for the determination of Total Tannin Content.
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0.5
0.4
Absorbance
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1 1.2
-0.1
Concentration
Figure 6: Quercetin calibration curve for the determination of Total Flavonoids Content.
n
a
a
c
e
r
s
t
t
i
i
Standard antibiotic
S. dysenteriae
P. aeruginosa
S. enteritidis
disc
S. pyogenes
V. cholera
Gram (+)
Gram (+)
P. species
S. aureas
Gram (-)
Gram (-)
Gram (-)
Gram (-)
Gram (-)
Gram (-)
Gram (-)
S. typhi
E. coli
Blank 0 0 0 0 0 0 0 0 0
Flucloxacillin
36 35 0 0 18 0 30 0 23
(30 μg/disc)
Sample
0 20.5 0 16.5 0 0 0 13 12.5
(250 μg/disc)
Sample
0 26.5 0 19.5 0 0 23 21.5 18
(500 μg/disc)
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40
35
Zone of inhibition
30
25 Blank
20 Extract
(250 μg/disc)
15 Extract
(500 μg/disc)
10 Flucloxacillin
(30 μg/disc)
0
S. S. aureus V. cholerae S. P. species S. P. S. typhi E. coli
pyogenes dysenteriae enteritidis aeruginosa
Bacterial strains
Figure 7: Antibacterial activity of B. orellana at two different doses against gram positive and gram negative bacteria in comparison with
Flucloxacillin.
Table 6: In vitro antibacterial activity of crude extract using Ciprofloxacin antibiotic disc as positive control.
Standard antibiotic disc
Antibacterial activity of
n
a
a
c
e
r
s
t
t
i
i
crude extract using
P. aeruginosa
S. dysenteriae
S. enteritidis
S. pyogenes
V. cholera
Gram (+)
Gram (+)
P. species
S. aureas
Gram (-)
Gram (-)
Gram (-)
Gram (-)
Gram (-)
Gram (-)
Gram (-)
S. typhi
E. coli
Blank 0 0 0 0 0 0 0 0 0
Ciprofloxa
cin
36 35 30 26 26 25 30 35 22
(30
μg/disc)
Sample
(250 0 21 0 16 0 0 0 13 12
μg/disc)
Sample
(500 0 27 0 20 0 0 22 22 19
μg/disc)
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Zone of inhibition 40
35
30
25 Blank
20 Extract
(250 μg/disc)
15 Extract
(500 μg/disc)
10 Ciprofloxacin
(30 μg/disc)
5
0
S. S. aureus V. cholerae S. P. species S. P. S. typhi E. coli
pyogenes dysenteriae enteritidis aeruginosa
Bacterial strains
Figure 8: Antibacterial activity of B. orellana at two different doses against gram positive and gram negative bacteria in comparison with
Ciprofloxacin.
% inhibition
Treatment
(mg/kg b.
writhing
Mean of
Dose
wt.)
Diclofenac Na
25 7.2 ± 0.58*
79.55
Extract 250 21 ± 0.71* 40.34
Extract 500 11.60 ± 0.51* 67.05
Values are expressed as mean ± SEM (standard error for mean), n = 5, *p< 0.01 vs. control.
Here, b. wt- body weight.
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90 79.55
80 67.05
70
% writhing inhibition
60
40.34
50
Negative control
40 Positive control
Extract (250 mg/kg b. wt)
30
Extract (500 mg/kg b. wt)
20
10
0
Negative control Positive control Extract (250 mg/kg b. Extract (500 mg/kg b.
wt) wt)
Treatment
Figure 9: % Writhing inhibition by the standard drug & extract (*** p < 0.01).
Time taken
Treatment
for death
(mg/mL)
Conc.
(min)
(min)
Negative control -- -- --
Albendazole 15 7.5 ± 0.18 16.23 ± 0.47
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45
39.23
40
33.16
35 30.08
30
Time (Minute)
25 22.52
20 16.23 Paralysis time
Death time
15
10
7.5
5
0
Standard Extract (25 mg/mL) Extract (50 mg/mL)
Treatment
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complexation. Alkaloids exert antimicrobial activity the cuticle of parasite, and cause death. The data
through intercalating into cell wall and DNA of parasites presented in the table and observations made thereof,
[29-31]. B. orellana was effective against both gram lead to the conclusion that the phytoconstituents, tannin
positive and gram negative bacteria but gram negative may be responsible for anthelmintic activity.
bacteria were most sensitive. Phytochemical groups CONCLUSION
present in B. orellana like polyphenols, flavonoids, This project work can be summarized into the
tannins and alkaloids may be responsible for antibacterial photochemistry and pharmacology of seeds of Bixa
activity. orellana. Preliminary phytochemical screening of B.
The phytochemical groups may exert analgesic property orellana seed extract revealed the presence of reducing
by inhibiting the synthesis, release, and/or antagonizing sugar, flavonoids, tannins, carbohydrates, alkaloids,
the action of pain mediators at the target sites. The saponin, terpenoids, proteins, steroids and acidic
identified phytochemical groups namely flavonoids, compounds which are valuable for pharmacological
tannins, terpenoids, gums and reducing sugar in B. activities. The results of the pharmacological
orellana seed extract may be responsible for analgesic investigation rationalize the uses of seeds of the plant in
activity both centrally and peripherally [32-34]. folk medicine. But more research is needed to find out its
Helminthiasis is a serious disease in human and poultry individual phytoconstituents in order to introduce this
farming in South-East Asia. Tannins, the secondary plant part in pharmaceutical industries for developing
metabolite, occurring in several plants have been semi-synthetic and synthetic drugs with similar or better
reported to show anthelmintic property by several therapeutic properties for the welfare of human being.
investigators [35]. Tannins, the Polyphenolic ACKNOWLEDGEMENT
compounds, are shown to interfere with energy The authors are grateful to the Khulna University
generation in helminthes parasites by uncoupling Research Cell for partially funding of this research.
oxidative phosphorylation or, binds to the glycoproteinon
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