Tufikul, et al.

Int J Pharm 2015; 5(4): 1058-1067 ISSN 2249-1848

International Journal of Pharmacy

Journal Homepage: [Link]

Research Article CODEN: IJPNL6

SCREENING OF IN-VITRO ANTIOXIDANT, BRINE SHRIMP LETHALITY

BIOASSAY AND ANTIMICROBIAL ACTIVITIES OF EXTRACTS OF BRIDELIA
RETUSA (L.) SPRENG. FRUIT

Tufikul Islam1*, Mohammad Raquibul Hasan1, Aumit Roy1, Md. Shafiqul Islam1, Md. Afaz
Uddin1, Md. Ariful Islam2, Md. Nuruzzaman Neon1, Md. Sohel Rana1
1
Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.
2
Department of Botany, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.

*Corresponding author e-mail: tufikulislam@[Link]

ABSTRACT

The objective of present study was to investigate the antioxidant, brine shrimp lethality and antimicrobial activities
of Bridelia retusa (L.) Spreng. fruit using n-hexane, ethyl acetate and methanol extracts. Several methods were used
for assessing antioxidant activity. In total phenol content determination methanolic extract showed highest value of
134.5 mg/g as Gallic Acid Equivalent (GAE). N-hexane extract showed highest total flavonoid content with
60.50±4.95 mg/gm as Quarcetin Equivalent (QE). Methanolic extract showed highest tannin content with
652.50±3.53 mg/gm as Tannic Acid Equivalent (TAE) and total anti-oxidant capacity with 236.00±36.78 mg/gm as
Ascorbic Acid Equivalent (AAE). Methanolic extracts showed good DPPH free radical scavenging activity with
IC50 of 168.757 µg/mL. Ethyl acetate extract showed highest reducing capacity in CUPRAC test. Methanolic
extracts showed good cytotoxic activity with IC50 value of 20 µg/ml in brine shrimp lethality bioassay. In
antimicrobial assay extracts showed poor activity. Further studies are needed to isolate active compounds.

Key Words: Antioxidant Activity, Free Radical Scavenging, Tannic Acid, CUPRAC, Flavonoids Equivalent Content
and Brine Shrimp Lethality Bioassay.

INTRODUCTION antibacterial agent [4, 5, 6]. The bark is used to treat

dysentery, rheumatism and as astringent and
Although the modern medicine has achieved contraceptive agents [4, 7, 8]. So the present study
tremendous development, plants are still playing an was designed to assess the antioxidant, cytotoxic and
important role in healthcare. Several plants have been antimicrobial activities which were previously
investigated for their antioxidant potential [1]. explored very little.
Bridelia retusa (L.) Spreng. is a medium-sized to
large deciduous tree that commonly grows in forests Antioxidants are substances that are used to
and open land [2]. This plant belongs to the deactivate or stabilize the free radicals like Reactive
euphorbiaceae family and the Bengali name is Oxygen Species (ROS) or other oxidants. These are
Kantakoi. It is found at the forests of Chittagong and involved in food and chemical material degradation
Chittagong Hill Tracts, Cox's Bazar, Mymensingh, and damage nucleic acids, proteins and lipids.
Sylhet and Dhaka in Bangladesh [2]. The plant is also Eventually they will cause oxidative stress and
found in China, India, Nepal, Sri Lanka and Sumatra leading to degenerative diseases such as cancer,
[3]. The fruit is fleshy drupe, size of a pea, purple- Alzheimer’s disease, Parkinson’s diseases and
black [2]. The fruits of the plant are edible [3]. some cardiovascular diseases. In recent years,
Different parts of the plant are used for their prevalence of the diseases result from oxidative stress
medicinal activities. The leaf is useful in treating has been increased [9]. In human body, an imbalance
urinary tract infection, wound healing and as between ROS and the inherent antioxidant capacity

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directed the use of dietary and /or medicinal serial extraction by Soxhlet apparatus at elevated
supplements. Investigation on medicinal plants, temperature (65°C) using n-Hexane, Ethyl Acetate
vegetables, and fruits has indicated the presence of and Methanol consecutively (500 mL of each
antioxidants such as phenolics, flavonoids, tannins, solvent). After each extraction the plant material was
and proanthocyanidins. The antioxidant contents of dried and used again for the next extraction.
medicinal plants may contribute to the protection Extraction was considered to be completed when the
against disease. The relationship between morbidity fruit materials become exhausted of their constituents
and mortality from degenerative disorders and the that were confirmed from cycles of colorless liquid
ingestion of natural antioxidants is inversely siphoning in the Soxhlet apparatus. The filtrates
proportional [10]. obtained were dried at temperature of 40±2°C to have
gummy concentrate of the crude extract. The extract
Cytotoxicity of a substance is the quality of being was kept in a suitable container with proper labeling
toxic to cells. Research interest on screening of and then stored in cold and dry place for further use
medicinal plants has intensified in recent years with a [17].
view to finding out potential cytotoxic compounds
for cancer chemotherapy [11, 12, 13, 14]. As a Phytochemical Screening: The freshly prepared
result, several potential lead compounds such as crude extract was qualitatively tested for the presence
vincristine, vinblastine, taxol, camptothecin, of chemical constituents i.e. carbohydrates through
podophyllotoxin, combretastatins, etc have been molisch’s test and fehling’s test, flavonoids,
isolated from plants and are currently successfully glucosides through general test for glycoside and
employed in cancer treatment [15]. In developing glucoside, steroids through liebermann-burchard’s
countries, synthetic drugs are expensive and test, saponins through frothing test, tannins through
inadequate for the treatment of disease and need to Ferric chloride and Potassium dichromate test,
search for new infection-fighting agents to control alkaloids through mayer’s test, hager’s test, wagner
microbial infections [16]. The antimicrobial activities test and dragendorff’s test. These phytochemicals
were determined by disc diffusion method. were identified from their respective characteristic
color changes as stated in the standard procedures
MATERIALS AND METHODS [18].

Chemicals: Ammonium molybdate, Methanol, Antioxidant Activity Evaluation

Sodium Phosphate (Na3PO4) and Folin-Ciocalteu Total phenol content determination: Total phenolic
reagent were purchased from Merck, Germany. content of the prepared n-hexane, ethyl acetate and
Sodium carbonate (Na2CO3), Potassium Acetate and methanol extracts was determined by using Folin–
H2SO4 (98%) were purchased from Merck (India) Ciocalteu Reagent (FCR) [19].One (1.0) mL of fruit
Limited. Gallic acid, Quercetin and 1, 1-diphenyl-2- extract (200 µg/mL) and the standard (gallic acid) of
picrylhydrazyl (DPPH) were purchased from Sigma different concentrations (6.25, 12.5, 25, 50, 100 and
Chemicals, USA. Aluminium Chloride and Ascorbic 200 µg/mL) were taken into marked six test tubes.
acid were purchased from SD Fine Chem. Ltd., All test tubes were marked accordingly. Five (5) mL
Biosar, India. Vincristine sulphate was collected from of Folin–Ciocalteu reagent solution (diluted to 10
Techno Drugs Ltd., Bangladesh. All chemicals and fold) were taken in the test tubes followed by the
reagents used were of analytical grade. addition of 4 mL of 7.5% sodium carbonate solution
in each. The test tubes were incubated at 20°C (30
Collection of Plant Material: The fruit of the plant minutes for standard solutions, and 1 hour for extract
was collected from Gazipur, Bangladesh and solution). Absorbance at 765 nm was measured using
identified by the taxonomist of the Department of a UV-Vis spectrophotometer (Shimadzu UV PC-
Botany, Jahangirnagar University, Savar, Dhaka, 1600) against a blank. Total phenol contents of the
Bangladesh. fractions were expressed as Gallic acid equivalents
(GAE) [20, 21].
Preparation of Plant material & Extraction
procedure: Fruits of the plant were first washed with Determination of Total Flavonoids Content: Total
water to remove adhering dirt and then cut into small Flavonoid was determined using the Aluminum
pieces and sun-dried for few days and then dried in a chloride colorimetric method described by Wang and
hot air oven (Size 1, Gallenkamp) at reduced Jiao [22]. One (1.0) mL of fruit extract (200µg/mL)
temperature (not more than 50°C). Dried fruits were and standard (Quercetin) of six different
grinded into coarse powder using high capacity concentrations (6.25, 12.5, 25, 50, 100 and 200
grinding mill. The powdered fruits were used for µg/mL) were taken into different marked test tubes.

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Then 3 mL of methanol was added to each of the test 50, 100, 200, 400 and 800 µg/mL) and 1.0 mL
tubes followed by 200 µL of 10% aluminium standard (ascorbic acid) of different concentrations
chloride solution and 200 µL of 1 M potassium (2.5, 5, 10, 20, 40 and 80 µg/mL) were taken in
acetate solution. Finally, 5.6 mL of distilled water different pre-marked test tubes. Then, 2 mL of
was added to the test tubes. After this the test tubes 0.004% methanolic DPPH solution was added to
were incubated for 30 minutes at room temperature to each test tube. All the prepared test tubes with their
complete the reaction. Absorbance of the solution contents were then incubated for 30 minutes at room
was measured at 415 nm using UV-Vis temperature. Absorbance of each of the incubated
spectrophotometer (Shimadzu UV PC-1600) against solutions was determined at 517 nm using UV-Vis
a blank. Total Flavonoid content of the extract was spectrophotometer (Shimadzu UV PC-1600) against
expressed as Quercetin equivalents (QE). a blank and IC50 value was calculated from the curve.
(Fig.: 5).
Determination of Total Tannin Content: The
tannins were determined by slightly modified Folin Cupric Reducing Antioxidant Capacity: Cupric
and Ciocalteu method [19]. The standard (Tannic Reducing Antioxidant Capacity of the fruit extracts
Acid) solution of six different concentrations (6.25, was determined following the method described by
12.5, 25, 50, 100 and 200 µg/mL) and the fruit Resat [Link]. al. [25]. Five hundred (500) µL solution
extract (200µg/mL) of 0.1 mL were taken in different of each plant extract and standard (Ascorbic Acid)
marked test tubes. Then 7.5mL of distilled water, 0.5 having different concentrations (12.5, 25, 50, 100,
mL of Folin Phenol reagent, 1 mL of 35% sodium 200, 400 and 800 µg/mL) were taken in different
carbonate solution were added and the volume was marked test tubes. One (1.0) mL of 0.01M
finally adjusted upto 10 mL with distilled water. The CuCl2.2H2O solution, 1.0 mL of ammonium acetate
mixture was shaken well, kept at room temperature buffer (pH 7.0), 1.0 mL of 0.0075 mL of neocaproin
for 30 minutes and absorbance was measured using solution and 600 µL of distilled water were added to
UV-Vis spectrophotometer (Shimadzu UV PC-1600) each test tubes and the final volume of the mixture
at 725 nm against a blank. Total Tannin content of was adjusted to 4.1 mL. The total mixtures were
the extracts was expressed as Tannic Acid Equivalent incubated for 1 hour at room temperature and the
(TAE). absorbance of the solutions were measured at 450 nm
using UV-Vis spectrophotometer (Shimadzu UV PC-
Determination of Total Antioxidant Capacity: Total 1600) against a blank.
antioxidant capacity of the fruit extracts was
determined by following the method described by Brine shrimp lethality bioassay: Lethal activity of
Prieto P et al., 1999 [23]. Three hundred micro-liters the fruit extracts of Bridelia retusa (L.) Spreng. was
(300µL) of extract (200µg/mL) and standard determined by Brine shrimp lethality bioassay
(ascorbic acid) of different concentrations (6.25, described by Meyer et al. [26]. Brine shrimp eggs
12.5, 25, 50, 100 and 200 µg/mL) were taken in (Artemia salina leach) were hatched in simulated
different marked test tubes and then 3 mL of reagent seawater (3.8% NaCl) with continuous oxygen
solution (a mixture of 3.3mL of concentrated 98 % supply for two days and got the nauplii. Stock
H2SO4, 0.381gm sodium phosphate and 0.494gm of solution of the sample was prepared by dissolving 20
ammonium molybdate prepared in a 100mL mg of extract in 400 µL of pure dimethylsulfoxide
volumetric flask adjusting the volume to 100 mL (DMSO) and adding sea water to make the total
with distilled water) was added to each test tubes. volume 20 mL. Thus the stock solution gained
These test tubes were then incubated at 95°C for 90 concentration of the extract as 1µg/µL. Then specific
minutes to complete the reaction. Absorbance of each volumes of stock solution was transferred into
of the incubated solutions, after cooling to room different test tubes so that the final concentration of
temperature, was measured at 695 nm using a UV- the extract becomes 6.25, 12.5, 25, 50, 100, 200, 400
Vis spectrophotometer (Shimadzu UV PC-1600) and 800μg/mL in the respective test tubes after
against a blank. Total antioxidant capacity of the volume adjustment to 5 mL with sea water. In the
extract was expressed as ascorbic acid equivalent control tubes 75µL and 150µL DMSO were taken
(AAE). and volume was adjusted to 5mL with sea water (as
in the sample tubes). Vincristine sulfate was used as
DPPH Free Radical Scavenging Assay: DPPH (1, 1- positive control and evaluated at very low
diphenyl-2-picrylhydrazyl) free radical scavenging concentration (10, 5, 1, 0.5, 0.25, 0.125 and
activity of the plant extract was determined following 0.06μg/mL). Using a Pasteur pipette 10 living nauplii
the method described by Braca et al. [24]. One (1.0) were put to each of the test tubes. After 24 hours the
mL fruit extract of different concentrations (12.5, 25, test tubes were observed and the number of nauplii

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survived in each test tube were [Link] mortality by inactivating the lipid free radicals [31]. The total
was corrected using Abott’s formula [27]. phenolic compounds content of the test solutions
Pt= [(Po-Pc)/ (100-Pc)] × 100 were calculated using the calibration curve of the
Where, Pt = Corrected mortality, Po= Observed standard (Fig. 1) of Gallic acid (y = 0.0106x +
mortality and Pc= Control mortality. 0.0507, R² = 0.9998). The results were expressed as
LC50 values of the test samples after 24 hours are gallic acid equivalents (GAE) per gram of the extract.
obtained by regression analysis. Methanolic extract of fruit of Bridelia retusa (L.)
Spreng was found to contain the highest amount of
Determination of Antibacterial Activity by Disc phenols 134.50±0.00 mg/gm GAE (Table 3). Phenol
Diffusion Method: The antimicrobial activity of contents of the extracts were found to the following
different extracts was determined by the disc order: methanol> ethyl acetate> n-hexane (Table 3).
diffusion method [28]. The bacterial strains used
for the experiment were collected as pure Determination of Total Flavonoids Content: The
cultures from Department of Microbiology of total Flavonoid content of the extracts were
Jahangirnagar University. Both Gram positive and calculated using the standard line (Fig. 2) of
gram negative organisms were taken for the test and Quercetin (y=0.0055x+0.0142, R² = 0.9973).
they are listed in the following Table 1. Solutions of Flavonoid content of the extracts was expressed as
known concentration (40µg/mL) of the test samples mg/gm Quercetin equivalent (QE). The n-hexane
were made by dissolving measured amount of the extract was found to have the highest total flavonoid
samples in calculated volume of solvents. Dried and content 60.5±4.950 mg QE/gm of the extract and the
sterilized filter paper discs were then impregnated methanolic extract was lowest 17.00±2.82 mg QE/gm
with 10µL of the test samples (400µg/ Disc) using (Table 3). The flavonoid content in plant extracts
micropipette. Discs containing the test material were depend on the polarity of solvent used in extract
placed on nutrient agar medium uniformly seeded preparation[32]. The flavonoid contents of the
with the test microorganisms. Standard antibiotic extracts were found to the following order: n-hexane
discs (Chloramphenicol 50 μg/disc) and blank discs > ethyl acetate> methanol (Table 3).
(impregnated with respective solvents 10µL) were
used as a positive and negative control. These plates Determination of Total Tannin Content: Total
were then incubated at 37ºC for 24 h allowing tannin content of the different extracts fruit of
maximum growth of the organisms. The test Bridelia retusa (L.) Spreng. was evaluated by the
materials having antibacterial activity inhibited the Folin method and was expressed as tannic acid
growth of the microorganisms and a clear, distinct equivalents (TAE) per gram of plant extract. Total
zone of inhibition was visualized surrounding the tannin capacity of the test samples was calculated
medium. The antimicrobial activity of the test agents using the standard curve of tannic acid (y = 0.0014x
was determined by measuring the diameter of zone of + 0.023; R2 = 0.9987) (Fig. 3). Methanolic extract
inhibition expressed in millimeter. was found to possess the highest Total tannin content
(652.50±3.53 mg/gm TAE); ethyl acetate extract also
RESULTS possesses good 650.00±7.07 mg/gm TAE (table 3).
Total tannin content of the extracts was found to the
Phytochemical screening: In the present study, following order: Methanol> Ethyl Acetate > n-
various qualitative tests were done to detect the Hexane (Table 3).
presence of different phytochemical compounds in
the n-hexane, ethyl acetate and methanolic extracts of Total antioxidant capacity assessment: Total
the fruits of Beidelia retusa (L.) Spreng. antioxidant capacity of Bridelia retusa (L.) Spreng.
Phytochemical constituents in the plants are known to fruit extracts was evaluated by the
be biologically active compounds and they are phosphomolybdenum method and was expressed as
responsible for different activities against diseases ascorbic acid equivalents (AAE) per gram of fruit
[29]. The results of the phytochemical testing are extracts. Total antioxidant capacity of the test
given in Table 2. samples was calculated using the standard line (Fig.
4) of ascorbic acid (y=0.0052x+0.0164, R² = 0.9908).
Antioxidant activity evaluation Methanolic extract of fruit of Bridelia retusa (L.)
Total phenol content determination: Phenolic Spreng. was found to possess the highest total
compounds of plants have been said to account for antioxidant capacity with 236.00±36.78mg/gm AAE
most of the antioxidant activities of plant extracts (Table 3). Total antioxidant capacity of the extracts
[30]. They show antioxidant activities by preventing was found to decrease in the following order:
decomposition of hydroperoxides into free radicals or Methanol> Ethyl Acetate > n-Hexane (Table 3).

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The total antioxidant activity of fruit extracts of Increase in the absorbance of the sample solution is
Bridelia retusa (L.) Spreng. was evaluated in the an indicator of reducing power of the sample [40].
present study from its ability to reduce Phosphate/Mo The standard ascorbic acid showed highest reducing
(VI) complex to Phosphate/Mo (V). Recently it is capacity. Among the extracts the ethyl acetate extract
reported that a highly positive relationship between of the fruits showed maximum reducing capacity that
total phenols and antioxidant activity appears to be is comparable to ascorbic acid (Fig. 6). Cupric
the trend in many plant species [33]. The statement Reducing Antioxidant Capacity of the extracts was
has been justified in the current study where the found to be decreased in the following order: Ethyl
methanol and ethyl acetate extracts of fruit of Acetate > Methanol > n- Hexane.
Bridelia retusa (L.) Spreng. showed significant total
antioxidant capacity (in term of ascorbic acid Brine shrimp lethality bioassay: All the extracts of
equivalent) (Table 3) with maximum phenol content fruit were subjected to Brine Shrimp lethality
(Table 3). bioassay for possible cytotoxic action. In this study,
methanol extract of fruits was found to be the most
DPPH Free Radical Scavenging Assay: The DPPH toxic to Brine Shrimp nauplii, with LC50 of
free radical scavenging assay is considered as a valid 20.00μg/ml, whereas anticancer drug vincristine
accurate, easy and economic method to evaluate sulphate showed LC50 value 0.699μg/ml. On the other
radical scavenging activity of antioxidants, since the hand, ethyl acetate extracts showed good and n-
radical compound is stable and need not be generated hexane extract showed low toxicity (table 4).
[34]. When DPPH accepts an electron donated by an
antioxidant compound, the DPPH is decolorized, For the determination of bioactivities of synthetic
which can be quantitatively measured from the compound as well as plant derivatives the brine
changes in absorbance at 517 nm. The IC50 values shrimp bioassay has been established as a safe,
of the different extracts of fruit of Bridelia retusa (L.) practical and economic method [41, 42]. According
Spreng. are presented in the Table 4. The ethyl to the national Cancer Institute (NCI, USA) , the
acetate fraction exhibited highest antioxidant activity relationship between the brine shrimp bioassay and
with an IC50 value of 141.954 g/mL compared to growth inhibition of human solid tumor cell lines (In
other fractions. The value is 16.654 g/mL for the vitro) is significant because it exhibits the importance
standard ascorbic acid. DPPH radical scavenging as a pre-screening tool for anti-cancer drug research
capacity of the extracts was found to decrease in the [43]. Extracts derived from natural resources which
following order: Ethyl Acetate > Methanol > n- have LC50 ≤ 1000μg/mL in brine shrimp bioassay
Hexane (Fig. 5). were claimed to contain bioactive principles [42].
Principle of brine shrimp toxicity for compound or
The lower the IC50 the higher the antioxidant property plant extract above 1000μg/ml is non-toxic, between
of a plant [35]. In the present study, extracts showed 500 & 1000μg/ml is weakly toxic, and below
DPPH radical scavenging activity in a similar manner 500μg/ml is toxic which were established as LC50
to that of the reference antioxidant ascorbic acid- values [44].
increasing activity with the increase in concentration
(Fig. 5). This free radical scavenging activity might In this study methanol and ethyl-acetate extracts
be due to the presence of phenols and flavonoids in showed very good LC50 values of 20.00 μg/mL and
the extracts. Saurabh K et al., found the IC50 value of 191.618 μg/mL respectively. According to the
48 g/mL and 69 g/mL for two samples of bark of National Cancer Institute (NCI), the LC50 value of ≤
methanolic extract of Bridelia retusa (L.) Spreng., 20μg/mL is considered to be very cytotoxic [45]. So
which is much less than that of the finding of the methanolic extract may be considered as very
present study [36]. This may suggest that the fruit cytotoxic. It was reported that toxicity of plant
part of the plant possesses higher antioxidant capacity extracts is attributed to the presence of different
than bark. types of secondary metabolites such as saponins,
terpenoids, steroids, tannins, alkaloids etc. [46].
Cupric Reducing Antioxidant Capacity: Reduction
of Cu2+ ion to Cu+ was found to rise with increasing Determination of Antibacterial Activity by Disc
concentrations of the different extracts. Reducing Diffusion Method: The result of antimicrobial
capacity of the component of plant extracts is a key screening of different extracts of fruit of Bridelia
indicator of antioxidant property [37]. Different retusa (L.) Spreng. has been presented in Table 5.
studies have indicated that the antioxidant activity is Among the extracts the ethyl acetate extracts of fruit
associated with the electron donation capacity [38, showed a slight activity against Escherichia coli and
39]. Pseudomonus aeruginosa. The standard,

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chloramphenicol, exhibited significant zone of retusa (L.) Spreng fruits possess good antioxidant
inhibition against all the test organisms. The ethyl property. N-hexane extracts also demonstrated good
acetate extract of fruits exhibited slight total flavonoid content and DPPH free radical
antimicrobial activity and it is probably attributed scavenging activity. The result suggests that the
to the presence of saponins, flavonoids and total methanolic extract possess overall good antioxidant
tannin content [47, 48] which were detected in and cytotoxic potential. Further well-structured
phytochemical screening (Table 2). studies are needed to isolate the bioactive compounds
responsible for these activities and to determine their
underlying molecular mechanism to find out novel
CONCLUSION lead compounds.
From the foregoing, it was observed that the
methanolic and ethyl acetate extracts of Bridelia

Table 1: Microorganisms used in antimicrobial assay

Gram Positive Bacteria Gram Negative Bacteria
Bacillus subtilis Escherichia coli
Salmonella typhi
Pseudomonus aeroginusa

Table 2: Results of Phytochemical Screening of the extracts

Phytochemical Group Of n-Hexane Ethyl Acetate Methanol
Compounds
Alkaloid ⁺ ⁺ ⁺
Carbohydrate ⁻ ⁺ ⁺
Flavonoid ⁻ ⁺ ⁺
Glucoside ⁺ ⁺ -
Glycoside ⁻ ⁺ ⁺
Saponin ⁻ ⁺ ⁺
Steroid ⁻ ⁻ ⁻
Tannin ⁻ ⁺ ⁺
[‘+’ sign indicates presence of phytochemical group of compounds while the ‘-’ sign indicates absence of
phytochemical group of compounds tested for]

Table 3: Total phenolic, flavonoid contents, Total Tannin Contents (mean ± SD) of extracts from fruit
extracts of B. retusa
Plant Extracts Total Phenolic Total Flavonoid Total Tannin Total Antioxidant
Content (mg/gm Content (mg/gm Content (mg/gm Capacity (mg/gm
GAE) QE) TAE) AAE)
Methanol 134.50±0.00 17.00±2.82 652.50±3.53 236.00±36.78
Ethyl Acetate 91.00±4.243 46.00±0.00 650.00±7.07 180.50±31.81
n-Hexane 3.50±1.414 60.50±4.95 42.50±3.53 24.50±16.26

Table 4: IC50 and LC50 values of the different extracts in DPPH scavenging assay and brine shrimp lethality
bioassay
DPPH Scavenging Assay Brine Shrimp Lethality Bioassay
Sample IC50 ( µg/mL) Sample LC50(µg/mL)
Methanol 168.757 Methanol 20.00
Ethyl Acetate 141.954 Ethyl Acetate 191.618
n-Hexane 381.609 n-Hexane 915.4545
Ascorbic Acid 16.654 Vincristine 0.699

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Table 5: Anti-microbial screening of different extracts of fruit of B. retusa.

Test Organisms Inhibition Zone Diameter (mm)
Chloranphenicol Methanol (400 Ethyl Acetate n-Hexane (400
(50µg/Disc) µg/Disc) (400 µg/Disc) µg/Disc)
Bacillus subtilis 23 _ _ _
Escherichia coli 32 _ 11 _
Salmonella typhi 39 _ _ _
Pseudomonus aeruginosa 23 _ 9 _

Figure 1: Calibration line of gallic acid standard

Figure 2: Calibration line of quercetin standard

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Figure 3: Calibration curve of tannic acid

Figure 4: Calibration curve of ascorbic acid

Figure 5: DPPH radical scavenging activity of the different extracts of fruit of B. retusa.

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Figure 6: Cupric Reducing Antioxidant Capacity Bridelia retusa (L.) Spreng. fruit.

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