Evaluation of in vitro antioxidant and b
Evaluation of in vitro antioxidant and
brine shrimp lethality activities of different
stem extracts of Zizyphus rugosa Lam
Md. Sazzad Hossain, Nizam
Uddin, A. F. M. Mahmudul Islam,
A. H. M. Nazmul Hasan, Md. Monir
Hossain, Mohammad Raquibul Hasan,
et al.
Journal of Food Measurement and
Characterization
ISSN 2193-4126
Volume 9
Number 3
Food Measure (2015) 9:454-462
DOI 10.1007/s11694-015-9253-4
1 23
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1 23
Author's personal copy
Food Measure (2015) 9:454–462
DOI 10.1007/s11694-015-9253-4
ORIGINAL PAPER
Evaluation of in vitro antioxidant and brine shrimp lethality
activities of different stem extracts of Zizyphus rugosa Lam
Md. Sazzad Hossain1 • Nizam Uddin1 • A. F. M. Mahmudul Islam1 •
A. H. M. Nazmul Hasan1 • Md. Monir Hossain1 • Mohammad Raquibul Hasan1
Md. Farhan Khalik1 • Md. Sohel Rana1
•
Received: 27 May 2014 / Accepted: 26 April 2015 / Published online: 6 May 2015
Springer Science+Business Media New York 2015
Abstract Zizyphus rugosa Lam (Z. rugosa Lam) (Family:
Rhamnaceae), has been regarded as a food and medicinal
plant. It is locally known as ‘‘Bon Boroi’’ or as ‘‘Jongli Boroi’’
in Bangladesh. Its stem, roots and fruits are used medicinally
for the treatment of carbuncle, syphilis, menorrhea, and ulcer
tongue. It is also used for dysentery in Laos, Burma, Thailand,
China (Hainan, Yunnan), Sri-Lanka and Vietnam. The
present study was designed to investigate antioxidant properties (through in vitro method) as well as brine shrimp
lethality and phytochemical group evaluation of stem part of
Z. rugosa Lam extracted with different solvents i.e. from non
polar to polar (petroleum ether [ ethyl acetate [ ethanol [
methanol [ water). Phytochemical investigation showed the
presence of alkaloids, flavonoid, glycosides and carbohydrates which provides evidence on good to moderate
antioxidant and good lethality properties of the subjected
plant. Ethyl acetate extract of stem was found to contain the
highest amount of phenols (97.188 ± 12.816 mg/g gallic
acid equivalent) and flavonoids (15.009 ± 0.385 mg/g
quercetin equivalent). In 2,2-diphenyl,1-picrylhydrazyl
(DPPH) free radical scavenging assay, among all the extracts
ethanolic stem extract showed the highest scavenging property (IC50 83.550 lg/ml) whereas standard drug ascorbic acid
showed (IC50 18.348 lg/ml). But in nitric oxide (NO) scavenging assay maximum scavenging of NO was found with
water extract of stem (IC50 5.975 lg/ml) comparatively
similar to standard ascorbic acid (IC50 5.934 lg/ml).
Methanolic stem extract was found to contain the greater
& Md. Sazzad Hossain
sazzad.phrm@gmail.com
1
Department of Pharmacy, Jahangirnagar University, Savar,
Dhaka 1342, Bangladesh
123
reducing power in reducing power capacity assessment
(correlation coefficient r = 0.99 and P \ 0.001). In brine
shrimp lethality bioassay (BSLB) among all extracts, ZSM
part showed good activity (LC50 40.43 lg/ml) whereas
standard anticancer drug vincristine sulphate showed high
toxicity (LC50 2.48 lg/ml). The overall findings provide
scientific basis for the use of Z. rugosa Lam stem extracts in
traditional medicine in the treatment of aforementioned diseases. Hence, the stem may serve as a new potential source of
medication.
Keywords Jongli Boroi Zizyphus rough In vitro
antioxidant Lethality Phytochemicals
Introduction
Natural products are considered as the prime source of
most of the active ingredients of modern medicines. Natural product, a chemical substance produced by a living
organism; a term used commonly in reference to chemical
substances found in nature that have distinctive pharmacological effects. When applied to drug discovery in ‘olden
times’ before the advent of high-throughput screening and
the post-genomic era: more than 80 % of drug substances
were natural products or inspired by a natural compound
[1]. Natural compounds provide a strategic starting to
synthesize new compounds, that have diversified structures
and often with multiple stereocenters [2, 3]. More than 100
natural-product-derived compounds are currently undergoing clinical trials and at least 100 similar projects are in
preclinical development. Most are derived from leads from
plants and microbial sources [4]. Most of the today’s
medicines are obtained directly from the natural source.
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Evaluation of in vitro antioxidant and brine shrimp lethality activities of different stem…
Medicinal plants are various plants that possess therapeutic
properties or exert beneficial pharmacological effects on
the animal body [5].
Zizyphus rugosa Lam (Z. rugosa Lam) appears as pulpy
white or pinkish in colour and has a mildly sweet taste. It is
the species of plant in the Rhamnaceae family. They are
found on the hills in bunches on thorny branches of the Z.
rugosa Lam trees. It is found in hills and mountains below
1400 m altitude and its bark and wood are used
medicinally for dysentery in Laos, China (Hainan, Yunnan), India, Burma, Sri Lanka, Thailand and Vietnam. Its
leaf size is larger than normal Plum. Leaves are elliptic,
often rounded from an oblique or cordate base, 5–13 cm
long, densely tawny–villous and paler beneath and Fruits
are drupe, globose or pear-shaped, about 2 cm long. Its
stem is commonly used for the treatment of diarrhea while
the flowers, together with leaves, are used in menorrhagia
[6]. Its bark, roots and fruits are also used medicinally for
the treatment of carbuncle, syphilis, menorrhea, and ulcer
tongue [7]. A new glycoside zizyphoside has been isolated
along with the betulicoleanolic, alphitolic and 2-ahydroxyrusolic acids; three flavonoids—kaempferol-40 methylether, luteolin and luteolin-7-O-glucoside have been
isolated from the barks of Z. rugosa Lam [8]. The cyclopeptide alkaloids of the plant show antibacterial as well
as antifungal activity [9].
This study aims to investigate antioxidant and brine
shrimp lethality activities of five different stem extracts of
Z. rugosa Lam using various in vitro assay models.
Methodology
Study design
The present protocol was designed to evaluate the phytochemical group, antioxidant and cytotoxic potentiality of
stem extract of Z. rugosa Lam extracted with different
solvent i.e., from non polar to polar i.e., petroleum
ether [ ethyl acetate [ ethanol [ methanol [ water.
Collection and identification of plant material
The whole plants of Z. rugosa Lam were collected from
Jahangirnagar University campus, Savar, Dhaka, Bangladesh in the month of October, 2013 and was identified by
Mr. Abdur Rahim, Technical officer, Department of Botany, Jahangirnagar University, Savar, Dhaka, Bangladesh
(DACB accession No. 38729) where voucher specimen has
been deposited for further reference. The specimen samples
were kept in the Laboratory of Natural Products Research
in the Department of Pharmacy, Jahangirnagar University,
Bangladesh.
455
Chemicals and reagents
DPPH (2,2-diphenyl,1-picrylhydrazyl), ascorbic acid, sodium nitroprusside, sodium phosphate, sulphanilamide,
phosphoric acid and naphthylethylenediamine were obtained from SD Fine Chem. Ltd. India. Ammonium molybdate was obtained from Merck, Germany. Ferric chloride and
neocaproine were obtained from Sigma Chemical Co.
Preparation of plant material and extraction
procedure
Stem or branches of the plants were first washed with water to
remove adhering dirt and then cut into small pieces and sundried for few days and then, dried in a hot air oven (size 1,
Gallenkamp) at reduced temperature (not more than 50 C).
After that stem parts were grinded into coarse powders using
high capacity grinding mill which were then stored in air-tight
container with necessary markings for identification and kept
in cool, dark and dry place for the investigation.
The powdered plant materials were used for extraction by
Soxhlet apparatus at elevated temperature (65 C) using petroleum ether, ethyl acetate, ethanol and methanol consecutively (500 ml of each solvent). After each extraction the
plant material was dried and used again for the next extraction.
Extraction was considered to be complete when the plant
materials become exhausted of their constituents that were
confirmed from cycles of colorless liquid siphoning in the
Soxhlet apparatus. After methanol extraction was completed
the plant materials were dried and soaked into distilled water
(1 L). The plant materials were kept in water for 7 days in
sealed container accompanying occasional shaking and string.
All five extracts of stem part were filtered individually through
fresh cotton bed. The filtrates obtained were dried at temperature of 40 ± 2 C to have gummy concentrate of the
crude extracts. Each extract was kept in suitable container
with proper labeling and stored in cold and dry place [10].
Phytochemical test
The freshly prepared crude extract was qualitatively tested
for the presence of chemical constituents i.e., carbohydrates, flavonoids, glucosides, steroids, saponins, tannins
and alkaloids. These were identified by characteristic color
changes using standard procedures [5].
Antioxidant activity evaluation
Total phenol content determination
Total phenolic contents of the fractions were determined by
Folin–Ciocalteu reagent (FCR) [11, 12]. 1.0 ml of each
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456
Md. S. Hossain et al.
plant extract (200 lg/ml), standard (gallic acid) of different
concentrations and 5 ml of FCR (diluted 10 fold) reagent
solution were taken in marked test tubes and 4 ml of 7.5 %
sodium carbonate solution was added. The test tubes were
incubated at 20 C (30 min for standard solutions and 1 h
for extract solution). Absorbance at 765 nm was determined using a UV–Vis spectrophotometer (Shimadzu UV
PC-1600) against blank. Total phenol contents of the
fractions were expressed as gallic acid equivalents (GAE).
ethylene diamine dihydrochloride (0.1 % w/v) instead of
1-naphthylamine (5 %). 4.0 ml of each fraction and standard (ascorbic acid) was added into 1.0 ml of sodium nitroprusside (5 mM) solution and incubated for 2 h at 30 C
to complete reaction. Then 2.0 ml solution was withdrawn
from the mixture and mixed with 1.2 ml of Griess reagent.
Absorbance of the solution was measured at 550 nm using
a spectrophotometer (Shimadzu UV PC-1600) against
blank and IC50 values were calculated.
Determination of total flavonoid content
Reducing power capacity assessment
Total flavonoid was determined using the aluminum chloride colorimetric method described by Wang and Jiao [13].
1.0 ml plant extracts (200 lg/ml) and standard (Quercetin)
were added to 3 ml of methanol and 200 ll of 10 % aluminium chloride solution, 200 ll of 1 M potassium acetate
solution and 5.6 ml of distilled water were added and then
incubated for 30 min at room temperature to complete the
reaction. Absorbance of the solution was measured at
415 nm using a spectrophotometer (Shimadzu UV PC1600) against blank. Total Flavonoid contents of the fractions were expressed as Quercetin equivalents (QE).
Reducing power capacity assessment of the plant extracts
was determined following the method described by Oyaizu
et al. [17]. 2.0 ml of each plant extracts or standard of
different concentration solutions were taken and 2.5 ml of
potassium ferricyanide [K3Fe(CN)6], 1 % solution was
added into each of test tubes. The test tubes were incubated
for 10 min at 50 C and 2.5 ml of trichloro acetic acid,
10 % solution was added. The resultant mixtures were
centrifuged at 3000 rpm for 10 min and 2.5 ml supernatant
solution was withdrawn from each of the mixtures and
mixed with 2.5 ml of distilled water. Then 0.5 ml of ferric
chloride (Fe3Cl), 0.1 % solution was added. The absorbances of the solutions were measured at 700 nm using
a spectrophotometer against a typical blank solution.
Determination of total antioxidant capacity
Total antioxidant capacity of the plant extract was determined following the method described by ascorbic acid
equivalents (AAE) [14]. 300 ll of each fraction (200 lg/
ml) and standard (ascorbic acid) in different concentrations
were taken in test tubes and 3 ml of reagent solution
(0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate) was added. The test tubes were
incubated at 95 C for 90 min to complete the reaction.
Absorbance of the solution was measured at 695 nm using
a spectrophotometer (Shimadzu UV PC-1600) against
blank after cooling to room temperature. Total antioxidant
capacity was expressed as the number of AAE.
DPPH free radical scavenging assay
DPPH free radical scavenging activity of the plant fractions
was determined following the method described by Braca
et al. [15]. Plant extract (1.0 ml) and standard (ascorbic
acid) was added to 2 ml of a 0.004 % methanol solution of
DPPH and incubated for 30 min. Absorbance at 517 nm
was determined and IC50 values were calculated.
Nitric oxide (NO) scavenging capacity assay
Nitric oxide (NO) radical scavenging was estimated on the
basis of Griess–Illosvoy reaction [16]. In this investigation,
Griess–Illosvoy reagent was modified by using naphthyl
123
Cupric reducing antioxidant capacity (CUPRAC)
Cupric reducing antioxidant capacity of the plant extracts was
determined following the method described by Resat et al.
[18]. 500 ll of each fraction and standard (ascorbic acid) in
different concentrations were taken in test tubes. 1.0 ml of
0.01 M CuCl22H2O solution and 1.0 ml of ammonium acetate buffer (pH 7.0) was added into the test tubes. Then 1.0 ml
of 0.0075 M of neocaproine solution and 600 ll of distilled
water was added into the test tubes. The total mixture was
incubated for 1 h at room temperature then the absorbance of
the solution was measured at 450 nm using a spectrophotometer (Shimadzu UV PC-1600) against blank. The molar
absorptivity of the CUPRAC method for each antioxidant was
found from the slope of the calibration line concerned.
Total alkaloid content determination
Total alkaloid content was determined by slightly modified
Fazel et al. [19] method. 4 mg of each plant extract was
dissolved in 4 ml 2 N HCl to get the concentration 1 mg/
ml. Then 5 times dilution was done by adding 1 ml of this
solution into 4 ml 2 N HCl to get the concentration of
stock solution 200 lg/ml. 1 ml stock solution of each plant
extract, 5 ml phosphate buffer and 5 ml BCG (Bromocresol green) solution taken in a separating funnel, shaken
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Evaluation of in vitro antioxidant and brine shrimp lethality activities of different stem…
well and 5 ml CHCl3 was added. The funnel was shaken
vigorously and the lower layer of the solution was collected
in a labeled test tube. The absorbance of the complex in
chloroform was measured at 470 nm. A typical blank solution contained the same solution mixture without plant
extract or standard. Total alkaloid content is expressed as
the number of equivalents of atropine (AE).
457
were performed to analyze the data set using Graph pad
prism version 5.00 (Graph Pad Software Inc., San Diego,
CA, USA) and SPSS version 16 (IBM software Inc, USA).
Results
Phytochemical group evaluation test
Total tannin content determination
The tannins were determined by slightly modified Folin
and Ciocalteu method [11]. 0.1 ml of the sample extract
(200 lg/ml) was added with 7.5 ml of distilled water.
0.5 ml of Folin Phenol reagent (diluted 10 fold) solution
was added into the test tubes. 1 ml of 35 % sodium carbonate solution was added into the test tubes. The volume
was adjusted up to 10 ml with distilled water. The mixture
was shaken well, kept at room temperature for 30 min. The
absorbance was measured at 725 nm. A typical blank was
prepared with water instead of the sample. A set of standard solutions of Tannic acid is treated in the same manner
as described earlier and read against a blank. The results of
tannins are expressed in terms of tannic acid in mg/g TAE
of extract.
Brine shrimp lethality bioassay (BSLB)
Lethal activity of the plant extract was determined by Brine
shrimp lethality bioassay described by Meyer et al. [20].
Brine shrimp lethality bioassay [20–22] is a rapid and
comprehensive bioassay for the bioactive compounds of
natural and synthetic origin. Brine shrimp eggs (Artemia
salina leach) are hatched in simulated seawater to get
nauplii. Sample solutions are prepared by dissolving the
test materials in pre-calculated amount of DMSO (dimethyl sulphoxide). Ten nauplii are taken in vials containing simulated seawater. The samples of different concentrations are added and the volume was adjusted up to
5 ml. Survivors were counted after 24 h. Vincristine sulphate is usually used as the reference cytotoxic drug. The
mortality was corrected using Abott’s formula [23]
Pt ¼ ½ðPo
Pc Þ=ð100
Pc Þ 100;
where, Po = observed mortality and Pc = control mortality. LC50 values of the test samples after 24 h are obtained by regression analysis.
Statistical analysis
Values were presented as mean ± SD (standard deviation).
IC50 and LC50 values were obtained with the help of Microsoft excel 2007. Pearson correlation analysis and one
way ANOVA followed by Tukey multiple comparison
In the present study, various qualitative tests were done to
detect the presence of various phytochemical compounds
in the petroleum ether, ethyl acetate, ethanol, methanol and
water extracts of the stem or branches part of Z. rugosa
Lam. Preliminary phytochemical screening of the crude
extracts of different parts of Z. rugosa Lam showed the
presence of different kind of phytochemical groups that can
be summarized in Table 1.
Antioxidant activity evaluation
Total phenol content determination
The total phenolic contents of the test fractions were calculated using the standard curve of Gallic acid and were
expressed as GAE per gram of plant extract. ZSEA was
found to contain the highest amount of phenols
(97.188 ± 12.816 mg/g) and ZSE showed the lowest
amount (32.500 ± 10.607 mg/g). Phenol contents of the
extracts were found to decrease in the following order:
ZSEA [ ZSPE [ ZSM [ ZSW [ ZSE (Table 2).
Total flavonoid content determination
The total flavonoid content was calculated using the standard
curve of Quercetin. Flavonoid contents of the test fractions
were found as mg/g Quercetin equivalent (QE). ZSEA was
found to contain the highest amount of Flavonoid
(15.009 ± 0.385 mg/g) and ZSE was found to contain the
lowest amount of flavonoid (0.293 ± 0.003 mg/g). Flavonoid contents of the extracts were found to decrease in the
following order: ZSEA [ ZSPE [ ZSM [ ZSW [ ZSE
(Table 2).
Total antioxidant capacity assessment
Total antioxidant capacity of Z. rugosa Lam stem extract was
evaluated by the phosphomolybdenum method and was expressed as AAE per gram of plant extract. ZSM was found to
contain the highest (37.500 ± 3.712 mg/g) and ZSPE was
found to contain the lowest (16.875 ± 4.773 mg/g) amount
of total antioxidant capacity. Total antioxidant capacities of
123
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–
?
?
–
–
–
absence
–
?
ZSEA
strong presence,
–
?
Presence,
?
ZSW
ZSM
ZSE
??
?
?
?
?
-
?
–
ZSPE
ZSPE Z. rugosa Lam, petroleum ether extract of stem part, ZSEA Z. rugosa Lam, ethyl acetate extract of stem part, ZSE Z. rugosa Lam, ethanol extract of stem part, ZSM Z. rugosa Lam,
methanol extract of stem part, ZSW Z. rugosa Lam, water extract of stem part
?
–
?
?
–
?
?
??
?
?
?
?
–
–
?
–
?
??
?
?
–
?
?
–
–
–
?
?
–
?
?
?
?
?
–
?
?
–
–
?
Glucoside
Fehling’s test
Molisch’s test
Glycoside
–
?
Dragendorff’s test
Wagner test
Mayer’s test
Hager’s test
–
Tannins
Steroids
Alkaloids
Flavonoids
Glycosides
Carbohydrates
Extracts
Table 1 Result of chemical group test of stem extracts of Z. rugosa Lam
123
?
Md. S. Hossain et al.
Saponins
458
the extracts were found to decrease in the following order:
ZSM [ ZSW [ ZSEA [ ZSE [ ZSPE (Table 2).
DPPH free radical scavenging assay
DPPH scavenging assay has been widely used to evaluate the
free radical scavenging capacity of antioxidant. The IC50
values of different extracts of Z. rugosa Lam are summarized
in the Table 3. ZSE was found to contain the lowest IC50 value
(83.550 lg/ml) that represents the highest scavenging property than the other tested extracts. The IC50 value of standard
drug ascorbic acid was 18.348 lg/ml. DPPH free radical
scavenging activity of the plant extracts of Z. rugosa Lam was
found to decrease in the following order: ZSE [ ZSM [
ZSW [ ZSEA [ ZSPE.
Nitric oxide (NO) scavenging capacity assay
Different extracts of the stem part exhibited dose dependent scavenging of nitric oxide. Maximum scavenging of
NO was found with ZSW with an IC50 value of 5.975 lg/
ml; the result was similar to standard ascorbic acid (IC50
value 5.934 lg/ml) can be summarized in the Table 3. NO
scavenging activity decreased in the following order;
ZSW [ ZSM [ ZSE [ ZSPE [ ZSEA that are summarized in the Table 3.
Reducing power capacity assessment
Reducing power of the tested extracts were assessed using
ferric to ferrous reduction activity as determined spectrophotometrically from the formation of Perl’s Prussian
blue color complex [24]. The standard ascorbic acid
showed the highest reducing capacity. The stem extracts of
Z. rugosa Lam showed reducing capacity. ZSM was found
to contain more reducing power than other extracts (correlation coefficient r = 0.99 and P \ 0.001) presented in
Fig. 1.
Cupric reducing antioxidant capacity (CUPRAC)
Reduction of Cu2? ion to Cu? was found to rise with
increasing concentrations of the different extracts. The
standard ascorbic acid showed the highest reducing capacity in CUPRAC method. Comparison graph of cupric
reducing antioxidant capacity (CUPRAC) between ascorbic acid and Z. rugosa Lam stem extracts are given in
Fig. 1.
Total alkaloid content determination
Total Alkaloid content of Z. rugosa Lam was determined by
Dragendroff’s method and was expressed as atropine
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Evaluation of in vitro antioxidant and brine shrimp lethality activities of different stem…
459
Table 2 Summarized anti-oxidant results of stem extracts of Z. rugosa Lam
Extract
Total phenol
(mg/g GAE)
ZSPE
78.125 ± 12.374b
12.136 ± 0.252c
ZSEA
97.188 ± 12.816
b
d
15.009 ± 0.385
32.500 ± 10.607
a
a
ZSM
76.250 ± 10.607
b
ZSW
b
ZSE
Total flavonoid
(mg/g QE)
0.293 ± 0.003
b
3.651 ± 0.135
a,b
78.125 ± 4.419
1.271 ± 0.041
Total tannin
(mg/g TAE)
Total antioxidant
(mg/g AAE)
Total alkaloid
(mg/g AE)
16.875 ± 4.773a
28.694 ± 0.005a
a,b
21.938 ± 5.038
87.600 ± 0.044b
a
154.622 ± 0.022c
a
57.596 ± 0.015a
a
221.026 ± 0.050d
a
63.291 ± 0.005a
24.062 ± 0.001
b
24.375 ± 1.061
30.705 ± 0.004
c
37.500 ± 3.712
25.747 ± 0.004
c
35.250 ± 5.834
38.135 ± 0.003
Values are presented as mean ± SD (n = 3). One way ANOVA followed by Tukey multiple comparisons was performed to analyze the data
sets. Values in same column with different superscripts are significantly different from one another (P \ 0.05)
Table 3 IC50 and LC50 values of the different extracts in DPPH, NO and Brine shrimp lethality bioassays
Extract
DPPH free radical
scavenging assay, IC50 (lg/ml)
Nitric oxide scavenging
capacity assay, IC50 (lg/ml)
Brine shrimp lethality
bioassay (BSLB) LC50 (lg/ml)
ZSPE
474,147
15.661
134.4
ZSEA
312.210
31.634
385.38
ZSE
83.550
8.945
535.39
ZSM
87.298
7.228
40.43
ZSW
282.430
5.975
235.11
Ascorbic acid
18,348
5.934
–
Vincristine sulphate
–
–
2.48
B
1.0
Ascorbic acid
ZSPE
ZSEA
ZSE
ZSM
ZSW
Absorbance
0.8
0.6
0.4
2.5
Ascorbic acid
ZSPE
ZSEA
ZSE
ZSM
ZSW
2.0
Absorbance
A
1.5
1.0
0.5
0.2
0.0
0.0
0
50
100
150
200
(Concentration µg/ml)
0
50
100
150
200
250
(Concentration µg/ml)
Fig. 1 a Cuprac reducing assessment of five different stem extracts
and standard. Values are presented as mean ± SEM (n = 3). Pearson
correlation analysis was performed between different concentrations
and absorbance of each stem extract and standard. For ZSPE,
correlation coefficient r = 0.98 and P \ 0.01; for ZSEA, r = 0.98
and P \ 0.01; ZSE, correlation coefficient r = 0.99 and P \ 0.0001;
for ZSM, correlation coefficient r = 0.98 and P \ 0.01; for ZSW,
correlation coefficient r = 0.95 and P \ 0.05; for ascorbic acid,
r = 0.999 and P \ 0.001. b Reducing power capacity assessment of
five different stem extracts and standard. Values are presented as
mean ± SEM (n = 3). Pearson correlation analysis was performed
between different concentrations and absorbance of each stem extract
and standard. For ZSPE, correlation coefficient r = 0.97 and
P \ 0.01; for ZSEA, r = 0.98 and P \ 0.01; ZSE, correlation
coefficient r = 0.99 and P \ 0.001; for ZSM, correlation coefficient
r = 0.99 and P \ 0.001; for ZSW, correlation coefficient r = 0.93
and P \ 0.001; for ascorbic acid, r = 0.98 and P \ 0.05
equivalents (AE) mg/g of plant extract. ZSW was found to
contain the highest amount of Alkaloid (38.135 ± 0.003 mg/g)
and ZSEA was found to contain the lowest amount of alkaloid (24.062 ± 0.001 mg/g). Alkaloid contents of five
different extracts did not find significantly (P [ 0.05).
Alkaloid contents of the extracts were found to decrease
in the following order; ZSW [ ZSE [ ZSPE [ ZSM [
ZSEA (Table 2).
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460
Total tannin content determination
Total Tannin content of Z. rugosa Lam was determined by
slightly modified Folin and Ciocalteu method [11] and was
expressed as tannic acid equivalent (TAE) mg/g of plant
extract. ZSM was found to contain the highest amount of
total tannin that can be expressed as tannic acid equivalent
(221.026 ± 0.050 mg/g). Tannin contents of the extracts
were found to decrease in the following order;
ZSM [ ZSEA [ ZSPE [ ZSW [ ZSE (Table 2).
Brine shrimp lethality bioassay (BSLB)
In this study methanol extract of stem part (ZSM) showed
most toxicity to brine shrimp nauplii, with LC50 value of
40.43 lg/ml whereas anticancer drug Vincristine sulphate
showed LC50 value 2.48 lg/ml described in the Table 3.
The order at which cytotoxic potential of the test samples
decreased was as follows: ZSM [ ZSPE [ ZSW [
ZSEA [ ZSE.
Discussion
Several phytochemical study reported that cyclopeptide
alkaloids [25, 26] and six flavones glycosides [27] and one
saponine were [28] isolated from root barks of Z. rugosa. In
continuation of this study, we have extracted this plant with
different solvents and a preliminary screening showed that
ZSPE, ZSEA and ZSM were rich in flavonoids. It can be
assumed that Z. rugosa Lam stem are rich in alkaloids,
flavonoids, tannins, carbohydrates, steroids, saponin which
may account for their various pharmacological activities.
Antioxidative properties of polyphenols arise from their
high reactivity as hydrogen or electron donors which can
stabilize and delocalize the unpaired electron (chainbreaking function) and from their potential to chelate metal
ions (termination of the Fenton reaction) [29]. Polyphenols
have been shown to block LDL oxidation, decrease the
formation of atherosclerotic plaques and reduce arterial
stiffness, leaving arteries more responsive to endogenous
stimuli of vasodilation [30–32]. This ability is believed to
be mainly due to their redox properties [33], which play an
important role in adsorbing and neutralizing free radicals.
The results strongly suggest that phenolics are important
components of the tested plant extracts Z. rugosa Lam stem
extract showed good to moderate amount of phenolic
content which is showed in gallic acid equivalent. Flavonoids play an important role in antioxidant system in
plants. The antioxidative properties of flavonoids are due to
several different mechanisms, such as scavenging of free
radicals, chelation of metal ions, such as iron and copper,
and inhibition of enzymes responsible for free radical
123
Md. S. Hossain et al.
generation [34]. Depending on their structure, flavonoids
are able to scavenge practically all known ROS. The stem
extract of Z. rugosa Lam has been shown to possess mild to
moderate amount of flavonoids. The total antioxidant activity of stem extracts of Z. rugosa Lam was evaluated
from their ability to reduce phosphate/Mo(VI) complex to
phosphate/Mo(V). According to recent reports, a highly
positive relationship between total phenols and antioxidant
activity appears to be the trend in many plant species [35].
Z. rugosa Lam stem extracts showed moderate content
which is showed in ascorbic acid equivalent. DPPH radical
scavenging is a popular and reliable method for screening
the free radical scavenging activity of compounds or antioxidant capacity of plant extracts [12, 36–38]. In DPPH
radical scavenging assays, the stem extracts of Z. rugosa
Lam showed good to moderate scavenging of DPPH
radicals in a way similar to that of the reference antioxidant
ascorbic acid. NO is generated in biological tissues by
specific nitric oxide syntheses (NOSs), which metabolizes
arginine to citrulline with the formation of NO via a five
electron oxidative reaction [39]. The toxicity and damage
caused by NO- and O2- is multiplied as they react to
produce reactive peroxynitrite (ONOO-), which leads to
serious toxic reactions with biomolecules as mentioned
above [40–42]. Our findings indicate that all of the five
plant extracts showed good NO scavenging activity. The
reducing power of the extracts of the selected plants might
be due to its hydrogen donating ability [43]. The antioxidant activity has been reported to be concomitant with
the development of reducing power [44]. Among the extracts ZSM exhibited the most reducing power in reducing
power capacity assessment. Reducing power is associated
with antioxidant activity and may serve as a significant
reflection of the antioxidant activity [35]. The standard
ascorbic acid showed the highest reducing capacity. The
stem extracts of Z. rugosa Lam showed reducing capacity.
It also showed to possess moderate amount of Alkaloids
which were also identified by phytochemical group
evaluation test. Moreover, the extracts also showed to
possess good to moderate amount of tannin content. These
phytochemicals may strengthen the antioxidant potential of
the stem.
The brine shrimp lethality bioassay has been considered
as a practical, safe and economic method for determination
of bioactivities of synthetic compound as well as plant
products [20]. In Brine Shrimp lethality bioassay all of the
samples showed considerable lethality. The stem extracts
of Z. rugosa Lam has showed good cytotoxic potentiality.
The toxicity of plants is principally contributed by the
presence of alkaloids, glycosides, steroids, tannins, phlobatannins, terpenoids and flavonoids [45–48]. However,
phenolics and flavonoids are also known to show cytotoxicity in Hoechst 33258 fluorescence assay by inhibiting
Author's personal copy
Evaluation of in vitro antioxidant and brine shrimp lethality activities of different stem…
cellular DNA in a concentration-dependent manner [49].
Different phytochemical group like alkaloids, glycosides,
steroids, tannins and flavonoids were found in our observation (Table 1). So the observed cytotoxic action may be
due to the presence of these phytochemical constituents,
although further studies are required to be confirmed.
Conclusion
The results from the experiments confirmed that the stem
extract of Z. rugosa Lam in general, possesses good to
moderate antioxidant and good brine shrimp lethality properties. Various phytochemical constituents like terpenoids,
glycosides and steroids present in the plant, which may be
responsible for the observed activities. The good toxicity
exerted by the extracts of Z. rugosa Lam in brine shrimp
lethality bioassay suggests the presence of bioactive principles in the plant. However, further studies are suggested to be
undertaken to understand the underlying mechanism of the
observed activities and to isolate, purify and characterize
active phytochemical compounds that are responsible for
these bioactivities in animal models and to carry out advance
research on cancer because the other plants of Rhamnaceae
family have significant cytotoxic and inhibitory activity on
tumors of various types of cancers. Besides, these extracts
can be used as promising source of natural antioxidants
which can work as stabilizing agent against oxidative deterioration in pharmaceutical applications.
Acknowledgments We are greatly thankful to Laboratory of Natural Products Research (LNPR), Department of Pharmacy, Jahangirnagar University to provide sufficient laboratory facilities to carry out
this research.
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brine shrimp lethality activities of different
stem extracts of Zizyphus rugosa Lam
Md. Sazzad Hossain, Nizam
Uddin, A. F. M. Mahmudul Islam,
A. H. M. Nazmul Hasan, Md. Monir
Hossain, Mohammad Raquibul Hasan,
et al.
Journal of Food Measurement and
Characterization
ISSN 2193-4126
Volume 9
Number 3
Food Measure (2015) 9:454-462
DOI 10.1007/s11694-015-9253-4
1 23
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Food Measure (2015) 9:454–462
DOI 10.1007/s11694-015-9253-4
ORIGINAL PAPER
Evaluation of in vitro antioxidant and brine shrimp lethality
activities of different stem extracts of Zizyphus rugosa Lam
Md. Sazzad Hossain1 • Nizam Uddin1 • A. F. M. Mahmudul Islam1 •
A. H. M. Nazmul Hasan1 • Md. Monir Hossain1 • Mohammad Raquibul Hasan1
Md. Farhan Khalik1 • Md. Sohel Rana1
•
Received: 27 May 2014 / Accepted: 26 April 2015 / Published online: 6 May 2015
Springer Science+Business Media New York 2015
Abstract Zizyphus rugosa Lam (Z. rugosa Lam) (Family:
Rhamnaceae), has been regarded as a food and medicinal
plant. It is locally known as ‘‘Bon Boroi’’ or as ‘‘Jongli Boroi’’
in Bangladesh. Its stem, roots and fruits are used medicinally
for the treatment of carbuncle, syphilis, menorrhea, and ulcer
tongue. It is also used for dysentery in Laos, Burma, Thailand,
China (Hainan, Yunnan), Sri-Lanka and Vietnam. The
present study was designed to investigate antioxidant properties (through in vitro method) as well as brine shrimp
lethality and phytochemical group evaluation of stem part of
Z. rugosa Lam extracted with different solvents i.e. from non
polar to polar (petroleum ether [ ethyl acetate [ ethanol [
methanol [ water). Phytochemical investigation showed the
presence of alkaloids, flavonoid, glycosides and carbohydrates which provides evidence on good to moderate
antioxidant and good lethality properties of the subjected
plant. Ethyl acetate extract of stem was found to contain the
highest amount of phenols (97.188 ± 12.816 mg/g gallic
acid equivalent) and flavonoids (15.009 ± 0.385 mg/g
quercetin equivalent). In 2,2-diphenyl,1-picrylhydrazyl
(DPPH) free radical scavenging assay, among all the extracts
ethanolic stem extract showed the highest scavenging property (IC50 83.550 lg/ml) whereas standard drug ascorbic acid
showed (IC50 18.348 lg/ml). But in nitric oxide (NO) scavenging assay maximum scavenging of NO was found with
water extract of stem (IC50 5.975 lg/ml) comparatively
similar to standard ascorbic acid (IC50 5.934 lg/ml).
Methanolic stem extract was found to contain the greater
& Md. Sazzad Hossain
sazzad.phrm@gmail.com
1
Department of Pharmacy, Jahangirnagar University, Savar,
Dhaka 1342, Bangladesh
123
reducing power in reducing power capacity assessment
(correlation coefficient r = 0.99 and P \ 0.001). In brine
shrimp lethality bioassay (BSLB) among all extracts, ZSM
part showed good activity (LC50 40.43 lg/ml) whereas
standard anticancer drug vincristine sulphate showed high
toxicity (LC50 2.48 lg/ml). The overall findings provide
scientific basis for the use of Z. rugosa Lam stem extracts in
traditional medicine in the treatment of aforementioned diseases. Hence, the stem may serve as a new potential source of
medication.
Keywords Jongli Boroi Zizyphus rough In vitro
antioxidant Lethality Phytochemicals
Introduction
Natural products are considered as the prime source of
most of the active ingredients of modern medicines. Natural product, a chemical substance produced by a living
organism; a term used commonly in reference to chemical
substances found in nature that have distinctive pharmacological effects. When applied to drug discovery in ‘olden
times’ before the advent of high-throughput screening and
the post-genomic era: more than 80 % of drug substances
were natural products or inspired by a natural compound
[1]. Natural compounds provide a strategic starting to
synthesize new compounds, that have diversified structures
and often with multiple stereocenters [2, 3]. More than 100
natural-product-derived compounds are currently undergoing clinical trials and at least 100 similar projects are in
preclinical development. Most are derived from leads from
plants and microbial sources [4]. Most of the today’s
medicines are obtained directly from the natural source.
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Evaluation of in vitro antioxidant and brine shrimp lethality activities of different stem…
Medicinal plants are various plants that possess therapeutic
properties or exert beneficial pharmacological effects on
the animal body [5].
Zizyphus rugosa Lam (Z. rugosa Lam) appears as pulpy
white or pinkish in colour and has a mildly sweet taste. It is
the species of plant in the Rhamnaceae family. They are
found on the hills in bunches on thorny branches of the Z.
rugosa Lam trees. It is found in hills and mountains below
1400 m altitude and its bark and wood are used
medicinally for dysentery in Laos, China (Hainan, Yunnan), India, Burma, Sri Lanka, Thailand and Vietnam. Its
leaf size is larger than normal Plum. Leaves are elliptic,
often rounded from an oblique or cordate base, 5–13 cm
long, densely tawny–villous and paler beneath and Fruits
are drupe, globose or pear-shaped, about 2 cm long. Its
stem is commonly used for the treatment of diarrhea while
the flowers, together with leaves, are used in menorrhagia
[6]. Its bark, roots and fruits are also used medicinally for
the treatment of carbuncle, syphilis, menorrhea, and ulcer
tongue [7]. A new glycoside zizyphoside has been isolated
along with the betulicoleanolic, alphitolic and 2-ahydroxyrusolic acids; three flavonoids—kaempferol-40 methylether, luteolin and luteolin-7-O-glucoside have been
isolated from the barks of Z. rugosa Lam [8]. The cyclopeptide alkaloids of the plant show antibacterial as well
as antifungal activity [9].
This study aims to investigate antioxidant and brine
shrimp lethality activities of five different stem extracts of
Z. rugosa Lam using various in vitro assay models.
Methodology
Study design
The present protocol was designed to evaluate the phytochemical group, antioxidant and cytotoxic potentiality of
stem extract of Z. rugosa Lam extracted with different
solvent i.e., from non polar to polar i.e., petroleum
ether [ ethyl acetate [ ethanol [ methanol [ water.
Collection and identification of plant material
The whole plants of Z. rugosa Lam were collected from
Jahangirnagar University campus, Savar, Dhaka, Bangladesh in the month of October, 2013 and was identified by
Mr. Abdur Rahim, Technical officer, Department of Botany, Jahangirnagar University, Savar, Dhaka, Bangladesh
(DACB accession No. 38729) where voucher specimen has
been deposited for further reference. The specimen samples
were kept in the Laboratory of Natural Products Research
in the Department of Pharmacy, Jahangirnagar University,
Bangladesh.
455
Chemicals and reagents
DPPH (2,2-diphenyl,1-picrylhydrazyl), ascorbic acid, sodium nitroprusside, sodium phosphate, sulphanilamide,
phosphoric acid and naphthylethylenediamine were obtained from SD Fine Chem. Ltd. India. Ammonium molybdate was obtained from Merck, Germany. Ferric chloride and
neocaproine were obtained from Sigma Chemical Co.
Preparation of plant material and extraction
procedure
Stem or branches of the plants were first washed with water to
remove adhering dirt and then cut into small pieces and sundried for few days and then, dried in a hot air oven (size 1,
Gallenkamp) at reduced temperature (not more than 50 C).
After that stem parts were grinded into coarse powders using
high capacity grinding mill which were then stored in air-tight
container with necessary markings for identification and kept
in cool, dark and dry place for the investigation.
The powdered plant materials were used for extraction by
Soxhlet apparatus at elevated temperature (65 C) using petroleum ether, ethyl acetate, ethanol and methanol consecutively (500 ml of each solvent). After each extraction the
plant material was dried and used again for the next extraction.
Extraction was considered to be complete when the plant
materials become exhausted of their constituents that were
confirmed from cycles of colorless liquid siphoning in the
Soxhlet apparatus. After methanol extraction was completed
the plant materials were dried and soaked into distilled water
(1 L). The plant materials were kept in water for 7 days in
sealed container accompanying occasional shaking and string.
All five extracts of stem part were filtered individually through
fresh cotton bed. The filtrates obtained were dried at temperature of 40 ± 2 C to have gummy concentrate of the
crude extracts. Each extract was kept in suitable container
with proper labeling and stored in cold and dry place [10].
Phytochemical test
The freshly prepared crude extract was qualitatively tested
for the presence of chemical constituents i.e., carbohydrates, flavonoids, glucosides, steroids, saponins, tannins
and alkaloids. These were identified by characteristic color
changes using standard procedures [5].
Antioxidant activity evaluation
Total phenol content determination
Total phenolic contents of the fractions were determined by
Folin–Ciocalteu reagent (FCR) [11, 12]. 1.0 ml of each
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456
Md. S. Hossain et al.
plant extract (200 lg/ml), standard (gallic acid) of different
concentrations and 5 ml of FCR (diluted 10 fold) reagent
solution were taken in marked test tubes and 4 ml of 7.5 %
sodium carbonate solution was added. The test tubes were
incubated at 20 C (30 min for standard solutions and 1 h
for extract solution). Absorbance at 765 nm was determined using a UV–Vis spectrophotometer (Shimadzu UV
PC-1600) against blank. Total phenol contents of the
fractions were expressed as gallic acid equivalents (GAE).
ethylene diamine dihydrochloride (0.1 % w/v) instead of
1-naphthylamine (5 %). 4.0 ml of each fraction and standard (ascorbic acid) was added into 1.0 ml of sodium nitroprusside (5 mM) solution and incubated for 2 h at 30 C
to complete reaction. Then 2.0 ml solution was withdrawn
from the mixture and mixed with 1.2 ml of Griess reagent.
Absorbance of the solution was measured at 550 nm using
a spectrophotometer (Shimadzu UV PC-1600) against
blank and IC50 values were calculated.
Determination of total flavonoid content
Reducing power capacity assessment
Total flavonoid was determined using the aluminum chloride colorimetric method described by Wang and Jiao [13].
1.0 ml plant extracts (200 lg/ml) and standard (Quercetin)
were added to 3 ml of methanol and 200 ll of 10 % aluminium chloride solution, 200 ll of 1 M potassium acetate
solution and 5.6 ml of distilled water were added and then
incubated for 30 min at room temperature to complete the
reaction. Absorbance of the solution was measured at
415 nm using a spectrophotometer (Shimadzu UV PC1600) against blank. Total Flavonoid contents of the fractions were expressed as Quercetin equivalents (QE).
Reducing power capacity assessment of the plant extracts
was determined following the method described by Oyaizu
et al. [17]. 2.0 ml of each plant extracts or standard of
different concentration solutions were taken and 2.5 ml of
potassium ferricyanide [K3Fe(CN)6], 1 % solution was
added into each of test tubes. The test tubes were incubated
for 10 min at 50 C and 2.5 ml of trichloro acetic acid,
10 % solution was added. The resultant mixtures were
centrifuged at 3000 rpm for 10 min and 2.5 ml supernatant
solution was withdrawn from each of the mixtures and
mixed with 2.5 ml of distilled water. Then 0.5 ml of ferric
chloride (Fe3Cl), 0.1 % solution was added. The absorbances of the solutions were measured at 700 nm using
a spectrophotometer against a typical blank solution.
Determination of total antioxidant capacity
Total antioxidant capacity of the plant extract was determined following the method described by ascorbic acid
equivalents (AAE) [14]. 300 ll of each fraction (200 lg/
ml) and standard (ascorbic acid) in different concentrations
were taken in test tubes and 3 ml of reagent solution
(0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate) was added. The test tubes were
incubated at 95 C for 90 min to complete the reaction.
Absorbance of the solution was measured at 695 nm using
a spectrophotometer (Shimadzu UV PC-1600) against
blank after cooling to room temperature. Total antioxidant
capacity was expressed as the number of AAE.
DPPH free radical scavenging assay
DPPH free radical scavenging activity of the plant fractions
was determined following the method described by Braca
et al. [15]. Plant extract (1.0 ml) and standard (ascorbic
acid) was added to 2 ml of a 0.004 % methanol solution of
DPPH and incubated for 30 min. Absorbance at 517 nm
was determined and IC50 values were calculated.
Nitric oxide (NO) scavenging capacity assay
Nitric oxide (NO) radical scavenging was estimated on the
basis of Griess–Illosvoy reaction [16]. In this investigation,
Griess–Illosvoy reagent was modified by using naphthyl
123
Cupric reducing antioxidant capacity (CUPRAC)
Cupric reducing antioxidant capacity of the plant extracts was
determined following the method described by Resat et al.
[18]. 500 ll of each fraction and standard (ascorbic acid) in
different concentrations were taken in test tubes. 1.0 ml of
0.01 M CuCl22H2O solution and 1.0 ml of ammonium acetate buffer (pH 7.0) was added into the test tubes. Then 1.0 ml
of 0.0075 M of neocaproine solution and 600 ll of distilled
water was added into the test tubes. The total mixture was
incubated for 1 h at room temperature then the absorbance of
the solution was measured at 450 nm using a spectrophotometer (Shimadzu UV PC-1600) against blank. The molar
absorptivity of the CUPRAC method for each antioxidant was
found from the slope of the calibration line concerned.
Total alkaloid content determination
Total alkaloid content was determined by slightly modified
Fazel et al. [19] method. 4 mg of each plant extract was
dissolved in 4 ml 2 N HCl to get the concentration 1 mg/
ml. Then 5 times dilution was done by adding 1 ml of this
solution into 4 ml 2 N HCl to get the concentration of
stock solution 200 lg/ml. 1 ml stock solution of each plant
extract, 5 ml phosphate buffer and 5 ml BCG (Bromocresol green) solution taken in a separating funnel, shaken
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Evaluation of in vitro antioxidant and brine shrimp lethality activities of different stem…
well and 5 ml CHCl3 was added. The funnel was shaken
vigorously and the lower layer of the solution was collected
in a labeled test tube. The absorbance of the complex in
chloroform was measured at 470 nm. A typical blank solution contained the same solution mixture without plant
extract or standard. Total alkaloid content is expressed as
the number of equivalents of atropine (AE).
457
were performed to analyze the data set using Graph pad
prism version 5.00 (Graph Pad Software Inc., San Diego,
CA, USA) and SPSS version 16 (IBM software Inc, USA).
Results
Phytochemical group evaluation test
Total tannin content determination
The tannins were determined by slightly modified Folin
and Ciocalteu method [11]. 0.1 ml of the sample extract
(200 lg/ml) was added with 7.5 ml of distilled water.
0.5 ml of Folin Phenol reagent (diluted 10 fold) solution
was added into the test tubes. 1 ml of 35 % sodium carbonate solution was added into the test tubes. The volume
was adjusted up to 10 ml with distilled water. The mixture
was shaken well, kept at room temperature for 30 min. The
absorbance was measured at 725 nm. A typical blank was
prepared with water instead of the sample. A set of standard solutions of Tannic acid is treated in the same manner
as described earlier and read against a blank. The results of
tannins are expressed in terms of tannic acid in mg/g TAE
of extract.
Brine shrimp lethality bioassay (BSLB)
Lethal activity of the plant extract was determined by Brine
shrimp lethality bioassay described by Meyer et al. [20].
Brine shrimp lethality bioassay [20–22] is a rapid and
comprehensive bioassay for the bioactive compounds of
natural and synthetic origin. Brine shrimp eggs (Artemia
salina leach) are hatched in simulated seawater to get
nauplii. Sample solutions are prepared by dissolving the
test materials in pre-calculated amount of DMSO (dimethyl sulphoxide). Ten nauplii are taken in vials containing simulated seawater. The samples of different concentrations are added and the volume was adjusted up to
5 ml. Survivors were counted after 24 h. Vincristine sulphate is usually used as the reference cytotoxic drug. The
mortality was corrected using Abott’s formula [23]
Pt ¼ ½ðPo
Pc Þ=ð100
Pc Þ 100;
where, Po = observed mortality and Pc = control mortality. LC50 values of the test samples after 24 h are obtained by regression analysis.
Statistical analysis
Values were presented as mean ± SD (standard deviation).
IC50 and LC50 values were obtained with the help of Microsoft excel 2007. Pearson correlation analysis and one
way ANOVA followed by Tukey multiple comparison
In the present study, various qualitative tests were done to
detect the presence of various phytochemical compounds
in the petroleum ether, ethyl acetate, ethanol, methanol and
water extracts of the stem or branches part of Z. rugosa
Lam. Preliminary phytochemical screening of the crude
extracts of different parts of Z. rugosa Lam showed the
presence of different kind of phytochemical groups that can
be summarized in Table 1.
Antioxidant activity evaluation
Total phenol content determination
The total phenolic contents of the test fractions were calculated using the standard curve of Gallic acid and were
expressed as GAE per gram of plant extract. ZSEA was
found to contain the highest amount of phenols
(97.188 ± 12.816 mg/g) and ZSE showed the lowest
amount (32.500 ± 10.607 mg/g). Phenol contents of the
extracts were found to decrease in the following order:
ZSEA [ ZSPE [ ZSM [ ZSW [ ZSE (Table 2).
Total flavonoid content determination
The total flavonoid content was calculated using the standard
curve of Quercetin. Flavonoid contents of the test fractions
were found as mg/g Quercetin equivalent (QE). ZSEA was
found to contain the highest amount of Flavonoid
(15.009 ± 0.385 mg/g) and ZSE was found to contain the
lowest amount of flavonoid (0.293 ± 0.003 mg/g). Flavonoid contents of the extracts were found to decrease in the
following order: ZSEA [ ZSPE [ ZSM [ ZSW [ ZSE
(Table 2).
Total antioxidant capacity assessment
Total antioxidant capacity of Z. rugosa Lam stem extract was
evaluated by the phosphomolybdenum method and was expressed as AAE per gram of plant extract. ZSM was found to
contain the highest (37.500 ± 3.712 mg/g) and ZSPE was
found to contain the lowest (16.875 ± 4.773 mg/g) amount
of total antioxidant capacity. Total antioxidant capacities of
123
Author's personal copy
–
?
?
–
–
–
absence
–
?
ZSEA
strong presence,
–
?
Presence,
?
ZSW
ZSM
ZSE
??
?
?
?
?
-
?
–
ZSPE
ZSPE Z. rugosa Lam, petroleum ether extract of stem part, ZSEA Z. rugosa Lam, ethyl acetate extract of stem part, ZSE Z. rugosa Lam, ethanol extract of stem part, ZSM Z. rugosa Lam,
methanol extract of stem part, ZSW Z. rugosa Lam, water extract of stem part
?
–
?
?
–
?
?
??
?
?
?
?
–
–
?
–
?
??
?
?
–
?
?
–
–
–
?
?
–
?
?
?
?
?
–
?
?
–
–
?
Glucoside
Fehling’s test
Molisch’s test
Glycoside
–
?
Dragendorff’s test
Wagner test
Mayer’s test
Hager’s test
–
Tannins
Steroids
Alkaloids
Flavonoids
Glycosides
Carbohydrates
Extracts
Table 1 Result of chemical group test of stem extracts of Z. rugosa Lam
123
?
Md. S. Hossain et al.
Saponins
458
the extracts were found to decrease in the following order:
ZSM [ ZSW [ ZSEA [ ZSE [ ZSPE (Table 2).
DPPH free radical scavenging assay
DPPH scavenging assay has been widely used to evaluate the
free radical scavenging capacity of antioxidant. The IC50
values of different extracts of Z. rugosa Lam are summarized
in the Table 3. ZSE was found to contain the lowest IC50 value
(83.550 lg/ml) that represents the highest scavenging property than the other tested extracts. The IC50 value of standard
drug ascorbic acid was 18.348 lg/ml. DPPH free radical
scavenging activity of the plant extracts of Z. rugosa Lam was
found to decrease in the following order: ZSE [ ZSM [
ZSW [ ZSEA [ ZSPE.
Nitric oxide (NO) scavenging capacity assay
Different extracts of the stem part exhibited dose dependent scavenging of nitric oxide. Maximum scavenging of
NO was found with ZSW with an IC50 value of 5.975 lg/
ml; the result was similar to standard ascorbic acid (IC50
value 5.934 lg/ml) can be summarized in the Table 3. NO
scavenging activity decreased in the following order;
ZSW [ ZSM [ ZSE [ ZSPE [ ZSEA that are summarized in the Table 3.
Reducing power capacity assessment
Reducing power of the tested extracts were assessed using
ferric to ferrous reduction activity as determined spectrophotometrically from the formation of Perl’s Prussian
blue color complex [24]. The standard ascorbic acid
showed the highest reducing capacity. The stem extracts of
Z. rugosa Lam showed reducing capacity. ZSM was found
to contain more reducing power than other extracts (correlation coefficient r = 0.99 and P \ 0.001) presented in
Fig. 1.
Cupric reducing antioxidant capacity (CUPRAC)
Reduction of Cu2? ion to Cu? was found to rise with
increasing concentrations of the different extracts. The
standard ascorbic acid showed the highest reducing capacity in CUPRAC method. Comparison graph of cupric
reducing antioxidant capacity (CUPRAC) between ascorbic acid and Z. rugosa Lam stem extracts are given in
Fig. 1.
Total alkaloid content determination
Total Alkaloid content of Z. rugosa Lam was determined by
Dragendroff’s method and was expressed as atropine
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Evaluation of in vitro antioxidant and brine shrimp lethality activities of different stem…
459
Table 2 Summarized anti-oxidant results of stem extracts of Z. rugosa Lam
Extract
Total phenol
(mg/g GAE)
ZSPE
78.125 ± 12.374b
12.136 ± 0.252c
ZSEA
97.188 ± 12.816
b
d
15.009 ± 0.385
32.500 ± 10.607
a
a
ZSM
76.250 ± 10.607
b
ZSW
b
ZSE
Total flavonoid
(mg/g QE)
0.293 ± 0.003
b
3.651 ± 0.135
a,b
78.125 ± 4.419
1.271 ± 0.041
Total tannin
(mg/g TAE)
Total antioxidant
(mg/g AAE)
Total alkaloid
(mg/g AE)
16.875 ± 4.773a
28.694 ± 0.005a
a,b
21.938 ± 5.038
87.600 ± 0.044b
a
154.622 ± 0.022c
a
57.596 ± 0.015a
a
221.026 ± 0.050d
a
63.291 ± 0.005a
24.062 ± 0.001
b
24.375 ± 1.061
30.705 ± 0.004
c
37.500 ± 3.712
25.747 ± 0.004
c
35.250 ± 5.834
38.135 ± 0.003
Values are presented as mean ± SD (n = 3). One way ANOVA followed by Tukey multiple comparisons was performed to analyze the data
sets. Values in same column with different superscripts are significantly different from one another (P \ 0.05)
Table 3 IC50 and LC50 values of the different extracts in DPPH, NO and Brine shrimp lethality bioassays
Extract
DPPH free radical
scavenging assay, IC50 (lg/ml)
Nitric oxide scavenging
capacity assay, IC50 (lg/ml)
Brine shrimp lethality
bioassay (BSLB) LC50 (lg/ml)
ZSPE
474,147
15.661
134.4
ZSEA
312.210
31.634
385.38
ZSE
83.550
8.945
535.39
ZSM
87.298
7.228
40.43
ZSW
282.430
5.975
235.11
Ascorbic acid
18,348
5.934
–
Vincristine sulphate
–
–
2.48
B
1.0
Ascorbic acid
ZSPE
ZSEA
ZSE
ZSM
ZSW
Absorbance
0.8
0.6
0.4
2.5
Ascorbic acid
ZSPE
ZSEA
ZSE
ZSM
ZSW
2.0
Absorbance
A
1.5
1.0
0.5
0.2
0.0
0.0
0
50
100
150
200
(Concentration µg/ml)
0
50
100
150
200
250
(Concentration µg/ml)
Fig. 1 a Cuprac reducing assessment of five different stem extracts
and standard. Values are presented as mean ± SEM (n = 3). Pearson
correlation analysis was performed between different concentrations
and absorbance of each stem extract and standard. For ZSPE,
correlation coefficient r = 0.98 and P \ 0.01; for ZSEA, r = 0.98
and P \ 0.01; ZSE, correlation coefficient r = 0.99 and P \ 0.0001;
for ZSM, correlation coefficient r = 0.98 and P \ 0.01; for ZSW,
correlation coefficient r = 0.95 and P \ 0.05; for ascorbic acid,
r = 0.999 and P \ 0.001. b Reducing power capacity assessment of
five different stem extracts and standard. Values are presented as
mean ± SEM (n = 3). Pearson correlation analysis was performed
between different concentrations and absorbance of each stem extract
and standard. For ZSPE, correlation coefficient r = 0.97 and
P \ 0.01; for ZSEA, r = 0.98 and P \ 0.01; ZSE, correlation
coefficient r = 0.99 and P \ 0.001; for ZSM, correlation coefficient
r = 0.99 and P \ 0.001; for ZSW, correlation coefficient r = 0.93
and P \ 0.001; for ascorbic acid, r = 0.98 and P \ 0.05
equivalents (AE) mg/g of plant extract. ZSW was found to
contain the highest amount of Alkaloid (38.135 ± 0.003 mg/g)
and ZSEA was found to contain the lowest amount of alkaloid (24.062 ± 0.001 mg/g). Alkaloid contents of five
different extracts did not find significantly (P [ 0.05).
Alkaloid contents of the extracts were found to decrease
in the following order; ZSW [ ZSE [ ZSPE [ ZSM [
ZSEA (Table 2).
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460
Total tannin content determination
Total Tannin content of Z. rugosa Lam was determined by
slightly modified Folin and Ciocalteu method [11] and was
expressed as tannic acid equivalent (TAE) mg/g of plant
extract. ZSM was found to contain the highest amount of
total tannin that can be expressed as tannic acid equivalent
(221.026 ± 0.050 mg/g). Tannin contents of the extracts
were found to decrease in the following order;
ZSM [ ZSEA [ ZSPE [ ZSW [ ZSE (Table 2).
Brine shrimp lethality bioassay (BSLB)
In this study methanol extract of stem part (ZSM) showed
most toxicity to brine shrimp nauplii, with LC50 value of
40.43 lg/ml whereas anticancer drug Vincristine sulphate
showed LC50 value 2.48 lg/ml described in the Table 3.
The order at which cytotoxic potential of the test samples
decreased was as follows: ZSM [ ZSPE [ ZSW [
ZSEA [ ZSE.
Discussion
Several phytochemical study reported that cyclopeptide
alkaloids [25, 26] and six flavones glycosides [27] and one
saponine were [28] isolated from root barks of Z. rugosa. In
continuation of this study, we have extracted this plant with
different solvents and a preliminary screening showed that
ZSPE, ZSEA and ZSM were rich in flavonoids. It can be
assumed that Z. rugosa Lam stem are rich in alkaloids,
flavonoids, tannins, carbohydrates, steroids, saponin which
may account for their various pharmacological activities.
Antioxidative properties of polyphenols arise from their
high reactivity as hydrogen or electron donors which can
stabilize and delocalize the unpaired electron (chainbreaking function) and from their potential to chelate metal
ions (termination of the Fenton reaction) [29]. Polyphenols
have been shown to block LDL oxidation, decrease the
formation of atherosclerotic plaques and reduce arterial
stiffness, leaving arteries more responsive to endogenous
stimuli of vasodilation [30–32]. This ability is believed to
be mainly due to their redox properties [33], which play an
important role in adsorbing and neutralizing free radicals.
The results strongly suggest that phenolics are important
components of the tested plant extracts Z. rugosa Lam stem
extract showed good to moderate amount of phenolic
content which is showed in gallic acid equivalent. Flavonoids play an important role in antioxidant system in
plants. The antioxidative properties of flavonoids are due to
several different mechanisms, such as scavenging of free
radicals, chelation of metal ions, such as iron and copper,
and inhibition of enzymes responsible for free radical
123
Md. S. Hossain et al.
generation [34]. Depending on their structure, flavonoids
are able to scavenge practically all known ROS. The stem
extract of Z. rugosa Lam has been shown to possess mild to
moderate amount of flavonoids. The total antioxidant activity of stem extracts of Z. rugosa Lam was evaluated
from their ability to reduce phosphate/Mo(VI) complex to
phosphate/Mo(V). According to recent reports, a highly
positive relationship between total phenols and antioxidant
activity appears to be the trend in many plant species [35].
Z. rugosa Lam stem extracts showed moderate content
which is showed in ascorbic acid equivalent. DPPH radical
scavenging is a popular and reliable method for screening
the free radical scavenging activity of compounds or antioxidant capacity of plant extracts [12, 36–38]. In DPPH
radical scavenging assays, the stem extracts of Z. rugosa
Lam showed good to moderate scavenging of DPPH
radicals in a way similar to that of the reference antioxidant
ascorbic acid. NO is generated in biological tissues by
specific nitric oxide syntheses (NOSs), which metabolizes
arginine to citrulline with the formation of NO via a five
electron oxidative reaction [39]. The toxicity and damage
caused by NO- and O2- is multiplied as they react to
produce reactive peroxynitrite (ONOO-), which leads to
serious toxic reactions with biomolecules as mentioned
above [40–42]. Our findings indicate that all of the five
plant extracts showed good NO scavenging activity. The
reducing power of the extracts of the selected plants might
be due to its hydrogen donating ability [43]. The antioxidant activity has been reported to be concomitant with
the development of reducing power [44]. Among the extracts ZSM exhibited the most reducing power in reducing
power capacity assessment. Reducing power is associated
with antioxidant activity and may serve as a significant
reflection of the antioxidant activity [35]. The standard
ascorbic acid showed the highest reducing capacity. The
stem extracts of Z. rugosa Lam showed reducing capacity.
It also showed to possess moderate amount of Alkaloids
which were also identified by phytochemical group
evaluation test. Moreover, the extracts also showed to
possess good to moderate amount of tannin content. These
phytochemicals may strengthen the antioxidant potential of
the stem.
The brine shrimp lethality bioassay has been considered
as a practical, safe and economic method for determination
of bioactivities of synthetic compound as well as plant
products [20]. In Brine Shrimp lethality bioassay all of the
samples showed considerable lethality. The stem extracts
of Z. rugosa Lam has showed good cytotoxic potentiality.
The toxicity of plants is principally contributed by the
presence of alkaloids, glycosides, steroids, tannins, phlobatannins, terpenoids and flavonoids [45–48]. However,
phenolics and flavonoids are also known to show cytotoxicity in Hoechst 33258 fluorescence assay by inhibiting
Author's personal copy
Evaluation of in vitro antioxidant and brine shrimp lethality activities of different stem…
cellular DNA in a concentration-dependent manner [49].
Different phytochemical group like alkaloids, glycosides,
steroids, tannins and flavonoids were found in our observation (Table 1). So the observed cytotoxic action may be
due to the presence of these phytochemical constituents,
although further studies are required to be confirmed.
Conclusion
The results from the experiments confirmed that the stem
extract of Z. rugosa Lam in general, possesses good to
moderate antioxidant and good brine shrimp lethality properties. Various phytochemical constituents like terpenoids,
glycosides and steroids present in the plant, which may be
responsible for the observed activities. The good toxicity
exerted by the extracts of Z. rugosa Lam in brine shrimp
lethality bioassay suggests the presence of bioactive principles in the plant. However, further studies are suggested to be
undertaken to understand the underlying mechanism of the
observed activities and to isolate, purify and characterize
active phytochemical compounds that are responsible for
these bioactivities in animal models and to carry out advance
research on cancer because the other plants of Rhamnaceae
family have significant cytotoxic and inhibitory activity on
tumors of various types of cancers. Besides, these extracts
can be used as promising source of natural antioxidants
which can work as stabilizing agent against oxidative deterioration in pharmaceutical applications.
Acknowledgments We are greatly thankful to Laboratory of Natural Products Research (LNPR), Department of Pharmacy, Jahangirnagar University to provide sufficient laboratory facilities to carry out
this research.
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