- Email: [email protected]
Antioxidant activity and DNA damage inhibition in vitro by a methanolic extract of Carissa carandas (Apocynaceae) leaves
Accepted Manuscript Title: In vitro antioxidant and DNA damage inhibition activity of methanolic extract of Carissa carandas (Apocynaceae) leaves Author: Khushbu Verma Divya Shrivastava Gaurav Kumar PII: DOI: Reference:
S1658-3655(14)00067-3 http://dx.doi.org/doi:10.1016/j.jtusci.2014.07.001 JTUSCI 87
To appear in: Received date: Revised date: Accepted date:
16-2-2014 1-7-2014 1-7-2014
Please cite this article as: K. Verma, D. Shrivastava, G. Kumar, In vitro antioxidant and DNA damage inhibition activity of methanolic extract of Carissa carandas (Apocynaceae) leaves, Journal of Taibah University for Science (2014), http://dx.doi.org/10.1016/j.jtusci.2014.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro antioxidant and DNA damage inhibition activity of methanolic extract of Carissa carandas (Apocynaceae) leaves
ip t
Khushbu Verma, Divya Shrivastava, Gaurav Kumar*
Ac ce p
te
d
M
an
us
cr
School of Life Sciences, Jaipur National University, Jaipur- 302025, Rajasthan, India
*Corresponding author Dr. Gaurav Kumar
Assistant Professor, School of Life Sciences, Jaipur National University, Jaipur- 302025, Rajasthan, India M: +91 98287 48869; Email: [email protected]
Page 1 of 25
Abstract The aim of this study was to investigate the antioxidant and DNA damage inhibition potential of methanolic extract of Carissa carandas leaves. Extract was found to exhibit significant
ip t
(p<0.05) dose dependent DPPH radical scavenging activity (IC50 value= 73.12µg/ml), total antioxidant activity, H2O2 scavenging activity (IC50 value 84.03µg/ml), reducing power
cr
activity. In addition, extract was found to exhibit complete protection of pBR322 plasmid DNA from free radicals mediated oxidative stress during DNA damage inhibition assay.
us
Antioxidant and DNA damage inhibition properties of C. carandas could be attributed to the
an
presence of high amount of phenolic compounds (84.00 mg GAE/gm dry weight of the extract) in the extract which was estimated by Folin-Ciocalteau assay. These observations
M
emphasize the high antioxidant and DNA damage inhibition potential of the C. carandas which can be further used to develop natural antioxidant compounds for therapeutic
te
d
applications.
Ac ce p
Keywords: Carissa carandas, antioxidant activity, DNA damage inhibition potential
Page 2 of 25
1. Introduction Free radicals are commonly known as reactive oxygen species (ROS), contains one or more unpaired electrons in their outer most orbital. Some of the common examples of ROS are
ip t
peroxyl radical (ROO-), superoxide anion (O2-), peroxyl radical (ROO•), ozone (O3), hydrogen peroxide (H2O2) and reactive hydroxyl radical (OH-) [1]. Free radicals are highly
cr
unstable, therefore they react with other molecules in their vicinity to obtain stability, and this initiates a chain reaction of free radical generation [1]. Excessive production of free radicals
us
results in the depletion of in vivo antioxidants and causes imbalance between free radicals and
an
antioxidant defence of body, therefore causing serious damage (oxidative stress). Almost 100 different serious diseases are known to associate with oxidative stress viz, cardiovascular
M
diseases, liver diseases, diabetes, cancer, neurodegenerative disease and aging [2, 3]. Antioxidants are usually reducing agents such as vitamins, carotenoids, polyphenols
d
and trace metals (Zn, Mo) which can effectively scavenge ROS and inhibit the chain reaction
te
by donating the electron to free radical. The antioxidant defence system of body, in support with dietary antioxidants protects the body from free radicals. However during oxidative
Ac ce p
stress, in vivo antioxidants are not sufficient to maintain homeostasis therefore; antioxidants could be given as supplements to the body. Consumption of additional antioxidant can significantly reduce the chances of getting free radicals associated diseases [4]. In recent literature, plants are regarded as a potential source of natural antioxidant
compounds [5-11]. Medicinal plants provide a safe, cost effective and eco friendly alternative to the chemical antioxidants which could be toxic on prolong exposure [12]. Hence in this study C. carandas Linn. leaves were screened for their antioxidant properties. C. carandas is a flowering armed shrub belonging to family Apocyneceae. The species is native to India and distributed in Sri Lanka, Indonesia, Malaysia, Myanmar and Pakistan. It grows throughout India and commonly known as Christ thorn and Karanda. It is dichotomously branched and
Page 3 of 25
contains milky latex. Leaves are glossy and dark green and light green on dorsal and ventral side respectively. Leaves are elliptic-oblong, notched at the base and tip is blunt. Flowers are white to rosy pink in color and possess a characteristic fragrance. It produce small, oval shape
ip t
fruits which are initially green in colour and turned to reddish purple when ripen [13]. Several medicinal and nutritional properties of C. carandas establish it as an
cr
economically important plant in the country. In India, C. carandas is traditionally used for the treatment of flatulence, poor digestion, acidity and wounds. Roots are used as
us
anthelmintic, stomachic and antiscorbutic agents and for the treatment of intestinal worm,
an
scabies and pruritus. It is also used as a medicine for high blood pressure due to its blood pressure decreasing property. Fruits of C. carandas is used for the treatments of malaria,
M
epilepsy, nerve disorder, relieve of body ache, fever, blood purifier, myopathic spams, dog bite, cough, colds, itches and leprosy. Ripen fruit of C. carandas are used for making
d
puddings, curries and pickles, whereas slightly under ripen fruits are used to make jelly [13-
te
15]. C. carandas has been extensively studied for its pharmacological properties and reported to show anticonvulsant [16], analgesic, anti-inflammatory and antipyretic [17], antibacterial
Ac ce p
and antifungal [18, 19], hepatoprotective [20], antioxidant [21], acute hypotensive [22] and anti-cancerous activities [23]. Most of these pharmacological studies have been conducted with roots, fruits and bark, however leaves are relatively less explored, therefore this study was designed to investigate phytochemical composition and antioxidant properties of C. carandas. In addition, C. carandas was first time evaluated for its DNA damage inhibition properties against free radical mediated DNA damage.
Page 4 of 25
2. Materials and Methods 2.1. Chemicals Sulphuric acid (H2SO4), Methanol, Ferric chloride (FeCl3), Potassium Ferricyanide (K3Fe
ip t
(CN)6), Trichloroacetic acid, Ascorbic acid, Hydrogen peroxide (H2O2), Sodium chloride (NaCl), Folin- Ciocalteau reagent and Gallic acid were purchased from SRL Pvt. Ltd.
cr
(Mumbai, India). 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Sodium carbonate (Na2CO3) was purchased from
us
Himedia Laboratories Pvt. Ltd. (Mumbai, India). All other chemicals used were of analytical
an
grade.
M
2.2. Plant Material
The leaves of C. carandas were collected from naturally growing population in the village
d
Jaitpura, Jaipur, Rajasthan, India during January 2014. The plant material was carried to the
te
Research Laboratory, Jaipur National University, Jaipur, Rajasthan, India. A voucher specimen was maintained in our laboratory for future reference (Accession number: JNU-
Ac ce p
JPR/RL/ CC/19.01.2014-1).
2.3. Plant processing
The collected leaves (100 gm) of C. carandas were washed thoroughly with sterilized distilled water to remove soil particles then fresh weight of leaves was observed. The leaves were oven dried at 50°C. Dried leaves were pulverized using the mechanical grinder to make delicate powder. The leave powder (10 gm) was soaked in methanol (10% w/v) and loaded on a shaker at a speed of 120 rpm for 24 hour at room temperature. Mixtures were filtered by using Whatman number 1 filter paper. The filtrate was concentrated at 40°C under reduced
Page 5 of 25
pressure (72 mbar) with a rotary evaporator and dried using lyophilizer. Dried extract was collected in an air tight receptacle and stored at room temperature for further use.
ip t
2.4. Phytochemical screening Phytochemical screening of the leaves of C. carandas was carried out by using the standard
cr
protocols [24]. The leaf extract was screened for the presence of total carbohydrates, reducing sugars, protein, alkaloids, phenolic compounds, flavonoids, tannins, glycosides, saponins, oil
2.5. Antioxidant activity
M
2.5.1. DPPH radical scavenging activity
an
us
and fats.
The extract was diluted in methanol to make 25, 50, 75 and 100 µg/ml dilutions. Two
d
milliliter of each dilution were mixed with 1 ml of DPPH solution (0.2 mM/mL in methanol)
te
and mixed thoroughly. The mixture was incubated in the dark at 20°C for 40 min. Absorbance was measured at 517 nm using UV-Vis spectrophotometer with methanol as
Ac ce p
blank. Ascorbic acid was used as experimental control. Experiment was performed in triplicates at each concentration [25]. The percentage scavenging of DPPH by the extracts was calculated according to the following formula:
Percentage DPPH radical scavenging= [(Ac - At) / Ac] × 100 Here,
Ac is the absorbance of the control (DPPH) At is the absorbance of test sample and standards
Page 6 of 25
2.5.2. Determination of total antioxidant activity A volume of 1 ml of the extract (125, 250, 500 and 1000 µg/ml) was mixed with 3 ml of the reaction mixture (containing 10 ml of concentrated H2SO4, 1.005 g of sodium phosphate
ip t
monobasic and 1.47 g of ammonium molybdate which was dissolved in 290 ml of water). The mixture was kept in water bath for one hour at 95º C. The solution containing 3 ml of
cr
reaction mixture and 1 ml of distilled water was used as blank and the absorbance was measured using UV-Vis spectrophotometer at 695 nm. Ascorbic acid was used as
an
us
experimental control. Experiment was performed in triplicates at each concentration [26].
2.5.3 Scavenging of hydrogen peroxide
M
The extract was diluted to make 25, 50, 75 and 100 µg/ml dilutions. Two millilitre of each dilution was mixed with 1.2 ml of hydrogen peroxide solution (40 mM in phosphate buffer,
d
pH 7.4) and incubated for 10 min. The absorbance of the solution was taken at 230 nm using
te
a UV–Visible spectrophotometer against blank solution containing the plant extract without H2O2. Ascorbic acid was used as experimental control. Experiment was performed in
Ac ce p
triplicates at each concentration [27].
The percentage scavenging of hydrogen peroxide of the extracts was calculated using the following equation:
% Scavenging of H2O2 = [(Ac – As)/Ac] × 100 Here,
Ac is the absorbance of the control
As is the absorbance in the presence of the sample and standards
Page 7 of 25
2.5.4. Reducing power activity The extract (1 ml) at different concentrations (125, 250, 500 and 1000 µg/ml) was mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% K3Fe(CN)6. The mixture was
ip t
incubated at 50°C for 20 min. A volume of 2.5 ml of Trichloroacetic acid (10%) was added to the mixture, and was centrifuged at 3000 rpm for 10 min in a cooling centrifuge. 2.5 ml of the
cr
supernatant was mixed with equal volume of distilled water and 0.5 ml Ferric chloride (0.1%). Absorbance was measured at 700 nm using a UV–Visible spectrophotometer.
us
Ascorbic acid was used as positive control. Experiment was performed in triplicates at each
an
concentration [28].
M
2.6. DNA damage inhibition efficiency
DNA damage inhibition by methanolic extract of leaves of C. carandas was tested by
d
photolysing H2O2 by UV radiation in the presence of pBR322 plasmid DNA and performing
te
agarose gel electrophoresis with the irradiated DNA [29]. A total of 1 µl aliquots of pBR322 plasmid DNA were taken in three microcentrifuge tubes. A quantity of 50 µg of extract was
Ac ce p
added to one tube. The remaining tubes were left untreated as the irradiated controls. An amount of 4 µl of 3% H2O2 was added to all the tubes, which were then placed directly on the surface of a UV transilluminator (300 nm) for 10 min at room temperature. After irradiation, 4 µl of tracking dye (0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol) was added. The samples in all the tubes were analyzed by gel electrophoresis on a 1% agarose gel (containing ethidium bromide) in TAE buffer (pH 8). Untreated non-irradiated DNA (C) was run along with untreated UV-irradiated DNA (R) and extract treated UVirradiated sample (S).
Page 8 of 25
2.7. Estimation of total phenolic content Total phenolic content of the methanolic extract of leaves of C. carandas was determined using the Folin-Ciocalteaureagent method [30]. The crude extract was diluted in distilled
ip t
water to obtain different concentrations (125, 250, 500 and 1000 µg). Extract (50 µL) was mixed with 2.5 ml of Folin-Ciocalteau reagent (1/10 dilution in purified water) and 2 ml of
cr
7.5% Na2CO3 (w/v in purified water). The mixture was incubated at 45°C for 15 min. The absorbance was measured at 765 nm. Na2CO3 solution (2 ml of 7.5% Na2CO3 in 2.55 ml of
us
distilled water) was used as blank. The results were expressed as gallic acid equivalence
an
(GAE) in µg. Each experiment was performed in triplicates at each concentration.
M
2.8. Statistical analysis
The results of DPPH radical scavenging activity, total antioxidant activity, Hydrogen
d
peroxide scavenging activity, reducing power activity and total phenolic content of the
te
methanolic extract of C. carandas leaves are expressed as mean ± standard deviation of the response of three replicates per sample. Statistical significance between the groups was
Ac ce p
analyzed by one-way ANOVA coupled with Tukey's post-hoc test at P<0.05. Statistical analysis was performed by using Microsoft Excel 2007 and GraphPad Prism 5.
3. Results and Discussion 3.1. Percentage yield
Hundred grams of fresh leaves of C. carandas were dried and the dried powder was extracted in water. The percentage yield of the plant extract was estimated with respect to fresh mass and dry weight. Extraction of C. carandas leaves with methanol resulted in 7.85% and 17.98% yield with respect to fresh mass and dry weight respectively.
Page 9 of 25
3.2. Phytochemical analysis The result of preliminary phytochemical analysis of the methanolic extract of C. carandas leaves showed the presence of total carbohydrates, proteins, phenolic compounds, flavonoids,
ip t
tannins, saponins, oil and fats, alkaloids and cardiac glycosides, whereas reducing sugars were found to be absent (Table 1). Results of phytochemical analysis are in agreement with
cr
the previous reports where C. carandas leaves have been reported to possess alkaloids, flavonoids, saponins, cardiac glycosides, triterpenoids, phenolic compounds and tannins [16].
us
Other studies have also reported the presence of steroids, flavonoids, tannins, phenolic
an
compound, terpenoids, saponins, alkaloid and glycosides as major phytochemical groups [17]. Presence of phenolic compounds and flavonoids enhance the chances of getting high
M
antioxidant activity in the extract as these compounds from plants and vegetable are
te
3.3. Antioxidant activity
d
extensively reported to show antioxidant properties.
In last few decades, C. carandas has been extensively studied for its pharmacological studies,
Ac ce p
however a very few studies have reported the antioxidant potential. Various solvent extracts prepared from the C. carandas roots have been reported to demonstrate significant dose dependent antioxidant activity in various antioxidant methods [21]. In one other study, aqueous extracts of C. carandas leaves has been reported to show significant antioxidant activity in MCF 7 cell lines (breast cancer cell line). Extract treatment was found to increase the levels of in vivo antioxidants viz, catalase, superoxide dismutase, glutathione transferase and glutathione on MCF-7 cancer lines [23]. In this study, methanolic extract of C. carandas was screened for its antioxidant properties by various in vitro methods viz, DPPH radical scavenging activity, total antioxidant activity, H2O2 scavenging activity and reducing power activity.
Page 10 of 25
3.3.1. DPPH radical Scavenging activity DPPH is a comparatively stable free radical, contains unpaired electron on its nitrogen molecule. DPPH gives a characteristic purple color in methanol which is reduced to yellow
ip t
after receiving a proton from an antioxidant compound. This reduction in the color of DPPH can be measured by using a UV Visible spectrophotometer (517 nm). In this study,
cr
methanolic extract of C. carandas leaves was evaluated for its DPPH radical scavenging activity in a dose dependent manner. Extract was found to exhibit significant (p<0.05) dose
us
dependent DPPH radical scavenging activity with IC50 value= 73.12µg/ml. The results are
an
represented as percentage inhibition of DPPH and expressed as mean ± standard deviation (n= 3). The DPPH radical scavenging activity of the extract was compared with the ascorbic
M
acid and reported in Figure 1.
d
3.3.2. Total antioxidant activity
te
The total antioxidant test is based on the reduction of molybdate (VI) to molybdate (V) at acidic pH by the extract and as well as the formation of green phosphate complex, which
Ac ce p
could be quantified spectrophotometrically at 695 nm. In the current study, methanolic extract of C. carandas leaves was evaluated for total antioxidant activity in a dose dependent manner. The extract showed significant (p<0.05) dose dependant increase in total antioxidant activity with an OD value of 1.39±0.21 (at 1000 µg/ml). The total antioxidant activity of the extract was compared with the ascorbic acid and reported in Figure 2.
3.3.3. H2O2 scavenging activity Hydrogen peroxide radical scavenging activity is a very useful method for the assessment of antioxidant property of the samples. Hydrogen peroxide is usually none reactive to cell but it may give rise to the hydroxyl radicals, therefore the compounds with can donate electron to
Page 11 of 25
hydrogen peroxide could reduce hydrogen peroxide in to water and thus blocks the generation of hydroxyl radicals and protect the cell. Electron donation potential of the sample is directly proportional to its antioxidant potential. In the present study, methanolic extract of
ip t
C. carandas leaves was evaluated for hydrogen peroxide radical scavenging activity. The extract exhibited significant (p<0.05) hydrogen peroxide scavenging activity in a dose
cr
dependent manner with IC50 value 84.03µg/ml. Hydrogen peroxide radical scavenging
us
activity of the extracts was compared with the ascorbic acid and reported in Figure 3.
an
3.3.4. Reducing power activity
Reducing power activity is based on the reduction of Fe (III) to Fe (II) by the electron-
to
bluish
green
color
in
the
M
donating potential of the compounds. Reduction of Fe (III) to Fe (II) ions produces a yellow reaction
mixture,
which
could
be
measured
d
spectrophotometrically at 700 nm. In the current study, methanolic extract of C. carandas
te
leaves was evaluated for reducing power activity. The extract showed significant (p<0.05) dose dependant increase in reducing power activity with an OD value of 1.73±0.04 at 1000
Ac ce p
µg/ml. The result for reducing power activity of methanolic extract of C. carandas leaves in comparison to ascorbic acid is reported in Figure 4.
3.4. DNA damage inhibition activity Free radicals are capable of both initiating and increasing several types of disease. Free radicals such as hydroxyl free radicals are well known to damage cellular DNA in humans. Partial damage in DNA may turn the cell in the cancerous cell which is a very severe health problem in humans. Plants with DNA damage inhibition potential could be used to protect the DNA from the free radicals mediated damage. Therefore in this study, DNA damage inhibition efficiency of the methanolic extract of C. carandas leaves was evaluated and
Page 12 of 25
reported in Figure 5, it shows the electrophoretic pattern of pBR322 DNA following UVphotolysis of H2O2 in absence (in controls C and R) and presence (samples S) of the extract. Control pBR322 (C) showed the bands of super coiled and open circular plasmid DNA in
ip t
agarose gel electrophoresis. UV-photolysis of H2O2 (R) damaged the entire DNA (no bands visible). The extract displayed considerable protective activity and showed the bands of super
cr
coiled and open circular plasmid DNA. The results infer that UV-photolysed H2O2 (3%) treatment of pBR322 obliterated the entire DNA (in R), while 50 µg of methanolic extract of
us
C. carandas leaves protected against the free radical mediated DNA damage (S). The results
an
of this experiment demonstrate the DNA damage inhibition potential of the methanolic extract of C. carandas leaves and provides a hope to use it in future as a cancer prevention
M
medicine. Earlier also some plants have been reported to exhibit DNA protective efficacy
te
3.5. Total phenolic content
d
towards free radical mediated DNA damage [11, 29].
Phenolic compounds are unarguably the major group of phytochemicals with antioxidant
Ac ce p
properties in plants. Phenolic compounds are produced as secondary metabolite in plant system to protect themselves from oxidative stress. These are ubiquitous in plants and one of the most abundant groups of phytochemical present in the plants and vegetables. In past, many groups have reported a strong positive correlation between the phenolic content and the antioxidant activity of the extracts [23, 31]. In the current study, phenolic content of the methanolic extract of C. carandas leaves was quantified using a biochemical method. Extract was found to possess a good amount of phenolic compounds (84.00 mg gallic acid equivalence/gm dry weight of the extract). Extract showed significant (p<0.05) dose dependant increase in phenolic compounds. Results for total phenolic content are expressed as mean ± standard deviation (n= 3) and reported in Figure 6.
Page 13 of 25
Conclusion The results obtained in the study signify that the methanolic extract of C. carandas leaves possess a variety of phytochemical compounds, which can efficiently defend the body from
ip t
oxidative stress caused by the free radicals, and thus can be used as a potent source of natural antioxidant compounds. This report also showed the DNA damage inhibition potential of the
cr
extract; which could be utilized to develop a cancer protective medicine. In future, further studies could be carried out to establish the antioxidant mechanism of methanolic extract of
an
as a source of safe and natural antioxidant compounds.
us
C. carandas leaves. With all these results, we conclude that C. carandas leaves can be used
M
Acknowledgements
The authors wish to thank the Management and Staff of Jaipur National University, Jaipur-
References
T.P.A. Devasagayam, J.C. Tilak, K.K. Boloor, K.S. Sane, S.S. Ghaskadbi, R.D. Lele,
Ac ce p
[1]
te
d
302025, Rajasthan, India for providing necessary facilities to carry out this study.
Free radicals and antioxidants in human health: Current status and future prospects, J. Assoc. Physicians India. 52 (2004) 794-804.
[2]
J.T. Hancock, R. Desikan, S.J. Neill, Role of reactive oxygen species in cell signaling pathways, Biochem. Soc. Trans. 29 (2001) 345-350.
[3]
A. Spector, Review: Oxidative stress and disease, J. Ocul. Pharmacol. Ther. 16 (2000)
193-201. [4]
F. Shahidi, P.D. Wanasundara, Phenolic antioxidants, Cri. Rev. Food Sci. Nutr. 32 (1992) 67-103.
Page 14 of 25
[5]
T. Stangeland, S.F. Remberg, K.A. Lye, Total antioxidant activity in 35 Ugandan fruits and vegetables, Food Chem. 113 (2009) 85-91.
[6]
A. Semiz, A. Sen, Antioxidant and chemoprotective properties of Momordica charantia
[7]
ip t
L. (bitter melon) fruit extract, Afr. J. Biotechnol. 6 (2007) 273-277. N. Ozsoy, A. Can, R. Yanardag, N. Akev, Antioxidant activity of Smilax excelsa L. leaf
[8]
cr
extracts, Food Chem. 110 (2008) 571-583.
G. Kumar, L. Karthik, K.B.V. Rao, Phytochemical composition and in vitro antioxidant
[9]
an
Asian Pac. J. Trop. Med. 6 (2013) 180-187.
us
activity of aqueous extract of Aerva lanata (L.) Juss. ex Schult. Stem (Amaranthaceae).
A. Jain, V. Ojha, G. Kumar, L. Karthik, K.B.V. Rao, Phytochemical composition and
M
antioxidant activity of methanolic extract of Ficus benjamina (moraceae) leaves. Research J. Pharm. and Tech. 6 (2013) 1184-1189.
d
[10] C.L. Priya CL, G. Kumar, L. Karthik, K.B.V. Rao, Phytochemical composition and in
te
vitro antioxidant activity of Achyranthes aspera Linn (Amaranthaceae) leaf extracts, Journal of Agricultural Technology. 8 (2012) 143-156.
Ac ce p
[11] S. Kalita, G. Kumar, L. Karthik, K.B.V. Rao, In vitro antioxidant and DNA damage inhibition activity of aqueous extract of Lantana camara L. (Verbenaceae) leaves.
Asian Pac. J. Trop. Biomed. 2 (2012) S1675-S1679.
[12] R. Kahl, H. Kappus, Toxicology of the synthetic antioxidants BHA and BHT in comparison with the natural antioxidant vitamin E. Z Lebensm Unters Forsch. 196 (1993) 329-338.
[13] V. Devmurari, P. Shivanand, N.P. Jivani, A review: Carissa Congesta: Phytochemical constituents, traditional use and pharmacological properties, Int. J. Chem. Sci. 8 (2010) 81-87.
Page 15 of 25
[14] S.N. Hasmah, A. Bhatt, C.L. Keng, Micropropagation of Asam Karanda (Carissa carandas Linn.), Pertanika J. Trop. Agric. Sci. 36 (2013) 89-98. [15] M. Rahmatullah, A. Noman, M.S. Hossan, M. Harun-Or-Rashid, T. Rahman, M.H.
ip t
Chowdhury, R. Jahan, A survey of medicinal plants in two areas of Dinajpur district, Bangladesh including plants which can be used as functional foods, American-Eurasian
cr
Journal of Sustainable Agriculture. 3 (2009) 862-876.
[16] K. Hegde, S.P. Thakker, A.B. Joshi, C.S. Shastry, K.S. Chandrashekhar,
us
Anticonvulsant activity of Carissa carandas Linn. root extract in experimental mice,
an
Trop. J. Pharm. Res. 8 (2009) 117-125.
[17] V.H. Bhaskar, N. Balakrishnan, Analgesic, anti-inflammatory and antipyretic activities
M
of Pergularia daemia and Carissa carandas, DARU. 17 (2009) 168-174. [18] C.K. Mishra, A.K. Pattnaik, A. Rani, D. Sasmal, R.K. Nema, Antifungal and
te
4 (2009) 564-568.
d
antibacterial activity of Carissa carandas Linn., International Journal of Plant Sciences.
[19] T. Agarwal, R. Singh, A.D. Shukla, I. Waris, In vitro study of antibacterial activity of
Ac ce p
Carissa carandas leaf extracts, Asian J. Plant Sci. Res. 2 (2012) 36-40.
[20] K. Hegde, A.B. Joshi, Hepatoprotective effect of Carissa carandas Linn root extract against CCl4 and paracetamol induced hepatic oxidative stress, Indian J. Exp. Biol. 47 (2009) 660-667.
[21] F. Aslam, N. Rasool, M. Riaz, M. Zubair, K. Rizwan, M. Abbas, T.H. Bukhari, I.H. Bukhari, Antioxidant, haemolytic activities and GC-MS profiling of Carissa carandas roots. International Journal of Phytomedicine 3 (2011) 567-578. [22] S. Shamim, S.I. Ahmad, Pharmacodynamic study on acute hypotensive activities of Carissa carandas extract in normal rats, Pak. J. Pharm. Sci. 25 (2012) 577-582.
Page 16 of 25
[23] Anti-cancerous and antioxidant potential of aqueous extracts of Annona reticulata, Podophyllum peltatum, Psidium guajava, Ananas comosus, Carissa carandas on MCF7 cancer cell line, Int. J. Int. Sci. Inn. Tech. Sec. 2 (2013) 15-19.
analysis, Fifth ed., Chapman and Hall, London, 1998.
ip t
[24] J.B. Harborne, Phytochemical methods. A guide to modern techniques of plant
cr
[25] G. Guha, V. Rajkumar, R.A. Kumar, L. Mathew, Aqueous extract of Phyllanthus amarus inhibits chromium(VI)-induced toxicity in MDA-MB-435S cells, Food Chem.
us
Toxicol. 48 (2010) 396-401.
an
[26] P. Prieto, M. Pineda, M. Aguilar, Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application
M
to the determination of vitamin E, Anal Biochem. 269 (1999) 337-341. [27] R.J. Ruch, S.J. Cheng, J.E. Klaunig, Prevention of cytotoxicity and inhibition of
d
intracellular communication by antioxidant catechins isolated from Chinese green tea,
te
Carcinogenesis. 10 (1989) 1003-1008.
[28] M. Oyaizu, Studies on products of the browning reaction, antioxidative activities of
Ac ce p
browning reaction products prepared from glucosamine, Jpn. J. Nutr. 44 (1986) 307315.
[29] G. Guha, V. Rajkumar, R.A. Kumar, L. Mathew, The antioxidant and DNA protection potential of Indian tribal medicinal plants, Turk. J. Biol. 35 (2011) 233-242.
[30] V.L. Singleton, R. Orthofer, R.M. Lamuela-Raventos, Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent, Methods Enzymol. 299 (1999) 152-178. [31] M. Maizura, A. Aminah, W.M.W. Aida, Total phenolic content and antioxidant activity of kesum (Polygonum minus), ginger (Zingiber officinale) and turmeric (Curcuma longa) extract. International Food Research Journal. 18 (2011) 529-534.
Page 17 of 25
Figure captions Figure 1. Percentage DPPH● radical scavenging potential of varying concentrations of methanolic extract of Carissa carandas leaves. Data is given in mean ± SD (n = 3 test;
ip t
p<0.05) with ascorbic acid equivalence Figure 2. Total antioxidant activity of varying concentrations of methanolic extract of
cr
Carissa carandas leaves. Data is given in mean ± SD (n = 3 test; p<0.05) with ascorbic acid equivalence
us
Figure 3. H2O2 scavenging potential of varying concentrations of methanolic extract of
an
Carissa carandas leaves. Data is given in mean ± SD (n = 3 test; p<0.05) with ascorbic acid equivalence
M
Figure 4. Reducing power activity of varying concentrations of methanolic extract of Carissa carandas leaves. Data is given in mean ± SD (n = 3 test; p<0.05) with ascorbic
d
acid equivalence
te
Figure 5. Effect of Carissa carandas leaves methanol extracts on the protection of plasmid DNA (pBR322) against oxidative damage caused by UV-photolysed H2O2.
Ac ce p
C=untreated non-irradiated DNA, R=untreated UV-irradiated DNA and aqueous extract treated DNA (S)
Figure 6. Total phenolic content of varying concentrations of methanolic extract of Carissa carandas leaves. Data is given in mean ± SD (n = 3 test; p<0.05) and expressed as gallic acid equivalence (GAE)
Page 18 of 25
Methods used
Result
Total carbohydrates
Molish’s test
++
Reducing sugars
Fehling’s test
-
Protein
Ninhydrin
++
Xenthoproteic test
++
Wangers test
+++
Mayer test Phenolic compounds
FeCl3 (5%) test Lead acetate test
+++
+++
M
AlCl3 test
+++
+++
Tannins
FeCl3 (0.1%) test
++++
Oil and fats
Spot test
+
Foam test
++
Brontrager’s test
++++
Saponins
Ac ce p
Glycosides
d
Alkaline reagent test
te
Flavonoids
cr
+++
an
Alkaloids
ip t
Phytochemicals
us
Table 1: Phytochemical analysis of methanolic extract of P. juliflora leaves
Here, +, ++, +++: present, -: not present
Page 19 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 1
Page 20 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 2
Page 21 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 3
Page 22 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 4
Page 23 of 25
ip t cr us an
Ac ce p
te
d
M
Figure 5
Page 24 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 6
Page 25 of 25
S1658-3655(14)00067-3 http://dx.doi.org/doi:10.1016/j.jtusci.2014.07.001 JTUSCI 87
To appear in: Received date: Revised date: Accepted date:
16-2-2014 1-7-2014 1-7-2014
Please cite this article as: K. Verma, D. Shrivastava, G. Kumar, In vitro antioxidant and DNA damage inhibition activity of methanolic extract of Carissa carandas (Apocynaceae) leaves, Journal of Taibah University for Science (2014), http://dx.doi.org/10.1016/j.jtusci.2014.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro antioxidant and DNA damage inhibition activity of methanolic extract of Carissa carandas (Apocynaceae) leaves
ip t
Khushbu Verma, Divya Shrivastava, Gaurav Kumar*
Ac ce p
te
d
M
an
us
cr
School of Life Sciences, Jaipur National University, Jaipur- 302025, Rajasthan, India
*Corresponding author Dr. Gaurav Kumar
Assistant Professor, School of Life Sciences, Jaipur National University, Jaipur- 302025, Rajasthan, India M: +91 98287 48869; Email: [email protected]
Page 1 of 25
Abstract The aim of this study was to investigate the antioxidant and DNA damage inhibition potential of methanolic extract of Carissa carandas leaves. Extract was found to exhibit significant
ip t
(p<0.05) dose dependent DPPH radical scavenging activity (IC50 value= 73.12µg/ml), total antioxidant activity, H2O2 scavenging activity (IC50 value 84.03µg/ml), reducing power
cr
activity. In addition, extract was found to exhibit complete protection of pBR322 plasmid DNA from free radicals mediated oxidative stress during DNA damage inhibition assay.
us
Antioxidant and DNA damage inhibition properties of C. carandas could be attributed to the
an
presence of high amount of phenolic compounds (84.00 mg GAE/gm dry weight of the extract) in the extract which was estimated by Folin-Ciocalteau assay. These observations
M
emphasize the high antioxidant and DNA damage inhibition potential of the C. carandas which can be further used to develop natural antioxidant compounds for therapeutic
te
d
applications.
Ac ce p
Keywords: Carissa carandas, antioxidant activity, DNA damage inhibition potential
Page 2 of 25
1. Introduction Free radicals are commonly known as reactive oxygen species (ROS), contains one or more unpaired electrons in their outer most orbital. Some of the common examples of ROS are
ip t
peroxyl radical (ROO-), superoxide anion (O2-), peroxyl radical (ROO•), ozone (O3), hydrogen peroxide (H2O2) and reactive hydroxyl radical (OH-) [1]. Free radicals are highly
cr
unstable, therefore they react with other molecules in their vicinity to obtain stability, and this initiates a chain reaction of free radical generation [1]. Excessive production of free radicals
us
results in the depletion of in vivo antioxidants and causes imbalance between free radicals and
an
antioxidant defence of body, therefore causing serious damage (oxidative stress). Almost 100 different serious diseases are known to associate with oxidative stress viz, cardiovascular
M
diseases, liver diseases, diabetes, cancer, neurodegenerative disease and aging [2, 3]. Antioxidants are usually reducing agents such as vitamins, carotenoids, polyphenols
d
and trace metals (Zn, Mo) which can effectively scavenge ROS and inhibit the chain reaction
te
by donating the electron to free radical. The antioxidant defence system of body, in support with dietary antioxidants protects the body from free radicals. However during oxidative
Ac ce p
stress, in vivo antioxidants are not sufficient to maintain homeostasis therefore; antioxidants could be given as supplements to the body. Consumption of additional antioxidant can significantly reduce the chances of getting free radicals associated diseases [4]. In recent literature, plants are regarded as a potential source of natural antioxidant
compounds [5-11]. Medicinal plants provide a safe, cost effective and eco friendly alternative to the chemical antioxidants which could be toxic on prolong exposure [12]. Hence in this study C. carandas Linn. leaves were screened for their antioxidant properties. C. carandas is a flowering armed shrub belonging to family Apocyneceae. The species is native to India and distributed in Sri Lanka, Indonesia, Malaysia, Myanmar and Pakistan. It grows throughout India and commonly known as Christ thorn and Karanda. It is dichotomously branched and
Page 3 of 25
contains milky latex. Leaves are glossy and dark green and light green on dorsal and ventral side respectively. Leaves are elliptic-oblong, notched at the base and tip is blunt. Flowers are white to rosy pink in color and possess a characteristic fragrance. It produce small, oval shape
ip t
fruits which are initially green in colour and turned to reddish purple when ripen [13]. Several medicinal and nutritional properties of C. carandas establish it as an
cr
economically important plant in the country. In India, C. carandas is traditionally used for the treatment of flatulence, poor digestion, acidity and wounds. Roots are used as
us
anthelmintic, stomachic and antiscorbutic agents and for the treatment of intestinal worm,
an
scabies and pruritus. It is also used as a medicine for high blood pressure due to its blood pressure decreasing property. Fruits of C. carandas is used for the treatments of malaria,
M
epilepsy, nerve disorder, relieve of body ache, fever, blood purifier, myopathic spams, dog bite, cough, colds, itches and leprosy. Ripen fruit of C. carandas are used for making
d
puddings, curries and pickles, whereas slightly under ripen fruits are used to make jelly [13-
te
15]. C. carandas has been extensively studied for its pharmacological properties and reported to show anticonvulsant [16], analgesic, anti-inflammatory and antipyretic [17], antibacterial
Ac ce p
and antifungal [18, 19], hepatoprotective [20], antioxidant [21], acute hypotensive [22] and anti-cancerous activities [23]. Most of these pharmacological studies have been conducted with roots, fruits and bark, however leaves are relatively less explored, therefore this study was designed to investigate phytochemical composition and antioxidant properties of C. carandas. In addition, C. carandas was first time evaluated for its DNA damage inhibition properties against free radical mediated DNA damage.
Page 4 of 25
2. Materials and Methods 2.1. Chemicals Sulphuric acid (H2SO4), Methanol, Ferric chloride (FeCl3), Potassium Ferricyanide (K3Fe
ip t
(CN)6), Trichloroacetic acid, Ascorbic acid, Hydrogen peroxide (H2O2), Sodium chloride (NaCl), Folin- Ciocalteau reagent and Gallic acid were purchased from SRL Pvt. Ltd.
cr
(Mumbai, India). 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Sodium carbonate (Na2CO3) was purchased from
us
Himedia Laboratories Pvt. Ltd. (Mumbai, India). All other chemicals used were of analytical
an
grade.
M
2.2. Plant Material
The leaves of C. carandas were collected from naturally growing population in the village
d
Jaitpura, Jaipur, Rajasthan, India during January 2014. The plant material was carried to the
te
Research Laboratory, Jaipur National University, Jaipur, Rajasthan, India. A voucher specimen was maintained in our laboratory for future reference (Accession number: JNU-
Ac ce p
JPR/RL/ CC/19.01.2014-1).
2.3. Plant processing
The collected leaves (100 gm) of C. carandas were washed thoroughly with sterilized distilled water to remove soil particles then fresh weight of leaves was observed. The leaves were oven dried at 50°C. Dried leaves were pulverized using the mechanical grinder to make delicate powder. The leave powder (10 gm) was soaked in methanol (10% w/v) and loaded on a shaker at a speed of 120 rpm for 24 hour at room temperature. Mixtures were filtered by using Whatman number 1 filter paper. The filtrate was concentrated at 40°C under reduced
Page 5 of 25
pressure (72 mbar) with a rotary evaporator and dried using lyophilizer. Dried extract was collected in an air tight receptacle and stored at room temperature for further use.
ip t
2.4. Phytochemical screening Phytochemical screening of the leaves of C. carandas was carried out by using the standard
cr
protocols [24]. The leaf extract was screened for the presence of total carbohydrates, reducing sugars, protein, alkaloids, phenolic compounds, flavonoids, tannins, glycosides, saponins, oil
2.5. Antioxidant activity
M
2.5.1. DPPH radical scavenging activity
an
us
and fats.
The extract was diluted in methanol to make 25, 50, 75 and 100 µg/ml dilutions. Two
d
milliliter of each dilution were mixed with 1 ml of DPPH solution (0.2 mM/mL in methanol)
te
and mixed thoroughly. The mixture was incubated in the dark at 20°C for 40 min. Absorbance was measured at 517 nm using UV-Vis spectrophotometer with methanol as
Ac ce p
blank. Ascorbic acid was used as experimental control. Experiment was performed in triplicates at each concentration [25]. The percentage scavenging of DPPH by the extracts was calculated according to the following formula:
Percentage DPPH radical scavenging= [(Ac - At) / Ac] × 100 Here,
Ac is the absorbance of the control (DPPH) At is the absorbance of test sample and standards
Page 6 of 25
2.5.2. Determination of total antioxidant activity A volume of 1 ml of the extract (125, 250, 500 and 1000 µg/ml) was mixed with 3 ml of the reaction mixture (containing 10 ml of concentrated H2SO4, 1.005 g of sodium phosphate
ip t
monobasic and 1.47 g of ammonium molybdate which was dissolved in 290 ml of water). The mixture was kept in water bath for one hour at 95º C. The solution containing 3 ml of
cr
reaction mixture and 1 ml of distilled water was used as blank and the absorbance was measured using UV-Vis spectrophotometer at 695 nm. Ascorbic acid was used as
an
us
experimental control. Experiment was performed in triplicates at each concentration [26].
2.5.3 Scavenging of hydrogen peroxide
M
The extract was diluted to make 25, 50, 75 and 100 µg/ml dilutions. Two millilitre of each dilution was mixed with 1.2 ml of hydrogen peroxide solution (40 mM in phosphate buffer,
d
pH 7.4) and incubated for 10 min. The absorbance of the solution was taken at 230 nm using
te
a UV–Visible spectrophotometer against blank solution containing the plant extract without H2O2. Ascorbic acid was used as experimental control. Experiment was performed in
Ac ce p
triplicates at each concentration [27].
The percentage scavenging of hydrogen peroxide of the extracts was calculated using the following equation:
% Scavenging of H2O2 = [(Ac – As)/Ac] × 100 Here,
Ac is the absorbance of the control
As is the absorbance in the presence of the sample and standards
Page 7 of 25
2.5.4. Reducing power activity The extract (1 ml) at different concentrations (125, 250, 500 and 1000 µg/ml) was mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% K3Fe(CN)6. The mixture was
ip t
incubated at 50°C for 20 min. A volume of 2.5 ml of Trichloroacetic acid (10%) was added to the mixture, and was centrifuged at 3000 rpm for 10 min in a cooling centrifuge. 2.5 ml of the
cr
supernatant was mixed with equal volume of distilled water and 0.5 ml Ferric chloride (0.1%). Absorbance was measured at 700 nm using a UV–Visible spectrophotometer.
us
Ascorbic acid was used as positive control. Experiment was performed in triplicates at each
an
concentration [28].
M
2.6. DNA damage inhibition efficiency
DNA damage inhibition by methanolic extract of leaves of C. carandas was tested by
d
photolysing H2O2 by UV radiation in the presence of pBR322 plasmid DNA and performing
te
agarose gel electrophoresis with the irradiated DNA [29]. A total of 1 µl aliquots of pBR322 plasmid DNA were taken in three microcentrifuge tubes. A quantity of 50 µg of extract was
Ac ce p
added to one tube. The remaining tubes were left untreated as the irradiated controls. An amount of 4 µl of 3% H2O2 was added to all the tubes, which were then placed directly on the surface of a UV transilluminator (300 nm) for 10 min at room temperature. After irradiation, 4 µl of tracking dye (0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol) was added. The samples in all the tubes were analyzed by gel electrophoresis on a 1% agarose gel (containing ethidium bromide) in TAE buffer (pH 8). Untreated non-irradiated DNA (C) was run along with untreated UV-irradiated DNA (R) and extract treated UVirradiated sample (S).
Page 8 of 25
2.7. Estimation of total phenolic content Total phenolic content of the methanolic extract of leaves of C. carandas was determined using the Folin-Ciocalteaureagent method [30]. The crude extract was diluted in distilled
ip t
water to obtain different concentrations (125, 250, 500 and 1000 µg). Extract (50 µL) was mixed with 2.5 ml of Folin-Ciocalteau reagent (1/10 dilution in purified water) and 2 ml of
cr
7.5% Na2CO3 (w/v in purified water). The mixture was incubated at 45°C for 15 min. The absorbance was measured at 765 nm. Na2CO3 solution (2 ml of 7.5% Na2CO3 in 2.55 ml of
us
distilled water) was used as blank. The results were expressed as gallic acid equivalence
an
(GAE) in µg. Each experiment was performed in triplicates at each concentration.
M
2.8. Statistical analysis
The results of DPPH radical scavenging activity, total antioxidant activity, Hydrogen
d
peroxide scavenging activity, reducing power activity and total phenolic content of the
te
methanolic extract of C. carandas leaves are expressed as mean ± standard deviation of the response of three replicates per sample. Statistical significance between the groups was
Ac ce p
analyzed by one-way ANOVA coupled with Tukey's post-hoc test at P<0.05. Statistical analysis was performed by using Microsoft Excel 2007 and GraphPad Prism 5.
3. Results and Discussion 3.1. Percentage yield
Hundred grams of fresh leaves of C. carandas were dried and the dried powder was extracted in water. The percentage yield of the plant extract was estimated with respect to fresh mass and dry weight. Extraction of C. carandas leaves with methanol resulted in 7.85% and 17.98% yield with respect to fresh mass and dry weight respectively.
Page 9 of 25
3.2. Phytochemical analysis The result of preliminary phytochemical analysis of the methanolic extract of C. carandas leaves showed the presence of total carbohydrates, proteins, phenolic compounds, flavonoids,
ip t
tannins, saponins, oil and fats, alkaloids and cardiac glycosides, whereas reducing sugars were found to be absent (Table 1). Results of phytochemical analysis are in agreement with
cr
the previous reports where C. carandas leaves have been reported to possess alkaloids, flavonoids, saponins, cardiac glycosides, triterpenoids, phenolic compounds and tannins [16].
us
Other studies have also reported the presence of steroids, flavonoids, tannins, phenolic
an
compound, terpenoids, saponins, alkaloid and glycosides as major phytochemical groups [17]. Presence of phenolic compounds and flavonoids enhance the chances of getting high
M
antioxidant activity in the extract as these compounds from plants and vegetable are
te
3.3. Antioxidant activity
d
extensively reported to show antioxidant properties.
In last few decades, C. carandas has been extensively studied for its pharmacological studies,
Ac ce p
however a very few studies have reported the antioxidant potential. Various solvent extracts prepared from the C. carandas roots have been reported to demonstrate significant dose dependent antioxidant activity in various antioxidant methods [21]. In one other study, aqueous extracts of C. carandas leaves has been reported to show significant antioxidant activity in MCF 7 cell lines (breast cancer cell line). Extract treatment was found to increase the levels of in vivo antioxidants viz, catalase, superoxide dismutase, glutathione transferase and glutathione on MCF-7 cancer lines [23]. In this study, methanolic extract of C. carandas was screened for its antioxidant properties by various in vitro methods viz, DPPH radical scavenging activity, total antioxidant activity, H2O2 scavenging activity and reducing power activity.
Page 10 of 25
3.3.1. DPPH radical Scavenging activity DPPH is a comparatively stable free radical, contains unpaired electron on its nitrogen molecule. DPPH gives a characteristic purple color in methanol which is reduced to yellow
ip t
after receiving a proton from an antioxidant compound. This reduction in the color of DPPH can be measured by using a UV Visible spectrophotometer (517 nm). In this study,
cr
methanolic extract of C. carandas leaves was evaluated for its DPPH radical scavenging activity in a dose dependent manner. Extract was found to exhibit significant (p<0.05) dose
us
dependent DPPH radical scavenging activity with IC50 value= 73.12µg/ml. The results are
an
represented as percentage inhibition of DPPH and expressed as mean ± standard deviation (n= 3). The DPPH radical scavenging activity of the extract was compared with the ascorbic
M
acid and reported in Figure 1.
d
3.3.2. Total antioxidant activity
te
The total antioxidant test is based on the reduction of molybdate (VI) to molybdate (V) at acidic pH by the extract and as well as the formation of green phosphate complex, which
Ac ce p
could be quantified spectrophotometrically at 695 nm. In the current study, methanolic extract of C. carandas leaves was evaluated for total antioxidant activity in a dose dependent manner. The extract showed significant (p<0.05) dose dependant increase in total antioxidant activity with an OD value of 1.39±0.21 (at 1000 µg/ml). The total antioxidant activity of the extract was compared with the ascorbic acid and reported in Figure 2.
3.3.3. H2O2 scavenging activity Hydrogen peroxide radical scavenging activity is a very useful method for the assessment of antioxidant property of the samples. Hydrogen peroxide is usually none reactive to cell but it may give rise to the hydroxyl radicals, therefore the compounds with can donate electron to
Page 11 of 25
hydrogen peroxide could reduce hydrogen peroxide in to water and thus blocks the generation of hydroxyl radicals and protect the cell. Electron donation potential of the sample is directly proportional to its antioxidant potential. In the present study, methanolic extract of
ip t
C. carandas leaves was evaluated for hydrogen peroxide radical scavenging activity. The extract exhibited significant (p<0.05) hydrogen peroxide scavenging activity in a dose
cr
dependent manner with IC50 value 84.03µg/ml. Hydrogen peroxide radical scavenging
us
activity of the extracts was compared with the ascorbic acid and reported in Figure 3.
an
3.3.4. Reducing power activity
Reducing power activity is based on the reduction of Fe (III) to Fe (II) by the electron-
to
bluish
green
color
in
the
M
donating potential of the compounds. Reduction of Fe (III) to Fe (II) ions produces a yellow reaction
mixture,
which
could
be
measured
d
spectrophotometrically at 700 nm. In the current study, methanolic extract of C. carandas
te
leaves was evaluated for reducing power activity. The extract showed significant (p<0.05) dose dependant increase in reducing power activity with an OD value of 1.73±0.04 at 1000
Ac ce p
µg/ml. The result for reducing power activity of methanolic extract of C. carandas leaves in comparison to ascorbic acid is reported in Figure 4.
3.4. DNA damage inhibition activity Free radicals are capable of both initiating and increasing several types of disease. Free radicals such as hydroxyl free radicals are well known to damage cellular DNA in humans. Partial damage in DNA may turn the cell in the cancerous cell which is a very severe health problem in humans. Plants with DNA damage inhibition potential could be used to protect the DNA from the free radicals mediated damage. Therefore in this study, DNA damage inhibition efficiency of the methanolic extract of C. carandas leaves was evaluated and
Page 12 of 25
reported in Figure 5, it shows the electrophoretic pattern of pBR322 DNA following UVphotolysis of H2O2 in absence (in controls C and R) and presence (samples S) of the extract. Control pBR322 (C) showed the bands of super coiled and open circular plasmid DNA in
ip t
agarose gel electrophoresis. UV-photolysis of H2O2 (R) damaged the entire DNA (no bands visible). The extract displayed considerable protective activity and showed the bands of super
cr
coiled and open circular plasmid DNA. The results infer that UV-photolysed H2O2 (3%) treatment of pBR322 obliterated the entire DNA (in R), while 50 µg of methanolic extract of
us
C. carandas leaves protected against the free radical mediated DNA damage (S). The results
an
of this experiment demonstrate the DNA damage inhibition potential of the methanolic extract of C. carandas leaves and provides a hope to use it in future as a cancer prevention
M
medicine. Earlier also some plants have been reported to exhibit DNA protective efficacy
te
3.5. Total phenolic content
d
towards free radical mediated DNA damage [11, 29].
Phenolic compounds are unarguably the major group of phytochemicals with antioxidant
Ac ce p
properties in plants. Phenolic compounds are produced as secondary metabolite in plant system to protect themselves from oxidative stress. These are ubiquitous in plants and one of the most abundant groups of phytochemical present in the plants and vegetables. In past, many groups have reported a strong positive correlation between the phenolic content and the antioxidant activity of the extracts [23, 31]. In the current study, phenolic content of the methanolic extract of C. carandas leaves was quantified using a biochemical method. Extract was found to possess a good amount of phenolic compounds (84.00 mg gallic acid equivalence/gm dry weight of the extract). Extract showed significant (p<0.05) dose dependant increase in phenolic compounds. Results for total phenolic content are expressed as mean ± standard deviation (n= 3) and reported in Figure 6.
Page 13 of 25
Conclusion The results obtained in the study signify that the methanolic extract of C. carandas leaves possess a variety of phytochemical compounds, which can efficiently defend the body from
ip t
oxidative stress caused by the free radicals, and thus can be used as a potent source of natural antioxidant compounds. This report also showed the DNA damage inhibition potential of the
cr
extract; which could be utilized to develop a cancer protective medicine. In future, further studies could be carried out to establish the antioxidant mechanism of methanolic extract of
an
as a source of safe and natural antioxidant compounds.
us
C. carandas leaves. With all these results, we conclude that C. carandas leaves can be used
M
Acknowledgements
The authors wish to thank the Management and Staff of Jaipur National University, Jaipur-
References
T.P.A. Devasagayam, J.C. Tilak, K.K. Boloor, K.S. Sane, S.S. Ghaskadbi, R.D. Lele,
Ac ce p
[1]
te
d
302025, Rajasthan, India for providing necessary facilities to carry out this study.
Free radicals and antioxidants in human health: Current status and future prospects, J. Assoc. Physicians India. 52 (2004) 794-804.
[2]
J.T. Hancock, R. Desikan, S.J. Neill, Role of reactive oxygen species in cell signaling pathways, Biochem. Soc. Trans. 29 (2001) 345-350.
[3]
A. Spector, Review: Oxidative stress and disease, J. Ocul. Pharmacol. Ther. 16 (2000)
193-201. [4]
F. Shahidi, P.D. Wanasundara, Phenolic antioxidants, Cri. Rev. Food Sci. Nutr. 32 (1992) 67-103.
Page 14 of 25
[5]
T. Stangeland, S.F. Remberg, K.A. Lye, Total antioxidant activity in 35 Ugandan fruits and vegetables, Food Chem. 113 (2009) 85-91.
[6]
A. Semiz, A. Sen, Antioxidant and chemoprotective properties of Momordica charantia
[7]
ip t
L. (bitter melon) fruit extract, Afr. J. Biotechnol. 6 (2007) 273-277. N. Ozsoy, A. Can, R. Yanardag, N. Akev, Antioxidant activity of Smilax excelsa L. leaf
[8]
cr
extracts, Food Chem. 110 (2008) 571-583.
G. Kumar, L. Karthik, K.B.V. Rao, Phytochemical composition and in vitro antioxidant
[9]
an
Asian Pac. J. Trop. Med. 6 (2013) 180-187.
us
activity of aqueous extract of Aerva lanata (L.) Juss. ex Schult. Stem (Amaranthaceae).
A. Jain, V. Ojha, G. Kumar, L. Karthik, K.B.V. Rao, Phytochemical composition and
M
antioxidant activity of methanolic extract of Ficus benjamina (moraceae) leaves. Research J. Pharm. and Tech. 6 (2013) 1184-1189.
d
[10] C.L. Priya CL, G. Kumar, L. Karthik, K.B.V. Rao, Phytochemical composition and in
te
vitro antioxidant activity of Achyranthes aspera Linn (Amaranthaceae) leaf extracts, Journal of Agricultural Technology. 8 (2012) 143-156.
Ac ce p
[11] S. Kalita, G. Kumar, L. Karthik, K.B.V. Rao, In vitro antioxidant and DNA damage inhibition activity of aqueous extract of Lantana camara L. (Verbenaceae) leaves.
Asian Pac. J. Trop. Biomed. 2 (2012) S1675-S1679.
[12] R. Kahl, H. Kappus, Toxicology of the synthetic antioxidants BHA and BHT in comparison with the natural antioxidant vitamin E. Z Lebensm Unters Forsch. 196 (1993) 329-338.
[13] V. Devmurari, P. Shivanand, N.P. Jivani, A review: Carissa Congesta: Phytochemical constituents, traditional use and pharmacological properties, Int. J. Chem. Sci. 8 (2010) 81-87.
Page 15 of 25
[14] S.N. Hasmah, A. Bhatt, C.L. Keng, Micropropagation of Asam Karanda (Carissa carandas Linn.), Pertanika J. Trop. Agric. Sci. 36 (2013) 89-98. [15] M. Rahmatullah, A. Noman, M.S. Hossan, M. Harun-Or-Rashid, T. Rahman, M.H.
ip t
Chowdhury, R. Jahan, A survey of medicinal plants in two areas of Dinajpur district, Bangladesh including plants which can be used as functional foods, American-Eurasian
cr
Journal of Sustainable Agriculture. 3 (2009) 862-876.
[16] K. Hegde, S.P. Thakker, A.B. Joshi, C.S. Shastry, K.S. Chandrashekhar,
us
Anticonvulsant activity of Carissa carandas Linn. root extract in experimental mice,
an
Trop. J. Pharm. Res. 8 (2009) 117-125.
[17] V.H. Bhaskar, N. Balakrishnan, Analgesic, anti-inflammatory and antipyretic activities
M
of Pergularia daemia and Carissa carandas, DARU. 17 (2009) 168-174. [18] C.K. Mishra, A.K. Pattnaik, A. Rani, D. Sasmal, R.K. Nema, Antifungal and
te
4 (2009) 564-568.
d
antibacterial activity of Carissa carandas Linn., International Journal of Plant Sciences.
[19] T. Agarwal, R. Singh, A.D. Shukla, I. Waris, In vitro study of antibacterial activity of
Ac ce p
Carissa carandas leaf extracts, Asian J. Plant Sci. Res. 2 (2012) 36-40.
[20] K. Hegde, A.B. Joshi, Hepatoprotective effect of Carissa carandas Linn root extract against CCl4 and paracetamol induced hepatic oxidative stress, Indian J. Exp. Biol. 47 (2009) 660-667.
[21] F. Aslam, N. Rasool, M. Riaz, M. Zubair, K. Rizwan, M. Abbas, T.H. Bukhari, I.H. Bukhari, Antioxidant, haemolytic activities and GC-MS profiling of Carissa carandas roots. International Journal of Phytomedicine 3 (2011) 567-578. [22] S. Shamim, S.I. Ahmad, Pharmacodynamic study on acute hypotensive activities of Carissa carandas extract in normal rats, Pak. J. Pharm. Sci. 25 (2012) 577-582.
Page 16 of 25
[23] Anti-cancerous and antioxidant potential of aqueous extracts of Annona reticulata, Podophyllum peltatum, Psidium guajava, Ananas comosus, Carissa carandas on MCF7 cancer cell line, Int. J. Int. Sci. Inn. Tech. Sec. 2 (2013) 15-19.
analysis, Fifth ed., Chapman and Hall, London, 1998.
ip t
[24] J.B. Harborne, Phytochemical methods. A guide to modern techniques of plant
cr
[25] G. Guha, V. Rajkumar, R.A. Kumar, L. Mathew, Aqueous extract of Phyllanthus amarus inhibits chromium(VI)-induced toxicity in MDA-MB-435S cells, Food Chem.
us
Toxicol. 48 (2010) 396-401.
an
[26] P. Prieto, M. Pineda, M. Aguilar, Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application
M
to the determination of vitamin E, Anal Biochem. 269 (1999) 337-341. [27] R.J. Ruch, S.J. Cheng, J.E. Klaunig, Prevention of cytotoxicity and inhibition of
d
intracellular communication by antioxidant catechins isolated from Chinese green tea,
te
Carcinogenesis. 10 (1989) 1003-1008.
[28] M. Oyaizu, Studies on products of the browning reaction, antioxidative activities of
Ac ce p
browning reaction products prepared from glucosamine, Jpn. J. Nutr. 44 (1986) 307315.
[29] G. Guha, V. Rajkumar, R.A. Kumar, L. Mathew, The antioxidant and DNA protection potential of Indian tribal medicinal plants, Turk. J. Biol. 35 (2011) 233-242.
[30] V.L. Singleton, R. Orthofer, R.M. Lamuela-Raventos, Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent, Methods Enzymol. 299 (1999) 152-178. [31] M. Maizura, A. Aminah, W.M.W. Aida, Total phenolic content and antioxidant activity of kesum (Polygonum minus), ginger (Zingiber officinale) and turmeric (Curcuma longa) extract. International Food Research Journal. 18 (2011) 529-534.
Page 17 of 25
Figure captions Figure 1. Percentage DPPH● radical scavenging potential of varying concentrations of methanolic extract of Carissa carandas leaves. Data is given in mean ± SD (n = 3 test;
ip t
p<0.05) with ascorbic acid equivalence Figure 2. Total antioxidant activity of varying concentrations of methanolic extract of
cr
Carissa carandas leaves. Data is given in mean ± SD (n = 3 test; p<0.05) with ascorbic acid equivalence
us
Figure 3. H2O2 scavenging potential of varying concentrations of methanolic extract of
an
Carissa carandas leaves. Data is given in mean ± SD (n = 3 test; p<0.05) with ascorbic acid equivalence
M
Figure 4. Reducing power activity of varying concentrations of methanolic extract of Carissa carandas leaves. Data is given in mean ± SD (n = 3 test; p<0.05) with ascorbic
d
acid equivalence
te
Figure 5. Effect of Carissa carandas leaves methanol extracts on the protection of plasmid DNA (pBR322) against oxidative damage caused by UV-photolysed H2O2.
Ac ce p
C=untreated non-irradiated DNA, R=untreated UV-irradiated DNA and aqueous extract treated DNA (S)
Figure 6. Total phenolic content of varying concentrations of methanolic extract of Carissa carandas leaves. Data is given in mean ± SD (n = 3 test; p<0.05) and expressed as gallic acid equivalence (GAE)
Page 18 of 25
Methods used
Result
Total carbohydrates
Molish’s test
++
Reducing sugars
Fehling’s test
-
Protein
Ninhydrin
++
Xenthoproteic test
++
Wangers test
+++
Mayer test Phenolic compounds
FeCl3 (5%) test Lead acetate test
+++
+++
M
AlCl3 test
+++
+++
Tannins
FeCl3 (0.1%) test
++++
Oil and fats
Spot test
+
Foam test
++
Brontrager’s test
++++
Saponins
Ac ce p
Glycosides
d
Alkaline reagent test
te
Flavonoids
cr
+++
an
Alkaloids
ip t
Phytochemicals
us
Table 1: Phytochemical analysis of methanolic extract of P. juliflora leaves
Here, +, ++, +++: present, -: not present
Page 19 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 1
Page 20 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 2
Page 21 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 3
Page 22 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 4
Page 23 of 25
ip t cr us an
Ac ce p
te
d
M
Figure 5
Page 24 of 25
ip t cr us
Ac ce p
te
d
M
an
Figure 6
Page 25 of 25