ISSN (Online) 2249-6084 (Print) 2250-1029

International Journal of Pharmaceutical and

Phytopharmacological Research (eIJPPR)
[Impact Factor 0.7826]

Research Article

Direct Nanodrop Spectrophotometric Quantification of DNA and

Evaluation of Antioxidant Activity of Cayratia trifolia
Kavi Gour* and Vidya Patni
Plant Pathology, Tissue Culture and Biotechnology Laboratory,
Department of Botany and P.G. Biotechnology, University of Rajasthan,
Jaipur-302004, India

*Corresponding Author:
Kavi Gour
Research Scholar
Department of Botany and P.G. Biotechnology,
University of Rajasthan, Jaipur-302004, India
Email: kavigour1985@gmail.com
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Abstract:
Medicinal plant utilization and conservation has attained global attention. Here we report a
new, simple nanodrop spectrophotometric quantification of DNA as well as purity of isolated
DNA sample with ND-1000 of Cayratia trifolia - a plant of family vitaceae. DNA was
isolated with good purity by CTAB method. Maximum DNA was found in in vitro callus
(78.5 ng/L) while minimum was found in in vivo root (38.4 ng/L). Maximum crude
content was found in in vivo methanolic extract (6.960.26 gm) and the total phenolic content
was found in in vivo and in vitro plant parts varied from 199.981.77 to 212.670.45
(mg/100gm). The antioxidant activity of the plant extracts and the standard were assessed on
the basis of the radical scavenging effect of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH)free radical activity. The tested extracts showed good antioxidant potential with IC50%
43.3960.52 in in vitro, 47.880.04 in in vivo methanolic extract as compared to other solvent
extracts. The present investigation underlies that Cayratia trifolia gave good purity of DNA,
which will be very useful for any necessary researches in the field of molecular biology as
well as potential sources of natural antioxidants for medicinal and commercial use and as an
easily accessible source of natural bioactive compounds.

Keywords: ND-1000, Cayratia trifolia, DNA, Antioxidant.

1. Introduction:
Since time immemorial man has been using plant extracts to protect himself against several
diseases and also to improve his health and life style. According to WHO 20,000 plant
species are used for medicinal and aromatic purpose (Vural, 2009). The new uses for
medicinal plants have been discovered and popularized. So, the applications of molecular
technology would increase and facilitate production of theses useful substances in medicinal
plants by studying their genetic profile. A range of method is available to assess the quality of
the isolated DNA which includes gel electrophorois, spectrophotometric analysis and
chromatographic techniques. (Verma et al., 2007).
Semi-arid regions of Rajasthan, (India), especially in the tribal and rural areas, are a
major reservoir of medicinal plants. Cayratia trifolia (Amerchotioo; Vitaceae) is a woody
and herbaceous climber which is commonly distributed in semi-arid regions of Asia, Europe,
and Africa. The plant has different biochemical constituents which may be beneficial for
humans. The extract of the plant, along with infusion of Trifolium seed, is used orally to
check blood sugar level. The paste of its tuberous root is generally applied for snakebites and
carnacules and also used orally with milk for the early recovery of fractured bones. (Gour et
al., 2012).
Evolution of antioxidants defences must have been closely associated with the
evolution of photosynthesis and of O2 - dependent electron transport mechanism. The
evolution of aerobic metabolic processes such as respiration and photosynthesis unavoidably
led to the production of reactive oxygen species (ROS) in mitochondria, chloroplast and
peroxisomes. A common feature among the different ROS types is their capacity to cause
oxidative damage to proteins, DNA, and lipids. (Pham-Huy et al., 2008, Rajarajeswari and
Pari, 2011).
To prevent the oxidative damage, plants possess antioxidative systems composed of
low molecular weight antioxidants such as ascorbate and glutathione and protective enzymes
such as superoxide dismutase, catalase and peroxidases (Alscher et al., 1991; Pastori et al.,
2000). As there are several evidences implicating oxidative reactions in the pathogenesis of
many diseases, the popular media and the health supplement industries at times appear to
suggest that the holy grail of maintaining optimum health throughout life can be achieved by
the simple expedient of consuming antioxidants.
Thus, there is now a huge range of antioxidant containing concoctions and capsules
available from commercial outlets. However, there have been concerns about synthetic
antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)
because of this reason, there is a growing interest in replacing synthetic compounds with
natural plant products as potential antioxidants.
A range of plants have been studied in recent years as potential sources of
antioxidants. viz.. Ionidium suffruticosum (Kumar et al., 2011), Coleus spicatus (Edward and
Padmaja, 2011), Ficus glomerata (Ahmed and Tariq, 2012), Pleurotus ostreatus (Hapsari et
al., 2012). Many antioxidant compounds, naturally occurring in plant sources, have been
identified as a free radical or active oxygen scavengers (Zheng and Wang, 2001; Baris et al.,
2006). Their possible activity as promoters of carcinogenesis. (Govind and Madhuri, 2011;
Habib et al., 2011) is well documented.
Therefore, a systematic examination of antioxidant properties of various plant extracts
is extremely important to validate the use of, e.g., flavonoids as preservatives in both the food
and pharmaceutical industries. Hence, the aim of this study was the quantitative analysis of
DNA content of Cayratia trifolia as well as anti-oxidant activity of the different extracts.

2. Materials and Methods:

2.1 For DNA quantification:
2.1.1 Sample preparation for DNA quantification:
The plant material Cayratia trifolia (root, stem, leaves) was collected from Amani
Shah Nala of Sodala, Jaipur (Rajasthan) India. A voucher specimen (RUBL 13846) was
deposited at the Herbarium of Department of Botany, University of Rajasthan, Jaipur. Callus
of the plant was obtained through nodal segment by using MS medium (Murashige and
Skoog, 1962) contain specific combinations of NAA and BAP (N0.5B0.5). All in vivo and in
vitro plant parts were air dried at room temperature for further procedure.
2.1.2 Reagents and Chemicals: The following chemicals and reagents were used: CTAB
buffer pH 5.0 100 ml, 1M tris pH 8.0 and 5x TEB buffer. CTAB, tris, NaCl, PVP 40,
Ethanol, EDTA and all other chemicals were obtained from Himedia, India.
2.1.3 Method of extraction and estimation: 200 mg plant material was macerated in 500 l
CTAB buffer. The mixture was incubated for 15 min 550C in a water bath. After incubation it
was centrifuged at 12000 rpm for 5 mins. Supernatant was mixed with chloroform: Iso amyl
alcohol (24:1) by inversion and re-centrifuged at 13000 rpm for 1 min. Upper aqueous phase
was transferred to a clean microcentrifuge tube, mixed with 50 l of 7.5 M Ammonium
acetate followed by 500 l of ice cold ethanol. Tube was inverted slowly to precipitate the
DNA or placed for 1 hr. at -200C. Mixture was again centrifuged at 13000 rpm for 1 min.
After centrifugation DNA pellets were allowed to dry for 15 mins. This mixture was
resuspended in sterile DNase free water containing RNase (10 g/ml) and incubated at 650C
for 20 mins and stored at 4 0C. Nucleic acid content was measured by ND-1000 ver. 3.3
Nanodrop spectrophotometer.
2.2 For Phenol content and antioxidant activity :
2.2.1 Chemicals used:
All the chemicals and growth regulators used were of high analytical grade. 2,2Diphenyle-1-picryl hydrazyl (DPPH) from Sigma Aldrich Co., St. Louis, USA. All solutions
were prepared in doubled distilled water. Stock solutions of the test extracts were prepared in
methanol, ethanol and petroleum ether. Appropriate blanks were used for individual assays.
2.2.2 Plant Material:
Fresh plant samples (leaf, stem and root) of the above mentioned plants were
collected and washed individually under running tap water to remove soil particles and other
debris. In vitro callus obtained on MS medium fortified with different growth regulators
(previous mentioned) was also used for the present study. The in vivo concoctions viz. leaf,
stem and root were dried in shade condition for 5 to 6 days, until they became crispy while
still retaining the green coloration. Dried plants were ground in electric blender to get fine
powder for further use.
2.2.3 Plant extraction Preparation:
The dried and powdered samples of the experimental plants (in vivo) and callus (in
vitro) obtained as mentioned earlier (each 20g) were extracted successively with ethanol,
methanol and petroleum ether (each 400 ml) for 25-26 hrs., using a Soxhlet apparatus. The
collected solutions were filtered through Whatman No-1 filter paper. The extracts were
evaporated to dryness under reduced pressure at 90 C in Rotary vacuum evaporator to obtain
the respective extracts and stored in freeze condition at 18 C until further analysis.
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2.2.4 Phenolic Estimation:

To estimate the total phenolic content of plant extracts the protocol of Bray et al.,
(1954) was followed.
2.2.5 DPPH assay:
The antioxidant activity of the plant extracts and the standard were assessed on the
basis of the radical scavenging effect of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH)free radical activity by modified method of Braca et al., (2002). The diluted working
solutions of the test extracts were prepared in methanol. Ascorbic acid was used as standard
in 1-1000 g/ml solution and the same concentrations were prepared of all the test solutions.
0.002% of DPPH was prepared in methanol and 1 ml of this solution was mixed with 1 ml of
sample solution and standard solution separately. Experiment was done in triplicate. These
solution mixtures were kept in dark for 30 min. and optical density was measured at 517 nm
using Cecil-Elect Spectrophotometer. The lower the absorbance of the reaction mixture,
higher the free radical scavenging activity. Methanol (1 ml) with DPPH solution (0.002%, 1
ml) was used as blank. The optical density was recorded and the inhibitory effect of DPPH
was calculated according to the following formula (Gupta et al., 2003):

Inhibition(%) =

(Absorbance control - Absorbance sample)

100
Absorbance control

IC50 represents the level where 50% of the radicals were scavenged by test samples.
3. Results and Discussion:
In the present study, we successfully obtained good yield of DNA from the different
in vivo and in vitro plant parts. Maximum amount of DNA concentration was achieved in
callus (78.5 ng/l) followed by leaf (73.6 ng/l), stem (65.1 ng/l) and root (38.4 ng/l). As
per results shown in Fig:1, nucleic acid content was higher in in vitro samples as compared to
in vivo samples. Good purity range was seen in Cayratia trifolia that was 1.80 (260/280 ratio)
from callus, 1.85 in leaf, 1.70 in stem and 1.76 in case of root.(Figure 1; A,B,C,D)

Fig. 1: Nucleic acid content in Cayratia trifolia (A) Laef (B) Stem (C) Root and (D) Callus.
The yield of extracts of plant using methanol, ethanol, petroleum ether for in vivo and
in vitro samples are shown in Table 1. Maximum crude content was found in in vivo
methanolic extract (6.960.26 gm) followed by in vitro methanolic extract (6.420.13 gm), in
vivo ethanolic extract (5.350.51 gm), in vitro ethanolic extract (5.130.17 gm), in vivo
petroleum ether extract (4.630.37 gm), in vitro petroleum ether extract (4.250.81 gm). The
variations in yield may be due to the polarity of the solvents used in the extraction process.
In case of Cayratia trifolia, the total phenol content in the tissues varied from
199.981.77 to 212.670.45 (mg/100gm). The amount of phenols was higher in the methanol
sample as compared to all other solvent extract plant samples (Table 1, Fig. 2)
Table 1: Phenolic content and IC50% of different extracts of Cayratia trifolia.
Solvent used
IC 50% of standard
Ascorbic acid 570.03
Methanol
Ethanol
Pet. Ether

Cayratia trifolia
Crude extract
(gm)

In vivo

6.960.26

In vitro
In vivo
In vitro
In vivo
In vitro

6.420.13
5.350.51
5.130.17
4.630.37
4.250.81

Phenol content
(mg/100gm)
210.450.32
212.670.45
205.321.89
208.090.59
199.981.77
203.540.54

IC50%
47.880.04
43.3960.52
83.460.21
52.380.36
132.460.41
116.820.12

PHENOLIC CONTENT
Crude extract (gm)
300

212.67

210.45

Phenol content (mg/100gm)

208.09

205.32

203.54

199.98

200
100

6.96

6.42

5.35

5.13

4.63

4.25

0
In vivo

In vitro

In vivo

Methanol

In vitro
Ethanol

In vivo

In vitro
Pet Ether

Fig. 2: In vivo and in vitro phenolic content of different extracts of Cayratia trifolia.
DPPH radical is commonly used as a substrate to evaluate antioxidant activity. It is a
stable free radical that could accept an electron or hydrogen radical to become a stable
molecule. The reduction of DPPH radical was determined by the decrease in its absorbance
induced by antioxidant at 517nm. Concentration of sample at which the inhibition percentage
reaches 50% is its IC50 value. It is calculated by optical density and inhibition percentage
with respect to concentration (Table 1,2; Figs. 3: A,B,C,D,E,F). IC50 value is negatively
related to the antioxidant activity, as it expresses the amount of antioxidant needed to
decrease its radical concentration by 50%. The lower the IC 50 value, the higher is the
antioxidant activity of the tested sample. In the present study, in Cayratia trifolia the order of
significant antioxidant activity was observed in the order of : In vitro methanol followed by
In vivo methanol, In vitro ethanol, In vivo ethanol, In vitro petroleum ether and In vivo
petroleum ether. (Table 1)
Table 2: Optical density and percent inhibition of various samples of Cayratia trifolia.

Plant
extracts

Methanolic

Ethanolic

Petroleum
ether

Concen O.D. of
tration
stan
(g/ml) -dard
10
50
100
200
400
600
800
1000
10
50
100
200
400
600
800
1000
10
50

0.673
0.567
0.475
0.291
0.13
0.108
0.094
0.06
0.673
0.567
0.475
0.291
0.13
0.108
0.094
0.06
0.673
0.567

O.D. of
in vivo
sample

O.D. of
in vitro
sample

%
inhibition
of
standard

%
inhibition
of in vivo
sample

%
inhibition
of in vitro
sample

0.724
0.58
0.491
0.472
0.365
0.233
0.18
0.16
0.714
0.612
0.436
0.416
0.28
0.106
0.087
0.043
0.76
0.64

0.754
0.63
0.447
0.403
0.346
0.315
0.216
0.184
0.745
0.53
0.386
0.36
0.337
0.211
0.154
0.03
0.783
0.604

39.532
49.056
57.322
73.854
88.319
90.296
91.554
94.609
39.532
49.056
57.322
73.854
88.319
90.296
91.554
94.609
39.532
49.056

34.95
47.888
55.885
57.592
67.205
79.065
83.827
85.624
35.849
45.013
60.826
62.623
74.842
90.476
92.183
96.136
31.716
42.497

32.255
43.396
59.838
63.791
68.912
71.698
80.592
83.468
33.063
52.38
65.318
67.654
69.721
81.042
86.163
97.304
29.649
45.732

100
200
400
600
800
1000

0.475
0.291
0.13
0.108
0.094
0.06

0.563
0.511
0.46
0.406
0.348
0.285

0.528
0.421
0.398
0.326
0.208
0.112

57.322
73.854
88.319
90.296
91.554
94.609

49.415
54.088
58.67
63.522
68.733
74.393

52.56
62.174
64.24
70.709
81.311
89.937

In all the plant samples, alcoholic (especially methanolic) extract showed better
antioxidant activity; this gives evidence that, methanolic preparation is more useful than the
petroleum ether one in medical approach. High precent of yield and high value of phenol
content in alcoholic extracts of all the plants species showed that phenolic constituents must
be responsible for antioxidant properties. The correlation between total phenolic content and
antioxidant capacity in the plant samples of different extracts are possible owing to the
presence of phenolic compounds including polyphenols/ flavonoids/ tannins. This correlation
is also in consonance with the report of Wang et al. (2011) in Schisandra chinensis,
Schisandra sphenanthera and Zaman et al. (2011) in Withania somnifera, Eclipta prostrata
and Gossypium herbaceum, Rezazadeh et al. (2012) in Cynara scolymus L.. Hence, it was
also observed that the alcoholic (especially methanolic) extracts showed vital amount of
antioxidant activity in in vitro as well as in in vivo plant samples as compared to other
extracts.

Figure 3: A-C: Optical density and concentration of methanolic, ethanolic and petroleum
ether extracts of Cayratia trifolia.
D-F: Percent inhibition of samples and concentration of methanolic, ethanolic and petroleum
ether extracts of Cayratia trifolia.
4. Conclusion:
The study concluded that ND-1000 Spectrophotometric determination of DNA gave
good result which is very useful and accurate for any necessary researches in the field of
molecular biology for the study of medicinal plants. The study also concluded that, all tested
samples of Cayratia trifolia which contained highest amount of flavonoid and phenolic
compounds, exhibited the maximum antioxidant activity. The in vitro samples were more
potently active than in vivo parts of all the experimental plant samples. The high scavenging
property may be due to hydroxyl groups existing in the phenolic compounds chemical
structure that can provide the necessary component as a radical scavenger. Further studies are
9

also needed to identify the antioxidative compounds of the plant in the management of human
diseases resulting from oxidative stress.
5. Acknowledgements:
The authors are thankful to Prof. Ashok K. Nagawat, Director, Center for Converging
Technologies University of Rajasthan for providing facilities to carry out this work.
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