j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 0 4 e8 0 9

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Original Article

Phytochemicals and antioxidants in leaf extracts of

Ginkgo biloba with reference to location, seasonal
variation and solvent system

Priyanka Sati a, Anita Pandey a,*, Sandeep Rawat a, Anju Rani b

a
Biotechnological Applications, G. B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal,
Almora 263 643, Uttarakhand, India
b
Department of Biotechnology, Graphic Era University, Dehradun 248002, Uttarakhand, India

article info abstract

Article history: Aims: To determine the influence of location, seasonal variation and solvent system in
Received 24 June 2013 production of phytochemicals and antioxidants from ginkgo leaves.
Accepted 3 September 2013 Methods: Total phenolic and flavonoid contents and antioxidant activity in ginkgo leaf
Available online 25 September 2013 extracts were estimated spectrophotometrically. Factorial analysis was performed to
correlate the influence of location, season and solvent on production of phytochemicals
Keywords: and antioxidants.
Ginkgo biloba (ginkgo) Results: Total phenolic and flavonoid contents as well as the antioxidants were estimated
Phytochemicals maximum in autumn. Among solvents, acetone/water extracts gave best results for
Phenolics phenolic and flavonoid contents while methanolic extracts were best for antioxidants.
Antioxidants Phenolic content, the predominant indicator of phytochemicals, showed significant cor-
Indian Himalayan Region (IHR) relation with antioxidant activity.
Conclusion: Factorial analysis among location, season and solvent with respect to the
phytochemicals and antioxidants, was found to be statistically significant. Presence of
phytochemicals along with the protective feature in the form of antioxidants is indicative
of the importance of this species in pharmacological industry.
Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.

1. Introduction food and pharmacological industry.2 Factors, such as

Medicinal plants are known potential source of many
phenolic compounds and antioxidants. Among these, poly-
phenols in particular, have been recognized for antioxidant
activity and many other health benefits.1 Phenolic and fla-
vonoids, as natural antioxidants and free radical scavengers,
have involved substantial interest due to their importance in
geographic location, age of the plant, season, associated
microflora, nutritional status, and environmental stress are
known to influence the secondary metabolite profile of a
particular plant species. Seasonal variation in trees, for
example from dormant to active phase, brings progressive
changes in traits like production of phytochemicals.3 Be-
sides, optimization of methods with respect to solvent

* Corresponding author. Tel.: þ91 5962 241041; fax: þ91 5962 241150.
E-mail addresses: anita@gbpihed.nic.in, anitapandey333@gmail.com (A. Pandey).
0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jopr.2013.09.001
 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 0 4 e8 0 9 805

system is important for determination or extraction of the

phytochemicals from any plant species.
A 70 ME EA n-B AW WE

60
Ginkgo biloba L. (family Ginkgoaceae), commonly known
50
as living fossil, harbors many beneficial medicinal proper-

mg/g dw
40
ties. Traditionally, it has been used on an extensive basis,
30
either as food or medicinal component, almost all over the
20
world. The leaf extract of ginkgo contains pharmaceutically
10
imperative flavonoids, glycosides and ginkgolides which
0
expand blood flow, act as antioxidant and mainly used as GB1 GB2 GB3 GB4 GB5 GB1 GB2 GB3 GB4 GB5
memory enhancer and anti-vertigo.4 The present study is Total phenolic content Total falvonoid content
focused on the evaluation of phytochemicals and antioxi-
dants in leaf extracts of ginkgo along with the factorial B 12
analysis among locations  seasons, seasons  solvents and
10
locations  solvents.
8

mg/g dw
6
2. Materials and methods
4

2
2.1. Plant material
0
GB1 GB2 GB3 GB4 GB5 GB1 GB2 GB3 GB4 GB5 GB1 GB2 GB3 GB4 GB5
Ginkgo leaves were collected in three seasons from five
ABTS DPPH FRAP
different locations referred as GB1 (Kalika, Almora), GB2
(Chaubatia, Almora), GB3 (Snow view, Nainital), GB4 (High Fig. 1 e Phytochemicals and antioxidants in ginkgo leaf
court, Nainital) and GB5 (Glenthorn, Nainital) in Uttarakhand, extracts at different locations. (A) total phenolic and
India.5 The leaves, dried at room temperature, were grounded flavonoid contents, and (B) antioxidant activity (ABTS,
to fine powder and stored at 4  C for further analysis. DPPH, FRAP).

2.2. Preparation of extracts

Dried leaf powder (10 g) was mixed with 25 ml methanol (ME),
ethyl acetate (EA), n-butanol (n-B), acetone/water (AW) (3:2)
and water (aqueous/WE), separately. The leaf extract was
stirred continuously for 24 h and then filtered. The filtrate was
centrifuged at 10,000 rpm for 10 min and the supernatant, was
stored at 4  C prior to use (within 2 days).
assay was conducted in triplicates. The value for each sample
was calculated as the mean  SD. Factorial analysis of vari-
ance and significant difference among means were tested by
two way ANOVA in replication. Correlation coefficients were
calculated using Microsoft Excel 2007.

2.3. Determination of total phenolic and flavonoid

contents

Total phenolic and flavonoid contents were determined by

FolineCiocalteu’s and aluminum chloride calorimetric
methods, respectively6,7 following quantification on the basis of
standard curve of gallic acid and quercetin. Results are presented
in milligrams (mg) gallic acid (GAE) and quercetin (QE) equiva-
lent, respectively, per gram of leaf sample on dry weight basis.

2.4. Determination of antioxidant activity (radical

scavenging (ABTS), antiradical (DPPH) and reducing power
(FRAP) assays)

Total antioxidant activity was measured by ABTS, DPPH and

FRAP assays following methods of Cai et al8 and Amarowicz
et al9,10 Standard curve of a range of concentrations of ascorbic
acid was prepared for quantification of antioxidant potential.
Results were expressed in milligram (mg) ascorbic acid equiv-
alent (AAE) per gram of leaf sample on dry weight basis.

2.5. Statistical analysis Fig. 2 e Phytochemicals and antioxidants in ginkgo leaf

extracts in different seasons. (A) total phenolic and
Determination of total phenolic and flavonoid contents and flavonoid contents, and (B) antioxidant activity (ABTS,
antioxidant capacity by ABTS, DPPH and reducing power DPPH, FRAP).
806 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 0 4 e8 0 9

extracts of GB3 and GB4, respectively. In WE, maximum con-

A 60 Spring Rainy Autumn
tent was for GB4 and minimum for GB1. GB3 gave maximum
50 value for n-B and GB5 for EA for total phenolic content
40 (Fig. 1A). Total flavonoids were higher in GB3 in ME and n-B,
mg/g dw

30 respectively, in comparison to GB2 and GB4. Higher flavonoid

content was in EA for GB4 and in WE for GB5 (Fig. 1A). Anti-
20
oxidant activity in ABTS was higher in ME and WE for GB2,
10
respectively. Subsequently, GB1 gave higher antioxidant ac-
0
tivity in EA and AW, respectively, while GB3 showed
ME EA n-B AW WE ME EA n-B AW WE
Total phenolic content Total falvonoid content
maximum antioxidants in n-B. Based on DPPH assay, GB3
exhibited highest values for antioxidants in n-B, AW and WE,
B 12 respectively. For GB1 and GB5, highest values were recorded in
EA and ME, respectively. In FRAP assay, GB5 showed higher
10
activity in AW and WE, respectively; GB3 in n-B; GB2 in EA and
8 GB1 in ME (Fig. 1B).
mg/g dw

6 Variations in phytochemicals arise due to the specific

4
environmental conditions, including both biotic and
abiotic.11,12 Generally, with increase in altitude, environ-
2
mental conditions such as UV radiation, temperature, rainfall,
0
moisture, etc., changes occur rapidly. The biochemicals
ME EA n-B AW WE ME EA n-B AW WE ME EA n-B AW WE
ABTS DPPH FRAP
measured in ginkgo leaf extracts, in the present study, are on
the higher side as compared to the earlier reports from other
Fig. 3 e Phytochemicals and antioxidants in ginkgo leaf countries.13,14 The 5 locations in the present study, falling
extracts in different solvents (A) total phenolic and between 1742 and 2260 m altitude representing temperate
flavonoid contents, and (B) antioxidant activity (ABTS, climatic conditions, are likely to be associated with the higher
DPPH, FRAP). contents of phytochemicals and antioxidants. Findings on
production of polyphenols and antioxidants, in respect to
environmental stress, have been linked to the defense
3. Results and discussion mechanism.15

3.1. Phytochemicals and antioxidants in ginkgo leaves 3.2. Phytochemicals and antioxidants in ginkgo leaves
at different locations in different seasons

Significant variations (p < 0.05) were observed in phyto- Total phenolic content in ginkgo leaf extracts varied signifi-
chemicals and antioxidants in leaf extracts of different loca- cantly with respect to season and organic solvent, being
tions in different solvents. In ME and AW, GB2 gave higher maximum in autumn (Fig. 2A). Phenolic content was excep-
phenolic content, while lower values were recorded in EA tionally higher in rainy and spring season in EA and n-B. Total

Table 1 e Analysis of variance for determining effect of solvents, seasons and their interaction with phytochemicals and
antioxidant activity in ginkgo leaf extracts (given as f value).
Location Source of variation df Phytochemicals Antioxidant activity

TPC TFC ABTS DPPH FRAP

GB1 Solvent (S) 4 691,936.67*** 405,216.72*** 90,893.87*** 13,656.23*** 12,737.04***

Season (S) 2 51,039.16*** 8289.85*** 24,412.81*** 3616.33*** 1129.70***
SS 8 53,096.14*** 13,357.34*** 21,752.97*** 993.04*** 491.58***
GB2 Solvent (S) 4 2,025,206.05*** 444,923.81*** 211,154.29*** 28.89*** 4065.47***
Season (S) 2 33,178.64*** 9466.39*** 5331.77*** 3.96*** 48.73***
SS 8 14,617.20*** 10,601.75*** 5790.39*** 12.78*** 36.30***
GB3 Solvent (S) 4 1,031,256.71*** 483,295.95*** 243,030.95*** 20,906.01*** 3732.14***
Season (S) 2 94,933.48*** 16,824.75*** 8065.06*** 4389.47*** 1641.31***
SS 8 116,135.97*** 27,375.31*** 15,780.63*** 910.08*** 595.57***
GB4 Solvent (S) 4 888,494.27*** 593,733.35*** 162,822.13*** 10,388.55*** 77,975.80***
Season (S) 2 44,739.59*** 56,428.10*** 10,839.66*** 808.30*** 3861.01***
SS 8 53,242.26*** 25,616.70*** 3396.54*** 1331.49*** 6377.25***
GB5 Solvent (S) 4 1,195,793.77*** 272,908.46*** 160,479.36*** 10,078.51*** 468,226.40***
Season (S) 2 4423.19*** 12,862.07*** 14,936.57*** 2754.08*** 42,617.60***
SS 8 29,119.36*** 15,369.38*** 21,628.06*** 1519.68*** 24,587.54***

df e degree of freedom; dw e dry weight; TPC e Total phenolic content; TFC e Total flavonoid content; Level of significance: *** e p < 0.001.
 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 0 4 e8 0 9 807

Importance of seasonal variation in accumulation of total

Table 2 e Correlation matrix between total phenolic and
flavonoid contents and antioxidant activity in ginkgo phenolic and flavonoid contents and antioxidants has been
leaves from 5 locations using different assays. recognized. Although a clear and regular trend due to seasonal
variation was not observed in the present study, the total
TPC TFC ABTS DPPH FRAP
phenolic content was relatively higher in autumn. Kobus et al13
TPC 1 reported higher level of polyphenols in October as compared to
TFC 0.737** 1
August. Besides, higher accumulation of phenolic and flavo-
ABTS 0.7167** 0.639** 1
noids during winter is likely to be attributed to the stress con-
DPPH 0.376* 0.1924 0.507** 1
FRAP 0.734** 0.614** 0.639** 0.602** 1 ditions such as temperature and plant growth stage. In general,
the secondary metabolites remain at low level in ginkgo during
TPC e Total phenolic contents; TFC e Total flavonoid contents;
spring and summer which are the initial stages for the growth of
Level of significance: * e p < 0.05; ** e p < 0.01.
shoots and leaves. Afterward, towards autumn and winter, as
the growth and metabolism become slower, the phytochemi-
cals tend to accumulate in higher amounts.
flavonoid content was higher in spring in 3 solvents, AW, WE
and n-B, during rainy season in ME and during autumn in EA
(Fig. 2A). Antioxidant activity performed by three assays 3.3. Optimization of solvent systems for extraction of
showed significant variation with respect to the seasons, phytochemicals and antioxidants
maximum being in ABTS and DPPH in autumn (Fig. 2B). In case
of FRAP, higher activity was recorded during spring followed The optimization experiments conducted for preference of
by autumn (Fig. 2B). solvent revealed that AW was the best solvent for extracting

A 14.0 y = 0.0963x + 6.0404

R² = 0.5128
12.0
10.0
ABTS

8.0
6.0
4.0
2.0
0.0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0

B 1.8 y = 0.0104x + 0.5332

1.6 R² = 0.1411
1.4
1.2
DPPH

1.0
0.8
0.6
0.4
0.2
0.0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0

C
5.0 y = 0.0439x + 0.4705
R² = 0.5384
4.0

3.0
FRAP

2.0

1.0

0.0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0

Total phenolic content

Fig. 4 e Linear relationship between total phenolic contents and antioxidants measured by (A) ABTS (B) DPPH and (C) FRAP
in ginkgo leaf extracts.
808 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 0 4 e8 0 9

phenolic content in all the three seasons; followed by
ME > WE > n-B > EA. Similarly, total flavonoid content was
recorded highest in AW during rainy and autumn followed by
ME during spring (Fig. 3A). Different solvent systems also
influenced the extraction of antioxidant activity in different
seasons. Antioxidant activity measured by ABTS assay was
highest in ME in rainy and autumn and in WE in spring. In
DPPH assay, the activity was recorded highest in WE in all the
seasons. Also, the reducing power assay showed higher anti-
oxidant activity in AW during all the seasons (Fig. 3B). Facto-
rial analysis exhibited that the solvents and seasons
individually and their interaction significantly (p < 0.001)
influenced the accumulation of phytochemicals and antioxi-
dant activity (Table 1). The best results obtained for extraction
of phytochemicals and antioxidants in AW and ME are in line
with the earlier reports.13,16 Phenolic compounds are often
linked with other biomolecules, such as polysaccharides,
proteins, etc., therefore, an appropriate solvent system is
required for their extraction. Polarity of different solvents is
likely to have significant consequence on polyphenolic con-
tent and antioxidant activity as well.17 Importance of solvent
system has also been reported in determination of antimi-
crobial activity5 in ginkgo leaf extracts.
Among the three assays used for determination of anti-
oxidant activity in the present study, ABTS gave best results
followed by DPPH and FRAP. ABTS is soluble in both aqueous
and organic solvents and having reducing properties of 2, 2-
azinobis-(3-ethylbenzoline sulphonate) radical, in which the
antioxidant activity can be précised due to the hydrophilic and
lipophilic nature of the compound. DPPH, possessing ability to
get dissolved only in organic solvent, ethanol in particular,
can be predicted as an imperative restriction while inter-
preting the role of hydrophilic antioxidants. Previous studies
have also indicated the merits of using ABTS assay in
assessing antioxidant potential of plant extracts.18 With re-
gard to the FRAP, the antioxidants reduce the ferric ion/ferri-
cyanide complex to the ferrous form, the Perl’s Prussian blue
complex. The reducing power is related to the presence of the
compounds, which apply their action by flouting the free
radical chain through donating hydrogen atom compounds.19
The reducing power of extracts prepared from ginkgo leaves
has been reported.20

A 14.0 y = 0.1713x + 6.5087

R² = 0.4082
12.0
10.0
8.0
ABTS

6.0
4.0
2.0
0.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

B 1.8 y = 0.0107x + 0.6841

1.6 R² = 0.037
1.4
1.2
DPPH

1.0
0.8
0.6
0.4
0.2
0.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

C 5.0 y = 0.0733x + 0.7463

4.5 R² = 0.3766
4.0
3.5
3.0
FRAP

2.5
2.0
1.5
1.0
0.5
0.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

Total flavonoid content

Fig. 5 e Linear relationship between total flavonoid contents and antioxidants measured by (A) ABTS (B) DPPH and (C) FRAP
in ginkgo leaf extracts.
 j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 8 0 4 e8 0 9
3.4. Correlations among total phenolic and flavonoid
contents and antioxidant activity
Correlation matrix exhibited significant positive relationship
between total phenolic and flavonoid contents and the antiox-
idant activity performed by all the three assays (Table 2). Linear
regression analysis revealed that total phenolic content con-
tributes 14.1e51.2% of radical scavenging property (r2 ¼ 0.141 for
DPPH and 0.512 for ABTS) and 53.8% of reducing property
(r2 ¼ 0.538) (Fig. 4AeC). Likewise, total flavonoid content con-
tributes 3.7e40% of radical scavenging property (r2 ¼ 0.037 for
DPPH and 0.408 for ABTS) and 37% of reducing property
(r2 ¼ 0.376) (Fig. 5AeC). Similar findings have been reported in
other Himalayan species as well where total phenolic content
and antioxidant activity correlate positively.18
4. Conclusion
The IHR harbors plethora of medicinal plants. While the nat-
ural habitat of ginkgo is in China, Japan, and Korea, some
established trees have been reported from the hilly areas of
IHR, maximum being in the state of Uttarakhand. Ginkgo
possesses high amounts of phenolic contents and high levels
of gallic acid equivalents. Ginkgo trees, being in limited num-
ber and growing under low temperature climatic conditions,
extend opportunity to make use of these trees for under-
standing the physiological aspects, such as accumulation of
phytochemicals, production of antimicrobials, with emphasis
on propagation and conservation of the species.5,21,22,23
Conflicts of interest
All authors have none to declare.
Acknowledgments
Director, GB Pant Institute of Himalayan Environment and
Development, Almora, is gratefully acknowledged for
extending the facilities. Council of Scientific and Industrial
Research and Ministry of Environment and Forests, Govt. of
India are thanked for financial support.
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