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Antioxidant and phytochemical analysis of volatile oil and extracts of Pinus wallichiana

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The present study aims to explore the chemical constituents and antioxidant potential of Pinus wallichiana essential oil and extracts. Chemical composition of the essential oil was analyzed by GC-MS and result showed that the oil contain three major monoterpene compounds viz. α-pinene (48.6%), β-pinene (45.6%) and limonene (5.6%). Antioxidant potential of Pinus wallichiana was analyzed by three basic methods; DPPH radical scavenging method, FRAP assay and Fe2+ ion chelating activity. Among leaf and bark extracts, best radical scavenging, activity was determined by aqueous extract (IC50 values 20.83±0.8µg/ml) of bark followed by its methanol extract (IC50 value 25.9±1.6µg/ml). The aqueous extract of bark also exhibited better reducing and chelating activity than leaf extracts. Essential oil showed moderate radical scavenging and chelating activity but negligible reducing activity. Phytochemical analysis of extracts determined that the aqueous and methanol extracts of bark contain rich amount of poly phenol and flavonoids, in comparison to the leaf extracts. A significant correlation between the antioxidant activity and these phytoconstituents of the extracts has been observed. The results of the present study concluded that the bark of Pinus wallichiana is a potential source of active antioxidant constituents that could be further explored and exploited for numerous commercial applications.
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Introduction
Oxidative stress and their adverse effect on human health has
become a serious problem. Under stress conditions, there is increase
in production of reactive species viz., hydrogen peroxide (H2O2),
superoxide ion (O2), and hydroxide radical (OH) during cellular
respiration than enzymatic antioxidants (e.g., superoxide dismutase
(SOD), glutathione peroxidase (GPx), and catalase) and non-enzymatic
antioxidants (e.g., ascorbic acid (vitamin C), α-tocopherol (vitamin
E), glutathione, carotenoids, and avonoids). This imbalance between
reactive oxygen species production and the antioxidant defence
could cause damage to cellular bio-molecules like carbohydrates,
proteins, lipids and DNA, that may leads to metabolic and genetic
alteration.1 A lack of antioxidants could facilitates the development
of degenerative diseases,2 including cardiovascular diseases, cancers,3
neurodegenerative diseases, Alzheimer’s disease4 and inammatory
diseases.5
Medicine plants are effective in treating various diseases caused
by oxidative stress. The medicinal properties of plants are because
to their biologically active metabolites like phenolic and avonoid
compounds, which are potential antioxidants.6,7 Positive correlation
between these phytochemicals in plant extracts and antioxidant
activity has been reported b many researchers.6,8,9
Pinus wallichiana commonly called Blue pine or Kail, is evergreen
coniferous tree native to Himalaya, grows in mountains at altitudes of
1800–4300m. it yields superior timber and especially used as joinery
wood. The wood yields oleoresin (by tapping) which by distillation
furnishes turpentine and rosin. It is a source of numerous valuable
bye products, such as gases, light and heavy tar oils, turpentine
pitch, pyroligneous acids, etc. The residue of wood is withdrawn as
charcoal. Pinus wallichiana essential oil produced by distillation is
used for medical purposes. Earlier, some efforts were made to use
leaves by reducing them to bre which was used for weaving into
medicinal underclothing, for the manufacture of coarse matting,
resembling coconut mating, for surgical dressings. During the recent
past, in many rural areas of Kashmir, blue pine wood pieces were used
to obtain a dark brown, viscous and sticky substance, called killam.
This substance was traditionally applied by farmers on their arms
and legs to protect them from insects (Khase) while working in water
logged paddy elds. Killam would stick rmly; thereby protecting
the exposed parts from insect bites (Khase), etc.10 So keeping the
immense traditional importance of Pinus wallichiana in view, the
present study aims to explore the phytochemicals and antioxidant
properties of different parts of Pinus wallichiana.
Material and methods
Plant material and Preparation of extracts
Spines and bark of Pinus wallichiana was collected from China
region of Bhaderwah, Jammu and Kashmir. Identication of the plant
material was done an expert taxonomist of Department of Botany,
University of Jammu, Jammu. Plant material (leaf and bark separately)
was shade dried was powdered in an electronic grinder for the
preparation of extracts. Three types of extracts were prepared in three
different solvents viz., water, methanol and chloroform. 200g of dried
powder was extracted thrice for each solvent. Resulting extracts were
pooled, ltered and evaporated by using rotary vacuum evaporator.
The slurry obtained was nally lyophilized to dried powder.11
MOJ Biol Med. 2019;4(2):3740. 37
© 2019 Sharma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and build upon your work non-commercially.
Antioxidant and phytochemical analysis of volatile oil
and extracts of Pinus wallichiana
Volume 4 Issue 2 - 2019
Pooja Sharma, Sahil Gupta, Nazli Bhatt,
Shajaat Hussain Ahanger, Divya Gupta,
Pramveer Singh, Roshani Lochan, Madhulika
Bhagat
School of Biotechnology, University of Jammu, India
Correspondence: Madhulika Bhagat, School of Biotechnology,
University of Jammu, Jammu, J&K-180006, India, Tel 011-
9419124018, Email
Received: November 23, 2018 | Published: April 30, 2019
Abstract
The present study aims to explore the chemical constituents and antioxidant
potential of Pinus wallichiana essential oil and extracts. Chemical composition of
the essential oil was analyzed by GC-MS and result showed that the oil contain
three major monoterpene compounds viz. α-pinene (48.6%), β-pinene (45.6%)
and limonene (5.6%). Antioxidant potential of Pinus wallichiana was analyzed by
three basic methods; DPPH radical scavenging method, FRAP assay and Fe2+ ion
chelating activity. Among leaf and bark extracts, best radical scavenging, activity
was determined by aqueous extract (IC50 values 20.83±0.8µg/ml) of bark followed
by its methanol extract (IC50 value 25.9±1.6µg/ml). The aqueous extract of bark
also exhibited better reducing and chelating activity than leaf extracts. Essential oil
showed moderate radical scavenging and chelating activity but negligible reducing
activity. Phytochemical analysis of extracts determined that the aqueous and methanol
extracts of bark contain rich amount of poly phenol and flavonoids, in comparison to
the leaf extracts. A significant correlation between the antioxidant activity and these
phytoconstituents of the extracts has been observed. The results of the present study
concluded that the bark of Pinus wallichiana is a potential source of active antioxidant
constituents that could be further explored and exploited for numerous commercial
applications.
Keywords: Pinus wallichiana, antioxidant, phytochemicals
MOJ Biology and Medicine
Research Article Open Access
Antioxidant and phytochemical analysis of volatile oil and extracts of Pinus wallichiana 38
Copyright:
©2019 Sharma et al.
Citation: Sharma P, Gupta S, Bhatt N, et al. Antioxidant and phytochemical analysis of volatile oil and extracts of Pinus wallichiana. MOJ Biol Med.
2019;4(2):3740. DOI: 10.15406/mojbm.2019.04.00111
Isolation and chemical analysis of essential oil
Fresh spines of Pinus wallichiana were subjected to hydro
distillation in a Clevenger type apparatus for 3-4h for isolation of
essential oil. The extracted oil was dried by adding anhydrous sodium
sulphate and stored at low temperature.12 Chemical composition of the
isolated essential oil was analyzed at Indian Institute of Integrative
Medicine (CSIR, India), Canal Road, Jammu, India. System used for
analysis is GC-MS 4000 (Varian, USA) with a Varian CP-SIL 8CB
column (30m×0.32mm i.d., 1μm lm thickness). Injector temperature
was 230°C. Oven temperature program used was holding at 60°C
for 5min, heating to 250°C at 3°C/min. Helium as a carrier gas used
at a constant ow of 1.0 ml/min and an injection volume used was
0.20μl. The Mass spectrometer scan parameters included electron
impact ionization voltage of 70 eV, a mass range of 40–500m/z. The
components present in essential oil were analyzed by comparing their
mass spectra with the compounds available in NIST05 (version 2.0)
library.13
DPPH radical scavenging activity
Antiradical activity of the extracts and essential oil was determined
by DPPH method. Essential oil was analyzed by method given by
Bozin et al.14 1ml of different concentrations of the test sample was
mixed with 1ml of a DPPH (90𝜇M, in methanol), and nal volume
was made to 4ml with methanol. The mixtures were kept at 25°C in
the dark for 1hour. The absorbance was measured at 517nm using a
UV-VIS spectrophotometer.
Free radical scavenging activity of Pinus wallichiana extracts
was determined by DPPH method given by Abe et al.15 with some
modications. 1ml of DPPH (0.5mM in methanol) solution was
mixed to 2 ml sample and to this 2ml of 0.1 M sodium acetate buffer
(pH 5.5) was added. The mixtures were well shaken and kept at room
temperature in the dark for 30min. The absorbance was measured at
517nm. The radical scavenging activity was calculated as a percentage
of DPPH radical discoloration, using the equation:
( )
00
% 100
S
Radical Scavenging Activity A A A

×
=
Where, A0 is the absorbance of the control and AS is the absorbance
of the test compound.
FRAP assay
Reducing power of Pinus wallichiana extracts was analyzed by
FRAP assay according to the method followed by Li et al.16 FRAP
reagent was prepared in acetate buffer (300mM) by adding 10mM
2,4,6-tri (2-pyridyl)-s-triazine (TPTZ) solution in 40mM HCl and
20mM FeCl3 solution in proportion of 10:1:1 (v/v), respectively.
FRAP reagent was prepared fresh at the time of use. 50µl of the
sample was added to 1.5ml of the FRAP reagent and after 3-4min,
absorbance was measured at 593nm. A standard curve was prepared
by using FeSO4 (100-2000µM) and the result was expressed as µmol
Fe (II)/gm dry weight of extract.
Chelation activity
The chelating power of the Pinus wallichiana Extracts on ferrous
ions was determined by the method given by Dinis et al.17 Test sample
(500µl) and 740µl of methanol were added to 20µl of 2mM FeCl2. The
reaction was initiated by the addition of 40µl of 5mM ferrozine into
the mixture. The reaction mixture was incubated at room temperature
for 10min and nally the absorbance was measured at 562nm. The
ratio of inhibition of ferrozine-Fe2+ complex formation was calculated
using the equation:
( )
00
% 100
S
chelating power I I I

×
= −
Where, I0 is the absorbance of the control and IS is the absorbance
of the test compound.
Total phenols and avonoids in extracts
Total phenolic content in the extracts was estimated by Folin-
Ciocalteu method.18 0.5ml of extract was mixed with 0.5ml of 1N
Folin-Ciocalteu reagent and 1ml of 20% Na2CO3. After 10 min of
incubation, the absorbance was measured at 750nm Gallic acid was
used as standard compound to estimate total phenolic content and was
expressed in mg GAE/g of the extract.
Total avonoid content of the extracts was estimated by AlCl3
colorimetric method.19 Plant extracts were diluted with distilled water
to a volume of 3.5ml and added 150µl of a 5% NaNO2 solution. After
5min, 300µl of 10% AlCl3 solution was added. After 6min, 300µl
of 1M NaOH and 550µl of distilled water were added. The mixture
was well shaken and absorbance was measured at 510nm in UV-VIS
spectrophotometer. Quercetin was used as standard compound to
estimate total avonoid content in extracts and was expressed in mg
GAE/g of the extract.
Results and discussion
Hydro-distillation of fresh needles of Pinus wallichiana yielded
1.2% light yellow coloured essential oil. Chemical composition of
the essential oil was analyzed by Gas Chromatography and Mass
Spectrometry (GC-MS). Analysis showed the presence of three major
components α-pinene (48.6%), β-pinene (45.6%) and limonene (5.6%)
accounting for 99.8% of total components of the essential oil. All the
three compounds found in essential oil belong to monoterpene class
of terpenoids. Dar et al.20 also analyzed the chemical composition of
the essential oil of Pinus wallichiana growing in high altitudes of
Kashmir, India. They found diverse range of compounds in essential
oil but still in agreement that β-pinene (46.8%) and α-pinene (25.2%)
are the major components of the essential oil. The difference in
chemical composition might be due to spatial variation as the plant
samples are from two different regions of Jammu and Kashmir.
Antioxidant activity of Pinus wallichiana was analyzed by three
different methods viz., DPPH radical scavenging activity, metal ion
chelating activity and FRAP assay. The results of antioxidant activity
have been given in Table 1. The DPPH radical scavenging activity was
determined on the basis of concentration providing 50% inhibition.
Among the leaf and bark extracts, highest antiradical potential was
observed in aqueous and methanol bark extracts with IC50 values
20.83±0.8µg/ml and 25.9±1.6µg/ml respectively. Chloroform extract
of both leaf and bark have shown negligible radical scavenging
activity. Essential oil isolated from leaf part showed moderate radical
scavenging activity with IC50 values 514.4±5.8µg/ml.
The ability of antioxidants to reduce Fe3+ to Fe2+ in the presence
of TPTZ, was determined in FRAP assay. Reducing potential of
Pinus wallichiana extracts and essential oil was expressed in μmol
Fe (II)/g dry weight of plant material. Strongest antioxidant activity
Antioxidant and phytochemical analysis of volatile oil and extracts of Pinus wallichiana 39
Copyright:
©2019 Sharma et al.
Citation: Sharma P, Gupta S, Bhatt N, et al. Antioxidant and phytochemical analysis of volatile oil and extracts of Pinus wallichiana. MOJ Biol Med.
2019;4(2):3740. DOI: 10.15406/mojbm.2019.04.00111
to reduce the Ferric ions (Fe3+) was observed in bark aqueous extract
(3495±11.3μmol Fe (II)/g) followed by bark methanol extract
(2530±6.3μmol Fe (II)/g) like in case of radical scavenging activity.
All leaf extracts and essential oil exhibited very poor reducing activity
in comparison to the bark extracts.
Table 1 Total Phenols, avonoids and antioxidant potential of Pinus wallichiana extracts
Extract
TPC (mg
GAE/g dry
weight)
TFC (mg QE/g
dry weight)
DPPH Assay (IC50
in µg/ml)
FRAP Assay
(µmol FeII /g)
%age Chelation
capacity at 500µg
(Leaves)
Methanolic
Aqueous
Chloroform
Essential oil
30.8±1.1 - 260.4±3.2 111.6±3.2 57±1.2
40±1.1 10±0.5 285.3±4.1 86.6±1.3 19±0.6
10±0.4 - - 26.6±2.1 34±0.4
- - 514.4±5.8 - 16±0.3
(Bark)
Methanolic
Aqueous
Chloroform
330±6.4 520±12.a7 25.9±1.6 2530±6.3 59±1.8
447±9.6 610±11.2 20.83±0.8 3495±11.3 44±1.4
30±0.5 - - 245±3.3 29±0.9
Data presented as mean±standard deviation; TPC, Total phenolic content; TFC, Total avonoid content; GAE, Gallic acid equivalent; QE, Quercetin equivalent.
Chelating property on metal ions is one of important mechanism
of antioxidant activity. Metal ion chelating capacity was determined
at 0.5mg/ml concentration. Methanol extract of both leaf and bark
of Pinus wallichiana showed a better chelating effect on ferrous
ions (59±1.8% & 57±1.2%). Aqueous extract of bark also showed
moderate chelating capacity with 47% at 0.5mg/ml. Essential oil
and chloroform extracts determined poor chelating capacity of fe2+
ions. Biological properties of phenolic and avonoid compounds
have already been known universally. Table 1 also represented total
phenol and total avonoid content in leaf and bark extracts of Pinus
wallichiana. Highest amount of poly phenols and avonoids was
observed in aqueous extract of bark (447±9.6mg GAE/g; 610±11.2
QE/g dry weight, respectively) followed by methanol extract of bark
(330±6.4 mg GAE/g; 520±12.7 QE/g dry weight). Leaf extracts
possess comparatively less poly phenol and avonoid content.
Among all the extracts, least content of poly phenol and avonoid
was observed in chloroform extracts of both leaf and bark. Many
studies have shown that the antioxidant activities in the plants are
associated with their phytochemicals contents. Naeem et al.21 reported
that the methanol extract of Pinus wallichiana (bark) growing in
Kuldana, Murree (Pakistan), contained a wide variety of avanols
in considerable amounts. Dar et al.20 analyzed that the essential oil
isolated from Pinus wallichiana growing in Kashmir (J&K), has
strong antiproliferative potential against ve human cancer cell lines
and good radical scavenging activity on DPPH radicals. Maimoona
et al.22 also reported antioxidant potential and phytochemicals in
different extracts of Pinus wallichiana growing in Pakistan and found
a positive correlation between the antioxidant properties and total
avonoid content. This may be due to their redox properties of the
phenolic and avonoids that make them good reducing, scavenging
and chelating agent.18,23 The results also revealed that there is
signicant correlation between the antioxidant activity (Reducing
activity, Radical scavenging activity, and chelating power) and the
phenolic/avonoid content of Pinus wallichiana extracts. Therefore,
it is possible that these phytochemical compounds are the major
contributor for the antioxidant activity of the Pinus wallichiana.
Conclusion
In conclusion, the present study explored the phyto constituent and
antioxidant properties of Pinus wallichiana growing in Bhaderwah
region of Jammu & Kashmir (India). GC-MS analysis showed that
its essential oil is a rich source of α-pinene and β-pinene and possess
moderate antioxidant activity. Bark of Pinus wallichiana determined
strong antioxidant activity and can serve as a valuable source of
natural antioxidant. A signicant correlation between antioxidant
activity and TPC/TFC were found indicating phenolic and avonoids
compounds are the major contributor of antioxidant potential of Pinus
wallichiana.
Acknowledgments
Authors would like to acknowledge the School of Biotechnology
and Department of Bio-informatics, University of Jammu, for their
support. The authors also wish to acknowledge Instrumentation
division, IIIM-Jammu, J&K (India) for their kind support.
Antioxidant and phytochemical analysis of volatile oil and extracts of Pinus wallichiana 40
Copyright:
©2019 Sharma et al.
Citation: Sharma P, Gupta S, Bhatt N, et al. Antioxidant and phytochemical analysis of volatile oil and extracts of Pinus wallichiana. MOJ Biol Med.
2019;4(2):3740. DOI: 10.15406/mojbm.2019.04.00111
Conicts of interest
Author declares that there are no conicts.
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... The percentage yield obtained in our study is same in case of PWNSEO and very close in case of PWNHEO to the previously reported study in which Maurya et al. 31 extracted essential oil of needles of Pinus wallchiana and yield was found to be 0.35%. Dambolena et al. 22 and Sharma et al. 1 revealed a greater yield of oils as compared to our study which were 0.97% and 1.2% respectively. ...
... 21 who reported the radical scavenging activity of essential of bark of Pinus wallchiana with IC50 value 58.4 μg/mL. Shrama et al. 1 also reported the radical scavenging activity of essential oil of Pinus wallchiana isolated from leaf part with IC50 value 514.4 μg/mL and this IC50 value is very much high than our findings for PWNSEO (41.74 µg /mL) and PWNHEO (33.15 µg /mL). Values are mean ± standard deviations of triplicate determinations. ...
... Antibacterial activity of P. wallachiana essential oil against tested bacterial strains 36 and antifungal e cacy of its essential oil against Fusarium verticillioides 37 , antimicrobial activity of its hydroalcoholic extracts against tested bacterial strains and fungi 38 , antibacterial activity against Acinetobacter baumannii 29 put forward its antimicrobial potential. Phytochemical studies reported antioxidant activity of P. wallachiana extracts that accounts for the presence of plentiful avanoids and polyphenols in their phytochemical composition 32,39 . Phenolic compounds i.e. chlorogenic acid, catechins, ferulic acid, caffeic acid are wellknown toxic compounds that are much faster concentrated in resistant varieties after their infection by the pathogen 40 . ...
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