# Antioxidant Activity of Essential Oil and Extracts ofValeriana jatamansi Roots

Authors: Sakshima Thusoo; Sahil Gupta; Rasleen Sudan; Jaspreet Kour; Sahil Bhagat; Rashid Hussain; Madhulika Bhagat
Journal: BioMed Research International
 (2014)

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## Abstract

Valeriana jatamansi is an indigenous medicinal plant used in the treatment of a number of diseases. In the present study, chemical composition of the essential oil was determined by GC-MS. Seven major components were identified in Valeriana jatamansi essential oil, namely, β-vatirenene, β-patchoulene, dehydroaromadendrene, β-gurjunene, patchoulic alcohol, β-guaiene, and α-muurolene. Methanolic, aqueous, and chloroform extracts of Valeriana jatamansi roots were also prepared and analyzed for their polyphenols and flavonoid content. Antioxidant activity of essential oil and different extracts of Valeriana jatamansi roots was determined by DPPH radical scavenging and chelation power assay. A linear correlation has been obtained by comparing the antioxidant activity and polyphenols and flavonoid content of the extracts. Results indicated that antioxidant activity of methanolic extract could be attributed to the presence of rich amount of polyphenols and flavonoid. Essential oil of Valeriana jatamansi roots showed moderate antioxidant activity.


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## Body

## 1. Introduction

Valeriana jatamansiJones syn.V. wallichiipopularly known as Indian Valerian (Mushkibala in Hindi/Kashmiri, Suganthdhawal or Tagara in Sanskrit) belongs to family Valerianaceae [1].Valerianais a major genus of the family Valerianaceae and is represented in all the temperate and subtropical areas of the world. In India, about 16 species and two subspecies have been reported [2].Valeriana jatamansi is a small, perennial dwarf, hairy, rhizomatous herb having thick roots covered with fibers. The plant grows at an altitude of 1220–3000 m [1].Valeriana jatamansi is regarded as an aphrodisiac, antispasmodic, tranquilizer, antiseptic, expectorant, febrifuge, nerve tonic, ophthalmic, sedative, and tonic useful in hysteria, cholera, snakebite, scorpion sting, asthma, and eurosis [3]. Roots are acrid and bitter which are used as carminative, laxative and are also used for curing blood diseases, burning sensation, cholera, skin disease, throat troubles, and ulcers [3]. The therapeutic properties of the plant are attributed to a class of compounds called valepotriates. The valepotriates are a group of monoterpenoids of iridoid type having epoxy group and β-acetoxy isovaleric acids [4]. The root ofV. jatamansi is a source of effective antileishmanial agent [5]. Root extract ofV. jatamansi also exhibits larvicidal and adulticidal activity against different mosquito species [6]. The aqueous and methanolic extracts of rhizomes possess anti-inflammatory activity. This could be attributed to the high amount of flavonoids and tannins in the plant [7]. The objective of this study was to verify the phytochemicals and antioxidant potential of both essential oil and extracts ofValeriana jatamansiroots.


## 2. Materials and Methods

### 2.1. Collection of Plant Material

Fresh plant material (roots) was collected from the high altitude of Patnitop in Jammu and Kashmir, India, and identified at the herbarium of the department of Botany, Jammu University. The material was shade dried and ground to a fine texture in a grinding machine.


### 2.2. Isolation of Essential Oil

Essential oil was extracted by hydrodistillation for 4 h using a Clevenger-type apparatus. The oil was stored at 4°C in the dark until analyzed.


### 2.3. Preparation of Extracts

The finely powdered roots were extracted with three different solvents on the basis of their polarity, that is, from nonpolar to polar. Chloroform, methanol, and water were used as solvents. 15 g of the given powdered plant material was mixed in 75 mL of each solvent and the mixture was stirred for 24 hours. The suspended mixture was filtered through whatman’s filter paper and filtrate was collected. This procedure was performed thrice to get three filtrates and residue. The filtrates were then dried at room temperature. Gummy solid thus obtained after evaporation of each of the solvents was labelled and stored for further use.


### 2.4. GC-MS Analysis of Essential Oil

Analysis of the oil using gas chromatography and mass spectrometry was carried out at Indian Institute of Integrative Medicine (CSIR, India), Canal Road, Jammu, India. GC-MS 4000 (Varian, USA) system with a HP-5MS agilent column (30 m × 0.25 mm i.d., 0.25μ film thickness) was used for analysis. Injector temperature was 280°C. Oven temperature programme used was holding at 50°C for 5 min, heating to 280°C at 3°C/min, and keeping the temperature constant at 280°C for 7 min. Helium was used as a carrier gas at a constant flow of 1.0 mL/min and an injection volume of 0.20 μL was employed.The MS scan parameters included electron impact ionization voltage of 70 eV, a mass range of 40–500 m/z. The identification of components of the essential oil was based on comparison of their mass spectra with those stored in NIST05 library or with mass spectra from literature [8].


### 2.5. Determination of Total Phenols and Flavonoids in Extracts

Total phenolic content was determined according to Folin-Ciocalteu method [9]. 0.5 mL of extract solution was mixed with 0.5 mL of 1 N Folin-Ciocalteu reagent. The mixture was kept for 5 min, followed by the addition of 1 mL of 20% Na2CO3. After 10 min of incubation at room temperature, the absorbance was measured at 750 nm using a spectrophotometer. The concentration of phenolic compounds was calculated according to the following equation obtained from the standard gallic acid:
(1)
Absorbance
=
0.0364
gallic
acid
(
μ
g
)
+
0.009
.Flavonoid content in the extract/fractions was determined by a colorimetric method [10]. Plant extracts were diluted with distilled water to a volume of 3.5 mL and 150 μL of a 5% NaNO2 solution. After 5 min, 300 μL of 10% AlCl3·H2O solution was added. After 6 min, 300 μL of 1 M NaOH and 550 μL of distilled water were added to prepare the mixture. The solution was mixed well and the absorbance was observed at 510 nm using UV-VIS spectrophotometer. The concentration of flavonoid compounds was calculated according to the following equation obtained from the standard quercetin graph:
(2)
Absorbance
=
0.001
quercetin
(
μ
g
)
+
0.032
.


### 2.6. DPPH Radical Scavenging Assay

The radical scavenging activity of extracts was determined with slight modifications in the method [11]. 1 mL from a 0.5 mM methanol solution of the DPPH radical was mixed to 2.0 mL sample and to this 2.0 mL 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 30 min. The absorbance was measured at 517 nm using a double beam UV-VIS spectrophotometer. Methanol was used as a negative control.The radical scavenging activity of essential oil was determined [12]. 1 mL of different concentrations of the essential oil or bioactive fraction was mixed with 1 mL of a 90 μM DPPH solution in methanol, and final volume was made to 4 mL with methanol. The mixtures were well shaken and kept at 25°C in the dark for 1 h. The absorbance was measured at 517 nm. Oil concentration providing 50% inhibition (IC50) was calculated from the graph by plotting inhibition% against oil concentration. BHT was used as reference.


### 2.7. Chelating Power on Ferrous (Fe2+) Ions

The chelating effect on ferrous ions ofValeriana jatamansiextracts was estimated by slight modifications in the method [13]. Different dilutions of extract/fractions were made to a volume of 3 mL with methanol. 60 μL of 2 mM FeCl2 was added. The reaction was initiated by the addition of 120 μL of 5 mM ferrozine into the mixture, which was then left at room temperature for 10 min before determining the absorbance of the mixture at 562 nm. The ratio of inhibition of ferrozine-Fe2+ complex formation was calculated using the following equation:
(3)
%
inhibition
=
(
[
absorbance
of
control
-
absorbance
of
test
sample
]
absorbance
of
control
)
×
100
.


### 2.8. Statistical Analysis

For all the experiments, three samples were analysed and all the assays were carried out in triplicates. The results were expressed as mean values with standard deviation.


## 2.1. Collection of Plant Material

Fresh plant material (roots) was collected from the high altitude of Patnitop in Jammu and Kashmir, India, and identified at the herbarium of the department of Botany, Jammu University. The material was shade dried and ground to a fine texture in a grinding machine.


## 2.2. Isolation of Essential Oil

Essential oil was extracted by hydrodistillation for 4 h using a Clevenger-type apparatus. The oil was stored at 4°C in the dark until analyzed.


## 2.3. Preparation of Extracts

The finely powdered roots were extracted with three different solvents on the basis of their polarity, that is, from nonpolar to polar. Chloroform, methanol, and water were used as solvents. 15 g of the given powdered plant material was mixed in 75 mL of each solvent and the mixture was stirred for 24 hours. The suspended mixture was filtered through whatman’s filter paper and filtrate was collected. This procedure was performed thrice to get three filtrates and residue. The filtrates were then dried at room temperature. Gummy solid thus obtained after evaporation of each of the solvents was labelled and stored for further use.


## 2.4. GC-MS Analysis of Essential Oil

Analysis of the oil using gas chromatography and mass spectrometry was carried out at Indian Institute of Integrative Medicine (CSIR, India), Canal Road, Jammu, India. GC-MS 4000 (Varian, USA) system with a HP-5MS agilent column (30 m × 0.25 mm i.d., 0.25μ film thickness) was used for analysis. Injector temperature was 280°C. Oven temperature programme used was holding at 50°C for 5 min, heating to 280°C at 3°C/min, and keeping the temperature constant at 280°C for 7 min. Helium was used as a carrier gas at a constant flow of 1.0 mL/min and an injection volume of 0.20 μL was employed.The MS scan parameters included electron impact ionization voltage of 70 eV, a mass range of 40–500 m/z. The identification of components of the essential oil was based on comparison of their mass spectra with those stored in NIST05 library or with mass spectra from literature [8].


## 2.5. Determination of Total Phenols and Flavonoids in Extracts

Total phenolic content was determined according to Folin-Ciocalteu method [9]. 0.5 mL of extract solution was mixed with 0.5 mL of 1 N Folin-Ciocalteu reagent. The mixture was kept for 5 min, followed by the addition of 1 mL of 20% Na2CO3. After 10 min of incubation at room temperature, the absorbance was measured at 750 nm using a spectrophotometer. The concentration of phenolic compounds was calculated according to the following equation obtained from the standard gallic acid:
(1)
Absorbance
=
0.0364
gallic
acid
(
μ
g
)
+
0.009
.Flavonoid content in the extract/fractions was determined by a colorimetric method [10]. Plant extracts were diluted with distilled water to a volume of 3.5 mL and 150 μL of a 5% NaNO2 solution. After 5 min, 300 μL of 10% AlCl3·H2O solution was added. After 6 min, 300 μL of 1 M NaOH and 550 μL of distilled water were added to prepare the mixture. The solution was mixed well and the absorbance was observed at 510 nm using UV-VIS spectrophotometer. The concentration of flavonoid compounds was calculated according to the following equation obtained from the standard quercetin graph:
(2)
Absorbance
=
0.001
quercetin
(
μ
g
)
+
0.032
.


## 2.6. DPPH Radical Scavenging Assay

The radical scavenging activity of extracts was determined with slight modifications in the method [11]. 1 mL from a 0.5 mM methanol solution of the DPPH radical was mixed to 2.0 mL sample and to this 2.0 mL 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 30 min. The absorbance was measured at 517 nm using a double beam UV-VIS spectrophotometer. Methanol was used as a negative control.The radical scavenging activity of essential oil was determined [12]. 1 mL of different concentrations of the essential oil or bioactive fraction was mixed with 1 mL of a 90 μM DPPH solution in methanol, and final volume was made to 4 mL with methanol. The mixtures were well shaken and kept at 25°C in the dark for 1 h. The absorbance was measured at 517 nm. Oil concentration providing 50% inhibition (IC50) was calculated from the graph by plotting inhibition% against oil concentration. BHT was used as reference.


## 2.7. Chelating Power on Ferrous (Fe2+) Ions

The chelating effect on ferrous ions ofValeriana jatamansiextracts was estimated by slight modifications in the method [13]. Different dilutions of extract/fractions were made to a volume of 3 mL with methanol. 60 μL of 2 mM FeCl2 was added. The reaction was initiated by the addition of 120 μL of 5 mM ferrozine into the mixture, which was then left at room temperature for 10 min before determining the absorbance of the mixture at 562 nm. The ratio of inhibition of ferrozine-Fe2+ complex formation was calculated using the following equation:
(3)
%
inhibition
=
(
[
absorbance
of
control
-
absorbance
of
test
sample
]
absorbance
of
control
)
×
100
.


## 2.8. Statistical Analysis

For all the experiments, three samples were analysed and all the assays were carried out in triplicates. The results were expressed as mean values with standard deviation.


## 3. Results and Discussions

### 3.1. GC-MS Analysis of Essential Oil

Hydrodistillation of roots ofValeriana jatamansi produced greenish yellow essential oil with the yield of 0.8%. Chemical composition of the essential oil was determined by gas chromatography and mass spectrometry (Table 1). Seven major components were identified in essential oilviz., β-vatirenene, β-patchoulene, Dehydroaromadendrene, β-gurjunene, patchoulic alcohol, β-guaiene and α-muurolene. The chemical analysis of essential oil shows thatV. jatamansi roots contain only sesquiterpenes in its essential oil.Table 1
Chemical composition of essential oil ofValeriana jatamansi roots analysed by GC-MS.
Compounds
Nature of compound
Amount in % age
β-patchoulene
Sesquiterpene
20.18
β-gurjunene
Sesquiterpene
13.0
β-vatirenene
Sesquiterpene
28.07
α-muurolene
Sesquiterpene
5.20
β-guaiene
Sesquiterpene
5.88
Dehydroaromadendrene
Sesquiterpene
15.92
Patchoulic alcohol
Sesquiterpene
11.72


### 3.2. Total Phenols and Flavonoids

Phenolic compounds have been reported to exhibit various biological activities like, antioxidant, antimicrobial etc. Total phenolic compounds in extracts were determined by Folin-Ciocalteu method and expressed as Gallic acid equivalents (GAEs). As shown in Table2, highest amount of phenolic compound was observed in methanolic extract (187.13 ± 6.8 mg GAEs/g), followed by aqueous extract (77.66 ± 2.1 mg GAEs/g). Least amount of phenolic compounds was observed in chloroform extract (9.89 ± 0.3 mg GAEs/g). Many studies have revealed that the phenolic contents in the plants are associated with their antioxidant activities probably due to their redox properties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers [9, 14].Table 2
Showing total phenol and flavonoid content of different extracts ofValeriana jatamansi roots.
S. no.
Test sample
Total phenols (mg GAEs/g dry wt.)
Total flavonoids (mg QEs/g dry wt.)
1
Methanolic extract
187.13 ± 6.8
257.69 ± 9.8
2
Aqueous extract
77.66 ± 2.1
452.30 ± 12.4
3
Chloroform extract
9.89 ± 0.3
25.38 ± 2.0
Data is represented as mean ± SD of three triplicate experiments.Total amount of flavonoids was expressed in quercetin equivalent (QE). Aqueous extract ofValeriana jatamansi had relatively high amount of flavonoids (452.30 ± 12.4 mg QEs/g) followed by methanolic extract (257.69 ± 9.8 mg QEs/g) and very less amount was observed in chloroform extract (5.38 ± 0.3 mg QEs/g).


### 3.3. DPPH Radical Scavenging Activity

DPPH assay has been extensively used for screening plant extracts because many samples can be accommodated in short period and are sensitive enough to detect active ingredients at low concentrations [15]. The antioxidant activity ofValeriana jatamansi roots essential oil and extracts is summarized in Table 3. Among all the test samples, methanolic extract ofValeriana jatamansi was found to be the most potent antioxidant (IC50 78 ± 2.9 μg/mL), followed by aqueous extract (IC50 values 154 ± 4.6 μg/mL). Essential oil ofValeriana jatamansi roots showed poor radical scavenging activity (IC50 values 876 ± 12.8 μg/mL), whereas chloroform extract showed negligible activity. BHT was taken as reference antioxidant (IC50 28 ± 0.8 μg/mL). A linear correlation has been obtained by comparing the antioxidant activity and polyphenols and flavonoid content of the extracts. The extracts containing good amount of phenols and flavonoids possess potential antioxidant activity. Previous studies have also reported positive correlation between phenolic and flavonoid content and DPPH radical scavenging activity of plant extracts [16]. The moderate antioxidant activity of essential oil could be attributed to the presence of sesquiterpenes.Table 3
Showing the antioxidant activity of the essential oil and different extracts ofValeriana jatamansi roots.
S. no.
Test sample
DPPH activity (IC50in μg/mL)
Chelation power on ferrous ions (% age at 100μg)
1
Essential oil
876 ± 12.8μg/mL
31%
2
Methanolic extract
78 ± 2.9μg/mL
76%
3
Aqueous extract
154 ± 4.6μg/mL
43%
4
Chloroform extract
—
12%
5
BHT
28 ± 0.8μg/mL
—
Data is represented as mean ± SD of three triplicate experiments.


### 3.4. Chelation Power

Chelation activity is also one of the important mechanisms of antioxidant activity. Result of chelation activity of the essential oil and different root extracts is shown in Table3. It is evident by the data that the methanolic extract ofValeriana jatamansi possesses good chelation activity (76%) followed by aqueous extracts (43%) and essential oil (31%) at 100 μg/mL concentration. Chloroform extract showed poor chelation activity (12%).


## 3.1. GC-MS Analysis of Essential Oil

Hydrodistillation of roots ofValeriana jatamansi produced greenish yellow essential oil with the yield of 0.8%. Chemical composition of the essential oil was determined by gas chromatography and mass spectrometry (Table 1). Seven major components were identified in essential oilviz., β-vatirenene, β-patchoulene, Dehydroaromadendrene, β-gurjunene, patchoulic alcohol, β-guaiene and α-muurolene. The chemical analysis of essential oil shows thatV. jatamansi roots contain only sesquiterpenes in its essential oil.Table 1
Chemical composition of essential oil ofValeriana jatamansi roots analysed by GC-MS.
Compounds
Nature of compound
Amount in % age
β-patchoulene
Sesquiterpene
20.18
β-gurjunene
Sesquiterpene
13.0
β-vatirenene
Sesquiterpene
28.07
α-muurolene
Sesquiterpene
5.20
β-guaiene
Sesquiterpene
5.88
Dehydroaromadendrene
Sesquiterpene
15.92
Patchoulic alcohol
Sesquiterpene
11.72


## 3.2. Total Phenols and Flavonoids

Phenolic compounds have been reported to exhibit various biological activities like, antioxidant, antimicrobial etc. Total phenolic compounds in extracts were determined by Folin-Ciocalteu method and expressed as Gallic acid equivalents (GAEs). As shown in Table2, highest amount of phenolic compound was observed in methanolic extract (187.13 ± 6.8 mg GAEs/g), followed by aqueous extract (77.66 ± 2.1 mg GAEs/g). Least amount of phenolic compounds was observed in chloroform extract (9.89 ± 0.3 mg GAEs/g). Many studies have revealed that the phenolic contents in the plants are associated with their antioxidant activities probably due to their redox properties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers [9, 14].Table 2
Showing total phenol and flavonoid content of different extracts ofValeriana jatamansi roots.
S. no.
Test sample
Total phenols (mg GAEs/g dry wt.)
Total flavonoids (mg QEs/g dry wt.)
1
Methanolic extract
187.13 ± 6.8
257.69 ± 9.8
2
Aqueous extract
77.66 ± 2.1
452.30 ± 12.4
3
Chloroform extract
9.89 ± 0.3
25.38 ± 2.0
Data is represented as mean ± SD of three triplicate experiments.Total amount of flavonoids was expressed in quercetin equivalent (QE). Aqueous extract ofValeriana jatamansi had relatively high amount of flavonoids (452.30 ± 12.4 mg QEs/g) followed by methanolic extract (257.69 ± 9.8 mg QEs/g) and very less amount was observed in chloroform extract (5.38 ± 0.3 mg QEs/g).


## 3.3. DPPH Radical Scavenging Activity

DPPH assay has been extensively used for screening plant extracts because many samples can be accommodated in short period and are sensitive enough to detect active ingredients at low concentrations [15]. The antioxidant activity ofValeriana jatamansi roots essential oil and extracts is summarized in Table 3. Among all the test samples, methanolic extract ofValeriana jatamansi was found to be the most potent antioxidant (IC50 78 ± 2.9 μg/mL), followed by aqueous extract (IC50 values 154 ± 4.6 μg/mL). Essential oil ofValeriana jatamansi roots showed poor radical scavenging activity (IC50 values 876 ± 12.8 μg/mL), whereas chloroform extract showed negligible activity. BHT was taken as reference antioxidant (IC50 28 ± 0.8 μg/mL). A linear correlation has been obtained by comparing the antioxidant activity and polyphenols and flavonoid content of the extracts. The extracts containing good amount of phenols and flavonoids possess potential antioxidant activity. Previous studies have also reported positive correlation between phenolic and flavonoid content and DPPH radical scavenging activity of plant extracts [16]. The moderate antioxidant activity of essential oil could be attributed to the presence of sesquiterpenes.Table 3
Showing the antioxidant activity of the essential oil and different extracts ofValeriana jatamansi roots.
S. no.
Test sample
DPPH activity (IC50in μg/mL)
Chelation power on ferrous ions (% age at 100μg)
1
Essential oil
876 ± 12.8μg/mL
31%
2
Methanolic extract
78 ± 2.9μg/mL
76%
3
Aqueous extract
154 ± 4.6μg/mL
43%
4
Chloroform extract
—
12%
5
BHT
28 ± 0.8μg/mL
—
Data is represented as mean ± SD of three triplicate experiments.


## 3.4. Chelation Power

Chelation activity is also one of the important mechanisms of antioxidant activity. Result of chelation activity of the essential oil and different root extracts is shown in Table3. It is evident by the data that the methanolic extract ofValeriana jatamansi possesses good chelation activity (76%) followed by aqueous extracts (43%) and essential oil (31%) at 100 μg/mL concentration. Chloroform extract showed poor chelation activity (12%).


## 4. Conclusions

Methanol extract of roots ofValeriana jatamansi possesses remarkable antioxidant activity as compared to its essential oil. Thus root extract ofV. jatamansi can prove beneficial in food and pharmaceutical industry.



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*Source: 614187-2014-04-06.xml*