Chemical Composition, Antioxidant and Antifungal Activity of Essential Oils of Pogostemon Amaranthoides from (Raya-Bajeta Valleys) Pithoragarh, Uttarakhand Himalayas, India.
Chemistry Lab, GIC Garkha, Uttarakhand, India
Submission: August 28, 2017;Published: October 23, 2018
*Corresponding author: Kundan Prasad, Chemistry Lab, GIC Garkha, Majhera, Pithoragarh, Uttarakhand, India
How to cite this article: Kundan P. Chemical Composition, Antioxidant and Antifungal Activity of Essential Oils of Pogostemon Amaranthoides from
002 (Raya-Bajeta Valleys) Pithoragarh, Uttarakhand Himalayas, India. J of Pharmacol & Clin Res. 2018; 6(4): 555691. DOI: 10.19080/JPCR.2018.06.555691
Pogostemon amaranthoides are wild edible vegetable.
Methods: The plant Pogostemon amaranthoides including leaves, stem, and flowers were extracted by hydro distillation method for 6 hours using Clevenger apparatus. Mineral content in plant was estimated by wet digestion method. Antioxidant activity was done by DPPH assay & ABTS assay.
Results: Total thirty-five compounds were identified constituting 81.26% of the total oil. The main compounds were β-Caryophyllene (15.48), Guaia-3,9-diene (8.27), β-Guaiene (7.15), (E)-, β-Ocimene (7.14), Germacrene B (6.91), β-Himachalene (6.55), β-Vetivenene (4.12) and α-Humulene (3.82). β-Carotene in Pogostemon amaranthoides was found to contain 259.63±1.1.34 mg.100g-1 on a dry weight basis. The free radical scavenging activity (DPPH assay) was 8.14±0.02mM AAE/100g. The inhibition for fungus Sclerotinia sclerotiorum was found to be 17.92-69.58%, Fusarium oxysporium was found to be 22.12- 54.58 % and Curvularia lunata was found to be 40.42-57.50% by the aromatic oil of Pogostemon amaranthoides.
Conclusion: The results data obtained in the present investigation suggest that an essential oil and whole plant possesses strong medicinal activities can be utilized for treatment of diseases and also used as healthy wild editable vegetable.
Keywords: β-Caryophyllene; GC-MS; ASS; Biochemical; phytochemicals and HPLC
Abbreviations: GC: MS gas chromatography/mass spectrometry; GC: FID gas chromatography/flame ionization detector; RI: retention index; HPLC: High performance Liquid chromatography; ABTS: Azinobis (3 benzylthiazole)-6- sulphonic acid; DPPH: Diphenyl-1-picrylhydrazyl; DRDO: Defence Research and Development Organisation; DARL: Defence Agriculture Research Laboratory; GBPNIHESD: Govind Ballabh Pant National Institute of Himalayan Environment & Sustainable Development; IIVR: Indian Institute of Vegetable Research; PDA: Potato Dextrose Agar
Pogostemon is a large genus from the family Lamiaceae. Pogostemon amaranthoides Benth leaves are improved blood. The common name of the plants are namnam in local people of Bajeta called it ena. Leaves and stem are the edible part of the plants as wild editable vegetables. The In most cases women suffer from anaemia following childbirth due to iron deficiency their vegetables cure them. Leaves are believed to have medicinal values to cure kidney problem (1). There is no more literature on this plant. Local people of Bajeta and this region its leaves and soft stem are used in increasing cow milk and its health after delivery. This is the first work on this plant. The use of medicinal plants by humans dates back thousands of years due to their medicinal and nutritional properties. Many natural compounds extracted from plants have important biological activities. Among these compounds, we highlight the essential oils, which are increasingly ttracting the attention of various segments of industry due to their multiple functions, especially antioxidant and antimicrobial activities Milene Aparecida Andrade et al. . Essential oils are marketed by various companies as raw material for various products with applications in perfumery, cosmetics, foods, and as adjuncts in medicines, among others. There are approximately 300 essential oils of commercial importance in the world. In the food industry, essential oils, besides imparting aroma and flavour to food, have important antioxidant activity, a property that further encourages its use Bizzo et al. .
Those important oils include a diffusion of risky molecules along with terpenes, terpenoids and phenol derived fragrant and aliphatic compounds, which might have bactericidal, antiviral, and fungicidal consequences. Terpenoids are the primary elements of
the important oils answerable for the aroma and flavour Nuzhat &
Vidyasagar . Medicinal and aromatics vegetation play a huge role
within the financial system of Morocco. As a part of a contribution
to the improvement of herbal Moroccan background, many kinds
of research are presently testing the efficacy of medicinal plant
extracts against human’s sicknesses or plant diseases or for
business cause Fadel et al. . Fusarium species are crucial plant
pathogens causing diverse ailments which encompass crown rot,
head blight, and scab on cereal grains (Nelson et al 1994), and
they’ll once in a while purpose infection in animals. Curvularia
lunata is a famous fungal plant pathogen which can motive
disorder in people and different animals. Sclerotinia sclerotiorum
is a plant pathogenic fungus and can cause a disease called white
mould if conditions are conducive. S. sclerotiorum can also be
known as cottony rot, watery soft rot, stem rot, drop, crown rot
and blossom blight. The present paper deals with the estimation
of antioxidants, aromatic oil, antioxidant and antifungal activity
and nutraceuticals of whole plants parts of medicinally and
nutritionally important plants Pogostemon amaranthoides. The
plants can use in pharmaceutical and nutrients raw material in
the formulation of many drugs and foods.
Arial parts of Pogostemon amaranthoides was collected in
the month of September 2006 to 2018 from Homtala (Bajeta,
Munsyari), Pithoragarh, India in the Kumaon Himalayas. The
plant was first identified in the Department of Botany, Kumaun
University, Nainital. The collected plant material was first washed
with cold water to remove the soil particles and then shade dried.
The dried material was finely powdered in the grinding machine
and weighed in an electrical balance.
Standard of xanthophyll, α-carotene, β-carotene and DL-
α-tocopherol was procured from Sigma Chemical Co. St Louis,
USA. Individual standard was accurately weighed, developed and
diluted with HPLC grade ethanol. Petroleum ether, methanol,
ethyl acetate and anhydrous sodium sulphate and other chemicals
and reagents used in this study was purchased Merck Chemical
Co. Mumbai, India. 1,1-Diphenyl-2-picrylhydrazyl (DPPH)
radical, gallic acid, ascorbic acid, chlorogenic acid, caffeic acid,
ρ-coumaric acid, 3-hydroxybenzoic acid, catechin and quercetin
was procured from Sigma-Aldrich (Steinheim, Germany). Sodium
carbonate, 2-(n-morpholino) ethanesulfonic acid (MES buffer),
potassium persulphate, ferric chloride, sodium acetate, potassium
acetate, aluminium chloride, glacial acetic acid and hydrochloric
acid from Qualigens (Mumbai, India), and 2,2_-azinobis(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,4,6-tri-2-
pyridyl-1,3,5-triazin (TPTZ), methanol and ethanol from Merck
Company (Darmstadt, Germany).
The plant Pogostemon amaranthoides including leaves, stem,
and flowers extracted by hydro-distillation method for 5 hours
using Clevenger apparatus. The oil was dried over anhydrous
sodium sulphate and stored at room temperature in a sealed
vial until analysis was performed. The percentage oil yield was
calculated based on the dry weight of the plant. The oil yield was
Essential oil analyses were performed by GC-MS and GC-FID
on a Shimadzu QP-2010 instrument, equipped with FID, in the
same conditions. The percentage composition of the oil sample
was computed from the GC peak areas without using correction
for response factors. The oil was analyzed using a Shimadzu GC/
MS Model QP 2010 Plus, equipped with Rtx-5MS (30m×0.25mm;
0.25mm film thickness) fused silica capillary column. Helium
(99.999%) was used as a carrier gas adjusted to 1.21ml/min at 69.0
K Pa, splitless injection of 1mL, of a hexane solution injector and
interface temperature was 270 0C, oven temperature programmed
was 50-2800 C at 30 C/min. Mass spectra was recorded at 70 eV.
Ion source temperature was 2300 C. The identification of the
chemical constituents was assigned on the basis of comparison
of their retention indices and mass spectra with those given in
the literature (Kundan and Deepak, 2018). Retention indices (RI)
were determined with reference to a homologous series of normal
alkanes, by using the following formula Kovats .
t1R – the net retention time (tR – t0)
t0 – the retention time of solvent (dead time)
tR – the retention time of the compound.
CN – number of carbons in longer chain of alkane
Cn– number of carbons in shorter chain of alkane
n - is the number of carbon atoms in the smaller alkane
N - is the number of carbon atoms in the larger alkane
The whole plant was dried in shade and powdered using
electrical grinder. The amount of total phenolic content was
estimated following Singleton et al.  with modification. The
reaction mixture contained 100 Dl of sample extract, 500 Dl
Folins-Ciocalteu’s reagent (freshly prepared), 2 ml of 20% Sodium
Carbonate and 5ml of distilled water. After 15min reaction at 450C
the absorbance at 650nm was measured using spectrophotometer.
The result was expressed as mg of Catechol equivalent per 100g
of dry weight.
The moisture content was estimated by dried in electrical oven
at 800 C for 24 hours and expressed on a percentage basis. The
dried leafs was powdered separately in electric mill to 60 mesh
size. The fine leaves powders so obtained was used for further biochemical and mineral analysis (three replication of each
parameter). The chlorophyll content in dry leaves powder was
estimated by method Singleton . Tannins content was estimated
as described by method Schanderl . Total carbohydrate content
in plant leaves was estimated by the Dubois et al. , Starch by
Hodge and Hofreiter . Total nitrogen was estimated by Micro-
Kjeldahl method, according to AOAC method, 1985. Crude protein
was calculated as Kjeldahl N x 6.25 (based on assumption that
nitrogen constitutes 16.0% of a protein). The content of crude
fat was estimated by AOAC method, 1970. Amylose content in
plant leave was estimated, as described method MeCready et al.
 Julians . Cellulose content was estimated as described
by method Updegraff . Crude fiber content was estimated as
described by methods (Maynard, 1978).
Ash content was estimated by AOAC method, 1985 and ash
insoluble content was estimated by method Peach et al.  and
Mishra R . Mineral content in plant was estimated by wet
digestion method. 1.0 g plant material was first digested with
conc. HNO3 (5ml each), followed by application of 15ml of triacid
mixture (HNO3, HClO4 and H2SO4, 10:4:1, v/v) heated at 2000
C and reduce to 1ml. The residue after digestion was dissolved
in double distilled water, filtered and diluted to 100ml. This
solution was used for the estimation of minerals. Macro minerals
viz., Na, K, Ca and Li was estimated by AIMIL, Flame Photometer
while micro elements viz. Fe, Cu, Mn, Zn and Co was estimated
by Atomic Absorption Spectrophotometer, model 4129, Electronic
Corporation of India Ltd. Phosphorous and sulpher content was
estimated by method Allen .
Ascorbic acid content was estimated by method Witham et al.
 with modification. Dry leaves powder (2.0 g) was extracted
with 4% oxalic acid and made up to 100 ml and centrifuged
at 10,000 rpm for a 10 minute. 5 ml supernatant liquid was
transferred in a conical flask, followed by addition of 10ml
4% oxalic acid and titrated against standard dye solution (2,
6-dichlorophenol indophenol) to a pink end point. The procedure
was repeated with a blank solution omitting the sample.
Dried plant material (1.0 g of each) was extracted with light
petroleum ether/methanol/ethyl acetate (1:1:1, V/V/V, 4 x 30ml)
until the extracts became colorless. The extract was mixed in a
250ml separating funnel, shaken vigorously and allowed to stand
for phase separation. Upper layer was collected in a 100ml flask
(Borosil India Co. Ltd.) and lower layer was shaken with 50ml
water and 50ml petroleum ether for phase separation. Upper layer
was mixed with the first extract. The organic extract was dried
over anhydrous sodium sulphate (10g), filtered and evaporated to
dryness in a Rotary Vacuum Evaporator under reduced pressure.
The residue was dissolved in light petroleum ether (5ml) and
filtered by 0.2μm membrane filter prior to HPLC analysis.
All the samples was analyzed using Shimadzu HPLC interfaced
with model SPD-10 AVP Variable wavelength (190-750nm) UVVis
detector, Column used was C18 Phenomenex® (150x4.60nm),
pore size 5μm with solvent system 8:2:40:50 (methanol, ethyl
acetate, acetonitrile and acetone), flow rate 0.7ml/min, run
time 20minutes and detector wavelength was 450nm. The HPLC
condition for the estimation DL-α-tocopherol was adopted as
described in Kurilich et al.  and Kundan & Deepak, 2018.
Free radical DPPH scavenging assay Brand-Williams et al.
 was slightly modified for the present study. DPPH (100μM)
was prepared in 80% (w/v) ethanol and 2.7ml mixed with 0.9ml
of sample extract and allowed to stand in the dark (22±10C,
20min). The reduction in the absorbance at 520nm was recorded
and results expressed in mM ascorbic acid equivalent per 100g
Total antioxidant activity was measured by improved ABTS
(ethylbenzothiazoline 6- sulphonic acid) radical scavenging
method Cai et al. ; Bhatt et. al. 2016). In brief, ABTS (7.0μM)
and potassium persulphate (2.45 μM) was added in amber
colored bottle for the production of ABTS cation (ABTS˙+) and
kept in the dark (16 h, 22±1°C). ABTS˙+ solution was diluted with
80% (v/v) ethanol till an absorbance of 0.700±0.05 at 734nm is
obtained. For sample analysis, 3.90ml of diluted ABTS˙+ solution
was added to 0.10 ml of methanolic extract and mixed thoroughly.
The reaction mixture was allowed to stand (22±1oC, 6min, dark)
and the absorbance was recorded at 734nm with respect to blank.
A standard curve of various concentrations of ascorbic acid is
prepared in 80% v/v methanol for the equivalent quantification
of antioxidant potential with respect to ascorbic acid. A result
was expressed in mM ascorbic acid equivalent per 100g (mM AAE
The foliage born and soil born fungi were obtained from the
Department of Plant Pathology, College of Agriculture, G. B. Pant
University of Agriculture & Technology, Pantnagar, India. The pure
culture of these pathogenic fungal species were maintained on
Potato Dextrose Agar (PDA) and stored at temperature below 40 C for further activity.
At first 200gm pealed potato was cut into fine pieces then it
was boiled in 500ml of distilled water for 30 minutes and filtered
through muslin cloth. 20gm of Agar-Agar was dissolved in 500ml
boiling water then potato extract was added in boiling mixture
and mixed thoroughly by stirring with glass rod. 20gm of dextrose
was added to the medium and transferred to about 200ml in each
500ml capacity flasks and were plugged with non-absorbent
cotton plugs. The pH of the medium was adjusted to 7.0±0.2 and
then allows the medium to sterilize at 15 lbs p.s.i (121.6°C) for
20 ml sterilized medium was poured into sterilized Petriplates.
5mm disc of fungal growth of each fungal was cut with the
help of cork borer from 10 days old culture growth on PDA. These
discs were placed onto sterilized PDA plate in a manner so that the
growth of the fungus touches the PDA in the plate and incubated
for 7 days at 25±2°C. After incubation for 7 days, radial growth
Poisoned Food Technique: Prepared Potato Dextrose Agar
medium and then add required amount of essential oil as to get
a final desired concentration and thoroughly mixed. Culture of
test fungus was multiplied growing on PDA medium for 7 days
at 25±2°C. Small disc of fungus culture was cut with sterile cork
borer and transferred aseptically in the centre of the Petri-dish
containing the medium having desired essential oil concentration.
Suitable checks, with the culture discs on PDA without essential oil
were maintained. Plates were incubated at 25±2°C and the fungal
colony diameter is measured at every 24h. The colony diameter
measured at each concentration of essential oil is compared with
check to evaluate the toxicity of essential oil towards the test
fungus. For essential oil, the different concentration of respective
essential oil was prepared by dissolving weighed quantity of
essential oil in a measured volume of sterilized distilled water. The
amount of solution to be added to PDA medium was calculated by
C1V1 = C2V2
C1 = Concentrations of stock solution (μg/ml)
C2 = Desired concentration (μg/ml)
V1 = Volume (ml) of the stock solution to be added
V2 = Measured volume (ml) of the PDA medium
The measured amount of each essential oil was added to
make a concentration of 25ppm, 50ppm, 100ppm, 250 ppm and
500ppm, separately and mixed thoroughly before plating. 20ml
toxicated medium with different treatment were poured in each
Pertiplate. After that, one 5 mm mycelial disc of 10days old culture
of each fungal isolate was inoculated separately and incubated
at 25 ± 2° C for 7 days. The radial growth was measured in mm.
by scale. Per cent inhibition were calculated by using following
formula Given by Mckinney :
The GC and GC-MS analyses of essential oil of Pogostemon
amaranthoides resulted in the identification of thirty-five
compounds (Table 1). The oil yield was (0.20%) by raw material
weight. Both, the major as well as minor constituents were
identified by their retention indices and comparison of their mass
spectra. Total fifty-seven compounds were identified constituting
81.26% of the total oil. The main compounds were β-Caryophyllene
(15.48), Guaia-3,9-diene (8.27), β-Guaiene (7.15), (E)-, β-Ocimene
(7.14), Germacrene B (6.91), β-Himachalene (6.55), β-Vetivenene
(4.12) and α-Humulene (3.82). The main minor compounds were
3-Octanone (0.05), β-Myrcene (0.06), Phenylacetaldehyde (0.10),
(+)-Camphor (0.10), Italicene (0.10), Camphene (0.11), α-Pinene
(0.14) and Cryptomeridiol (0.14). The presence of β-Caryophyllene
(15.48) show good source of natural β-Caryophyllene (Figure 1).
The amounts of certain nutrients in Pogostemon amaranthoides
are presented in (Table 2). Fat, protein and total carbohydrate
content in Pogostemon amaranthoides was found to be 2.61±0.41,
18.87±0.67 and 13.80±1.05 g.100g-1 respectively on dry weight
basis respectively (Figure 2). Starch, Amylose and Amylopectin
content in Pogostemon amaranthoides was found to be
27.46±0.65, 6.46±0.48 and 21.59±0.97 g.100g-1 respectively. The
energy content of plants was determined by multiplying the crude
protein, crude lipid and total carbohydrate content by the factor 4,
9 and 4 respectively Osborne & Voogt . The calorific values of
the plant leaves were found 154.17 K.Cal.100g1.
The cellulose, crude fiber and moisture content were found
3.50±0.31, 35.44±1.10 and 66.65±0.59g.100g-1 respectively. The
mineral content was found 9.46±0.37 g.100g-1 on dry weight basis.
Silica was found 3.54±0.49 g.100g-1 and acid soluble ash was found
5.91±0.29 g.100g-1. The content of chlorophyll-a and chlorophyll-b
in aerial parts of plants were found 105.40±1.00 and 74.00±0.64
mg.100g-1 on dry weight basis. The mineral content of Pogostemon
amaranthoides is presented in Table 3. The contents of Sodium,
Potassium, Calcium and Lithium Pogostemon amaranthoides was
found 336.59±0.39, 8621.47±0.57, 271.47±0.42 and 66.49±0.39
mg.100g-1 respectively on dry weight basis (Figure 3). The
contents of Nitrogen, Phosphorus and Sulphur Pogostemon
amaranthoides was found 3020.86±0.80, 2164.16+0.73 and
264.25±0.54 mg.100g-1 respectively on dry weight basis. The
micronutrients contents of Iron, Copper, Manganese, Zinc and
Cobalt in aerial parts of plants were found 86.67±0.44, 3.73±0.48,
12.79±0.35, 8.46±0.35 and 0.00 respectively on dry weight basis.
Antioxidant content in Pogostemon amaranthoides is presented
in Table 4. Total phenolics in Pogostemon amaranthoides was
found to contain 127.67±0.82 mg.100g-1 on a dry weight basis.
Xanthophyll in Pogostemon amaranthoides was found to contain
22.33±0.03 mg.100g-1 on a dry weight basis. α-Carotene in
Pogostemon amaranthoides was found to contain 259.63±1.34
mg.100g-1 on a dry weight basis. β-Carotene in Pogostemon amaranthoides
was found to contain 259.63±1.34 mg.100g-1 on a dry
weight basis (Figure 4). The content of Vitamin C in Pogostemon
amaranthoides was found to be 70.33±0.33mg.100g-1. DL-α-tocopherol
in Pogostemon amaranthoides was found to contain
4.48±0.03mg.100g-1 on the dry weight basis. The essential oil
showed good DPPH and ABTS radical scavenging activity (Figure
5). Antioxidant activity of plants Pogostemon amaranthoides analyzed
(Table 5). The free radical scavenging activity (DPPH assay)
was 8.14±0.02mM AAE/100g recorded in Pogostemon amaranthoides
aromatic oil. Total antioxidant activity (ABTS assay) was
found (6.71±0.01 mM AAE/100g) in Pogostemon amaranthoides
aromatic oil. This activity is significant, especially since this essential
oil are composed mainly of monoterpenes and sesquiterpenes
hydrocarbons and oxygenated ones which have a moderate
activity compared to phenolics and vitamin C. This result might be
related to the antioxidant activity of our essential oil.
All the concentration of plant aromatic oils had shown activity
against test fungal organisms. Effect of different essential oils
on the growth and inhibition (%) of the test pathogen- Sclerotinia
sclerotiorum are shown in Table 6. The growths of fungus Sclerotinia
sclerotiorum are presented in Figure 6 against Pogostemon
amaranthoides essential oil. The results showed that increase in
concentration of aromatic oils increased zone of inhibition. The
inhibition for fungus Sclerotinia sclerotiorum was found to be
17.92- 69.58 % by the aromatic oil of Pogostemon amaranthoides.
All the concentration of plant aromatic oils had shown activity
against test fungal organisms. Effect of different essential oils on
the growth and inhibition (%) of the test pathogen- Fusarium oxysporum
are shown in Table 7. The growths of fungus Fusarium
oxysporum are presented in Figure 7 against Pogostemon amaranthoides
essential oil. The inhibition for fungus Fusarium oxysporium
was found to be 22.12- 54.58 % by the aromatic oil of Pogostemon
amaranthoides. All the concentration of plant aromatic oils
had shown activity against test fungal organisms. The growths
of fungus Curvularia Lunata are presented in Figure 8 against
Pogostemon amaranthoides essential oil. The inhibition for fungus
Fusarium oxysporium was found to be 40.42-57.50% by the
aromatic oil of Pogostemon amaranthoides. The results of growth
and % of inhibition presented in Table 8. All the essential oils had
low amounts of phenolic compounds but showed good antioxidant
activity. The diversified mono- and sesquiterpenoids present
in the complex mixture of essential oils might be responsible for
the good antioxidant activity because of synergetic effects of the
constituents. This can be evidenced by a report which says that
antioxidant capacity is affected by other bioactive compounds and
could involve synergistic effects Sanchez et al. .
The essential oil and antioxidant phytochemical from Pogostemon
amaranthoides showed a qualitative and quantitative makeup
of constituents [23,24]. The plants oils are show good antifungal
activity. Clinically, this herb can be a good source of herbal
medicine for the treatment of diseases indigenously. The study
will also help to generate a database of species which can be exploited
scientifically and judiciously in the future by local people
and so that ecological balance is maintained [25,26]. The results
obtained in the present study suggest that the essential oil of
Pogostemon amaranthoides possesses medicinally active compounds.
This is the first report on the plants Pogostemon amaranthoides
at high altitudes of Kumaon Himalayas. Raya Bajeta Valley
is store of medicinal plants so there is great need to investigation
on these medicinal plants [27,28].
The authors are thankful to Dr H K Pandey and Dr Rawat,
Scientist D and Head, Herbal Medicine Division, DRDO (DARL),
Pithoragarh for providing laboratory facilities to work on this
aspect. We are grateful to Professor Y.P.S. Pangti, Department of
Botany, Kumaun University, Nainital for the identification of Plant.
The authors are grateful to AIRF, Jawaharlal Nehru University,
New Delhi for the Gas Chromatography coupled with Mass
Spectrometry (GC-MS). The authors are grateful to Professor
Ganga Bisht, Head of Department of Chemistry, K U, Nainital for
providing the necessary facilities and Dr. I. D. Bhatt Scientist-D,
GBPNIHESD, Kosi-Katarmal, Almora for provide antioxidant
activity. Authors are also grateful to Dr. Jagdeesh Singh, Principal
Scientist, IIVR- Varanasi for HPLC analysis to work on this aspect.
Thanks are due to the department of Plant pathology bio control
lab, G B Pant University of Agriculture and Technology Pantnagar
for antifungal activity determination.