BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, December 2017.
Vol. 14(4), p. 1413-1428
Isolation, Purification and Application of
Secondary Metabolites From Lichen Parmelia Perlata
K. Leela and C. Anchana Devi*
PG & Research Department of Biotechnology, Women’s Christian college,
College road, Chennai - 600006, Tamilnadu, India.
http://dx.doi.org/10.13005/bbra/2587
(Received: 07 October 2017; accepted: 16 November 2017)
Lichens are composite algae having a symbiotic association with a fungal partner. They
produce numerous secondary metabolites, which play an important role in pharmaceutical and
in other industrial applications. The Secondary metabolites produced by lichens are found to
be 80% more when compared to that produced by other organisms. Not much work has been
carried out on lichens due to the difficulty in their cultivation but still it emerges as a potential
source in developing therapeutically important drugs which are widely beneficial in all fields
of application. The Present study was aimed to isolate, purify and determine the applications of
secondary metabolites from Lichen Parmelia perlata. The presence of these compounds were
detected and purified by thin layer chromatography and column chromatography using specific
solvent systems. The purified fractions were then identified by Gas chromatography-Mass
spectrometry (GC-MS). The compounds were then subjected to application oriented studies such
as antimicrobial activity, antioxidant activity and antidiabetic activity. Not much work have
been carried out on the isolation of a specific glycoside and alkaloid compound from Lichen
Parmelia perlata, so this study was an attempt to explore the applications of these individual
compounds which could prove beneficial to the mankind for different purposes.
Keywords: Parmelia perlata, Secondary metabolites, Thin Layer Chromatography,
Column chromatography, Gas Chromatography-Mass Spectrometry.
Lichens are considered to be a
symbiotic association between a fungal and a
photosynthetic partner which is usually an algae
or Cyanobacterium. They are widely distributed
in all terrestrial habitats and are found to grow
inside rocks, on wood, soil, on other lichens, on
glass, metals and plastics (Joneson and Lutzoni,
2009). Lichens produce numerous secondary
metabolites which are deposited on the surface
of fungal hyphae in the form of tiny crystals and
these metabolites have enormous applications and
are widely utilized for different purposes. Usnic
acid is an active metabolite found in the genus
of Usnea with applications in pharmaceutical
preparation, antiviral, antimicrobial and analgesic
activity (Proksa and Proksova, 1999). Pulvinic acid
is another metabolite isolated from various lichens
and fungi which are found to possess antioxidant
properties (Fournet et al., 1997). Lichens thus play
an important role and hence are widely utilized for
different applications such as in cosmetics, food,
in diagnosis of diseases such as eczema, arthritis
and respiratory disorders etc.
*Corresponding author E-mail: dr.anchanababu@gmail.com
This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike
4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted Non
Commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Published by Oriental Scientific Publishing Company © 2017
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LEELA & DEVI, Biosci., Biotech. Res. Asia, Vol. 14(4), 1413-1428 (2017)
Parmelia perlata (Huds) Ach (family:
Parmeliaceae) is a species of lichen that is generally
referred to as “Stone flower or Charila” (Kritikar
KR and Basu BD, 1987). It is generally distributed
on the surface of walls, old trees and also found
extensively in hilly areas of Indian subcontinent.
They are also used as spice to improve the flavour
of food (Bhattarai T et al., 1999). Parmelia
perlata was found to contain numerous secondary
metabolites such as atranorin, salazinic acid,
Protolichesterinic acid, lecanoric acid and are used
as bioindicators of heavy metal pollution (Momoh
MA and Adikwu MU, 2008). Parmelia perlata
was found to possess innumerable applications
in medical sciences such as antiemetic, analgesic
and antipyretic activities. Due to its important
medicinal properties it is widely utilized in treating
boils, inflammations, sores, seminal weakness
and amenorrhoea. A compound gyrophoric acid
isolated from Parmelia species was found to be
an inhibitor of growth of human keratinocytes and
was also reported to exhibit antimitotic, antitumor
activities (K. Muller., 2001).
Secondary metabolites are organic
molecules that do not play a role in growth and
development of an organism. They are widely
synthesized by various natural sources such as
bacteria, fungi, algae, plants and animals. The
secondary metabolites are mainly classified on
the basis of their biosynthetic origin. They play
an important role in medicinal, colorant, aromatic
and functional foods. They are classified into three
main groups: Terpenes, Phenolic compounds and
Nitrogen containing compounds. In the current
study two different secondary metabolites have
been targeted and isolated they are Glycosides and
Alkaloids.
Glycosides are organic compounds which
on hydrolysis yield one or more sugar moieties
along with the non sugar moiety. Sugar moiety is
referred to as glycon and non sugar moiety is called
as aglycone. Glycoside is classified into different
groups based on chemical nature of Non-sugar
moiety, based on the nature of sugar moiety, based
on linkage between glycon and aglycone portion
and based on their therapeutic nature. (Fig 1)
Alkaloids are naturally occurring
compounds containing nitrogen atoms. The name
was derived from word alkaline which denotes a
nitrogen containing base. These compounds are
produced by a number of organisms such as bacteria,
fungi, plants as well as animals. The alkaloids are
also widely utilized as medications, recreational
drugs etc. Alkaloids are generally classified into
the following groups such as pyridine, pyrrolidine,
Tropane, Indolizidine, Quinoline, Isoquinoline,
Phenanthrene, Phenethylamine, Purine and
Terpenoid group. (Fig 2)
Thus in the present study the antimicrobial,
antioxidant and antidiabetic applications of
Glycoside and Alkaloid compounds isolated and
purified from the Lichen Parmelia perlata were
determined.
MATERIALS AND METHODS
Sample Preparation
Dried samples of Lichen Parmelia perlata
were obtained from Chennai, India and were
authenticated based on the morphological and
relevant keys given in the literature (Aswathi DD,
1988). The sample was cleaned, washed under tap
water and distilled water to remove the dirt, it was
dried and powdered using a mixer. The powdered
lichen sample was stored in clean bottle for further
analysis.
Extraction of Samples: (Rashmi S. and Rajkumar
H.G, 2014)
The crude extract from the Lichen samples
were obtained by means of cold extraction method
using methanol as solvent. About 50g of the
powdered lichen sample was added to 500ml of
methanol in a conical flask, covered with aluminum
foil and kept on a rotary shaker for 3 days at room
temperature. The solution was filtered with the
help of Whatman No.1 filter paper and the filtrate
obtained was evaporated. The dried extracts were
then stored for further experiments. The yield of
respective crude extract was calculated as:
Percentage yield (%) = (dry weight of
extract/dry weight of samples) × 100.
Identification, Isolation and Purification of
Secondary metabolites
Thin layer chromatography
Thin-layer chromatography (TLC) is a
chromatographic technique used for separating
different components from a mixture. It is generally
carried out on a thin sheet of plastic, glass or an
aluminium foil which is coated with an absorbent
LEELA & DEVI, Biosci., Biotech. Res. Asia, Vol. 14(4), 1413-1428 (2017)
material. The adsorbent could be cellulose, silica or
aluminium oxide (alumina). The layer of adsorbent
material is called as stationary phase. The sample
is spotted on the plate and the solvent or mixture of
solvents acts as a mobile phase via capillary action.
TLC plate is cut and using a pencil line is drawn
about 1cm from one end of the edge. Markings
are made and the samples were spotted on to the
plate using a capillary tube for each spot. The spots
should be air dried. The TLC chamber consists of a
glass jar with a lid and the solvent mixture is added
to chamber. The Mobile phase used for isolation
of glycosides are Toluene: methanol: glacial
acetic acid: water = 7:4:3:1 and for alkaloids are
Butanol: acetic acid: water = 4:1:3 (Viseshni R
et al., 2017). The TLC plates were placed carefully
in the TLC chamber. The solvent mixture should be
below the spot on the plate and the chamber is kept
closed. As the solvent moves up due to capillary
movement, the compounds present in the extract
are separated. The TLC plates are then removed
Fig. 1. Classification of Glycosides
Fig. 2. Classification of Alkaloids
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from the chamber once the solvent reaches an
approximate level in the plate and is kept for drying.
The dried plates are observed for spots by using
a suitable spraying reagent. For glycosides Iodine
chamber is used while for alkaloids Dragendroff’s
reagent is used for identification (Kumar et al.,
2007) (Gurpreet kaur et al., 2014). The formation
of brown band indicates the presence of glycosides
and the formation of reddish orange band indicates
the presence of alkaloids. The Rf value is calculated
using the formula:
Rf = Distance travelled by the solute / Distance
travelled by the solvent
Column chromatography: (G.K.Jayaprakasha
et al., 1998)
Silica gel (100 - 200 mesh) was chosen as
the stationary phase. The column was packed with
silica gel using methanol once packed the crude
residue from methanol extract was transferred
onto the bed of silica gel. The column was run by
using mobile phase (Toluene: methanol: glacial
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acetic acid: water in the ratio of 35:20:15:10) for
glycosides and (Butanol: acetic acid: water in
the ratio of 40:10:30) for alkaloids. The fractions
were collected at an interval of 5ml each and are
monitored by means of thin layer chromatography.
The fractions obtained were stored and utilized for
the identification of individual compounds present
in the sample by means of Gas chromatography –
Mass spectrometry (GC-MS).
Confirmatory test: (Sibi G et al., 2013)
Glycosides: Keller –Killani Test: To 1ml of
sample add 1ml of glacial acetic acid and 1ml of
concentrated Sulphuric acid. Appearance of reddish
brown colour at the junction of 2 layers indicates
the presence of glycosides.
Alkaloids: Dragendroff’s test: To 2ml of sample
2-3 drops of Dragendroff’s reagent is added.
Appearance of orange red coloured complex
indicates the presence of alkaloids
Identification of Secondary metabolites
Gas chromatography – Mass spectrometry (GCMS): (Ruthiran papitha et al., 2017)
It is an analytical method used for
identifying different substances within a test
sample. It also helps in identifying trace elements
in a sample. The Clarus 680 GC was used for the
Table 1. Rf Values of the Glycoside and
Alkaloid bands of Lichen
S.no
Compounds
Rf Values
1
2
Glycoside
Alkaloid
0.325, 0.20, 0.15
0.60, 0.526
Fig. 3a: TLC for Lichen Glycoside
analysis and a fused silica column was employed
and packed with Elite-5MS (5% biphenyl 95%
dimethylpolysiloxane, 30 m × 0.25 mm ID ×
250¼m df) and the components were separated
using Helium as carrier gas at a constant flow of 1
ml/min. The injector temperature was set at 260°C
during the chromatographic run. The 1¼L of
extract sample was injected into the instrument and
the oven temperature was as maintained as follows:
60 °C (2 min); followed by 300 °C at the rate of 10
°C min”1; and 300 °C, where it was held for 6 min.
The mass detector conditions were: transfer line
temperature 240 °C; ion source temperature 240
°C; and ionization mode electron impact at 70 eV,
a scan time 0.2 sec and scan interval of 0.1 sec. The
fragments were obtained from 40 to 600 Da. The
spectrums of the components were compared with
the database of spectrum of known components
stored in the GC-MS NIST (2008) library.
Applications
Antimicrobial activity
The antibacterial activity of Purified
glycoside and Purified alkaloid fractions of
Lichen (Parmelia perlata) were screened against
both Gram positive and Gram negative bacteria
such as Staphylococcus aureus, Streptococcus
spp, Escherichia coli, Klebsiella pneumoniae,
Salmonella spp and Pseudomonas aeruginosa
while the antifungal activity was carried out against
Aspergillus spp and Candida albicans.
Agar well diffusion method: (K.Nanthini Devi
et al., 2012)
The stock cultures were maintained at 4°C
on the nutrient agar slant slopes. Nutrient broth
Fig. 3b. TLC for Lichen Alkaloid
Fig. 3. Thin Layer chromatography for detection of Glycoside and Alkaloid compounds
LEELA & DEVI, Biosci., Biotech. Res. Asia, Vol. 14(4), 1413-1428 (2017)
was prepared for about 50ml and a loop-full of
stock cultures were transferred to 50ml of nutrient
broth and were incubated at 37°C for 24 hours.
The strains were inoculated in nutrient broth for
24 hours. Potato dextrose broth was prepared for
the growth of fungal strains and incubated for 48
hours. About 250ml of Muller Hinton agar medium
(MHA) was prepared for antibacterial activity
while Potato Dextrose agar medium (PDA) was
prepared for antifungal activity and poured into
the petriplates and was allowed to solidify. Once
solidified the bacterial cultures and the fungal
cultures were swabbed onto the agar medium using
a sterile cotton swab .The wells were punctured
Table 2. Rf value of the Purified
Glycoside and Alkaloid fractions of
Lichen Parmelia perlata
S.no
1
2
Compounds
Rf Value
Glycoside
Alkaloid
0.125
0.526
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using a sterile cork borer. Different concentrations
of samples (20, 40, 60, 80mg/ml) were dispensed
into each well using a micropipette. The petriplates
were then incubated at 37°C for 24 hours for
bacteria and 37°C for 3-4 days for fungi and
observed for the zone of inhibition. The diameter
of the zone of inhibition was measured in mm.
Antioxidant activity
The antioxidant potential of Purified
glycoside and Purified alkaloid fractions of lichen
(Parmelia perlata) were determined by 3 methods:
Total antioxidant capacity (TAC) assay, Hydrogen
Peroxide (H2O2) scavenging assay and reducing
power assay
Total antioxidant capacity assay
About 1ml of the test sample is added
to 3ml of freshly prepared phosphomolybdenum
reagent and was incubated for about 90 minutes
in water bath at 95ºC .It was then cooled to room
temperature and absorbance was measured at
695nm in a spectrophotometer.1ml methanol
without extract was used as blank. Ascorbic acid
was used as the standard (Prieto et al., 1999).The
Fig. 4a. Purified Glycoside fraction I
Fig. 4b. Purified Glycoside fraction II
Fig. 4c. Purified Alkaloid fraction I
Fig. 4d. Purified Alkaloid fraction II
Fig. 4. Purified Glycoside and Alkaloid fractions obtained through column chromatography
LEELA & DEVI, Biosci., Biotech. Res. Asia, Vol. 14(4), 1413-1428 (2017)
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Total antioxidant capacity (TAC) can be calculated
using the formula:
Total antioxidant capacity (TAC) = (A of Control
– A of test) / A of Control × 100
Hydrogen peroxide scavenging assay
Solution of Hydrogen peroxide (40mM)
was prepared in phosphate buffer (pH 7.4).1ml
of the test sample was added to 3ml of Hydrogen
peroxide solution and was incubated at room
temperature for 10 minutes and the absorbance
was determined at 230nm in a spectrophotometer.
Ascorbic acid was used as the standard. Phosphate
buffer without hydrogen peroxide served as blank
(Ruch et al., 1989).It can be calculated using the
formula:
Fig. 5a. Confirmatory test for Glycoside
Fig. 5b. Confirmatory test for Alkaloid
Fig. 5. Confirmatory test performed for the purified Glycoside and Alkaloid fractions I
Fig. 6a. GC-MS Chromatogram of Lichen Glycoside fraction showing various peaks at different retention time
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Fig. 6b. GC-MS spectrum for Lichen Glycoside (Methyl 4-Methoxy Salicylate)
Fig. 6c. GC-MS Chromatogram of Lichen Alkaloid fraction showing various peaks at different retention time
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Fig. 6d. GC-MS Spectrum for Lichen Alkaloid (N-(1-Cyclohexen-1-yl) Piperidine)
Fig. 6. GC-MS analysis of Glycoside and Alkaloid compounds from Lichen Parmelia perlata
Table 3. Antibacterial activity of Lichen Glycoside and Alkaloid fractions at different concentrations
S.no Organism
1
2
3
4
5
6
E.coli
Streptococcus spp.
Klebsiella Pneumoniae
Staphylococcus aureus
Salmonella typhimurium
Pseudomonas aeruginosa
Zone Of Inhibition (In Mm)
Lichen Glycoside
Lichen Alkaloid
Concentration (mg/ml)
Concentration (mg/ml)
20
40
60
80
C
20
40
60
80
C
6
7
5
4
3
3
8
8
5
5
4
5
% scavenged (H2O2) = (A of Control – A of test /
A of Control) × 100
Reducing power assay
In this method 2.5ml of sample was mixed
with 2.5ml of phosphate buffer (0.2M, pH 6.6) and
2.5ml of 1% Potassium ferricyanide (10mg/ml).
The mixture was incubated at 50ºc for 20 minutes
9
10
10
7
6
5
10
14
12
9
7
6
0
0
0
0
0
0
2
3
2
2
1
1
5
5
4
5
2
3
8
7
5
6
3
4
12
10
5
8
5
5
0
0
0
0
0
0
then rapidly cooled and mixed with 2.5ml of 10%
trichloroacetic acid and centrifuged at 6500 rpm
for 10 minutes. About 2.5ml of supernatant was
diluted with 2.5ml of distilled water and then 0.5ml
of 0.1% ferric chloride was added and allowed to
stand for 10 minutes. The absorbance was read
spectrophotometrically at 700nm (Ferreira et al.,
2007).
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Fig. 7a. Antibacterial activity of Lichen Glycoside fractions
Fig. 7b. Antibacterial activity of Lichen Alkaloid fractions
Fig. 7. Antibacterial activity of Lichen Glycoside and Alkaloid fractions at different concentrations
Table 4. Antifungal activity of Lichen Crude extract,
Glycoside and Alkaloid fractions
S.no Organism
1
2
Aspergillus niger
Candida albicans
Zone Of Inhibition (In Mm)
Lichen crude
Lichen
Lichen
Extract
Glycoside
Alkaloid
10
11
Antidiabetic activity: (Murugesan S et al., 2016)
The antidiabetic activity of Purified
glycoside and Purified alkaloid fractions of lichen
(Parmelia perlata) were determined by means of
alpha amylase inhibition assay.
Alpha amylase inhibition assay
About 1ml of the sample was added
to 1ml starch solution and was incubated for 10
minutes at room temperature.0.5ml of the prepared
enzyme solution was added to the mixture and
was incubated at 25ºc for about 10 minutes. The
reaction was then terminated by the addition of
1ml of colorimetric reagent and was kept in water
bath for 5 minutes and cooled to room temperature.
This was further diluted by adding 10ml of
distilled water and the absorbance of the mixture
was measured at 540nm in colorimeter. Sample
without extract served as blank. The % inhibition
was calculated using the formula:
% inhibition = (A of Control – A of test / A of
Control) × 100
22
15
15
11
Control
0
0
RESULTS AND DISCUSSION
Extraction
The Lichen (Parmelia perlata) sample
was extracted by means of cold extraction method
using methanol. Methanol has a polarity index
of 5.1 and was found to be capable of dissolving
polar compounds. Therefore methanol was highly
preferred and the solvent was also reported to have
been used by other authors for their extraction
purposes (Hafizur Rahman et al., 2014). The
Percentage yield of Lichen crude extract was found
to be 2.56 % and it has been reported to be the
total yield for about 50 grams of the dry weight of
the sample (A.Mastan et al., 2014). The work by
Fadwa Ali Mohamed Abdullah and Mona M.A.
Abdalmageed, 2016 have reported the extraction
of Lichen Parmelia perlata using petroleum ether
solvent and the total yield was found out to be 0.27
%
LEELA & DEVI, Biosci., Biotech. Res. Asia, Vol. 14(4), 1413-1428 (2017)
Identification of secondary metabolites – Thin
Layer Chromatography
Thin layer chromatography is a technique
used for the identification of secondary metabolites
in a sample by using the solvents in different
ratios. In this study thin layer chromatography was
employed to identify the presence of Glycosides
and Alkaloid compounds in the Lichen sample.
For Glycosides the mobile phase used is Toluene:
methanol: glacial acetic acid: water = 7:4:3:1 and
the bands were visualized by placing it in the iodine
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chamber while for Alkaloids the mobile phase used
is Butanol: acetic acid: water = 4:1:3 and the bands
were visualized by spraying it with Dragendroff’s
reagent (Fig 3). The solvent systems were used
based on the paper Viseshni R et al., 2017
The R f values obtained for the TLC
detection of glycoside compound was found to be
0.325, 0.20, 0.15 (Ravindra C. Sutar et al., 2014)
Similarly the Rf values obtained for the TLC
detection of alkaloid compound was found to be
0.60, 0.526 (Karthikeyan S et al., 2013) (Table 1).
Fig. 8. Antifungal activity of Lichen crude extract, Glycoside and Alkaloid fractions
Fig. 9. Total antioxidant capacity (TAC) assay for Lichen extract and purified fractions
Fig. 10. Hydrogen peroxide scavenging assay for Lichen extract and purified fractions
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Isolation of Secondary Metabolite fractions
using Column chromatography
Column chromatography is performed in
order to purify the individual compounds present
in the sample from a mixture of compounds. The
purified fractions collected by means of Column
chromatography were sent for GC-MS (Gas
chromatography - Mass spectrometry) analysis in
order to identify the major secondary metabolites
or the compounds present in the purified sample.
The Stationary phase is Silica gel (100 – 200 mesh)
while Mobile phase used for Glycosides - Toluene:
methanol: glacial acetic acid: water = 35:20:15:10
and for Alkaloids – Butanol: acetic acid: water =
40:10:30.
Totally two glycoside and two alkaloid
fractions were obtained through column
chromatography. Among both the fractions the
Glycoside fraction I and Alkaloid fraction I (Fig
4a and 4c) was subjected to confirmation by
performing Keller-Killani test and Dragendroff’s
test to detect the presence of glycoside and alkaloid
compounds in that particular fraction and TLC was
also carried out and the corresponding Rf Values
were recorded.
The Rf values obtained for the TLC of
individual purified fractions were found to be 0.125
and 0.526 (Table 2). The values were compared
with reference using various literatures and are
considered to be glycoside and alkaloid compounds
respectively (Ravindra C. Sutar et al., 2014) (S.K.
Reshmi et al., 2010)
The appearance of Reddish brown ring at
the junction of two layers indicates the presence of
Glycoside and the formation of orange red coloured
complex indicates the presence of Alkaloid in the
Lichen sample (Fig 5).
Fig. 11. Reducing power assay for Lichen extract and purified fractions
Fig. 12. Antidiabetic activity of the Lichen crude extract and purified fractions
LEELA & DEVI, Biosci., Biotech. Res. Asia, Vol. 14(4), 1413-1428 (2017)
Gas Chromatography - Mass Spectroscopy
(GC-MS)
The Purified fractions (Glycoside fraction
I and Alkaloid fraction I) were then subjected
to identification by Gas chromatography-Mass
spectrometry. It is an analytical method used
for identifying different compounds within
a test sample. In this study Glycoside and
alkaloid compounds isolated through column
chromatography were identified by means of GCMS technique. The acquisition parameters followed
for GC-MS analysis: Oven: Initial temp 60°C for
2 min, ramp 10°C/min to 300°C, hold 6 min, Total
Run Time: 32.00 mint InjA auto=260°C, Volume=1
¼L , Split=10:1,Flow Rate: 1 mL/mint, Carrier
Gas=He, Column=Elite-5MS (30.0m, 0.25mmID,
250¼m df).
Lichen Glycoside
GC-MS analysis revealed the presence of
7 different compounds in the fraction at different
retention times such as 3,5-dihydroxytoluene
(14.933), Benzoic acid,2,4-dihydroxy-6-methyl-,
methyl ester (16.754), Methyl 4-Methoxy
Salicylate (16.924), Acetic acid,(3,4-Dimethyl-5oxo-2(5H)-Furanylidene)-, Methyl ester (18.264),
1-Methyl Heptyl Trans-2,2-Dimethyl-3-(2-methyl1-propenyl) cycloprop (19.585), 1-Hexyl-2Nitrocyclohexane (21.171), 6-Nitroundec-5-ene
(21.366) respectively (Fig 6a).
Among the obtained compounds Methyl
4-Methoxy Salicylate with retention time 16.924
was identified to be a glycoside and the GC-MS
spectrum for the corresponding glycoside is also
given in the Fig 6b. This compound is found to
play an important role in pharmaceutical industry
as an analgesic and as an antiseptic agent. It
is used as a flavouring agent and also in anti
herbivore defense. It is also widely employed in
immunohistochemistry (Hoang Le Tuan Anh et
al., 2017).
Lichen Alkaloid
GC-MS analysis revealed the presence of
9 different compounds in the fraction at different
retention times such as 1,4-Benzenediol,2-Methyl(17.544), 2,5,9-Tetradecatriene,3,12-Diethyl
(18.089), N-(1-Cyclohexen-1-yl) Piperidine
(18.325), Pyrrolidine, 1-(1- Cyclohexen-1-yl)(18.710), P- Dodecyloxybenzaldehyde (19.935), NNoneyl Succinic anhydride (19.970), 7-Antiacetyl 3,3- dimethylbicyclo (2.2.2) octan-2-one (20.250),
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1-naphthalenepropanol, alpha-ethyldecahydro2,4- dihydroxy-AL (20.435), 2-oxabicyclo (4.4.0)
Decan – 10 - one, 1,3,7,7-Tetramethyl- (1R,3R,6R)
(30.149) (Fig 6c).
Among the obtained compounds N-(1Cyclohexen-1-yl) Piperidine with retention time
18.325 was identified to be an alkaloid and the GCMS spectrum for the corresponding alkaloid is also
given in the Fig 6d. This compound is known as the
best representative structural element in alkaloids.
It is widely used in the synthesis of organic
compounds and also in chemical degradation
reactions. It is also employed as an antidepressant
agent in pharmaceuticals (Rui Yan et al., 2014).
The compounds identified through GCMS analysis were then subjected to application
oriented studies wherein the antimicrobial,
antioxidant and antidiabetic applications of the
purified Glycoside and Alkaloid fractions of
Lichen Parmelia perlata were assessed.
Antimicrobial activity
The antibacterial activity of the Lichen
glycoside and alkaloid fractions were determined
against both the Gram positive and Gram
negative bacteria such as Staphylococcus aureus,
Streptococcus spp, Escherichia coli, Klebsiella
pneumoniae, Salmonella spp and Pseudomonas
aeruginosa were used for the study.
The glycoside and alkaloid fractions
were taken at different concentrations (20, 40, 60,
80mg/ml) and were tested for their antibacterial
activity against six bacterial organisms. The zone
of inhibition was found to increase with increasing
concentration of the compounds. Both the glycoside
and alkaloid fractions showed good antibacterial
activity against all the six bacterial organisms
but among the two fractions glycoside fraction
was found to be comparatively better showing
an increased zone of inhibition than the alkaloid
fraction (Table 3). A study by Musaddique Hussain
et al., 2014 has reported the antimicrobial potential
of lichen Parmelia perlata extracts against different
pathogenic microbes wherein the crude extract
was found to have shown maximum inhibition
against E.coli with zone of inhibition around
21.75mm followed by Pseudomonas aeruginosa
(21mm), Staphylococcus aureus (20.25mm),
Streptococcus pneumoniae (19.50mm) and
Klebsiella pneumoniae (17.90mm) respectively.
The antifungal activity of Lichen glycoside
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and alkaloid fractions were also determined against
2 fungi Aspergillus spp and Candida albicans.
The glycoside and alkaloid fractions
were tested for their antifungal activity against
2 fungal organisms wherein both the fractions
showed a good zone of inhibition against the
fungal organisms but comparatively glycoside
fraction was found to show much better activity
than the alkaloid fraction .The lichen crude extract
also exhibited good antifungal activity but was
found to be comparatively low than that of the
individual purified fractions (Table 4). A study by
Musaddique Hussain et al., 2014 has also reported
the antifungal potential of lichen Parmelia perlata
extracts against 2 pathogenic fungal organisms
wherein the crude extract was found to have shown
maximum inhibition against Candida albicans
with inhibition zone around 14.25mm followed
by Aspergillus niger with zone around 13.50mm
respectively.
Antioxidant activity
The antioxidant activity of the Lichen
crude extract, Purified glycoside and alkaloid
fractions were carried out by means of three assays:
Total antioxidant capacity (TAC) assay, Hydrogen
peroxide scavenging (H2O2) assay and reducing
power assay.
Total antioxidant capacity (TAC) assay
It is a widely employed technique for
detecting the antioxidant potential of the biological
samples. The amount of free radicals scavenged
by the test solution can be measured. This assay
is carried out by means of phosphomolybdenum
method. This technique is based on the principle
of reduction of Phosphate -Mo (VI) to Phosphate
- Mo (V) by the test sample and the immediate
formation of bluish green coloured Phosphate/Mo
complex at an acidic pH. It has several advantages
such as low cost, increased rate of reaction and this
method can also be performed using automated or
manual methods (Rohan Sharadanand Phatak and
Anup Subash Hendre, 2014)
The Total antioxidant capacity (TAC) of
the lichen crude extract and the individual purified
fractions were determined. In case of TAC assay
both glycoside and alkaloid compounds showed
good antioxidant percentage but on comparison
alkaloid compound was found to have shown a
good % of activity than the crude extract and the
glycoside fraction (Fig 9). A study by A.Mastan et
al., 2014 has reported the total antioxidant capacity
of methanolic and aqueous extracts of 4 different
lichen samples such as Cladonia fimbriata,
Permilopsis ambigua, Punctelia subrudecta and
Evernia mesomorpha by phosphomolybdenum
method wherein the antioxidant percentage was
found to be higher in the methanolic extracts of
lichen samples compared to that of the aqueous
extract.
Hydrogen peroxide scavenging assay
Hydrogen peroxide is considered to be
a weak oxidizing agent. Inside the cell hydrogen
peroxide reacts with Fe2+ or Cu2+ ion and gets
converted into hydroxyl radical which could cause
toxic effects and thereby leads to cell damage. So
it is essential to prevent the formation of hydroxyl
radical by preventing the accumulation of hydrogen
peroxide within the cell. This assay is based on the
principle of decrease in absorbance of hydrogen
peroxide upon oxidation of hydrogen peroxide.
The ability of the extracts to scavenge the hydrogen
peroxide is determined (Priyanka B et al., 2013).
The Hydrogen peroxide scavenging assay
for the lichen crude extract and the individual
purified fractions were carried out and determined.
In hydrogen peroxide scavenging assay the crude
extract as well as the purified fractions showed
good scavenging % but on comparing the activity
of both glycoside and alkaloid compounds
glycosides were found to have shown a better %
of scavenging than the alkaloid and crude extract
(Fig 10). A study by S.Suganya, 2015 has reported
the Hydrogen peroxide scavenging activity of the
Petroleum ether, ethyl acetate, ethanol and aqueous
extracts of lichen Parmotrema grayanum at
different concentrations such as 20, 50, 100, 200µg/
ml. Among all the extracts ethanolic extract of the
lichen sample was found to have shown increasing
scavenging percentage at all concentrations
followed by petroleum ether extract respectively.
Reducing power assay
It is a widely used technique and is based
on the principle that the substances with reduction
potential react with Potassium ferricyanide (Fe3+)
to form Potassium ferricyanide (Fe2+) which again
reacts with ferric chloride to form ferric-ferrous
complex that has an absorption at 700nm.The
presence of reducing compound in the extract
will tend to cause the conversion of ferric form
to ferrous form .By measuring the absorbance
LEELA & DEVI, Biosci., Biotech. Res. Asia, Vol. 14(4), 1413-1428 (2017)
of blue colour formed at 700nm it is possible to
determine the concentration of Fe3+ ion .Thus the
reducing ability of the crude extract and individual
purified fractions were determined (P. Jayanthi and
P.Lalitha.,2011).
The Reducing power assay was carried
out for the lichen crude extract and the individual
purified Glycoside and Alkaloid fractions. In
reducing power assay the absorbance of the
individual samples were analysed wherein the
absorbance of alkaloid fraction was found to be
comparatively higher than that of the glycoside
fraction and the crude extract but overall the crude
extract as well as both the purified fractions showed
an increased activity (Fig 11). High absorbance
of the extract as well as fractions indicates potent
reducing power. A study by A.Mastan et al., 2014
has reported the reducing power assay for the
methanolic and aqueous extracts of 4 different
lichen samples such as Cladonia fimbriata,
Permilopsis ambigua, Punctelia subrudecta
and Evernia mesomorpha wherein the value of
absorbance was found to vary from 0.12 to 1.98.
All the 4 lichen samples were found to have
shown increased absorbance but among theses
lichen extracts aqueous extract of lichen Evernia
mesomorpha was found to have shown better
antioxidant activity with an increased absorbance.
Antidiabetic activity
The antidiabetic activity of the crude
extract and purified Glycoside as well as Alkaloid
fractions of Lichen Parmelia perlata were
determined by means of alpha amylase inhibition
assay. It is based on the principle of in vitro
hydrolysis of starch in the presence of ±-amylase
enzyme. This process is quantified using iodine
which gives blue colour on reaction with starch.
The decrease in the intensity of blue colour
indicates the breakdown of starch by the enzyme
into monosaccharides. If the extracts possess an
increased activity then the intensity of blue colour
will be more or higher thus the intensity of blue
colour developed is directly proportional to the
alpha amylase inhibitory activity (Sheikh et al.,
2008).
The antidiabetic activity of Lichen
Parmelia perlata were determined by means of
alpha amylase inhibition assay in which the %
inhibition of the crude extracts as well as the
1426
purified fractions were analysed. The crude extract
as well as the purified compounds showed good
inhibition % but among the two fractions glycoside
fraction was found to possess higher antidiabetic
activity compared to that of the alkaloid fraction
and the crude extract (Fig 12). A study by Rashmi
shivanna et al., 2015 has reported the antidiabetic
activity of methanol and ethyl acetate extracts of
2 lichen samples namely Flavoparmelia caperata
and Physcia aipolia at different concentrations
such as 5, 10, 15mg/ml using alpha amylase
inhibition assay wherein the methanolic extract of
Flavoparmelia caperata were found to have shown
49% inhibition followed by Physcia aipolia with
46% inhibition.
CONCLUSION
The Present study was carried out in order
to explore the potential of individual secondary
compounds such as glycoside and alkaloid
isolated from Lichen Parmelia perlata in different
therapeutic applications such as antimicrobial,
antioxidant and antidiabetic activity. A number
of lichen compounds are being screened and
extracted to test their pharmacological potential
so that these compounds can also be employed
in developing therapeutically important drugs.
Since the compounds are derived from natural
sources they do not cause any health impacts or
don’t pose any threat to the environment. The use
of drugs derived from these natural sources could
also help in treating a number of diseases due to
the accumulation of bioactive compounds. Many
of the compounds derived from these natural
sources have been found to possess numerous
pharmacological properties. Not much work has
been reported before on the isolation of glycoside
and alkaloid compounds from lichen so this study
was an attempt to identify the presence of such
beneficial compounds in Lichen that could prove
to be useful for different applications.
ACKNOWLEDGEMENT
The authors are highly thankful to Dr.
Lillian Jasper, Principal, Women’s Christian
College and the Department staff members for the
needful support.
1427
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