Orchids, generally considered as the most beautiful creation of the Mother
Nature, comprise a group of flowering plants and are known to the natives since
ancient times. The use of orchids in the system of indigenous medicine dates
back to vedic period. The therapeutic use are in practice in different traditional
methods of medicines like Ayurveda, Siddha and Unani (Rao,
1998). Traditional therapeutic uses of a number of orchids and their phytochemical
constituents like alkaloids, flavonoids, terpenes, carbohydrates and glycosides
have been documented in ethanobotanical literatures (Singh
and Duggal, 2009). There are unmistakable references of orchids as medicinal
constituents in ancient Sanskrit literature like Charaka Samhita,
Nighantus and Amarakosha by Charaka, Sushruta and Vagbhata,
respectively (Hegde, 1984). In Ayurvedic system, Ashtawarga,
a group of eight drugs, is used for preparation of tonics such as Chyavanprash
and consists of four orchid species out of which Flickingeria nodosa
is also one among them (Kaushik, 1983). They are called
as Purusharathna in Kannada. In Charak Samhita, it is known as Jeewanti
means the life promoter (Rao, 1998). In ayurvedic and
traditional medicines it is believed that it can cure tridosha (Vatta, Pittha
and Kapha; Rao, 1998).
Antioxidants are the compounds that can delay or inhibit the oxidation of biomolecules
by regulation of oxidative chain reactions (Arya and Yadav,
2011). The antioxidative effects are mainly due to secondary metabolite
components like phenolic components. The phenolic compounds have antioxidative
activity mainly due to their redox potential, which plays an important role
in absorbing and neutralizing free radicals (Butkhup and
Samappito, 2011; Coulidiati et al., 2011).
Many researchers have investigated powerful antioxidants from natural herbal
sources to prevent the reactive oxygen species related disorders in human as
well as replace the synthetic compounds (Alam et al.,
2012). So the present investigation was started with an aim to evaluate
the phytochemical components and in vitro free radical scavenging activity
of cold and hot successive pseudobulb extracts of medicinally important orchid
Flickingeria nodosa (Dalz.) Seidenf.
MATERIALS AND METHODS
Preparation of the plant material: The pseudobulb stem plant material was collected from the natural habitat and rinsed with distilled water to remove the runaway dust particles. The water was removed by blotting over a filter paper. The plant materials were shade dried and powdered. Ten grams of powdered plant materials was weighed, taken in a muslin cloth and made into packets. The packets were used for the successive extraction by using 5 solvents namely petroleum ether, chloroform, acetone, ethanol and water.
Cold successive extraction: Ten grams of powdered plant material made into packet was soaked in petroleum ether and incubated at 25°C on an orbital shaker at 100 rpm for 24 h. The solvent was decanted into collection bottle and the fresh solvent was added repeatedly till all the plant metabolites were leached out. Then the packet containing the plant material was dried and the extraction was carried out with next successive solvents. The successive extracts were dried by using rotary vacuum evaporator. The dried extracts were used for further studies.
Hot successive extraction: Ten grams of powdered plant material made into packet was placed in the soxhlet extraction apparatus basket which is a vessel with perforated sides and bottom so that liquid can fall through it. When gentle heat is applied to the main flask, the solvent begins to evaporate and the solvent vapours reach the cold condenser at the top of the flask and begin to liquefy on the condenser. The re-condensed solvent on the sides of the condenser begins flowing down the sides of the condenser and begins dripping off of drip points on the end of the condenser. This solvent drips into the top of the soxhlet basket. The solvent flows through the basket and out of the holes in the bottom of the basket carrying the extract with it into the bottom of the flask. The extract laden solvent falling from the soxhlet basket is dark in color and as it becomes clearer, one can know that the plant material is leached out and the process is finished. Then the packet containing the plant material was dried and the extraction was carried out with next successive solvents. The successive extracts were dried by using rotary vacuum evaporator. The dried extracts were used for further studies.
Qualitative phytochemical screening: The different qualitative chemical
tests were performed for establishing phytochemical profile of ten extracts
obtained from cold and soxhlet successive extractions. The following tests were
performed on all the extracts to detect various phytoconstituents present in
them (Raaman, 2006).
Detection of alkaloids: The dried extracts were dissolved in few mL of 0.1N HCl and used for all the following tests.
Mayers test (Evans, 1997): One milliliter
of sample was taken and 1-2 drops of Mayers reagent was added from the
sides of the test tubes. The appearance of white precipitate indicates the presence
of alkaloids in the sample.
Wagners test (Wagner, 1993): One milliliter
of sample was taken and 2-3 drops of Wagners reagent was added from the
sides of the test tubes. The appearance of reddish-brown precipitate indicates
the presence of alkaloids in the sample.
Hagers test (Wagner et al., 1996):
One milliliter of sample was taken and 1-2 mL of Hagers reagent was added
into the test tubes. The appearance of yellow precipitate indicates the presence
of alkaloids in the sample.
Dragendroffs test (Waldi, 1965): One milliliter
of sample was taken and 1-2 mL of Dragendroffs reagent was added into
the test tubes. The appearance of yellow precipitate indicates the presence
of alkaloids in the sample.
Detection of Saponins by foam test (Kokate, 1999):
Fifty milligram of extract was diluted with distilled water and volume was made
upto 20 mL in a measuring cylinder. It was shaken well for 15 min. The appearances
of layer of foam of about 2 cm indicate the presence of saponins.
Detection of phytosterols by Liebermann-Burchards method (Finar,
1986): Sample was dissolved in 2 mL of acetic anhydride and 1-2 drops
of concentrated H2SO4 was added from the sides of the
test tube. The array of change in colours from purple to green indicates the
presence of phytosterols.
Detection of phenolic compounds
Ferric chloride (Mace, 1963): The extract was dissolved
in 5 mL of distilled water and few drops of neutral 5% FeCl3 solution
were added. The appearance of dark green colour indicates the presence of phenols.
Gelatin (Evans, 1997): The extract was dissolved
in 5 mL of distilled water and 2 mL of 1% gelatin was added. Appearance of white
precipitate indicates the presence of phenols.
Folin-Ciocalteau reagent (Sadasivam and Manickam, 1997):
The extract was dissolved in 3 mL of water, 0.5 mL of Folin-Ciocalteau reagent
was added and incubated for 3 min and 2 mL of 20% Na2CO3
solution was added. Appearance of blue coloured complex indicates the presence
Detection of flavonoids by aluminium chloride method (Zhishen
et al., 1999): A 0.5 mL of extract was mixed with 2 mL of distilled
water and subsequently with 0.15 mL of 5% NaNO2 solution. After 6
min, 0.15 mL of a 10% AlCl3 solution was added and allowed to stand
for 6 min, then 2 mL of 4% NaOH solution was added to the mixture. Immediately
distilled water was added and then the mixture was incubated at RT for 15 min.
Pink colour indicates the presence of flavonoids.
Detection of Glycosides by Borntragers test (Evans,
1997): Fifty milligram of extract was hydrolyzed with concentrated hydrochloric
acid for 2 h on a water bath and filtered. To 2 mL of filtered hydrolysate,
3 mL of chloroform was added and shaken, chloroform layer is separated
and 10% ammonia solution was added to it. Pink colour indicates the presence
Estimation of alkaloids (Sreevidja and Mehrotra, 2003):
The calibration curve was obtained with Bismuth nitrate pentahydrate stock solution.
Series dilutions of the stock solution were made by pipetting out from 200 to
1000 μL and the volume was made upto 1 mL with distilled water, 5 mL of
3% thiourea solution was added to it. The absorbance value of the yellow solution
was measured at 435 nm against colorless reagent blanks.
A 5 mL amount of the extract/solution was taken and the pH was maintained at 1.2-2 with dilute HCl. A 2 mL amount of Dragendroffs Reagent (DR) was added to it and the precipitate formed was centrifuged. The centrifugate was checked for complete precipitation by adding DR. After centrifugation, the centrifugate was decanted completely and meticulously. The precipitate was further washed with alcohol. The filtrate was discarded and the residue was then treated with 2 mL of 1% disodium sulfide solution. The brownish black precipitate formed was then centrifuged. Completion of precipitation was checked by adding 2 drops of disodium sulfide. The residue was dissolved in 2 mL concentrated nitric acid. This solution was diluted to 10 mL in a standard flask with distilled water; 1 mL was then pipetted out and 5 mL of 3% thiourea solution was added to it and absorbance was measured at 435 nm. Two milliliter of nitric acid was diluted to 10 mL in a standard flask with distilled water; 1 mL was then pipetted out and 5 mL thiourea solution was added to it and which serves as a blank.
Estimation of phytosterols by libermann-burchard method (Finar,
1986): Prepare standard cholesterol solutions (1 mg mL-1)
in chloroform. Pipette out different aliquots of standard cholesterol ranging
from 0.2-1.0 mL in test tubes. Add 2 mL of acetic anhydride and 1-2 drops of
concentrated H2SO4. Make up the volume to equal quantity
using chloroform. Incubate in dark for 15 min. Prepare the blank with 1 mL chloroform
and 2 mL acetic anhydride. Sample was dissolved in 1 mL of chloroform and 2
mL of acetic anhydride was added to it, absorbance was measured at 640 nm.
Estimation of total flavonoid content
Aluminium chloride method (Zhishen et al., 1999):
Different aliquots (0.2-1 mL) of standard Quercetin (100 μg mL-1)
solution were taken in a series of test tubes. Five hundred micro liter of extract
was mixed with 2 mL of distilled water and subsequently with 0.15 mL of 5% NaNO2
solution. After 6 min, 0.15 mL of a 10% AlCl3 solution was added
and allowed to stand for 6min and then 2 mL of 4% NaOH solution was added to
the mixture. Immediately distilled water was added to bring the final volume
of 5 mL and then the mixture was thoroughly mixed and allowed to stand at RT
for 15 min. Absorbance of the mixture was determined at 510 nm.
Estimation of total phenols (Sadasivam and Manickam, 1997):
The extracts were dissolved in 5 mL of distilled water. Different aliquots (0.2-1
mL) of standard Catechol (50 μg mL-1) solution were taken in
a series of test tubes. The volume in each tube was made up to 5 mL with water.
0.5 mL of Folin-Ciocalteau reagent was then added to each test tube and mixed
well. After 3 min, 2 mL of 20% Na2CO3 solution was added
to each tube and mixed thoroughly. The tubes were kept in the boiling water
for exactly one minute and then cooled. The absorbance against a reagent blank
was taken at 650 nm.
Total antioxidant capacity
Phosphomolybdenum assay (Prieto et al., 1999):
One hundred micro liter of the extract dissolved in DMSO was mixed in the eppendorf
tube with 1 mL of total antioxidant reagent (0.6 M sulphuric acid, 28 mM sodium
phosphate and 4 mM ammonium molybdate). Different aliquots (20-100 μg mL-1)
of standard ascorbic acid were taken into series of eppendorf tubes and the
volume was made up to 0.1 mL with DMSO and 1 mL of total antioxidant reagent
was added. The eppendorf tubes were capped and incubated in a thermal block
at 95°C for 90 min. Cooled to room temperature and the absorbance measured
at 695 nm against black.
To evaluate the antioxidant properties of different phytochemical extracts:
Free radical scavenging activity on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) (Blois,
1958) Standard ascorbic acid Sample extracts (1 mg mL-1) at various
concentration (10-50 μg mL-1) were taken and the volume was
adjusted to 100 μL with methanol. Five milliliter of a 0.1 mM methanolic
solution of DPPH was added and shaken vigorously. The tubes were allowed to
stand for 20 min at 27°C. The absorbance of the sample was measured at 517
nm. Methanol serves as a blank and prepared DPPH serves as a control and the
experiment was performed in triplicate. Radical scavenging activity was expressed
as the inhibition percentage of free radical by the sample and was calculated
using the formula:
ABTS (2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)) radical cation
decolorization assay (Re et al., 1999): ABTS
radical cation (ABTS+) was produced by reacting ABTS (7 mM) with
2.45 mM ammonium persulfate and the mixture was allowed to stand in dark at
room temperature for 12-16 h before use. Standard ascorbic acid and sample extracts
(10 mg mL-1) at various concentrations (200-1000 μg mL-1)
were taken and the volume was adjusted to 500 μL with DMSO and 500 μL
of DMSO serves as blank. Three hundred micro liter of ABTS solution was added;
the final volume was made up with ethanol to make 1 mL and incubated in dark
for 30 min at RT. The absorbance was read at 745 nm and the experiment was performed
in triplicate. Radical cation decolorization activity was expressed as the inhibition
percentage of cations by the sample and was calculated using the formula:
Nitric oxide scavenging assay (Sreejayan and Rao, 1997):
Different concentrations of standard ascorbic acid and sample extract (200-1000
μg mL-1) dissolved in DMSO were taken and the volume was adjusted
to 1.5 mL with sodium nitroprusside (5 mM) in phosphate buffer saline and incubated
at 25°C for 30 min. After 30 min, it was diluted with 1.5 mL of Griess reagent
(1% sulphanilamide, 2% orthophosphoric acid and 0.1% napthyl ethylene diamine
dihydrochloride). The absorbance of the chromphore formed during diazotization
of the nitrite with suplhanilamide and subsequent coupling with naphlethylene
diamine was measured at 546 nm along with a control. The percentage inhibition
of nitric oxide generated was measured by comparing the absorbance values of
control and test sample using following formula:
Hydroxyl radical scavenging activity (Klein et al.,
1991): The scavenging activity of extracts on hydroxyl radical was measured
according to the method of Klein et al. (1991).
Various concentrations (20-100 μg mL-1) of extracts (1 mg mL-1)
and standard ascorbic acid (1 mg mL-1) were added with 1 mL of iron-EDTA
solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 mL of EDTA solution
(0.018%) and 1.0 mL of diemthyl sulphoxided (DMSO) (0.85% v/v in 0.1 M phosphate
buffer, pH 7.4). The reaction was initiated by adding 0.5 mL of ascorbic acid
(0.22%) and incubated at 80-90°C for 15 min in a water bath. After incubation
the reaction was terminated by the addition of 1 mL of ice-cold TCA (17.5% w/v).
Three milliliters of Nash reagent (75 g of ammonium acetate, 3 mL of glacial
acetic acid and 2 mL of acetyl acetone were mixed and raised to 1 liter with
distilled water) was added and left at room temperature for 15 min. The reaction
mixture without sample was used as control. The intensity of the color formed
was measured spectroscopically at 412 nm against reagent blank. The% hydroxyl
radical scavenging activity (HRSA) is calculated by the following formula:
IC50 value: IC50 values (concentration of sample required to scavenge 50% of free radicals) were calculated from the regression equation, prepared from the concentration of the samples and percentage inhibition of free radical formation. Ascorbic acid was used as positive control and all tests were carried out in triplicate.
Statistical analysis: The experiments were set up in a completely randomized design. All values obtained from the mean replicates were averaged. The data were analyzed in relation to the variance and presented as mean±standard error (SE). Analysis of variance was conducted by two way ANOVA and the mean were compared by Tukey HSD test. All statistical analysis was performed at 1% significance level using IBM SPSS Statistics (version 20) by IBM.
Qualitative phytochemical screening: The different qualitative chemical tests were performed for establishing phytochemical profile of ten extracts obtained from cold and soxhlet successive extractions. Phytochemical screening was performed for all the extracts which revealed the presence of alkaloids, flavonoids, phenols and phytosterols in different extracts (Table 1).
Quantitative estimation of phytochemicals: The quantitative estimations
of the phytochemicals, which were qualitatively detected in the pseudobulb stem
revealed the presence of high alkaloid content (50.5 μg mL-1)
in hot chloroform extract (Fig. 1). The presence of high phenol
content (225.84 μg mL-1) was recorded in cold acetone extracts
(Fig. 2). High content of phytosterols (24 μg mL-1)
was recorded in hot acetone extract (Fig. 3).
||Concentration of alkaloids (μg mL-1) present
in different extracts
||Concentration of phenols (μg mL-1) present
in different extracts
||Concentration of phytosterols (μg mL-1) present
in different extracts
|| Phytochemical screening of ten extracts of Flickingeria
|C: Cold extract, S: Soxhlet hot extract, PE: Petroleum ether,
C: Chloroform, A: Acetone, E: Ethanol ,W: Aqueous
|| Quantitative estimation of Phytochemical
|*Mean of 3 replications, SE: Standard error
||Concentration of Flavonoid (μg mL-1) present
in different extracts
High content of flavonoids (657.80 μg mL-1) was recorded in
cold chloroform extract (Fig. 4). High total antioxidant content
(34.95 μg mL-1) was recorded in cold acetone extracts (Fig.
5). The present study reveals that the cold extraction is more effective
for phenols, flavonoids and total antoxidants while hot extraction is more effective
for alkaloids and phytosterols (Table 2).
Free radical scavenging activity assay
DPPH free radical scavenging activity assay: Free radical scavenging
potential of extracts at different concentrations was tested by DPPH method.
||Concentration of total antioxidant content (μg mL-1)
present in different extracts
Highest percentage of scavenging activity and IC50 values were found
to be 43.68% and 46.49 μg mL-1 respectively for cold acetone
extract. The standard ascorbic acid showed the percentage of scavenging activity
and IC50 values as 60.878% and 21.04 μg mL-1 (Table
3-4) (Fig. 6).
ABTS radical scavenging assay: Radical cation decolorization activity
of extracts at different concentrations was tested by ABTS method. Highest percentage
of scavenging activity and IC50 values were found to be 73.11% and
12.7 μg mL-1 respectively for cold acetone extract.
||Percentage of scavenging activity of different extracts against
|Mean of 15 replicate. Mean values with different superscripts
(a,b,c,d,e,f,g,h,I,j and k) differ significantly at
p<0.01 by Tukey (HSD) test
||DPPH scavenging activity of different extracts and its IC50
||ABTS scavenging activity of different extracts and its IC50
The standard ascorbic acid showed the percentage of scavenging activity and
IC50 values as 52.082% and 55.66 μg mL-1 (Table
3-4) (Fig. 7).
|| IC50 value of 10 extracts different antioxidant
|Values with different superscripts (a,b,c,d,e,f,g,h,I,j
and k) differ significantly
||Nitric oxide radical scavenging activity of different extracts
and its IC50 (μg mL-1)
Nitric oxide scavenging assay: The extracts at different concentrations
were tested for nitric oxide radical scavenging activity. Highest percentage
of activity and IC50 values were found to be 90.352% and 34.66 μg
mL-1, respectively for cold water extract. The standard ascorbic
acid showed the percentage of scavenging activity and IC50 values
as 41.647% and 46.45 μg mL-1 (Table 3-4)
Hydroxyl radical scavenging activity assay: The extracts at different
concentrations were tested for hydroxyl radical scavenging activity. Highest
percentage of activity and IC50 values were found to be 28.9480%
and 0.54 μg mL-1, respectively for hot chloroform extract. The
standard ascorbic acid showed the percentage of scavenging activity and IC50
values as 41.7237% and 20.37 μg mL-1 for ascorbic acid (Table
3-4) (Fig. 9).
Based on the ethnobotanical literature, Flickingeria nodosa has a great
medicinal importance for having high amount of secondary metabolites (Kaushik,
||Hydroxyl radical scavenging activity of different extracts
and its IC50 (μg mL-1)
In the present investigation, hot and cold successive extraction was carried
with different solvents (Petroleum ether, chloroform, acetone, ethanol and water),
revealing the presence of alkaloids, flavonoids and phenols, which agrees with
the earlier findings of Chhajed et al.(2008).
The extracts obtained from the present study revealed the presence of phytosterols,
which was not reported earlier. The cold extraction was more effective for extracting
phenols and flavonoids, whereas hot extraction was more effective for alkaloids
and phytosterol extraction.
Free radicals are produced under certain environmental conditions and during
normal cellular functions in the body. These molecules are missing an electron,
giving them an electric charge. To neutralize this charge, free radicals try
to withdraw an electron from, or donate an electron to, a neighbouring molecule.
The newly created free radical, in turn, looks out for another molecule and
withdraws or donates an electron, setting off a chain reaction that can damage
hundreds of molecules. Antioxidants halt this chain reaction. Some antioxidants
are themselves free radicals, donating electrons to stabilize and neutralize
the dangerous free radicals. Other antioxidants work against the molecules that
form free radicals, destroying them before they can begin the domino effect
that leads to oxidative damage (Matill, 1947). In the
present investigation, total antioxidant activity was determined with different
antioxidant assays. High amount of scavenging activity with an evident IC50
was seen with the cold acetone and cold water extracts when compared to that
of hot extracts (Chhajed et al., 2008). As compared
to the hot extracts, cold extracts have given a better activity. This is because
the bioactive component present in the extracts might be thermolabile, which
might lose its activity when extracted under heat. In the present investigation,
high amount of DPPH scavenging activity, ABTS scavenging activity and nitric
oxide scavenging activity with an evident IC50 value was observed.
The DPPH involves in their hydrogen-donating ability, ABTS chemistry involves
direct generation of ABTS radical mono cation with no involvement of any intermediary
radical and nitric oxide involves in reduction of nitrogen-free radicals. Hence,
the more scavenging activity directly relates their antioxidant capacity. Thus
the cold extracts are more reducing agent for reactive oxygen species and nitric
oxide-free radical. The cold acetone extract has given good scavenging activity
against DPPH and ABTS radical and cold water extract has given good scavenging
activity against nitric oxide radical. These extracts have shown high antioxidant
activity due to the presence of phenolic compounds which agrees with the findings
of Materska and Perucka, (2005). The percentage of hydroxyl
radical scavenging activity does not show significant difference in result for
different extracts. The IC50 value for hot chloroform extract was
found to be the best as compared to other extracts.
The plant can be a source material to herbal drug industry since it is a reservoir of phytochemical components that can be used for the development of therapeutic phytomedicine for the therapy and treatments.
We acknowledge Dr. R. Chenraj Jain, President, Jain University Trust., Dr. N Sundararajan, Vice Chancellor, Jain University., Prof. K.S. Shantamani, Chief Mentor, Jain University and Dr. S Sundara Rajan, Director, CASB-Jain University, Bangalore for providing financial assistance, the necessary laboratory facilities and support. We also thank the staff at Genohelix Biolabs for technical support.