INTRODUCTION
Ormocarpum cochinchinense that belonging to Leguminaceae is a small
herb found all over different parts of Tamil Nadu. It is an efficient bone fracture
healer popular only to a few villagers in Tamil Nadu and hence it got its name
as “Elumbotti”. This herb has been used as source of medicine by human
from ancient time to the present day. Most of the medicines in practice were
formulated based on plant derived metabolites (Samy et
al., 2008). India is one of the countries which contain a lot of traditional
knowledge in terms of herbal medicines their effect against various diseases
(Bisht and Badoni, 2009). The scientific evidence was
found to be lacking due to the traditional information that was kept confidential
by the village vaidyas (Vedavathy, 2001).
The specific therapeutic effect of herbal plants contains several bioactive
compounds. The radical scavenging activity of the medicinal plants has thrown
light on their investigation for scientific empowerment (Hazra
et al., 2010). Antioxidants are applied in the food industry as well
as in the cosmetic industry as the functional ingredient to prevent oxidative
damages (Elzaawely and Tawata, 2012). The bioactive
compounds are of very much interest for the scientific advancement through natural
antioxidants (Jayaprakasha and Rao, 2000). Since application
of antioxidants become broad in various areas it is necessary to develop different
types of novel antioxidative agents. The plant source of natural antioxidants
are more favoured due to their eco-friendly nature (Adisa
et al., 2011; Sivakumar and Panneerselvam, 2011a,
b). However, there is no scientific proof justifying
the traditional use of O. cochinchinense leaves in the treatment of “bone
healing”. The present investigation was made to study the biochemically
active natural products, observed antioxidative activities in the different
solvent extracts prepared from the leaves of Ormocarpum cochinchinense.
MATERIALS AND METHODS
Plant materials: The leaves of O. cochinchinense collected from
local area of Villupuram District, Tamil Nadu, India during October-December-2012.
Preparation of plant extract: In green leaves were dried powdered using
with a mechanical grinder and stored in a jib lack cover. Leaf powder (1.25
kg) was refluxed with different solvents, Dimethyl sulfoxamide (DMSO), Ethyl
acetate (EtoAc), ethanol (EtOH), methanol (MeOH) and chloroform (CHCl3)
for five days. The total filtrate was concentrated to dryness, in hot
air oven at 32°C to render five solvent extract for investigation.
Chemicals: All were purchased from SD fine chemical company Mumbai and
all chemicals were of analytical grade.
Phytochemical screening: The phytochemical analysis of the (DMSO, EtoAc,
EtOH, MeOH, CHCl3 of O. cochinchinense leaf was carried out
by standard methods as described in Evans (2000), Harborne
(1998) and Ghani (2003). Specifically, the extract
was screened for the presence of secondary metabolites (flavonoids, saponins,
glycosides, steroids, alkaloids, resins, tannins, terpenoids and acidic compounds
and macronutrient carbohydrate, reducing sugar).
Chemical group tests of the extract: Testing different chemical groups
present in the extract were performed the through phytochemical studies (Evans,
2000). In each test 10% (w/v) solution of extract was taken unless otherwise
mentioned in individual test.
Active-principle analysis
Test for flavonoids: Two hundred milligram each of the extract from
different solvent was heated with 10 mL of ethyl acetate in boiling water for
3 min. The mixture was filtered and the filtrate was used for the following
tests:
| • |
Ammonium test: Four mililiter of the filtrate was shaken
with 1 mL of dilute ammonia solution (1%). The layers were allowed to separate.
A yellow coloration at the ammonia layer indicated the presence of flavonoids |
| • |
Ammonium chloride test: Four mililiter of the filtrates was shaken
with 1 mL of 1% aluminum chloride solution and observed for light yellow
coloration. A yellow precipitate indicated the presence of flavonoids |
Test for glycosides: Dilute sulphuric acid (5 mL) was added to 0.1 g
of the test extract in a test tube and boiled for 15 min in a water bath. It
was then cooled and neutralized with 20% potassium hydroxide solution. A mixture,
10 mL of equal parts of Fehling’s solution A and B were added and boiled
for 5 min. A more dense red precipitate indicated the presence of glycoside.
Test for steroids and terpenoids: Nine mililiter of ethanol was added
to extract and refluxed for a few minute and filtered. Each of the filtrates
was concentrated to 2.5 mL in a boiling water bath. Five mL distilled water
was added to the concentrated solution, the mixture was allowed to stand for
1 h and the waxy matter was filtered off. The filtrate was extracted with 2.5
mL of chloroform using a separating funnel. To 0.5 mL of the chloroform extract
in a test tube, 1 mL of concentrated H2SO4 was carefully
added to form a lower layer. A reddish brown interface showed the presence of
steroids.
A quantity, 0.5 mL of the chloroform extract was evaporated to dryness on a
water bath and heated with 3 mL of concentrated H2SO4 for
10 min on a water bath. A grey colour indicated the presence of terpenoids.
Test for alkaloids: A quantity (0.2 g) of the sample solvent was boiled
with 5 mL of 2% HCl on steam bath. The mixture was filtered and 1 mL filtrate
was treated with 2 drops of the following reagents:
| • |
Dragendorff’s reagent: A red precipitate was formed
indicating presence of alkaloids |
| • |
Wagner’s reagent: A reddish-brown precipitate was formed indicating
presence of alkaloids |
| • |
Hager’s reagent: A yellow precipitates was formed indicating
the presence of alkaloids |
Test for saponins: A quantity of 500 mg of the extract solvent was boiled
with 5 mL of distilled water for 5 min. The mixture was filtered while still
hot and the filtrate was used for the following test:
| • |
Frothing test: A quantity, 1 mL of the filtrate was
diluted with 4 mL distilled water. The mixture was shaken vigorously and
then observed on standing for a stable froth |
Test for tannins: A quantity, 2 g of the extract solvent was boiled
with 5 mL of 45% ethanol for 5 min. The mixture was cooled and filtered. The
filtrate was subjected to the following tests:
| • |
Lead-acetate test: A 1 mL of the filtrate was added
to 3 drops of the lead-acetate solution. A cream gelatinous precipitate
indicates the presence of tannins |
| • |
Ferric chloride test: A quantity (1 mL) of the filtrate was diluted
with distilled water and added 2 drops of ferric chloride. A transient greenish
to black colour indicates the presence of tannins |
Test for acidic compounds: A quantity, 0.1 g of the extract was placed
in a clear dry test tube and sufficient water added. These were warmed differently
in a hot water bath and cooled. A piece of water-wetted litmus paper was dipped
into the filtrate and observed for colour change. Acidic compounds turn blue
litmus paper red.
Test for resins: Two tests were carried out to detect the presence of
resins in the plant part extract under investigation:
| • |
Precipitate test: A quantity, 0.2 g of the extract
was treated with 15 mL of 96% ethanol. The alcoholic extract was then poured
into 20 mL of distilled water in a beaker. A precipitate occurring indicates
the presence of resins |
| • |
Colour test: A quantity, 12 mg of the extract was treated with
chloroform and extracts concentrated to dryness. The residues were re-dissolved
in 3 mL of acetone and 3 mL of concentrated HCl was added. The mixture were
now heated differently in a water bath for 30 min. Pink colour which changed
to magenta red, indicated the presence of resins |
Macronutrients analyses: The test which would be shown subsequently,
were carried out to determine the presence of macronutrients in the herb O.
cochinchinense leaf.
Test for protein
Burette test: A quantity, 2 mL of the extract was put in a test tube
and 5 drops of 1% hydrated copper sulphate and 2 mL of 40% sodium hydroxide
were added and the test-tube was shaken vigorously to mix the contents. A purple
coloration showed the presence of proteins (presence of two or more peptide
bonds).
Test for carbohydrate: A quantity, 0.1 g of the extract was shaken vigorously
with water and then filtered. To the aqueous filtrate, few drops of molisch
reagent were added, followed by concentrated H2SO4 (1
mL) was carefully added to form a layer below the aqueous solution. A brown
ring at the interface indicated the presence of carbohydrates.
Test for reducing sugar: A quantity, 0.1 g of the extract was shaken
vigorously with 5 mL of distilled water and filtered. To the filtrate equal
volumes of Fehling solution A and B were added and shaken vigorously. A brick
red precipitate indicated the presence of reducing sugars.
In vitro test for antioxidant activity
Free radical scavenging activity by DPPH method: Quantitative Assay was
performed on the basis of the modified method of Gupta et
al. (2003). Stock solutions (10 mg mL-1) of the plant extracts
were prepared in different solvent from which serial dilutions were carried
out to obtain concentrations of 10, 20, 30, 40, 50, 100 and 500 mg mL-1.
A quantity (2 mL) of diluted solutions were added to 2 mL of a 0.004% ethanol
solution of DPPH, mixed and allowed to stand for 30 min for reaction to occur.
The absorbance was determined at 517 nm and from these values corresponding
percentage of inhibition were calculated. The experiment was performed in duplicate
and average absorption was noted for each concentration. Ascorbic acid was used
as positive control.
DPPH free radical scavenging activity was determined by the method described
by Choi et al. (2007) and Desmarchelier
et al. (1997). Plant extract (0.1 mL) was added to 3 mL of a 0.004%
MeOH solution of DPPH. Absorbance at 517 nm was determined after 30 min and
percentage inhibition was calculated:
where, A0 is the absorbance of the control and A1 is
the absorbance of the extract/ standard. IC50 value was calculated
from the equation.
Statistical analysis: Statistical analysis of the results was performed
by student’s t-test for independent samples. Values of p<0.001 were
considered significant.
RESULTS
Phytochemical and macronutrient screening: The phytochemical screening
of DMSO from the O. cochinchinense leaves indicated the presence of alkaloids,
steroids, glycosides, saponins, tannins, acidic compounds, resins and coumerines
but not flavanoids, acidic compounds, resins and coumerins. Quantitatively,
alkaloids were more whereas resins were least among the detected secondary metabolites
(Table 1). Furthermore, only carbohydrates (in moderate abundance)
were found to be present in the DMSO of O. cochinchinense (Table
2).
Ethyl acetate (EtoAc) extract of O. cochichinense leaves indicated the
presence of flavanoids, steroids, glycosides, saponins, tannins, acidic compounds,
resins and coumerines but not detected alkaloids. Quantitatively, flavanoids
were more whereas resins were least among the detected secondary metabolites
(Table 1). Furthermore, only carbohydrates (in moderate abundance)
were found to be present in the ethyl acetate of O. cochinchinense (Table
2).
Ethanol extract of O. cochichinense leaves indicated the presence of
flavanoids, alkaloids, steroids, glycosides, saponins, tannins, acidic compounds,
resins and coumerines but not fats and oils. Quantitatively, alkaloids were
more whereas resins were least among the detected secondary metabolites (Table
1). Furthermore, only carbohydrates (in moderate abundance) were found to
be present in the ethanol of O. cochinchinense (Table 2).
Phytochemical screening of methanol from the O. cochinchinense leaves
indicates the presence of flavanoids, steroids, glycosides, saponins, tannins,
acidic compounds, resins and coumerines but not detected alkaloids and acidic
compounds. Quantitatively, flavanoids were more whereas resins were least among
the detected secondary metabolites (Table 1). Furthermore,
only carbohydrates (in moderate abundance) were found to be present in the methanol
of O. cochinchinense (Table 2).
| Table 1: |
Results of phytochemical analyses on O. cochinchinense
leaves using the different solvent extracts |
 |
| -: Absent, +: Low in abundance, ++: Moderate in abundance,
+++: High in abundance and Nd: Not detected |
| Table 2: |
Results of macronutrient analyses on O. cochinchinense
leaves using the test solvent extracts |
 |
| -: Absent, +: Low in abundance, ++: Moderate in abundance,
+++: High in abundance and Nd: Not detected |
Chloroform extract of O. cochichinense leaves indicates the presence
of alkaloids, steroids, glycosides, saponins, tannins, acidic compounds, but
not detected resins, coumerines fats and oils. Quantitatively, alkaloids were
more whereas resins were least among the detected secondary metabolites (Table
1). Furthermore, only carbohydrates (in moderate abundance) were found to
be present in the ethanol of O. cochinchinense (Table 2).
In vitro antioxidant activity
DPPH free radical scavenging activity: Dimethyl sulfoxamide extract of
O. cochichinense showed potential antioxidant activity where the IC50
was 13.05±0.39 μg mL-1 (p<0.001), as compared that
of ascorbic acid (IC50 6.10±0.18 μg mL-1)
(p<0.001) which is well known antioxidant (Table 3). The
extract caused an increase in DPPH free radical scavenging activity (% inhibition)
as increasing dose (Table 4). This table showed most potent
inhibitor with a (IC50 83.36±0.43 μg mL-1)
which was comparable to ascorbic acid (IC50 85.12±1.25 μg
mL-1).
Ethyl acetate (EtoAc) extract of O. cochichinense showed potential
antioxidant activity where the IC50 was 14.08±0.42 μg
mL-1 (p<0.001), as compared that of ascorbic acid (IC50 7.18±0.21
μg mL-1) (p<0.001) which is well known antioxidant (Table
3). Ethyl acetate extract caused an increase in DPPH free radical scavenging
activity (% inhibition) as increasing dose (Table 4). EtoAc
extract most potent the hydroxyl radical inhibition tests (IC50 83.33±0.40
μg mL-1) which was comparable to (IC50 84.68±1.20
μg mL-1) ascorbic acid.
Ethanol extract of O. cochichinense showed potential antioxidant activity
where the IC50 was 15.02±0.45 μg mL-1 (p<0.001),
as compared that of ascorbic acid (IC50 8.15±0.24 μg
mL-1) (p<0.001) which is well known antioxidant (Table
3). The ethanol extract caused an increase in DPPH free radical scavenging
activity (% inhibition) as increasing dose (Table 4). As the
ethanol extract exhibited considerable free radical inhibition properties are
(IC50 82.89±0.41 μg mL-1) which was comparable
to (IC50 84.18±1.31 μg mL-1) ascorbic acid.
| Table 3: |
DPPH free radical scavenging activity of O. cochinchinense
leaves using the different solvent extract |
 |
| Table 4: |
DPPH radical scavenging activity of O. cochinchinense
leaves using the different solvent extract |
 |
| Values represent the Mean±SED, No. of readings in each
group = 3 |
| Table 5: |
DPPH radical scavenging activity of O. cochinchinense
leaves using the different solvent extract |
 |
| Values represent the Mean±SED, No. of readings in each
group = 3 |
Table 4 and 5 indicated that hydroxyl radical
was scavenged from ethanol extract in a dose dependent manner. The other solvent
extracts appeared to have relatively higher or lower activities.
Methanol extract of O. cochichinense showed potential antioxidant activity
where the IC50 was 16.12±0.48 μg mL-1 (p<0.001),
as compared that of ascorbic acid (IC50 9.10±0.27 μg
mL-1) (p<0.001) which is well known antioxidant (Table
3). Methanol extract caused an increase in DPPH free radical scavenging
activity (% inhibition) as increasing dose (Table 5). This
table showed most potent the hydroxyl radical scavenge inhibition test (IC50
84.73±0.40 μg mL-1) which was comparable to (IC50
85.76±1.32 μg mL-1) ascorbic acid.
Chloroform extract of O. cochichinense showed potential antioxidant
activity where the IC50 was 14.88±0.44g mL-1 (p<0.001),
as compared that of ascorbic acid (IC50 8.18±0.23 μg
mL-1) (p<0.001) which is well known antioxidant (Table
3). The extract caused an increase in DPPH free radical scavenging activity
(% inhibition) as increasing dose (Table 5). The CHCl3
extract are free radical inhibition tests (IC50 83.13±0.42
μg mL-1) which was comparable to (IC50 85.68±1.25
μg mL-1) ascorbic acid.
DISCUSSION
The preliminary phytochemical screening of the chemical constituents of different
solvents from the O. cochinchinense plants showed that the leaves generally
contain the major secondary metabolites in moderate abundance. In higher plants
the flavonoids becomes inseparable with the antioxidant potentials that could
cure heart diseases and also cance (Noroozi et al.,
1998; Al-Humaid et al., 2010). Vitamin A,
C and E and flavonoids from plant sources are antioxidants in diet (Pietta,
2000). Thus, the absence of flavonoids in the DMSO extract of O. cochinchinense
leaves might limit the solvent choice of DMSO in the extraction of active medicinal
ingredients from O. cochinchinense leaves. Hence, the moderate abundance
of alkaloids in the leaves of O. cochinchinense appears to support the
efficacy of the use of the leaves in ethno-medicinal practice. The absence of
alkaloids in the (EtoAc) extract of O. cochinchinense leave limit the
solvent choice of (EtoAc) in the extraction of active medicinal ingredients
from O. cochinchinense leaves.
The presence of flavonoids from ethanol extracts of O. cochinchinense leaves
observed in the present study also attests to the possible efficacy of therapeutic
use of O. cochinchinense leaves. They are related to sex hormones and
could, by serving as potent starting material in the synthesis of sex hormones,
ensure such hormonal balance (Okwu, 2001). This could
in addition of the noted high carbohydrate content of O. cochinchinense leaves
that could provide useful energy, be highlighting the possible reproductive
benefits from the prospective use of O. cochinchinense leave as nutraceutical
beverage. The absence of acidic compounds from methanol extract of O. cochinchinense
leaves might limit the solvent choice of MeOH in the extraction of active medicinal
ingredients from O. cochinchinense leaves. The absence of flavonoids,
acidic compounds, resins, coumerins in the CHCl3 extract of O.
cochinchinense leaves might limit the CHCl3 solvent choice of
extraction and active medicinal ingredients from O. cochinchinense leaves.
The phytochemical analysis of the extract showed the presence of flavonoids,
alkaloids, saponins, sterols, terpenoids, resins and sugar. These constituents
may be responsible for antioxidant potentiality of O. cochinchinense.
CONCLUSION
The present study reveals that different solvent extracts of O. cochinchinense
leaves possesses significant of phytochemical screening and antioxidative activity.
Now research is continued to isolate lead compound from these extracts and also
to develop a potent formulation for treatment of bone healing activities.
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