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FREE RADICAL SCAVENGING ACTIVITY AND SECONDARY METABOLITES IN PASSIFLORA FOETIDA L.
AJPRHC
Research Article
IN VITRO FREE RADICAL SCAVENGING ACTIVITY AND SECONDARY METABOLITES IN PASSIFLORA
FOETIDA L.
P. JOSEPH ASIR, S. PRIYANGA, S. HEMMALAKSHMI , K.DEVAKI*
Department of Biochemistry, Karpagam University, Coimbatore - 641 021 , India
* Author for correspondence: dr.devaki.bc@gmail.com
This paper is available online at www.jprhc.in
ABSTRACT:
Investigation of the total phenol content, total flavanoid content, in vitro lipid peroxidation and radical scavenging activities from
Passiflora foetida L. Extraction from various solvents were used for the estimations. Among the five extracts, the flavanoid and
phenol content was found to be high in aqueous and ethanol extract of leaf when compared to root extract. Different
concentrations of plant extracts were used (100 – 500 µg/ml) and results showed strong reducing power at the concentration of
500µg/ml and the activity increases in dose dependent manner as to that of standard BHT. That the highest free radical
scavenging activity was found in ethanolic extract of leaf of Passiflora foetida L. Consumption of P. foetida L. will reduce the
free radical formation and afford protection against reactive oxygen species produced during diabetes which may be due to the
presence of phenol and flavanoids.
Keywords: Total flavanoid, total phenols, antioxidants, lipid peroxidation, Passiflora foetida L.
INTRODUCTION:
Medicinal plants have played a significant role in various ancient traditional systems of medicine. They are rich sources of
bioactive compounds and thus serve as an important raw material for drug production and have become a target for the search of
new drugs1. Natural bioactive compounds like phenols and flavonoids are the important secondary metabolites in plants having
intrinsic properties that affect appearance, taste, odor and oxidative stability of plant based foods. These compounds also posses
biological properties like antioxidant, anti-aging, anti-carcinogen, protection from cardiovascular, immune/autoimmune diseases
and brain dysfunctions viz. Parkinson’s, Alzheimer’s, Huntington’s diseases, etc 2. Like in case of laxatives, blood thinners,
antibiotics and antimalaria medications, contain ingredients from plants. Moreover the active ingredients of Taxol, vincristine,
and morphine isolated from foxglove, periwinkle, yew, and opium poppy, respectively3. The plants are the invaluable, incredible
and traditional sources for the curability of various diseases in the form of medicines. Plants are the main source of drugs that
being used from the ancient times as a herbal remedies for the health care, prevention and cure of various diseases and ailments4.
Plants secondary metabolites have been implicated for most of the plants therapeutic activites5. Though different types of oral
hypoglycemic agents are available along with insulin for the treatment of diabetes, there is an increased demand by patients to
use the natural products with antidiabetic activity.
Passiflora is the largest genus in the Passifloraceae family and comprises nearly 500 species. The mostly available wild species
are P. edulis, P. incarnata, P. leschenaultia, P. mollissima and P. subpelta. Passiflora foetida L. (Stinking passion flower) is
South American origin, which has been spread to many tropical areas in India. It is found in riverbeds, dry forest floors, covering
the top thorny shrubs and also growing near hamlets6. It is commonly called as Mupparisavalli, Siruppunaikkalli in Tamil,
Tellajumiki in Telugu, Kukkiballi in Kannada and Chadayan, Poochapazham in Malayalam7.
The ethanobotanical views of P.foetida L. suggest that decoction of leaves and fruits are used to treat asthma, leaves and root
decoction is emmenagogue, used in hysteria and leaf paste is applied on the head for giddiness and headache and skin disease.
Traditionally, the plant has been used for its properties like antiproliferative, sedative, anti-anxiety, antibacterial, leishmanicidal,
antispasmodic, emetic, dressing for wounds and antiulcer8. The major phytoconstituents of this plant are alkaloids, phenols,
glycosides, flavonoids and cyanogenic compounds, passifloricins, polypeptides and alpha-pyrones6.
MATERIALS AND METHODS
Collection, identification and powdering of Passiflora foetida L.
The leaves, root, fruit peel (both ripened & un ripened) & seed of Passiflora foetida L. were collected from in and around
Karpagam University, Coimbatore, Tamilnadu and authenticated by Dr.M. Palanisamy, Botanical Survey of India, Tamilnadu
Agricultural University Campus, Coimbatore. The Voucher No is BSI/SRC/5/23/2012-13/Tech-108. All the parts of plant were
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washed well with water. They were air dried at 250C for 10 days in the absence of sunlight and powdered coarsely using a mixer.
They were then weighed and kept in an airtight container and stored in refrigerator for future use.
Preparation of plant extracts Twenty grams of powdered plant material were mixed with 100 mL of various solvents
(petroleum ether, chloroform, ethyl acetate, ethanol and distilled water). The plant extracts were prepared by using soxhlet
extraction and an orbitory shaker apparatus. After extraction the samples were collected and stored in a vial for further studies.
Estimation of total flavonoids
The flavonoid content was examined by adopting the method developed by Ordon et al.,9 Briefly, 0.5 mL of 2% AlCl 3 in
ethanol solution was added to 0.5 mL of sample solution. After one h incubation at room temperature, yellow colour was
developed. This was measured at 420 nm with a UV-visible spectrophotometer. A standard graph was prepared using the
quercetin and the total flavonoid content was expressed as quercetin equivalent (mg/g).
Estimation of total phenols
Total phenolic content of the ethanolic extract of E. alsinoides was measured based on the Folin-Ciocalteu assay10. Briefly, 0.5
mL of the ethanolic extract was first mixed with 2.5 mL of distilled water, and then 0.5 mL of Folin-ciocalteu reagent was added.
After 3 min, 2 mL of 20% sodium carbonate was added and mixed thoroughly. The tubes were incubated in a boiling water bath
for exactly 1 min. It was then cooled and the absorbance was measured at 650 nm using a spectrophotometer against the reagent
blank. Total phenolic content was expressed as mg gallic acid equivalents (GAE)/g fresh weight.
Reducing power
Various concentrations of the extracts (100, 200, 300, 400 and 500 mg/ml) were prepared. To all the extracts in test tubes 2.5ml
of sodium phosphate buffer was added followed by 2.5 ml of 1% potassium ferricyanide [K3 Fe (CN) 6] solution. The contents
were vortexed well and then incubated at 50ºC for 20 minutes. After incubation, 2.5ml of 10% trichloroacetic acid (TCA) was
added to all the tubes and centrifugation was carried out at 3000 g for 10 minutes. To 5ml of the supernatant, 5 ml of deionised
water was added. To this about 1 ml of 1% ferric chloride was added to each test tube and incubated at 35ºC for 10 minutes. The
reducing power of the extract was linearly proportional to the concentration of the sample. Increased absorbance of the reaction
mixture indicated increased reducing power. Butylated Hydroxyl Toluene (BHT) was taken as reference standard 11.
DPPH radical scavenging assay
Various concentrations (100-500 µg/ml) of samples and ascorbic acid were taken in different test tubes. The volume was adjusted
to 500 µl by adding methanol. 5 ml of 0.1 mM methanolic solution of DPPH was added to these test tubes and vortexed. The
tubes were allowed to stand at room temperature for 20 minutes. The control was prepared as above without any extract and
methanol was used for the baseline correction. Changes in the absorbance of the samples were measured at 517 nm. The radical
scavenging activity was expressed as the inhibition percentage. The inhibition percentage was calculated using the following
formula:
% Radical scavenging activity = [Control OD – Sample OD)/ Control OD] × 100.
Ascorbic acid was taken as reference standard. The percentage inhibition versus concentration was plotted and the
concentration required for 50% inhibition of radicals was expressed as IC50 value12.
Inhibition of in vitro lipid peroxidation
Rat liver was processed to get 10% homogenate in cold phosphate buffered saline, pH 7.4 using homogeniser and centrifuged.
The degree of lipid peroxidation was assayed by estimating the Thio Barbituric Acid Reactive Substance (TBARS) by using the
standard methods with minor modifications. Different concentrations of extracts (100 -500µg /ml) were added to 0.1 ml of liver
homogenate. Lipid peroxidation was initiated by the addition of 100µl of 15mM ferrous sulphate solution to the 3 ml of reaction
mixture. After 30 minutes, 100 µl of this mixture was taken in a tube containing 1.5 ml of 10 % TCA, mixed well and kept for 10
minutes. The tubes were then centrifuged and supernatants were separated and mixed with 1.5 ml of 0.67 % TBA in 50 % acetic
acid. Then the mixture was heated for 30 minutes in a boiling water bath. The intensity of the pink coloured complex formed was
measured at 535 nm. The results were expressed as n moles of MDA formed/ mg protein13.
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RESULTS AND DISCUSION
Total flavanoid content (TFC) of P. foetida L:
Flavanoids are group of polyphenolic compounds with known properties which include free radical scavenging, inhibition of
hydrolytic and oxidative enzymes and anti-inflammatory action. The mechanisms of action of flavanoids are through scavenging
or chelating process14. The flavanoids have the ability to scavenge free radicals; superoxide and hydroxyl radicals by singleelectron transfer15. Consumption of flavanoids may be beneficial because they interact with various biological systems and show
anti-inflammatory, hypolipidemic, hypoglycemic and antioxidant activities16. Flavanoids can affect the functions of cells linked
to inflammatory processes, acting on enzymes and pathways involved in anti-inflammatory processes17.
Flavanoid content of different extracts of Passifolora foetida L. was studied and the results were represented in figure 1a. Among
the five extracts, the flavanoid content was found to be high in aqueous and ethanolic extract of leaf (0.936mg/g, 0.960 mg/g of
quercetin equivalent respectively) & root (0.528mg/g, 0.544 mg of quercetin equivalent/g respectively) whereas, less amount of
flavanoid content was present in petroleum ether extract of plant parts when compare to other extracts.
Figure 1(a): Estimation of total flavanoid content of P. foetida L:
Values are expressed as Mean±SD of three individual experiments
Figure 1(b): Estimation of total phenol content of P. foetida L:
Values are expressed as Mean±SD of three individual experiments
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Total phenol content (TPC) of P. foetida L:
Phenolics compounds are commonly found in both edible and non-edible plants and they have multiple biological effects.
Phenolic compounds are widely distributed in plants, which have gained much attention, due to their antimutegenic, antitumor,
antioxidant activities and free radical-scavenging abilities, which potentially have beneficial implication for human health.The
total phenolic content of different extract of Passiflora foetida L. was studied. In that aqueous and ethanolic extracts of P. foetida
L. showed high amount of total phenol content when compare to other extracts. Among the five extracts, the phenol content was
found to be high in aqueous and ethanolic extract of leaf (1.960mg/g) & root (1.768mg/g, 1.960 mg/g of gallic acid equivalent
respectively) whereas, less amount of phenol content was recorded in petroleum ether extract of plant parts when compare to
other extracts (Figure 1b). Our results are supported by6 who reported that the most of the phenolic content about 9.3% was found
in the ethanolic extract of root of P. foetida L.
In vitro radical scavenging assays of Passiflora foetida L.
The following free radical scavenging assays were performed by using different concentration of aqueous and ethanolic extract of
Passiflora foetida L. which ranges from 100- 500 µg/ml and IC50 value was calculated.
Reducing power
Reducing power Fe (III) reduction is often used as an indicator of electron donating activity, which is an important mechanism of
phenolic antioxidant action, can be strongly correlated with other antioxidant properties18. In the reducing power assay, the
presence of reductants (antioxidants) in tested samples would result in the reduction of Fe3+/ ferricyanide complex to the ferrous
form. The Fe2+ can therefore be monitored by measuring the formation of Perl’s Prussion blue at 700 nm 19. The reducing power
ability of ethanolic and aqueous extract of P. foetida L. was shown in figure 2a & 2b. Different concentrations of plant extracts
were used (100 – 500 µg/ml) and the ethanolic extract of root (1.12 µg/ml) showed strong reducing power at the concentration of
500µg/ml and the activity increases in dose dependent manner when compared with that of standard BHT. Similar results were
reported by6.
DPPH radical scavenging assayDPPH scavenging is widely used to test the free radical scavenging activity of several natural
products20. The DPPH radical scavenging activity of various parts of P. foetida L. was measured and the results are represented
in the table 3(a) and 3(b). Different concentrations were used (100 – 500 µg/ml) and the percent inhibition of the ethanolic extract
of root and aqueous extract of unripe peel was found to be maximum when compared with the standard ascorbic acid. In6
reported that the highest free radical scavenging activity was found in ethanolic extract of leaf of Passiflora foetida L. The
highest radical scavenging activity in leaves may be due to its more phenolic content of P. foetida L. leaf. Phenols actively
remove the freeradicals due to the presence of their free OH group and afford protection against oxidative damage21.
Percentage inhibition of invitro lipid peroxidation with ethanolic & aqueous extract of Passiflora foetida L.
Lipid peroxidation is a free radical mediated process, which has been implicated in a variety of disease states. It involves the
formation and propagation of free radicals, the uptake of oxygen and rearrangement of double bond in unsaturated lipids, which
eventually result in destruction of membrane lipids8. Increased MDA content is an important indicator of lipid peroxidation22.
The excessive generation of free radicals leads to peroxidative changes that ultimately result in enhanced lipid peroxidation. In
figure 4a & 4b, P. foetida L. showed significant (P < 0.01) reduction in vitro lipid peroxidation. Some diseases have been
reported to be associated with increased disruption of membrane lipids leading to subsequent formation of peroxide radicals. The
process of lipid peroxidation generates hydroperoxides, aldehydes and other free radical intermediates which can react with
essential proteins, enzymes and nucleic acids and render them inactive. Elevated lipid peroxidation and poor antioxidant systems
have suggested that a lack of antioxidant defense is responsible for the elevated lipid peroxidation in erythrocytes23. Since the
extract is able to inhibit the lipid peroxidation, it is assumed that the consumption of P. foetida L. will reduce the free radical
formation and afford protection against reactive oxygen species produced during diabetes. This is also due to presence of phenol
& flavanoid content in leaf and root of ethanolic and aqueous extract of P. foetida L.
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Figure 2 (a): Reducing power assay of ethanolic extract of Passiflora foetida L.
Values are expressed as Mean±SD of three individual experiments
Figure 2 (b): Reducing power assay of aqueous extract of Passiflora foetida L.
Values are expressed as Mean±SD of three individual experiments
Figure 3(a): DPPH radical scavanging activity of ethanolic extract of Passiflora foetida L:
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Values are expressed as Mean±SD of three individual experiments
Figure 3(b): DPPH radical scavanging activity of aqueous extract of P. foetida L:
Values are expressed as Mean±SD of three individual experiments
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Figure 4(a): % inhibition of invitro lipid peroxidation with ethanolic extract of
P. foetida L:
Values are expressed as Mean±SD of three individual experiments
Figure 4(b): % inhibition of invitro lipid peroxidation with aqueous extract of P. foetida:
Values are expressed as Mean±SD of three individual experiments
CONCLUSION
In conclusion aqueous and etanolic extract of Passiflora foetida L. leaf & root recorded high amount of total phenol and
flavanoid whereas less amount of phytochemicals were found in petroleum ether extract. A strong reducing power was noted in
ethanolic extract of P. foetida L. root whereas less activity in aqueous extract of P. foetida L. unripe peels. The highest DPPH
free radical scavenging activity was found in ethanolic extract of root and aqueous extract of unripe peel of Passiflora foetida L.
when compared with other parts of plant. The ethanolic extract of leaf and aqueous extract of P. foetida L. root showed maximum
percent inhibition of lipid peroxidation when compare to other parts of P. foetida L.
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ACKNOWLEDGEMENT
We, the authors are thankful to our Chancellor, Advisor, Vice Chancellor and Registrar of Karpagam University for providing
facilities and encouragement.
CONFLICT OF INTEREST STATEMENT
We declare that we have no conflict of interest.
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