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DOI: 10.4103/0974-8520.159010
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Abstract
Background: Opuntia elatior Mill.(Nagaphani) fruits are traditionally recommended as
an expectorant, remedy for whooping cough, asthma, gonorrhea, ulcers, tumors, in the
treatment of diarrhea and syphilis. Many of these diseases are allied with oxidative stress
caused by free radicals. Thus, current research is directed towards finding naturallyoccurring
antioxidants of plant origin. Aim: To evaluate antioxidant potential of hydroalcoholic
extract of the O. elatior fruits(HAOE) and its fractions. Materials and Methods: Using
1,1diphenyl2picrylhydrazyl(DPPH) and nitric oxide radical scavenging assay, total polyphenolic,
flavonoid (FA), flavanone (FO) contents and degree of polymerization in relation with
its antioxidant activity were examined. Results: The experimental data indicated that
the HAOE, ethyl acetate (EAOE) and butanol (BFOE) soluble fractions have shown
significant antioxidant activity. The highest polyphenolic, FA, FO contents and degree of
polymerization were found in EAOE. The scavenging potential was in the order of Ascorbic
Acid > EAOE > BFOE > HAOE > BIOE, where ascorbic acid was used as a positive control.
The increased antioxidant potential of EAOE and BFOE fractions over HAOE extract may be
attributed to the purification achieved by fractionation of the extract which in turn resulted
in an increase in the degree of polymerization and segregation of secondary metabolites.
Conclusion: The fruit of O. elatior can be used as the best alternative for synthetic
antioxidants.
Key words: Antioxidant activity, flavanone, flavonoid, Opuntia elatior, polymerization
and anisaldehyde(Analytical grade, S. D. Fine in the same way without the FolinCiocalteus phenol reagent.
Chemicals Pvt. Ltd., Vadodara, Gujarat, India), Gallic acid was used as a reference for constructing a standard
1,1diphenyl2picrylhydrazyl(DPPH)(SigmaAldrich Chemie, curve(20100mg/ml). The results were expressed as mg of
Steinheim, Germany), precoated silica gel G 60 F254 thin layer gallic acid equivalents(GAE)/g of extract. All determinations
chromatography(TLC) aluminum plates(20cm20cm, were performed in triplicate.
0.2mm thick)(Merck Ltd., Germany) and AR grade chemicals
were used. Determination of flavonoids
Flavonoid content was determined by the aluminum
Plant material, preparation of the extract and its chloride method.[7,8] Briefly, to 1ml of test
fractionation solution(1mg/ml), 1.5ml of 95% alcohol, 0.1ml of 10%
The plant was collected from the local region of Nagpur, aluminum chloride hexahydrate(AlCl3.6H2O), 0.1ml of 1
botanically identified and authenticated at Department of M sodium acetate(CH3COONa) and 2.3ml of distilled
Botany, Rashtrasant Tukadoji Maharaj, Nagpur University, water were added. After incubation at room temperature for
Nagpur, Maharashtra, India. Avoucher specimen(specimen 40min, absorbance of the reaction mixture was measured
no.9786) has been deposited for future reference. at 435nm against corresponding blank, prepared in the same
manner without adding AlCl3. Rutin was used as a reference
The fresh fruits were pulverized and macerated with standard(20100mg/ml), and results were expressed as mg of
hydroalcoholic(ethanolic) solvent(7:3). The hydroalcoholic rutin equivalents(REs)/g of extract. All determinations were
extract(HAOE) was concentrated in a rotary vacuum evaporator performed in triplicate.
to yield a dark reddishbrown mass(yield: 10.5%w/w). For
fractionation, HAOE was triturated with silica(1:3), loaded to Determination of total flavanones
Soxhlet assembly and extracted by ethyl acetate to yield ethyl The modified DNPH method was used for determination of
acetate soluble fraction(EAOE; yield: 0.3%w/w). The ethyl FOs.[9] Naringin was used as the reference standard. Twenty
acetate insoluble portion was further extracted with saturated milligrams of naringin was dissolved in methanol and then
nbutanol(7:3) to yield nbutanol soluble fraction(BFOE; diluted to 5002500 g/ml. One milliliter of each of the diluted
yield: 26%w/w) and nbutanol insoluble fraction(BIOE; yield: standard solutions was reacted separately with 2ml of 1%
70%w/w). The HAOE and these three broad fractions, that is, DNPH reagent and 2ml of methanol at 50C for 50min. After
EAOE, BFOE and BIOE were subjected to phytochemical and cooling at room temperature, the reaction mixture was mixed
antioxidant screening.[3] with 5ml of 1% KOH in 70% methanol and incubated at room
temperature for 2min. Then, 1ml of the mixture was taken,
Phytochemical screening mixed with 5ml of methanol and centrifuged at 1000g
The HAOE and its broad fractions were screened for
for 10min to remove the precipitate. The supernatant was
the presence of unsaturated sterols, triterpenes, tannins,
collected and adjusted to 25ml. The absorbance of the
flavonoids(FA), saponins, carbohydrates and/or sugars with
supernatant was measured at 495nm. All test samples were
TLC. Thin layer plates precoated with silica gel G were used
similarly reacted with DNPH for determination of FOs. The
and development was carried out in the optimized solvent
mean of three readings was used, and the results were expressed
system Toluene: Ethyl acetate: Methanol(5:3:2, v/v/v).
as mg of naringin equivalents(NE)/g of extract.
After the development of chromatogram in the solvents,
plates were dried and sprayed with anisaldehydesulfuric acid, In vitro antioxidant assays
ferric chloride, and the panisidine hydrochloride reagent
1,1diphenyl2picrylhydrazyl radical scavenging assay
for the detection of saponins, tannins, carbohydrate and/or
The free radical scavenging activity was evaluated by the
sugars, respectively. While, detection of FAs, protein/amino
DPPH assay.[10] In its radical form, DPPH has absorbance
acids and unsaturated sterols was carried out using AlCl3,
maxima at 517nm, but upon reduction by an antioxidant,
hydroxylamineferric chloride, ninhydrin, and vanillinsulfuric
the absorbance decreases. Briefly, 1ml of 0.25 mM solution of
acid reagent, respectively, and visualization was carried out
DPPH in methanol was added to 1ml of HAOE/EAOE/BFOE/
under visible and UV light(: 366nm).[4]
BIOE solution in methanol(20100 g/ml). After 20min, the
Hydroalcoholic extract and its fractions were also quantified for absorbance was measured at 517nm. Ascorbic acid was used
the presence of important secondary metabolites such as total as a positive control. The percentage DPPH decolorization
polyphenol(TP), FA, and flavanone(FO) compounds using of the sample was calculated by the following equation. All
following spectroscopic methods. determinations were made in triplicates.
the reaction mixture containing nitrite was removed, mixed with The FO content was examined by DNPH method found
4ml of Griess reagent(0.1%w/v, N(1naphthyl) ethylenediamine in between 0.3 and 2.2 NE mg/g of extract[Table1]. FO
dihydrochloride and sulfanilic acid[0.33%w/v; 1:1]) and allowed content was determined from linear regression equation of
to stand for 30min. Aredviolet colored chromophore was naringin(y=0.0024x+0.0188, r2 = 0.981)[Figure2].
formed in diffused light. The absorbance of these solutions was
The flavones, flavonols, and isoflavones formed complexes only
measured at 540nm against the corresponding blank solution
with aluminum chloride, while FOs strongly reacted only with
in triplicates. The percentage NO inhibition of the sample was
DNPH, so the contents determined by the two methods were
calculated by the equation.
added up to obtain the total FA content(TFA).[12]
% of NO inhibition =([AcontrolAextract]/Acontrol) 100
Degree of polymerization
Where A is the absorbance. The FA and FO contents represented 23.18%(w/w) and
2.38%(w/w) of the TP in HAOE respectively and similar
Observations and Results pattern was observed in all its fractions, suggesting that the
extracts are very complex, contain many other polyphenols
Phytochemical screening such as coumarins, phenolic acids, and tannins. The degree
Qualitative phytochemical screening revealed the presence of of polymerization of the polyphenols present in the samples is
carbohydrates, steroid, FA, proteins and tannins in HAOE extract. substantial and can be estimated by the ratio between the TP
EAOE, BFOE fractions have shown the presence of carbohydrates, and TFA contents. The highest degree of polymerization was
sterols, FAs and tannins. However, BIOE showed the prominent observed in EAOE fraction(4.210.061), and it varies from
presence of carbohydrates and very minute color intensity was 1.83 to 4.21[Table1].
found in the case of FAs and tannins screening[Figure1].
In vitro antioxidant assay
Determination of total polyphenol, flavonoids, Antioxidant activity was carried out using DPPH and NO assay.
Analysis of the free radical scavenging activities of the extracts
f l ava n o n e c o m p o u n d s , a n d d e g r e e o f revealed a concentrationdependent antiradical activity resulting
polymerization from the reduction of DPPH, NO radicals to nonradical form.
The TP content(mg/g) was found in a range of The extract HAOE and fractions EAOE and BFOE have shown
11.0672.83 GAE mg/g of extract and the highest significant antioxidant activity.
content of polyphenolic compounds was found in EAOE
fraction(72.830.111 GAE mg/g of extract)[Table1]. 1,1diphenyl2picrylhydrazyl method
Polyphenol content was determined from linear regression The DPPH radical is considered to be a model for a lipophilic
equation of gallic acid and expressed as GAE of radical. Achain in lipophilic radicals was initiated by the lipid
extract(y=0.010x +0.005, r2 = 0.992)[Figure2]. autoxidation. DPPH is a stable free radical at room temperature
The FA content varied from 5.72 to 15.06 RE mg/g of and accepts an electron or hydrogen radical to become a stable
extracts[Table1]. FA content was ascertained from diamagnetic molecule. The reduction capability of DPPH was
linear regression equation of Rutin(y=0.005x +0.043, determined by the decrease in its absorbance at 517nm, which
r2 = 0.974)[Figure2]. is induced by antioxidants. Positive DPPH test suggests that the
samples were free radical scavengers.
While comparing with the standard, amongst the extract and
its fractions, EAOE has shown most prominent antioxidant
activity with the IC50 value 44.520.531 g/ml. The
a b c d
Figure 1: Graphical representation of phytochemical screening
for the extracts/fractions. (a) Sprayed with ferric chloride
solution at visible light; (b) sprayed with vanillin-sulfuric acid
solution at visible light; (c) sprayed with p-anisidine-hydrochloride
solution at visible light; (d) sprayed with aluminum trichloride Figure 2: Graphical representation of standard linearity curves
solution at 366 nm for gallic acid, rutin, and naringin
Table1: Antioxidant potential and contents of TP, FA, FO in HAOE extract and its fractions of Opuntia elatior fruit
Extract/ DPPH assay NO assay TP(GAE mg/g FA(RE mg/g FO(NE mg/g TFAa Polymerization
fractions IC50 value IC50 value of extract) of extract) of extract) degreeb
HAOE extract 64.140.18 81.430.70 44.000.10 10.200.01 1.050.00 11.250.03 3.910.09
EAOE fraction 44.520.53 51.080.19 72.830.11 15.060.09 2.200.00 17.260.12 4.210.06
BFOE fraction 57.280.21 75.270.28 60.200.60 12.600.14 1.900.00 14.50.4 4.150.00
BIOE fraction 136.360.31 143.400.18 11.060.08 5.720.02 0.300.05 6.020.10 1.830.11
Results are meansSD of three replicates: GAE, RE and NE, respectively; aTFA is determined by adding FA content with FO content; bThe estimation of the polymerization degree was
calculated by the ratio between TP and TFA. SD: Standard deviation,TP:Total phenolics, FA: Flavonoid, FO: Flavanones, HAOE: Hydroalcoholic extract, DPPH: 1,1 diphenyl2picrylhydrazyl,
NO: Nitric oxide, GAE: Gallic acid equivalents, RE: Rutin equivalents, NE: Naringin equivalents, TFA: Total flavonoid
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