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Comparative antioxidant and bioavailability studies of Vitamin C in Phyllanthus emblica Linn. and its combinations with Piper nigrum Linn. and Zingiber officinale Roscoe

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  • East West Group of Institutions Bangalore

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Phyllanthus emblica Linn. (amla) is used in Ayurveda, the ancient Indian system of medicine and its major constituent is vitamin C which has effective free radical scavenging property. The purpose of this study was to evaluate the in vitro antioxidant activity and the bioavailability profile of vitamin C in amla and its combinations with piperine and ginger in comparison to synthetic vitamin C using New Zealand rabbits. In vitro antioxidant activity studies of synthetic vitamin C, amla, amla with piperine and amla with ginger were carried out using different models such as 2,2-Diphenyl-1-picrylhydrazyl, Nitric Oxide, Hydrogen peroxide scavenging methods, Total reductive capability and Oxygen Radical Absorbance Capacity estimation. The study results showed that synthetic vitamin C, amla, amla with piperine and amla with ginger possess significant in vitro antioxidant activity. For bioavailability studies, synthetic vitamin C, amla, amla with piperine and amla with ginger 100 mg/kg, were administered orally and the serum samples were analyzed by HPLC at 0, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours. Bioavailability studies revealed that amla with piperine combination has higher concentration of vitamin C when compared to synthetic vitamin C. This is probably due to presence of piperine, which is a bioavailability enhancer. The present study supports the fact that amla with piperine combination can be an alternative to synthetic vitamin C. © 2016, Faculdade de Ciencias Farmaceuticas (Biblioteca). All rights reserved.
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*Correspondence: Vanita Somasekhar. KLE University’s College of Pharmacy.
Rajajinagar - P.B.No. 1062, II Block. Bengaluru - 560010, Karnataka, India.
E-mail: vanitasom@gmail.com
Article
Brazilian Journal of
Pharmaceutical Sciences
vol. 52, n. 1, jan./mar., 2016
<http://dx.doi.org/10.1590/S1984-82502016000100005
Comparative antioxidant and bioavailability studies of Vitamin C in
Phyllanthus emblica Linn. and its combinations with Piper nigrum
Linn. and Zingiber officinale Roscoe
Vanita Somasekhar1,*, Purnima Ashok1, Sri Adibatla Renuka Kameswari1,
Ramaswamy Rajendran2, Rajpreet Singh1
1KLE University’s College of Pharmacy, Bengaluru, Karnataka, India, 2Green-Chem Herbal Extracts and Formulations,
Bengaluru, Karnataka, India
Phyllanthus emblica Linn. (amla) is used in Ayurveda, the ancient Indian system of medicine and its
major constituent is vitamin C which has eective free radical scavenging property. The purpose of this
study was to evaluate the in vitro antioxidant activity and the bioavailability prole of vitamin C in amla
and its combinations with piperine and ginger in comparison to synthetic vitamin C using New Zealand
rabbits. In vitro antioxidant activity studies of synthetic vitamin C, amla, amla with piperine and amla
with ginger were carried out using dierent models such as 2,2-Diphenyl-1-picrylhydrazyl, Nitric Oxide,
Hydrogen peroxide scavenging methods, Total reductive capability and Oxygen Radical Absorbance
Capacity estimation. The study results showed that synthetic vitamin C, amla, amla with piperine and
amla with ginger possess signicant in vitro antioxidant activity. For bioavailability studies, synthetic
vitamin C, amla, amla with piperine and amla with ginger 100 mg/kg, were administered orally and the
serum samples were analyzed by HPLC at 0, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours. Bioavailability studies
revealed that amla with piperine combination has higher concentration of vitamin C when compared to
synthetic vitamin C. This is probably due to presence of piperine, which is a bioavailability enhancer.
The present study supports the fact that amla with piperine combination can be an alternative to synthetic
vitamin C.
Uniterms: Phyllanthus emblica Linn. Vitamin C/antioxidant activity/in vitro study. Vitamin C/
bioavailability. Vitamin C/synthetic/antioxidant activity. Ginger. Piperine.
Phyllanthus emblica Linn. (amla) é utilizada na medicina Ayurveda, medicina da Índia antiga e seu
principal constituinte é a vitamina C, que possui propriedade sequestrante de radicais livres. O propósito
deste estudo foi avaliar a atividade antioxidante in vitro e o perl de biodisponibilidade da vitamina
C na amla e suas combinações com piperina e gengibre em comparação com a vitamina C sintética,
utilizando coelhos da Nova Zelândia. Os estudos de atividade antioxidante in vitro de vitamina C
sintética, amla, amla com piperina e amla com gengibre foram realizados utilizando-se diferentes modelos
para sequestrantes, como 2,2-difenil-1-picrilidrazil, óxido nítrico, peróxido de hidrogênio, capacidade
redutiva total e a estimativa da capacidade de absorvância do radical oxigênio. Os resultados do estudo
mostraram que vitamina C sintética, amla, amla com piperina e amla com gengibre possuem atividade
antioxidante in vitro signicativa. Para os estudos de biodisponibilidade, administraram-se oralmente
vitamina C sintética, amla, amla com piperina e amla com gengibre 100 mg/kg e as amostras de soro foram
analisadas por CLAE em 0, 1, 2, 3, 4, 6, 8, 10, 12 e 24 horas. Os estudos de biodisponibilidade revelaram
que a associação de amla com piperina tem maior concentração de vitamina C, quando comparada com
a vitamina C sintética. Este efeito é provavelmente devido à presença de piperina, que é intensicador
de biodisponibilidade. O presente estudo apoia o fato de que a associação de amla e piperina pode ser
uma alternativa para a vitamina C sintética.
Unitermos: Phyllanthus emblica Linn. Vitamina C/atividade antioxidante/estudo in vitro. Vitamina C/
biodisponibilidade. Vitamina C/sintética/atividade antioxidante. Gengibre. Piperina.
V. Somasekhar, P. Ashok, S. A. R. Kameswari, R. Rajendran, R. Singh
36
INTRODUCTION
Cell damage caused by free radicals is a major
contributor to ageing and to degenerative diseases such as
cancer, cardiovascular disease, cataracts, immune system
decline and brain dysfunction (Percival, 1998).
One line of defense against free radical damage is
the presence of antioxidants. Antioxidant means “against
oxidation”. An antioxidant is a molecule stable enough
to donate an electron to a rampaging free radical and
neutralize it, thus reducing its capacity to damage. Some
such antioxidants, including glutathione, ubiquinol and
uric acid are produced during normal metabolism in
the body, other lighter antioxidants are found in the diet
and the best known are vitamin E, vitamin C and the
carotenoids.
Vitamin C (ascorbic acid) is a water soluble organic
compound involved in many biological processes
(Gazdik et al., 2008). It is one of the most ubiquitous
vitamins ever discovered and plays a paramount role
as an antioxidant and a free radical scavenger, able to
moderate the oxidative stress eects of various diseases
(Karslen et al., 2005). It has been found in fruits and
vegetables like citrus fruits, pepper, cabbage, spinach,
strawberries, tomatoes, turnip and other leafy vegetables.
The estimated average requirement and recommended
dietary allowance of ascorbic acid are 100 mg and 120 mg
per day respectively.
Ascorbic acid helps in the metabolism of cholesterol,
contributes to the synthesis of the amino acid, protects the
DNA of cell from damage and acts as a potential scavenger
of free radicals.
Phyllanthus emblica Linn. (amla) has been used in
Ayurveda and its major constituent is vitamin C which
has eective free radical scavenging property (Khopde et
al., 2001). The petroleum extract of Piper nigrum Linn.
(P) (Black pepper) has been reported to have antioxidant
activity (Singh et al., 2008). Zingiber ocinale Roscoe(G)
(Ginger) has high content of antioxidants which makes
it a free radical scavenger (Kikuzaki, Nakatani, 1993;
Kikuzaki et al., 1994). Hence, the present study was
carried out to evaluate the in vitro antioxidant activity of
amla and its combinations with piperine and ginger when
compared with synthetic vitamin C.
Many analytical techniques including sensors and
biosensors have been suggested for detection of ascorbic
acid in varied types of samples. HPLC combined with
UV-visible detector is the most common method for
identication of antioxidant vitamins in biological uids
(Zhao et al., 2004). The accepted gold standard method
of measuring vitamin C in serum or plasma is high
performance liquid chromatography (HPLC) (Emadi-
Konjin et al., 2005).
Since there are no reports on the bioavailability
studies of the combinations of amla with piperine and
ginger, this study proposes to investigate the same in
rabbits.
MATERIAL AND METHODS
Material
Phosphoric acid (HPLC grade) was obtained from
Spectrochem Pvt. Ltd., Mumbai. Methanol (HPLC grade)
and HPLC water were obtained from Central Drug House
Ltd., Gujarat. Monobasic potassium phosphate (Sd ne-
chem Limited, Mumbai) and perchloric acid (Merck
specialities. Pvt. Ltd., Mumbai) were used in the study.
2,2-Diphenyl-1-picrylhydrazyl (DPPH), fluorescein,
2,2’-azobis(2-amidino-propane) dihydrochloride
(AAPH) and trolox were obtained from Sigma-Aldrich
Inc., USA. Potassium ferricyanide, trichloroacetic acid,
ferric chloride, sodium nitroprusside, sulphanilamide,
N-(1-naphthyl) ethylene diamine dihydrochloride,
potassium dihydrogen phosphate, sodium hydroxide,
sodium chloride, sodium dihydrogen phosphate and
disodium hydrogen phosphate were obtained from Sd
ne-chem Limited, Mumbai, India. O–phosphoric acid
was obtained from Ranbaxy Fine Chemicals Limited,
Mumbai. Hydrogen peroxide was obtained from V.L.
Products, Mumbai.
Amla (A), piperine (P) and ginger (G) were
dry aqueous, alcoholic and hydroalcoholic extracts
respectively. All samples including synthetic vitamin C
were obtained as gift samples from M/s Green Chem
Herbal Extracts and Formulations, Domlur, Bengaluru.
Amla with piperine (A+P) was a mixture of amla (99.8 g)
and piperine (0.2 g). Amla with ginger (A+G) was a
mixture of amla (95.5 g) and ginger (4.5 g).
Animals
New Zealand rabbits of either sex with a body
weight of approximately 2 kg were procured from
registered breeder M/s Shri Venkateshwara Enterprises,
Bengaluru. Animals were housed in animal house facility
of KLE University’s College of Pharmacy, Bengaluru.
All the animals were housed according to CPSCEA
guidelines under standard animal house conditions. All
the animals were maintained in hygienic conditions with
food and water ad libitum. All animals were acclimatized
to laboratory condition for a week before commencement
Comparative antioxidant and bioavailability studies of Vitamin C in Phyllanthus emblica Linn. 37
of experiment. The study was approved by Institutional
Animal Ethics Committee (Reg. No.626/02/a/CPCSEA).
In vitro studies of antioxidant activity
DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical
scavenging activity
The free radical scavenging activity can be measured
using 2,2-diphenyl-1-picrylhydrazyl by the method of
McCune and Johns (McCune, Johns, 2002). 0.1 mM
solution of DPPH in methanol was prepared, 1 mL of this
solution was mixed with 1 mL of solution of test extract/
standard antioxidant and 1 mL of methanol at dierent
concentrations in the range of 15-75 µg/mL. The mixture
was incubated for 10 min in dark. After 10 min, absorbance
of the mixture was measured at 517 nm using Ultraviolet-
Visible Spectrophotometer (Shimadzu UV-1700 PC
spectrophotometer).
The % scavenging activity was calculated using the
following equation:
% SA = (A0 - A1/ A0) × 100
where % SA= percentage scavenging activity, A0 =
absorbance of control, A1 = absorbance of sample/
standard.
Scavenging of hydrogen peroxide
The free radical scavenging activity was determined
by using hydrogen peroxide (Ruch et al., 1989). Dierent
concentrations of the extract and standard in the range
2-10 µg/mL were prepared in distilled water and 0.6 mL
of hydrogen peroxide solution (40 mM) prepared in
phosphate buffer (pH 7.4) was added to make a final
volume of 4 mL. Absorbance of hydrogen peroxide at
230 nm was measured after 10 min against a blank solution
containing phosphate buer without hydrogen peroxide
and the percentage scavenging activity was calculated.
Total reduction capability
Total reduction capability was estimated using
the method of Gulcin (Gulcin et al., 2005). Different
concentrations of test/standard antioxidant (15-75 µg/mL)
in 1 mL of distilled water was mixed with 2.5 mL phosphate
buer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide
(1%). The mixtures were incubated at 50°C for 20 min.
2.5 mL trichloroacetic acid (10 %) was added to the mixture
and was centrifuged for 10 minutes at 1000 × g. 2.5 mL of
upper layer was mixed with 2.5 mL distilled water and 0.5
mL ferric chloride (0.1%) and the absorbance was measured
at 700 nm using UV-Visible spectrophotometer (Shimadzu
UV-1700). Higher absorbance of the reaction mixture
indicates greater reducing power.
Nitric oxide scavenging activity
Nitric oxide scavenging activity was determined
according to Sumanont et al. (2004). Nitric oxide radicals
were generated from sodium nitroprusside solution in
phosphate buer saline (PBS) at physiological pH (7.4).
Sodium nitroprusside solution (100 mM, 0.2 mL), with
1 mL of test/standard antioxidant solution and 1.8 mL of
PBS was mixed in dierent concentrations (2-10 µg/mL).
The mixture was incubated at 25°C for 180 minutes. 1 mL
of incubated solution was mixed with 1 mL of Griess
reagent (Equal portions of 1% sulphanilamide and 0.1%
N-(1-Naphthyl) ethylene diamine dihydrochloride in 2%
H3PO4). Absorbance was measured at 540 nm using UV
visible spectrophotometer (Shimadzu UV-1700) and the
percentage inhibition was calculated.
Oxygen Radical Absorbance Capacity assay
The Oxygen Radical Absorption Capacity (ORAC)
assay is a method which measures the loss of uorescein
uorescence over time due to peroxyl-radical formation
by the breakdown of AAPH (2,2’-azobis-2-methyl-
propanimidamide dihydrochloride). Trolox [6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid], a water
soluble vitamin E analog serves as a positive control
inhibiting uorescein decay in a dose dependent manner
(DeLang, Glazer, 1989; Cao et al., 1993).
Bioavailability studies
Experimental design and treatment
All the extracts were suspended in water and
administered orally to overnight fasted animals in the
dose of 100 mg/kg body weight, selected on the basis of
acute toxicity studies (OECD guidelines). Bioavailability
studies were carried out in 16 rabbits, divided into four
groups. Group 1: ascorbic acid, Group 2: amla, Group 3:
amla and piperine, Group 4: amla and ginger.
Instrumentation
The HPLC instrument used consisted of Merck
Hitachi LaChrom chromatographic system equipped
with Hitachi pump L-7110, Rheodyne universal injector
7725 and L-7400 Hitachi UV-visible detector. The
chromatographic studies were performed using Thermo
scientic ODS hypersil 5 µm, 250 × 4.6 mm i.d. column,
at ambient temperature. The mobile phase consisted of
30 mM monobasic potassium phosphate (pH 3.6) and
methanol in the ratio 82.5:17.5 (v/v) and the ow rate was
V. Somasekhar, P. Ashok, S. A. R. Kameswari, R. Rajendran, R. Singh
38
1 mL/min. Chromatograms were recorded at 250 nm and
the injection volume was 20 µL.
Sample collection
The bioavailability studies were done by drawing
blood samples without the addition of anticoagulant from
rabbit’s marginal ear vein at 0, 1, 2, 3, 4, 6, 8, 10, 12, 24 h.
Serum was separated by centrifugation at 8500 rpm for
10 min and estimation of vitamin C was done by using
HPLC (Ghosh et al., 2009). Perchloric acid (25 mL of
0.1 M) and 55 mL of distilled water were added to a 20 mL
aliquot portion of serum. Addition of acid was needed to
maintain the stability of ascorbic acid (Karatepe, 2004).
Standard solution preparation
The stock solution of SVC was 100 µg/mL in mobile
phase and all dilutions subsequently were made in mobile
phase. The serum was spiked with standard solution of
vitamin C to conrm the peak (Scartezzini et al., 2006).
Calibration curves
Standard solutions of SVC in the concentration
range 0.5-25 µg/mL were prepared and injected into the
HPLC system. The analyte peak area values were plotted
against the corresponding concentrations of the analyte
and the calibration curve was constructed by means of the
least square method.
Sample analysis
An aliquot of the sample was injected into the HPLC
system in triplicate. The area of SVC peaks obtained after
injecting the extract into the HPLC was interpolated on
the calibration curve.
Statistical analysis
Data were expressed as mean ± SEM. Statistical
dierences between means were determined by One-way
ANOVA followed by Dunnett’s post hoc test. Values of
p < 0.05 were considered as signicant.
RESULTS
In vitro antioxidant activity studies
DPPH radical scavenging activity
All the test compounds (SVC, A, A+P, A+G)
produced DPPH scavenging activity in the concentration
range of 15-75 µg/mL and it was found to increase with
increase in concentration (Figure 1). The scavenging
eect was found to be decreasing in the order of SVC >
A+G > A > A+P at the concentration of 75 µg/mL. There
was significant free radical scavenging produced by A
(p < 0.05), A+P (p < 0.001), A+G (p < 0.05) combinations
compared to SVC. The IC50 values of SVC, A, A+P, A+G
were found to be 38.14 ± 0.01, 38.30 ± 0.03, 38.44 ± 0.04
and 38.29 ± 0.01 respectively (Table I).
Hydrogen Peroxide scavenging activity
The hydrogen peroxide scavenging activity of all
samples and standard are summarized in Figure 2. The
activity was found to decrease in order of A+P > A > SVC
> A+G at a concentration of 2 μg/mL. A+P combination
produced maximum scavenging of hydrogen peroxide
when compared to SVC. The IC50 values of SVC, A, A+P,
A+G were found to be 1.16 ± 0.01, 1.15 ± 0.01, 1.13 ±
0.001 and 1.17 ± 0.01 respectively (Table I).
Total reduction capability
It was found that the total reduction capability
increased with increase in concentration from
15-75 µg/mL for all samples tested but none of the samples
showed signicant scavenging activity in this method.
Nitric Oxide scavenging activity
Effect of nitric oxide scavenging activity was
found to decrease in the order SVC > A+G > A+P > A
at a concentration of 10 μg/mL. Capability to scavenge
nitric oxide was found to be concentration dependant at
all concentrations from 2-10 µg/mL. Maximum inhibition
was produced at concentration 10 µg/mL and the results
are summarized in Figure 3. There was a significant
scavenging activity produced by A and A+P when
compared to SVC (p < 0.001) and the IC50 values for SVC,
A, A+P, A+G were found to be 6.35 ± 0.08, 7.24 ± 0.36,
6.76 ± 0.25 and 6.49 ± 0.42, respectively, as shown in
Table I.
Determination of antioxidant capacity of samples
FIGURE 1 - Free radical scavenging activity of different
concentrations of SVC, A, A+P, A+G by DPPH method.
Comparative antioxidant and bioavailability studies of Vitamin C in Phyllanthus emblica Linn. 39
by Oxygen Radical Absorbance Capacity (ORAC) assay
The antioxidant capacity of these compounds was
as follows: A+P >A> SVC >A+G .
Bioavailability studies
A good linearity was found from 0.5 to 25 μg/mL
of SVC and the linear regression equation was y = 25155x
- 15522 (r= 0.9975), where y is the peak area and x is the
concentration of Vitamin C expressed as μg/mL (Figure 4).
The HPLC method was validated and data shown in
Table III.
The presence of vitamin C was detected at 3.31 min.
Comparisons were made between the standard and
sample chromatograms. Larger area indicates larger
amount of vitamin C. Little interferences were detected
in the chromatogram due to contaminants. By comparing
the AUC, higher bioavailability was observed for A+P,
followed by A, SVC and A+G (Table IV).
DISCUSSION
The amla fruit contains more than 80% water. It also
has protein, carbohydrate, bre, minerals and vitamins.
It also contains gallic acid which is a potent polyphenol.
Amla restores the vitality and rejuvenates all bodily
systems. It is a rich source of vitamin C and has been
used as a powerful antioxidant agent which also boosts
immunity.
Vitamin C is important for human beings as it is
necessary for the synthesis of intercellular cement “collagen”.
TABLE I - IC50 values of SVC, A, A+P, A+G in hydrogen peroxide, DPPH and nitric oxide. All values are Mean ± SEM (n=3)
IC50 values ± SEM (µg/mL) for free radical scavenging activity
Test/ standard group Hydrogen peroxide DPPH Nitric oxide
SVC 1.16 ± 0.01 38.14 ± 0.01 6.35 ± 0.08
A1.15 ± 0.01 38.30 ± 0.03 7.24 ± 0.36
A+P 1.13 ± 0.001 38.44 ± 0.04 6.76 ± 0.25
A+G 1.17 ± 0.01 38.29 ± 0.01 6.49 ± 0.42
FIGURE 2 - Hydrogen peroxide scavenging activity.
FIGURE 3 - Free radical scavenging activity of different
concentrations of SVC, A, A+P, A+G by nitric oxide radical
scavenging method.
TABLE II - Antioxidant capacity of SVC, A, A+P and A+G by
ORAC Assay
Sl. No Sample Name ORAC value (TE/g)
1 SVC 2465
2 A 2651
3A+P 3150
4A+G 1875
FIGURE 4 - Calibration curve of Vitamin C.
V. Somasekhar, P. Ashok, S. A. R. Kameswari, R. Rajendran, R. Singh
40
Collagen is responsible for keeping the cells of the body
together. Hence, vitamin C helps to preserve the normal
immune function and promotes rejuvenation of cells.
Recent reports indicate that increased dietary
intake of antioxidant-rich foods decreases the incidence
of human diseases. However, synthetic antioxidants,
such as butylated hydroxytoluene (BHT) and butylated
hydroxyanisole (BHA) that have been widely used as
antioxidants in the food industry may be responsible for
liver damage and carcinogenesis. For this reason, the
use of natural antioxidants with lesser side eects, are
preferred. This work focuses on the antioxidant activity of
selected natural product, amla, alone and in combination
with piperine and ginger, compared with synthetic
vitamin C for their beneficial antioxidant potential.
Chemical investigations have indicated that amla is rich
in tannins, alkaloids, phenolic compounds, aminoacids,
carbohydrates, vitamin C, quercetin and chebulagic acid
(Khan, 2009). Piper nigrum is called the king of spices
and is one of the oldest spices which contains volatile
oil, crystalline alkaloids, piperine, piperidine, piperitine,
piperolein A, piperolein B and resins (Manoj et al., 2004).
The reported chemical constituents of Zingiber ocinale
are cineole, geraniol, citralgingerols, vitamins like
thiamine and vitamin C (Kalpagam et al., 2003).
Hydrogen peroxide initiates lipid peroxidation
weakly. However, it is able to produce active oxygen
species by generating highly reactive hydroxyl radical
through the Fenton reaction (Powers, Jackson, 2008). All
the test samples (SVC, A, A+P, A+G) were signicantly
dierent in terms of antioxidant potency. A+P showed
the highest scavenging activity which may be due to the
terpenoids which are powerful compounds with enormous
ability to mop up cell or damage free radicals followed by
A, SVC and A+G.
The DPPH radical is a lipophilic and relatively stable
nitrogen centred free radical that can accept an electron to
become a stable diamagnetic molecule (Yoganandam et
al., 2010; Bharathi et al., 2010). The eect of antioxidants
on DPPH radical scavenging is due to their hydrogen
donating ability where DPPH radical serves as the
oxidizing substrate which can be reduced by an antioxidant
compound to its hydrazine derivative. From the results it
is evident that the test compounds are acting as hydrogen
donors and A+G combination possesses highest DPPH
radical scavenging activity when compared to other
FIGURE 5 - Concentration of vitamin C vs Time plot in SVC,
A, A+P, A+G.
TABLE III - Analytical parameters of the HPLC procedure for Vitamin C quantitation
Parameter Vitamin C
Linearity range 0.5 to 25 μg/mL
Regression equation y = 25155x - 15522
Correlation coecient (r) 0.9975
Analyte Concentration (μg/mL) 5 15 25
Repeatability (R.S.D%)a (Intra-day precision) 1.89 0.78 1.01
Intermediate precision (R.S.D%)a (Inter-day precision) 1.96 0.97 1.13
Limit of Detection (LOD) 0.03μg/mL
Limit of Quantication (LOQ) 0.1μg/mL
an = 3
TABLE IV - Results of Bioavailability studies
Pharmacokinetic parameter SVC A A+P A+G
AUC 131.8 1218 2630 79.09
tmax (h) 10 12 10 1
Cmax (μg/mL) 79.794 226.989 465.880 48.537
Comparative antioxidant and bioavailability studies of Vitamin C in Phyllanthus emblica Linn. 41
samples which may be due to the presence of gingerol in
ginger, one of the polyphenols and vitamin C in amla as
its active principle (Kishk, Sheshetawy, 2010).
Oxygen reacts with the excess NO to generate free
radicals, nitrite and peroxy nitrite anions (Marcocci et
al., 1994) and quenching of these free radicals measures
the antioxidant potential of a test compound. As A+G
combination showed radical scavenging activity which
was comparable with SVC, this combination can be an
alternative to SVC.
The evaluation of reducing capability is based on the
principle that, increase in the absorbance of the reaction
mixture by the sample/standard increases the reductive
capability (Koksal et al., 2011). Owing to their reducing
capabilities, antioxidant compounds cause the reduction
of ferric (Fe3+) form to the ferrous (Fe2+) form. Prussian
blue colored complex is formed by adding FeCl3 to the
ferrous (Fe2+) form. All the test compounds under study
were found to increase the absorbance in a concentration
dependent manner, but none were found to have signicant
reducing capability.
The ORAC assay has become a valuable and popular
method to determine the potential antioxidant activities
of various compounds and biological samples because
it measures the scavenging capacity against peroxyl
radicals which are one of the most common reactive
oxygen species in the body. This method is superior to
other methods for two reasons. First, the ORAC assay
system uses an area-under-curve (AUC) technique thereby
combining into a single quantity both inhibition time and
inhibition degree of free radical action by an antioxidant.
Second, dierent free radical generators or oxidants can
be used in the ORAC assay (Cao et al., 1997).
ORAC is a uorescence method using AAPH which
produces peroxyl radicals by undergoing spontaneous
decomposition. This method is more sensitive than the
spectrophotometric assay as it requires a much lower nal
standard concentration than the spectrophotometric assay
(Cao, Prior, 1998).
Our aim was to compare SVC with A and its
combinations for their antioxidant potential and we found
that A+P combination showed highest ORAC value
followed by A, SVC and last was A+G. This may be due
to in vitro antioxidant activity of piperine (Mittal, Gupta,
2000).
Since, the second objective of our study was to
evaluate plasma concentration of vitamin C in dierent
combinations of amla, it was necessary to estimate the
antioxidant potential of all compounds under study
like SVC, A, A+P and A+G. As the in vitro antioxidant
studies revealed that A+P combination showed maximum
antioxidant potential, it was desirable to conrm the same
by in vivo studies with HPLC estimation of serum samples
for vitamin C in rabbits.
Numerous assays for ascorbic acid have been
employed and they can be divided into three categories
- enzymatic, spectrophotometric and chromatographic
assays. Enzymatic and spectrophotometric assays are often
inuenced by interferences leading to overestimation of
ascorbic acid in biological samples, and the necessity of
modern high performance liquid chromatographic (HPLC)
methods for the determination of vitamin C in biological
samples have been established (Mittal, Gupta, 2000).
Vitamin C is highly sensitive to factors such as light,
heat and pH. A slight change in the mobile phase, solvents
and temperature during detection can give false result
that would lead to change in retention time. Further the
dierences in solvent refractive index cause an unstable
chromatographic baseline. Selected solvents such as
methanol and monobasic potassium phosphate were used
as they were found to give best results for the estimation
of vitamin C (Hanachi, Golkho, 2009).
In the present study, HPLC estimation of vitamin
C revealed that A+P combination has the maximum
bioavailability compared to other samples tested (Fig. 5,
Table IV). This could be due to the presence of piperine
which is used as a bioavailability enhancer and contributing
for the increased vitamin C concentration shown by the
combination A+P (Gohil, Mehta, 2009). Since piperine
enhances the bioavailability of vitamin C present in
amla, when used in combination, this combination may
be suggested as the best source of vitamin C supplement.
To conclude, antioxidant potential of A+P
combination was conrmed to be the best when compared
to amla alone and SVC as revealed by in vivo studies in
rabbits.
CONCLUSION
When compared with the other combinations tested,
the A+P combination exhibited the highest concentration
of vitamin C both in vivo and in vitro. This may be due
to presence of piperine in pepper which enhances the
bioavailability of vitamin C from amla and can be an
alternative to synthetic vitamin C.
ACKNOWLEDGEMENTS
The authors would like to thank the Principal,
KLE University’s College of Pharmacy, Bengaluru for
providing the necessary facilities to carry out the study.
V. Somasekhar, P. Ashok, S. A. R. Kameswari, R. Rajendran, R. Singh
42
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Received for publication on 16th April 2015
Accepted for publication on 14th March 2016
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