Total antioxidant capacity (TAC) of fresh leaves of Kalanchoe pinnata

Abstract

Herb Kalanchoe pinnata is grown as weeds in the tropical countries like India. It has a wide range of active constituents which has potent medicinal properties. The main objective was to overview the total antioxidant capacity of the herb Kalanchoe pinnata. Antioxidant activities were evaluated in terms of total phenolics content, total antioxidant activity, and reducing power. Different studies were carried out by comparing Kalanchoe pinnata extract with antioxidant references such as gallic acid; ascorbic acid. All these antioxidant activities increased with increasing concentrations in a dose dependent manner. It was found to be significant and valuable.

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Journal of Pharmacognosy and Phytochemistry 2014; 2 (5): 32-35
ISSN 2278-4136
JPP 2014; 2 (5): 32-35
© 2013 AkiNik Publications
Received: 09-11-2013
Accepted: 25-11-2013
Rohan Sharadanand Phatak
Directorate of Research, Krishna
Institute of Medical Sciences
University, Karad-415110,
Maharashtra, India,
Anup Subhash Hendre
Department of Biochemistry, Krishna
Institute of Medical Sciences Karad-
415110, Maharashtra, India
Correspondence
Rohan Sharadanand Phatak
Directorate of Research, Krishna
Institute of Medical Sciences
University, Karad-415110,
Maharashtra, India,
Email: phatak.rohan1983@gmail.com
Total antioxidant capacity (TAC) of fresh
leaves of Kalanchoe pinnata
Rohan Sharadanand Phatak, Anup Subhash Hendre
ABSTRACT
Herb Kalanchoe pinnata is grown as weeds in the tropical countries like India. It has a wide range of
active constituents which has potent medicinal properties. The main objective was to overview the
total antioxidant capacity of the herb Kalanchoe pinnata. Antioxidant activities were evaluated in
terms of total phenolics content, total antioxidant activity, and reducing power. Different studies were
carried out by comparing Kalanchoe pinnata extract with antioxidant references such as gallic acid;
ascorbic acid. All these antioxidant activities increased with increasing concentrations in a dose
dependent manner. It was found to be significant and valuable.
Keywords: Antioxidant, Kalanchoe pinnata, total antioxidant capacity
1. Introduction
Kalanchoe pinnata is a perennial herb belonging to family Crassulaceae which grows as weeds
in the tropical countries like India [1, 2]. It has two more Latin synonyms such as Kalanchoe
pinnata Pers, Bryophyllum calycinum Salisb. It has different commons like miracle plant or air
plant, panfuti (Hindi), life plant, love plant, air plant (Mexican), Good luck or resurrection plant,
Zakhm-e-hayat, Canterbury bells, Cathedral bells, parnabija etc. [1, 3, 4]. It is astringent, sour in
taste, sweet in the post digestive effect and has hot potency. The plant grows all over India in
hot and moist areas, especially in Bengal.
Hydroalcoholic extract of Kalanchoe pinnata was optimized in ratio of methanol to water (1:1,
v/v) which may be superior to its ethanolic extract to observe the exact antioxidant nature of the
herb Kalanchoe pinnata [2, 5].
On revising the reviews and the studies of the herb Kalanchoe pinnata till the date, a majority of
studies has been worked on its phytochemical profile of different active principles rich in
alkaloids, triterpenes, glycosides, flavonoids, cardenolides, steroids, bufadienolides and lipids,
pharmacognostic studies, pharmacological activities such as antileishmanial, hepatoprotective,
nephroprotective, neuropharmacological, antimutagenic, anti-ulcer, antibacterial, antidiabetic,
immunosuppressive, antihypertensive, analgesic, anti-inflammatory, wound healing, uterine
contractility, insecticidal, fungitoxic, phytotoxic activities[1] and other in-vitro activities.
However studies on in-vitro antioxidant activities in the different important antioxidant
parameters have not been done in terms of total antioxidant capacity by using novel methods
such as CUPRAC. Thus in the present study, an effort has been made to overview the total
antioxidant capacity of the herb Kalanchoe pinnata.
2. Material and Methods
Chemicals: Folin & Ciocalteu’s phenol reagent, Ammonium molybdate, Cupric chloride
(CuCl2), Neocuproine, Ammonium acetate, Potassium Ferricyanide, Trichloroacetic acid, Ferric
chloride (FeCl3) were purchased from Loba chemicals Ltd.
Collection of plant: Plant material was collected in September 2013 and authenticated from the
Dept. of Botany, Yashwantrao Chavan College of Sciences, Karad on October 2013.
Extraction: Fresh leaves of Kalanchoe pinnata were chopped into small pieces by hand and put
into a conical flask. Volume of methanol to water was in ratio of 80 ml: 80 ml was added to the
conical flask and covered with a cotton plug on the mouth of conical flask. It was kept in
maceration for 15 days at 4 C in order to maximize the extraction. After 15 days it was filtered
through Whatman filter paper and transferred to a suitable container and kept for analysis.

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2.1 Folin-Ciocalteu reagent (FCR) assay
Phenolics content assay was performed in the method of Mello et
al. [6].
Procedure
Hydroalcoholic extract of Kalanchoe pinnata in different
concentration range from 100 l to 500 l were added to each test
tube containing of 900 l to 500 l distilled water respectively;
and 500 l of Folin-Ciocalteu reagent solution. 500 l of 100
mg/ml sodium carbonate was added after 5min. These tubes were
kept aside for 2 hrs. Absorbance was measured at 765 nm. The
concentrations of phenolic compounds in Kalanchoe pinnata
extract were expressed as gallic acid equivalents (GAEs). All
assays were conducted in triplicate and its mean was calculated.
2.2 Ferric ion reducing antioxidant power assay (FRAP)
Ferric ions reducing power was measured according to the
method of Oyaizu with a slightest modification [7].
Procedure
Hydroalcoholic extract of Kalanchoe pinnata in different
concentrations ranging from 100 l to 500 l were mixed with 2.5
ml of 20 mM phosphate buffer and 2.5 ml 1%, w/v potassium
ferricyanide, and then the mixture was incubated at 50 °C for 30
min. Afterwards, 2.5 ml of 10%, w/v trichloroacetic acid and 0.5
ml 0.1%, w/v ferric chloride were added to the mixture, which
was kept aside for 10 min. Finally, the absorbance was measured
at 700 nm. Ascorbic acid was used as positive reference standard.
All assays were run in triplicate way and averaged.
2.3 Cupric ion reducing capacity assay (CUPRAC)
Cupric ion reducing capacity was measured in accordance to the
method of Apak [8].
Procedure
1 ml 10 mM cupric chloride, 1 ml 7.5 mM neocuproine and 1 ml
1 M ammonium acetate buffer of pH 7 solutions were added to
test tubes containing 2 ml of distilled water. Hydroalcoholic
extract of Kalanchoe pinnata in different concentration ranging
from 100 l to 500 l were added to each test tube separately.
These mixtures were incubated for half an hour at room
temperature and measured against blank at 450 nm. Ascorbic acid
was used as positive reference standard. All methods were
repeated in triplicate in order to get mean value.
2.4 Phosphomolybdenum Assay (PM)
Total antioxidant activity was estimated by phosphomolybdenum
assay [9].
Preparation of Molybdate Reagent Solution
1ml each of 0.6 M sulfuric acid, 28 mM sodium phosphate and 4
mM ammonium molybdate were added in 20 ml of distilled water
and made up volume to 50 ml by adding distilled water.
Procedure
Hydroalcoholic extract of Kalanchoe pinnata in different
concentration ranging from 100 l to 500 l were added to each
test tube individually containing 3 ml of distilled water and 1 ml
of Molybdate reagent solution. These tubes were kept incubated
at 95 C for 90 min. After incubation, these tubes were
normalized to room temperature for 20-30 min and the
absorbance of the reaction mixture was measured at 695 nm.
Mean values from three independent samples were calculated for
each extract. Ascorbic acid was used as positive reference
standard.
3. Statistical analysis
Results were given as mean±standard deviation of 3 replicates.
The results are expressed as mean values and standard deviation
(SD). The results were analyzed using one-way analysis of
variance (ANOVA) followed by Tukey's HSD test with α=0.05.
This treatment was carried out using SPSS v.16.0 (Statistical
Program for Social Sciences) software.
4. Results
The reducing capacity of antioxidant was coined in a single
measure as “Total Antioxidant Capacity” (TAC) [10]. TAC of this
hydroalcoholic extract was estimated based on its reducing
capacity by different methods such as FRAP, CUPRAC, and PM
assays. Absorbance reflects directly to the reducing power in the
methods of FRAP, CUPRAC, PM.
4.1 FCR assay
As phenolics contribute to the reducing capacity so we have
focused on the phenolics concentration determined by FCR
method. FCR assay was used to quantify the reducing capacity of
antioxidant. It was suggested to define as FCR method better
rather than as total phenolics content of antioxidant [10].
Table 1: FCR Assay
Absorbance at 765nm
Volume(l) KE GA
100 0.446±0.05 0.163±0.01
200 0.766±0.006 0.314±0.01
300 1.284±0.27 0.433±0.04
400 1.471±0.12 0.610±0.03
500 1.857±0.37 0.792±0.03
Values are Mean±SD (n=3); Kalanchoe pinnata Extract - KE; Gallic
Acid – GA
4.2 FRAP assay
FRAP measures the reducing potency of extract and standard
antioxidant. Higher absorbance indicates higher reducing potency.
Table 2: FRAP Assay
Absorbance at 700nm
Volume(l) KE AA
100 0.411±0.06 0.316±0.01
200 0.430±0.06 0.357±0.02
300 0.436±0.07 0.401±0.003
400 0.517±0.13 0.436±0.02
500 0.684±0.16 0.450±0.01
Values are Mean±SD (n=3); Kalanchoe pinnata Extract - KE; Ascorbic
Acid - AA
4.3 CUPRAC assay
CUPRAC assay is based on the utilization of the copper (II)-
neocuproine [Cu (II)-Nc] reagent as the chromogenic oxidizing
agent [8].
Table 3: CUPRAC Assay
Absorbance at 450nm
Volume(l) KE AA
100 0.977±0.06 0.471±0.004
200 1.509±0.04 0.537±0.01
300 2.046±0.08 0.593±0.008
400 2.309±0.03 0.647±0.01
500 2.731±0.02 0.707±0.02
Values are Mean±SD (n=3); Kalanchoe pinnata Extract - KE; Ascorbic
Acid - AA

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Journal of Pharmacognosy and Phytochemistry
4.4 PM assay
PM assay is based on the reduction of Phosphate-Mo (VI) to
Phosphate Mo (V) by the sample and subsequent formation of a
bluish green colored phosphate/Mo (V) complex at acid pH. The
phosphomolybdenum method is routinely applied in the
laboratory to evaluate the total antioxidant capacity of plant
extracts [9].
Table 4: PM Assay
Absorbance at 695nm
Volume(l) KE AA
100 0.362±0.02 0.143±0.01
200 0.753±0.05 0.333±0.04
300 1.124±0.03 0.529±0.02
400 1.451±0.03 0.871±0.02
500 1.764±0.07 1.135±0.01
Values are Mean±SD (n=3); Kalanchoe pinnata Extract - KE; Ascorbic
Acid – AA
Figure 1 portrays the FCR along with different total antioxidant
capacity methods which evaluates the increase in concentration of
phenolics increases reducing capacity of antioxidant. CUPRAC
assay being a novel method which shows superiority among
assays. Studies in combination of different reducing power
methods help to understand the exact nature of antioxidant.
Fig 1: FCR and Total Antioxidant Capacity Assays Kalanchoe pinnata Extract –
KE; Ascorbic Acid – AA, Gallic Acid – GA
5. Discussion
Antioxidant capacity assays may be broadly classified as single
electron transfer (SET) and hydrogen atom transfer (HAT) based
assays. Majorities of HAT assays are kinetics based and involve a
competitive reaction scheme in which antioxidant and substrate
compete for free radicals thermally generated through the
decomposition of azo compounds. SET assays measure the
capacity of an antioxidant in the reduction of an oxidant which
changes colour when reduced. SET assays are easier than HAT
assays. SET assays like PM, CUPRAC, FRAP were selected to
analyze the reduction capacity. These methods are involving in
the mechanism of single electron transfer system. In this system
electron from oxidized antioxidant transferred to the substrate by
inhibiting oxidation of oxidant.
FRAP, CUPRAC and PM methods are based on the redox
antioxidant reaction. FRAP [8] and CUPRAC as a novel method [9]
is to assess the reduced concentration of ferric and cupric ions
respectively.
FRAP assay include the simultaneous use of ferricyanide and
ferric ions as chromogenic oxidants supplied more favorable
redox conditions for a greater variety of antioxidants.
1. FRAP assay gives an immediate result of a large range
of individual antioxidants in dose-response manner.
2. Higher degree of color formation indicates the more
reducing power of analyte.
3. Simple, reproducible analysis.
CUPRAC involves both of complexometric and redox reactions.
It has specific features distinct from FRAP.
1. The redox reaction giving rise to a colored chelate of Cu
(I)-Nc is relatively not affected by many parameters such
as air, sunlight, solvent type, and pH.
2. The CUPRAC reagent is reasonably selective, stable,
easily accessible, and sensitive comparing with the
FRAP method.
3. The reaction is carried out at normal pH as opposed to
acidic pH of FRAP.
PM assay measures the reduction degree of Mo (VI) to Mo (V).
1. PM assay is a quantitative method to investigate the
reduction reaction rate among antioxidant, oxidant and
molybdenum ligand. It involves in thermally generating
auto-oxidation during prolonged incubation period at
higher temperature.
2. It gives a direct estimation of reducing capacity of
antioxidant.
3. It is distinctive from FRAP and CUPRAC assays as it
remains intact irrespective of concentration of free metal
ions.
4. Unlike CUPRAC and FRAP, it forms a green
phosphomolybdenum complex without induction of free
metal ions solution. So it shows uniqueness among in-
vitro antioxidant assays.
Fenton reaction involves the creation of free radicals by
transition-metal ions such as iron and copper that are present in
vivo by donating or accepting free electrons via intracellular
reactions. Although most intracellular iron is in ferrous (+2 ion)
form, superoxide ions can convert it to the ferric (+3) form to take
part in Fenton reaction. Metal-Catalyzed Oxidation (MCO)
systems catalyze the reduction of intracellular iron which is in the
form of Fe (III) to Fe (II) mostly through superoxide ions and of
O2 to H2O2. These products react at metal-binding sites on the
protein to produce active oxygen species (viz; OH, ferryl ion)
which alters the nature of proteins at the metal-binding site and
cause DNA and protein damage [11,12].
6. Conclusion
On the basis of results obtained from different antioxidant
capacity assays, the hydroalcoholic extract of Kalanchoe pinnata
has shown a significant total antioxidant capacity. The reducing
capacity and reduction capacity of free oxidative metallic ions
such ferric and cupric ions by hydroalcoholic extract of
Kalanchoe pinnata can be approximated through PM assay,
through two methods namely FRAP and CUPRAC assays. The
reducing capacity depends on phenolic contents which can be
estimated with the help of FCR assay. Over viewing the reducing
capacity, the use of Kalanchoe pinnata might contribute a certain
level of health protection against oxidative damages. With the
established antioxidant activity of this extract, the specific
isolation of the active components in the hydroalcoholic extract of
Kalanchoe pinnata and characterization should be further
investigated.

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