Antioxidant Property of the Root of Asparagus
Gonoclados Baker (Liliaceae)
Ranajit Damodare Tijare, Varadharajan Madhavan, *Anita Murali, S. N. Yoganarasimhan
Faculty of Pharmacy, M. S. Ramaiah University of Applied Sciences, Bangalore 560 054
* Contact Author e-mail: [email protected]
Abstract
The present investigation was aimed at evaluating the antioxidant potential of the alcohol and aqueous extracts of root
of Asparagus gonoclados Baker by in vitro and in vivo methods. The ability of extracts to scavenge DPPH and superoxide
rdaicals was evaluated by in vitro methods. In vivo evaluations were done by CCl4 induced hepatotoxicity. Hepatotoxicity
was induced in Wistar rats by administration of CCl4 1.5 mL/kg i.p. following extract treatment for 15 days. Total proteins,
catalase and SOD were estimated in liver and MDA was estimated in brain. The studies showed that the alcohol and
aqueous extracts at dose levels of 200 and 400 mg/kg exhibited significant increase in catalase and superoxide levels (p <
0.01) in CCl4 intoxicated rats. Simultaneously, the levels of MDA in brain were significantly decreased (p < 0.01) in the
extract treated groups when compared with the positive control. Preliminary phytochemical screening of the extracts was
carried out to detect the presence of various phytoconstituents. HPTLC fingerprint profiles of the detected phytoconstituents
were also obtained. Acute toxicity studies revealed that both the alcohol and aqueous extracts were safe up to the dose of
3000 mg/kg on oral administration.
Key Words: Asparagus Gonoclados, Catalase, Superoxide Dismutase, Malondialdehyde
1.
2.2 Preparation of Extracts
INTRODUCTION
Shatavari is a well known drug in Ayurveda [1] and its accepted
botanical source is Asparagus racemosus L [2]. However, many
other species of Asparagus L. including Asparagus gonoclados
Baker are used as Shatavari [3, 4]. Root tuber of Shatavari (A.
racemosus) is considered as one of the Rasayana (adaptogenic)
drugs, having cooling, diuretic, emollient, rejuvenating, and
stomachic properties [5]. It is useful in the treatment of nervous
system disorders, dyspepsia, diarrhea, dysentery, tumors,
inflammation, tuberculosis, epilepsy and fatigue [6].
A. gonoclados contains phytoconstituents like apigenin,
kaempferol, rutin and chalcone glycoside, anthocymin and
malvin4. The diuretic, galactogogue, antiulcer and antioxidant
properties of A. racemosus have been reported [7-10].
Pharmacognostical studies on root tubers of A. gonoclados have
also been reported [11]. In the present work, the effect of alcohol
and aqueous extracts of root tuber of A. gonoclados on certain
liver antioxidant enzymes and brain antioxidant status have been
investigated besides assessing the free radical scavenging of the
extracts.
Alcohol extract was prepared by soxhlation and aqueous extract
was prepared by cold maceration.
2.3 Animals
Swiss albino mice of either sex, in the weight range of 18 – 25 g,
were used for acute toxicity studies and albino rats of Wistar
strain, of either sex in the weight range of 180 – 200 g were used
for the in vivo antioxidant activity studies. Animals were
maintained in accordance with CPCSEA regulations and the
study protocol was approved by the Institutional Animal Ethics
Committee of M. S. Ramaiah College of Pharmacy.
2.4 Phytochemical Analysis
Preliminary phytochemical analysis and HPTLC studies were
carried out on the methanol and aqueous extracts [14, 15].
2.5 In Vitro Antioxidant Activity Studies
DPPH and super oxide scavenging assays were performed [16,
17] to evaluate free radical scavenging ability of the extracts.
2.6 Acute Toxicity Studies
2.
The acute toxicity study was carried out following Ghosh [18].
METHODOLOGY
2.7 In Vivo Antioxidant Activity
2.1 Plant Material
The plant material was collected from the forests situated
between Madikeri and Sakleshpur, during March 2006. The plant
material was identified and authenticated by Dr. S.N.
Yoganarasimhan, Taxonomist and Research coordinator
following local floras [12, 13].
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49
Albino rats of Wistar strain weighing 180 – 200 g of either sex
were used for the study. The animals were divided in to 7 groups
containing 6 animals each. Group I was maintained as normal
control. Group II was maintained as positive control and
administered with CCI4 at a dose of 1.5 mL / kg, i.p. Group III
was administered with standard Vit E at a dose of 50 mg/kg body
weight, orally. Group IV and V animals were administered with
alcohol extract at doses 200 and 400 mg/kg respectively orally.
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Group VI and VII were administered with aqueous extract at
doses 200 and 400 mg/kg respectively orally. The treatment
schedule was once daily for 15 days. On 15th day, 6 h after the
last dose of extract, CCl4 (1.5 ml/kg) was administered to all
animals by i.p. injection.
Six hours after CCl4 administration, all the animals were
sacrificed, for the isolation of liver and brain. The liver was
isolated, perfused with ice cold saline to remove contaminated
blood. The isolated liver was divided into 2 parts for preparation
of homogenates. A 5 % w/v homogenate of liver was prepared in
0.15 M potassium chloride and centrifuged at 8000 rpm for 10
min. The supernatant thus obtained was used for estimation of
total proteins [19] and catalase [20]. Another 5 % w/v
homogenate of liver was prepared using 0.25 % w/v solution of
sucrose in 5M phosphate buffer, centrifuged at 8000 rpm for 10
min. The supernatant was used for estimation of super oxide
dismutase [21].
Brain was isolated and homogenized, for the estimation of MDA.
The isolated brain was washed with ice cold potassium chloride
solution (1.15%w/v) and sliced. The sliced rat brain was used for
preparing homogenate in the ratio, of 0.75:9.25 ml (Brain:
Potassium chloride (1.15%)). The brain homogenate was used for
the estimation of MDA [9].
2.8 Statistical Analysis
The values of all groups were compared with positive control
(CCl4 treated) and data expressed as Mean values ± S.E.M and
tested with one way ANOVA followed by Dunnett multiple
comparison test for in vivo and in vitro antioxidant activity.
3.
with the absorption spectrum obtained from the corresponding
band in alcohol and aqueous extracts (Fig. 1).
Alcohol and aqueous extracts of A. gonoclados were subjected to
in vitro antioxidant activity studies. Both extracts exhibited
significant free radical scavenging effects against DPPH and
superoxide radicals (Table 1).
Acute toxicity studies revealed that both the alcohol and aqueous
extracts were safe up to the dose of 3000 mg/kg on oral
administration. The in vivo antioxidant activity studies on the
root tubers showed that the alcohol and aqueous extracts in the
dose levels of 200 and 400 mg/kg exhibited significant increase
in catalase and superoxide levels (p < 0.01) in CCl4 intoxicated
rats. Simultaneously, the levels of MDA in brain were
significantly decreased (p < 0.01) in the extract treated groups
when compared with the positive control (Table 2).
A. gonoclados contains phenolic compounds, flavonoids
saponins, alkaloids, glycosides and tannins. HPTLC
fingerprinting revealed the presence of Shatavarin IV in alcohol
and aqueous extracts of A. gonoclados. It was reported that
saponins “Shatavarin I-IV”, phenolic compounds and alkaloids
are present in A. Racemosus [10], which also possesses
antioxidant properties [9, 22]. Further, the phytochemical
constituents of A. gonoclados (present study) and A. racemosus
are also similar; the present findings thus support the earlier
findings on in vitro and in vivo studies [23, 24].
4.
CONCLUSION
A. gonoclados can be a good substitute for A. racemosus, which
is the accepted botanical source of Shatavari.
RESULTS AND DISCUSSION
Preliminary phytochemical analysis revealed carbohydrates,
flavonoids, glycosides, phenolic compounds, saponins, alkaloids
and tannins in alcohol and aqueous extracts; proteins, amino
acids, gums and mucilage in aqueous extract. HPTLC
fingerprinting of Shatavarin IV in alcohol and aqueuous extracts
was developed. Alcohol extract revealed 9 phytoconstituents
while aqueous extract revealed 7 phytoconstituents. The peak
with Rf 0.34 in both extracts correspond to that of standard
Shatavarin IV (Rf 0.35). The specificity was confirmed by
overlaying the spectra of standard Shatavarin IV (λ max 426 nm),
ACKNOWLEDGEMENTS
The authors thank Gokula Education Foundation for providing
facilities to carry out this research work.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
Table 1. In vitro antioxidant activity of alcohol and aqueous extracts of A. gonoclados
IC50 (µg /ml) DPPH
Ascorbic
IC50 (µg /ml) Superoxide anion
Ascorbic
Alcohol ext
Aqueous Ext
acid
Alcohol Ext
Aqueous Ext
acid
150.61±1.065
171.27±0.95
3.24± 0.19
516.66±0.78
996.66±0.13
>1000.0
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Fig 1. Spectrum showing λmax at 425 nm for Shatavarin IV standard and in extract of Asparagus gonoclados
Table 2. In vivo antioxidant activity of alcohol and aqueous extracts of A. gonoclados
Values expressed as Mean  SEM. Results obtained by comparison with positive control.
ONE WAY ANOVA p value 0.01; Dunnett multiple comparison test ** p < 0.01
Catalase
m/min/mg
SOD
U/mg
proteins
Total protein
mg/ml
64.385  1.326
49.483 2.884
0.691  0.012
1874.75  9.428
36.758  0.980
31.665  0.635
1.383  0.047
1208.35  1.449**
60.343  0.551**
51.145  1.935**
0.766  0.015**
Alc Extract (200
mg/kg)
1285.013  0.565**
42.896  0.691**
41.008  1.851**
0.82  0.029**
Alc Extract (400
mg/kg)
1291.076  43.43**
55.061  0.582**
43.821  2.279**
0.78  0.026**
Aq Extract (200
mg/kg)
1283.63  3.604**
43.22  0.750**
38.665  2.151**
0.816 0.0.023**
Aq Extract (400
mg/kg)
1262.048  3.554**
56.41  0.815**
39.856  2.334**
0.773  0.021**
Groups
Normal control
Positive control
(CCl4 ip)
Standard,
(Vit E, 50 mg/kg)
MDA
NMOL/
100mg
991.253 
1.58
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