Professional Documents
Culture Documents
www.ijcps.com
International Journal of
Chemical and Pharmaceutical Sciences
2010, Dec., Vol.1 (2)
ISSN: 0976-9390
ABSTRACT
The present investigation was carried out to investigate the anti-tumor activity,
Antioxidant activity of the ethanolic extract of Amorphophallus Paeonifolius tubers against 7,12dimethyl benz (a) anthracene (DMBA) induced mammary tumour in rats.The Pharmacognostic
and Phytochemical studies over Amorphophallus Paeonifolius were studied first and the antioxidant activity of the same were studied In-vitro and In-vivo methods finally the plant extract
was evaluated for mammary tumour activity by chemical induced tumour.The results shows total
Flavanoids compound in Ethanolic tuber extract of Amorphophallus Paeonifolius was found to
be 8.8 g/100g calculated as Quercetin equivalent, effect of Ethanolic extract on RBC, WBC, Hb
& Neutrophils as a-P< 0.001,b-P<0.01,c-P<0.05, ns-non significant, Effect of AP-extract on
tumor latency and tumour burden were found as extremely significant at P<0.001. One-way
ANOVA Tukey method was followed for statistics. It was concluded that ethanolic extract of
Amorphophallus Paeonifolius has shown significant antitumor and antioxidant effect in animals.
Keywords: Amorphophallus Paeonifolius, Anti-Oxidant, Mammary tumour
1. INTRODUCTION
Cancer is a class of diseases in which
a group of cells display uncontrolled growth
(division beyond the normal limits),
invasion (intrusion on and destruction of
adjacent tissues), and sometimes metastasis
(spread to other locations in the body via
lymph or blood). These three malignant
properties of cancers differentiate them from
benign tumors, which are self-limited, and
do not invade or metastasis. [1, 2] Tumors can
be malignant (cancerous) or benign (non
Research Paper
one way or another from natural sources,
including plants, marine organisms and
micro-organisms. Indeed, these sources have
played, and continue to play, a dominant
role in the discovery of leads for the
treatment of most human diseases. [3]
The search for anticancer agents
from plant sources started in 1950s with the
discovery and development of vinca
alkaloids, vincristine, vinblastine and the
isolation of cytotoxic podophyllotoxin.
These discoveries in turn lead to discovery
of taxanes and camptothecins but their
development into clinically active agent
spanned period of 30 years from 1960s to
1990s.[4,5]
The
plant
Amorphophallus
paeonifolius
(AP)
synonym
Amorphophallus campanulatus belongs to
the family of Araceae Elephant foot yam is
basically a crop of Southeast Asian origin. It
grows in wild form in the Philippines,
Malaysia, Indonesia, and other Southeast
Asian countries. In India it is grown mostly
in Kerala, Andhra Pradesh, Maharashtra and
Orissa. A stout herbaceous plant""with
underground hemispherical depressed dark
brown corm; leaves compound, large,
solitary, petiole stout, mottled, 60-90 cm
long, leaflets 5-12.5 cm long of variable
width and the corms contain betulinic acid,
-sitosterol, stigmasterol, triacontane and sitosterol palmitate. Besides these, glucose,
galactose, rhamnose and exylose are also
present. The corms are irritant due to the
composition of arginine, histidine, leucine,
isoleucine,
lysine,
methionine,
phenylalanine, threonine, tryptophan, and
valine.
They are useful in vitiated conditions
of vata and kapha, rthralgia, ephantiasis,
tumours, inflammations, haemorrhoids,
haemorrhages, vomiting, cough, onchitis,
asthma, anorexia, dyspepsia, flatulence,
www.ijcps.com
colic constipation, helminthiasis hepatopathy,
splenopathy, amenorrhoea, dysmenorrhoea,
seminal weakness, fatigue, anemia and general
debility. [5- 8]
2. MATERIALS AND METHODS
Plant materials [tubers] were locally
collected and authenticated by Botanical
Survey of India (BSI), Southern circle,
Coimbatore, Tamilnadu
2.1 Extraction of tuber of Amorphophallus
paeonifolius
The
tuber
of
Amorphophallus
Paeonifolius was cut into pieces of around 5
mm, shade dried and ground to powder. 300
gm of powdered tuber was extracted with
petroleum ether to remove fatty material for 10
cycles. The material was air dried and then
extracted with chloroform as said above, the
tuber material was subjected to dry and finally
ethanolic extraction was done. The extract were
concentrated to dryness and stored in vaccum
dessicator.
2.2 Phytochemical analysis of ethanolic
extracts of Amorphophallus Paeonifolius
The photochemical analysis of the plant
with standard chemical tests was shown that
extract
contains
alkaloids,
glycosides,
carbohydrates , steroids and sterols, Tannins,
proteins, peptides or amino acids and aromatic
amino acid
2.2.1. Determination of Total Flavanoids
Flavones and flavanols in the ethanolic
extracts of Amorphophallus Paeonifolius were
estimated as Quercetin equivalent. Quercetin
was used to make the calibration curve [10, 20,
30, 40, 50, 60, 70, 80, 90 and 100g/ml in
99.9% ethanol (v/v)]. The standard solutions or
extracts (0.5 ml) were mixed with 1.5 ml of
95% ethanol (v/v), 0.1 ml of 10% aluminium
chloride, 0.1 ml of 1 mol/l sodium acetate and
2.8 ml water. The volume of 10% aluminum
chloride was substituted by the same volume of
41
Research Paper
distilled water in blank. After incubation at
room temperature for 30 min, the
absorbance of the reaction mixture was
measured at 415 nm. The mean of three
readings was used and the total Flavanoids
content was expressed in milligrams of
Quercetin equivalents 190.2 mg/g extract.
The coefficient of determination was
r2 = 0.9985. [9]
2.3. In vitro anti-oxidant activity
The in vitro anti-oxidant activity of
the extract was done by various available
methods as follows
2.3.1. DPPH (Diphenylpicrylhydrazyl)
Radical scavenging activity
The
scavenging
activity
of
Amorphophallus Paeonifolius extract was
measured in terms of hydrogen donating or
radical scavenging ability using the stable
radical DPPH Blois et al., (1958).[10] 0.1Mm
solution of DPPH in ethanol was prepared
and 1.0ml of this solution was added to 3 ml
of extract solution and standard in water at
different concentrations (10-100g/ml). 30
min later absorbance was measured at
517nm. Lower absorbance of the reaction
mixture indicated higher free radical
scavenging activity. The capability to
scavenge the DPPH radical was calculated
using the following equation
www.ijcps.com
trichloroaceticacid (10%w/v) was added to the
mixture, which was then centrifuged for 10 min
at 1000rpm. The 2.5 ml of supernatant was
mixed with distilled water (2.5ml) and Fecl3
(0.5ml, 0.1% w/v), and mixed. Absorbance was
measured at 700nm in a spectrophotometer.
Higher absorbance of the reaction mixture
indicated greater reductive potential.
2.3.3. Superoxide anion scavenging activity
assay
The
scavenging
activity
of
Amorphophallus
Paeonifolius
towards
superoxide anion radicals was measured by the
method of Ni-shimiki et al.,(1972). [12] and
slightly modified. About 1ml of nitroblue
tetrazolium solution (156m in 100Mm
phosphate buffer, pH 7.4) and 0.1ml of
different concentrations of extract and standard
in water were mixed. The reaction was initiated
by
adding
100l
of
phenazinemethosulphate(PMS)
solution
(60M) in 100Mm phosphate buffer(pH7.4) to
the mixture. The reaction mixture was
incubated at room temperature for 5 min and
the absorbance at 560nm was measured against
reagent blank in spectrophotometer. (Quercetin
was used as standard).
2.3.4. ABTS radical scavenging activity
assay
The ABTS radical scavenging activity
of the extract was measured by Rice-Evans
et.al. (1997).[13]ABTS radical cation (ABTS+)
was produced by reacting ABTS solution
(7mm) with 2.45 Mm ammonium persulphate
and the mixture were allowed to stand in dark
at room temperature for 12-16hrs before use.
Different concentration (20-100g/ml) of
ethanolic extract and standard (0.5ml) were
added to 0.3ml of ABTS solution and the
ethanol to make 1ml. Quercetin was used as
standard. The absorbance was read at 745nm.
The experiment was performed in triplicate.
2.4. Pharmacological Evaluation
42
Research Paper
2.4.1. Acute toxicity Studies
Healthy adult Wistar albino mice of
either sex, starved overnight were divided
into five groups (n=6) and were orally fed
with
the
Ethanolic
extract
of
Amorphophallus Paeonifolius in increasing
dose levels of 100, 500, 1000, 2000mg/kg
body weight respectively and they were
observed for behavioral, neurological and
autonomic profiles and after a period of 24
and 72 hr they were observed for any
lethality.
2.4.2. Induction of cancer
The cancer was inducted by using
chemical induction method using 7, 12Dimethyl Benz (A) Anthracene(DMBA)
DMBA was purchased from sigma
chemicals, Mumbai, India. 120 mg DMBA
was dissolved in 24 ml olive oil. It forms a
yellow colour solution. DMBA solution was
given to animals orally with the help of oral
feeding needle at a dose of 25 mg/kg.
2.5. In-vivo anti-oxidant activity
All the experimental animals were
killed by cervical decapitation after the
experimental period. For the estimation of
non-enzymic and enzymic antioxidants,
tissue (liver and kidney) was minced and
homogenized (10% w/v) in 0.1 M phosphate
buffer (Ph 7.0) and centrifuged for 10 min
and the resulting supernatant was used for
enzyme assays.
2.5.1. Enzymatic Anti-oxidant activity
2.5.1.1. Estimation of catalase activity
Catalase activity was measured by
method described by Sinha et.al.(1972).[14]
The reaction mixture (1.5ml, vol) contained
1.0ml of 0.01M phosphate buffer (pH7.0),
0.1ml of tissue homogenate and 0.4ml of
2M H2O2.The reaction was stopped by the
addition of 2.0ml of dichromate-acetic acid
reagent (5% pot. dichromate and glacial
www.ijcps.com
acetic acid were mixed in 1:3 ratio). Then the
absorbance was read at 620nm; CAT activity
was
expressed
as
Mol
of
H2O2
consumed/min/mg protein.
2.5.1.2. Estimation of Superoxide dismutase
(SOD) Activity
The activity of superoxide dismutase
was assayed by the method of Kakkar
et.al..(1984).[15] Breifly in a test tube; 0.5ml of
supernatant tissue homogenate was taken. To
this 1.5ml of carbonate buffer(pH10.2),0.5ml of
0.1Mm EDTA and 0.4ml of epinephrine was
added and the OD was taken at
480nm.Epinephrine was added just before
taking the OD. This activity is to be expressed
as units/min/mg protein.
2.5.1.3.
Estimation
of
peroxidase (GPx) Activity
Glutathione
Research Paper
reagent and 3ml of phosphate buffer (pH
8.0). Then the absorbance was read at
412nm. The values were expressed as
mg/100 g tissue.
2.6. Anti tumor activity
2.6.1. Experimental design for treatment
oriented study
Experimental rats were divided into
5 groups of six animals each and received
the following treatment for 90 days. Group
I- Control rats given only Saline (p.o.),
Group II- Rats given DMBA only 25 mg/kg
(p.o.), Group III- Rats treated with DMBA +
AP tuber extract 50 mg/kg (p.o.), Group IVRats treated with DMBA + AP tuber extract
100mg/kg (p.o.), Group V- Rats treated with
DMBA + TAM 10 mg/kg (p.o.)
2.6.2. Hematological studies
In this 90 days study both group of
animals were treated with respective extracts
and standard drugs via oral route. Body
weight was taken every week till 17th week.
After the completion of treatment, blood
was collected for hematological parameters
estimation like RBC, WBC (Total and
differential count by Improved Neubauers
counting chamber method using Haeyems
fluid) Hb (by sahlis Haemoglobinometer
method) and Neutrophil by Improved
Neubauers counting chamber method.
2.6.3. Bio chemical parameters
Aliquot of blood was collected and
centrifuged at 5000 rpm for 5 min to
separate the serum. This serum was used for
estimation of SGOT by Optimized UV- test
according to IFCC(International Federation
Of Clinical Chemistry and Laboratory
Medicine), SGPT by Kinetic UV test,
according to the International Federation of
Clinical
Chemistry
and
Laboratory
Medicine, serum creatinine by Jaffe Method
(modified), serum urea by Urease-GLDH
enzymatic UV test method .
www.ijcps.com
2.6.4. Estimation of protein
Procedure described by Lowery et.al.
(1951). [18] was used for protein estimation. The
method was based on the biuret reaction,
formation of a protein-copper complex and
reduction of phosphomolybdo tungstate reagent
(Folin-ciocalteu phenol reagent) by tyrosine
and tryptophan residues of protein to form a
coloured product.
2.7. Statistical Analysis
The data for various parameters were
analyzed using analysis of variance (ANOVA)
Tukey, followed by compared of all pairs of
column.
3. RESULTS AND DISCUSSION
Table 1: Determination of Total Flavanoids.
Concentration
Sample
Quercetin as
standard
Ethanolic
tuber
extract
of
Amorphophallus
Paeonifolius
(g/ml)
Absorbance
10
0.2651
20
0.3663
30
0.5014
40
0.8116
50
0.9934
250
0.4942
Total
Flavanoids
compound
in
Ethanolic tuber extract of Amorphophallus
Paeonifolius was found to be 8.8 g/100g
calculated as Quercetin equivalent
Preliminary phytochemical screening
indicated the presence of alkaloids and
flavanoids in Amorphophallus Paeonifolius
tuber extract. flavanoids (8.8 g/100 g)
calculated as Quercetin equivalent. Flavanoids
have been shown to possess anti-mutagenic and
anti-malignant effects. Moreover, flavanoids
have an antitumor role through their effects on
signal transduction in cell proliferation and
angiogenesis. The cytotoxic and antitumor
44
Research Paper
www.ijcps.com
DPPH radical
scavenging activity
ABTS radical
scavenging activity
assay
Superoxide anion
scavenging activity
assay
Quercetin Standard
Concentration(g/ml)
% Inhibition
Concentration(g/ml)
% Inhibition
10
35.40
10
42.75
20
45.15
20
55.11
30
53.06
30
65.18
40
53.42
40
66.33
50
53.48
10
70.56
60
54.78
20
72.19
20
65.35
20
81.03
40
67.47
40
81.85
60
69.81
60
82.04
80
71.79
80
82.56
100
73.58
100
86.96
40
61.93
40
87.76
60
62.24
60
87.99
80
62.40
80
88.66
100
63.07
100
88.76
Research Paper
www.ijcps.com
control
DMBA only
DMBA +
AP-Tuber
extract(50mg/kg)
DMBA +
AP-Tuber
extract(100mg/kg)
DMBA +
Tamoxifen
(10mg/kg)
Liver
317.470
287.910a
443.727.8 a
602.521.21 a
483.329.63 a
Kidney
279.1337.55
189.438.11a
540.7830.8 a
717.6933.9 a
827.0222.88 a
Estimation
of
Superoxide
Dismutase (SOD)
Activity
Liver
40.848.56
4.222.31a
31.633.35 a
53.424.02 a
63.827.17 a
Kidney
36.65.92
16.351.85
ns
73.411.28 a
Estimation
of
Glutathione
peroxidase (GPx)
activity
Liver
206.0428.88
97.4111.7a
132.6443.89 a
290.6617.09 a
230.948.27 a
Kidney
269.7111.47
87.3713.12a
227.2339.98 a
260.4536.80 a
291.596.00 a
Estimation
of
Catalase Activity
30.293.36
24.229.20
Estimation
of
reduced
glutathione (GSH)
Liver
Kidney
851.47105.2
837.9594.7
358.3659.47a
400.7468.84
907.66200.9c
1041.6155.69
1034.0344.52 a
1015.20166.8 a
1161.12112.4 a
1136.8693.07
Research Paper
www.ijcps.com
control
DMBA
only
DMBA +
AP-Tuber
extract
(50mg/kg)
DMBA +
AP-Tuber
extract
(100mg/kg)
DMBA +
Tamoxifen
(10mg/kg)
6.950.13
13.260.36
13.260.36
14.090.14 a
10.130.71 a
10.130.71 a
7.100.12 a
12.430.27 a
12.50.44 b
7.080.22 a
13.430.75 a
13.430.75 a
7.210.40 a
12.760.10a
12.760.17a
8.960.18
17.130.18 a
9.260.31 a
9.030.54b
8.90.17 a
78.662.251
13122.68
0.6330.051
38.560.459
112.3328.78 a
225.6627.07 a
0.7330.051 a
47.54.588 a
87.669.893c
140.3318.99 a
0.7330.051 a
30.934.519 a
747.797 a
148.6618.64 a
0.70.001 a
322.968a
88.663.141ns
154.667.607a
0.6660.051 a
32.265.643 a
Research Paper
www.ijcps.com
DMBA only
DMBA
+
extract(50mg/kg)
DMBA
+
extract(100mg/kg)
DMBA + Tamoxifen
(10mg/kg)
INCIDENCES
(%)
TUMOR
LATENCY
TUMOR
BURDEN
AP-Tuber
6/6
3/6
100
50
50.63
5.830.84a
2.50.83
10.63a
AP-Tuber
2/6
33.33
6.331.08a
0.660.52 a
1/6
16.66
8.161.36a
0.330.41a
Table: 6 Effect of Extract on The Level of Total Protein In Liver And Kidney
Experimental Rats.
Group
(n=6)
Kidney
control
DMBA
only
DMBA
+
AP-Tuber
extract
(50mg/kg)
DMBA
+
AP-Tuber
Extract
(100mg/kg)
DMBA
+
Tamoxifen
(10mg/kg)
0.870.04
0.820.02
0.690.01a
0.430.08a
0.840.02 b
0.690.01a
0.880.04 a
0.780.02 a
0.940.02 a
0.760.02 a
Research Paper
oxidative degradation of glycated protein
shown in table no 6.
4. CONCLUSION
The
ethanolic
extract
of
Amorphophallus Paeonifolius has shown
significant antitumor and antioxidant effect in
animals. This may be due to presence of
flavanoids
in
ethanolic
extract
of
Amorphophallus Paeonifolius. The present
preliminary investigation suggests that
Amorphophallus Paeonifolius tuber stimulates
both cellular and humoral immunity. Further
studies have elucidate the exact antitumor
mechanism of Amorphophallus Paeonifolius
tuber.
5. REFERENCE
1. UK cancer incidence statistics by age.
Cancer Research UK (January 2007).
info.cancerresearchuk.org/cancerstats
/incidence/age/. Retrieved 2007: 6 -25.
2.
www.ijcps.com
Encyclopedia of life support system
(EOLLS).
7. Graham JG, Quinn ML, Fabricant DS
and Farnsworth NR. Plants used against
cancer-an extension of the work of
Jonathan
Hartwell.
Journal
of
biological Science. 2000; 73:347-377.
8. Ramankutty C,Vasudevan Nair R.
Indian
medicinal
plants:
A
compendium of 500 species, Orient
Blackswan .1996;1:132.
9. Nataraj HN, Murthy RLN and
Ramachandra
Setty
S.
Invitro
Quantification of Flavonoids and
Phenolic content
of Suran,
International Journal of ChemTech
Research. 2009; 1: 1063-1067.
10. Blois MS, Zhao XY.Antioxdant
determinations by the use of a stable
free radical, Nature. 1958; 181: 11991200.
11. Oyaizu A ,llori O. Studies on product of
browning reaction prepared from
glucose amine Jpn J Nutr., 1986; 44:
307-315.
12. Ni-shimiki Rao NA and Yagi K. The
occurrence of superoxide anion in the
reaction
of
reduced
phenazine
methosulfate
and
molecular
oxygen.Biochem Biocommun 1972;
46: 849-853.
13. Rice-Evans CA and Miller NJ.The
relative contributions of ascorbic acid
and Phenolic antioxidants to the total
antioxidants to the activity of orange
Journal of Food Chem. 1997; 60: 331337.
14. Sinha BB,Peterson GA, Whitny RR.
Nuclear change Distribution of isotone.
Pairs Phys., 1972; Rev C6:1657-1663.
15. Kakkar P, Awasthi S and Viswanathan
PN. Effect of anesthetic ether on lipid
49
Research Paper
www.ijcps.com
peroxidation
and
superoxide
dismulatase isozymes of young and
adult rat brain. Ind J Exp Biol., 1984
27: 647-649.
16. Rotruck JTAL, Pope HE and Ganther
AB Swanson Biochemical role as
acomponent of glutathione peroxidase. J
science., 1973; 179: 588-590.
17. Ellman GL ,Fiches FT Quantitative
determination of peptides by sulfhydryl
groups Arch, Biochem Biophys., 1959;
82: 70-72.
18. Lowry OH, Rosembrough NJ Farr AL
Protein measurement with the folin
phenol reagent. Journal of biological
chemistry. 1951; 193(1): 267-275.
50