Preliminary Phytochemical Screening, Free radical
Scavenging and Antimicrobial activities of Citrus auranticum fruit
bio-mass
SL Munne*, DV Parwate and VN Ingle
Department
of Chemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, 440 033 (M.S)
*Corresponding Author E-mail: sonalimunne07@rediffmail.com
ABSTRACT
The present study was undertaken to evaluate
antioxidant activity and antimicrobial activity of methanol extracts of gamma
irradiated and unirradiated Citrus auranticum fruit bio-mass. In-vitro
antioxidant activity has been investigated by 1,1-Diphenyl, 2-picryl-hydrazyl
free radical scavenging method. The extracts were subjected to antibacterial
(Staphylococcus aureus, Escherichia coli, Salmonella typhi) and
antifungal (Aspergillus niger and Candida albicans) screening by disc
diffusion method. Preliminary phytochemical investigation was done and
different phytoconstituents present were identified.
KEYWORDS: Citrus
auranticum, Antibacterial,
Antifungal, Free radical scavenging, Gamma radiation
INTRODUCTION:
The Citrus auranticum Linn. [Family – Rutaceae] is
a species found in India. The fruits are sour, bitter, astringent,
thermogenic, laxative, appetiser, stomachic, digestive, anthelmintic and
antiscorbutic and are useful in vitiated conditions of Pitta and kapha, cough,
bronchitis, dyspepsia, nausea, flatulence, colic, helminthiasis scabies and
anaemia1. To our knowledge there were no scientific reports on the
antimicrobial and antioxidant activities of Citrus auranticum fruit
bio-mass. Preliminary phytochemical screening was performed as per standardized
procedure2.
MATERIALS AND METHODS:
Plant material: – Fruits of Citrus auranticum were collected
from National Research Center for Citrus (NRCC), Nagpur and authenticated by
Department of Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur,
India.
Preparation of extract:
The peels, seeds and juice of the fruit were
removed. The solid bio-mass was washed with distilled water filtered at
suction pump and dried at room temperature. The dried solid bio-mass was then
powdered using grinder to have uniform size. It was divided into two parts
1).
In this bio-mass was irradiated with gamma
radiations (60Co source GC-900 at a dose of ~ 0.7 kGy/hour) for 48
hrs. 2) The bio-mass was used without exposure to radiation. Both of these
materials were defatted with petroleum ether (60-80 0C) followed by
methanol using Soxhlet extraction process. The solvent was evaporated under
reduced pressure at 50 0C and the extract thus obtained was
used directly for phytochemical screening and for the assessment of antioxidant
activity through in vitro method and for antimicrobial activity.
Preliminary phytochemical screening was performed
as per standardized procedure and various phytoconstituents present in the
extracts were identified 3,4.
Antimicrobial activity:
The extract were subjected to antibacterial (E.coli,
S.aureus, S.typhi ) and antifungal (C.albicans and A. niger ) screening.
The antimicrobial screening was performed by disc diffusion method. Muller
Hinton Agar medium was prepared and sterilized by an autoclave in an aseptic
room. The agar medium was poured into sterilized petridishes to a uniform
depth of 4 mm and then allowed to solidify at room temperature. After
solidification the test organism were inoculated with the help of sterilized
swab soaked in a bacterial culture. This provides the uniform surface growth
of bacterium and is used for antibacterial sensitivity studies. The sterilized
Whatman filter paper discs (6mm) containing sample were immersed in plate
extracts and placed over the solidified agar in such a way that there was no
overlapping of zone of inhibition. Plates were kept at room temperature for
half an hour for the diffusion of the sample into the agar media. Then that
petridishes were incubated at 370C for 16 to 18 hrs. After
incubation period was over the zone of
TABLE 1. Antimicrobial activity of methanol extracts of
gamma irradiated and unirradiated of Citrus auranticum fruit bio-mass
|
Sr. No
|
Name of the organism
|
Zone of inhibition (mm)
|
|
Standard
|
DMSO
|
MEUCA
|
MERCA
|
|
200mg/ml
|
400mg/ml
|
200mg/ml
|
400mg/ml
|
|
1)
|
Escherichia coli
|
16
|
_
|
7
|
8
|
7
|
8
|
|
2)
|
Staphylococcus aureus
|
24
|
_
|
8
|
12
|
8
|
10
|
|
3)
|
Salmonella typhi
|
20
|
_
|
8
|
10
|
8
|
11
|
|
4)
|
Aspergillus niger
|
18
|
_
|
7
|
10
|
6
|
8
|
|
5)
|
Candida albicans
|
22
|
_
|
8
|
12
|
7
|
10
|
MEUCA – methanol extract of unirradiated C.auranticum,
MERCA – methanol extract of radiated C.auranticum, DMSO – Dimethyl
sulfaoxide
Table 2. DPPH radical scavenging activity of
extract
|
Sr. No
|
Extract
|
Concentration ( µg/ ml ) and % inhibition
|
IC 50 (mg/ml)
|
|
20
|
40
|
60
|
80
|
100
|
|
(1)
|
MEUCA
|
39.83±0.21
|
44.41± 0.14
|
48.03± 0.24
|
51.03±0.12
|
53.11±0.43
|
75.61
|
|
(2)
|
MERCA
|
29.76 ± 0.16
|
32.81 ±0.25
|
35.84± 0.22
|
39.32±0.13
|
45.93±0.64
|
_
|
|
(3)
|
Std.(Vit- C)
|
26.47± 0.03
|
38.57±0.03
|
52.63± 0.025
|
60.40±0.03
|
66.57±0.01
|
54.30
|
MEUCA – methanol extract of unirradiated C.auranticum,
MERCA – methanol extract of radiated C.auranticum
inhibition produced by various extracts of gamma irradiated
and unirradiated of Citrus auranticum fruit bio-mass with different
organism in different plates were measured5,6. Erythromycin and
Griseofulvine (50µg/disc) was used as standard for antibacterial and antifungal
activity respectively. The observed zones of inhibition are presented in Table
1
Antioxidant activity: 7,8
The free radical scavenging activity of the
extract was determined by using 1,1-Diphenyl, 2-picryl-hydrazyl free radical
scavenging method. The free radical scavenging activity of different extracts
of bio-mass of unirradiated and gamma irradiated Citrus auranticum and
L-ascorbic acid ( Vitamin C ) was measured in terms of hydrogen donating or
radical-scavenging ability using the stable radical DPPH. About 0.1 mM
solution of DPPH in ethanol was prepared and 1.0 ml of this solution was added
to 3.0 ml of extract solution in water at different concentrations
(20-100μg/ml). Thirty minutes later, the absorbance was measured at
λmax= 517 nm. Lower absorbance of the reaction mixture
indicates higher free radical scavenging activity. The capability to scavenge
the DPPH radical was calculated using the following equation:
(Acont - Atest)
X 100
(Acont)
Where,
Acont is the absorbance of the
control reaction
Atest is the absorbance in
presence of the extracts.
The antioxidant activities of extracts are
expressed as IC50. The IC50 value is defined as the
concentration ( μg /ml ) of extracts that inhibits the formation of DPPH
radicals by 50 %. The results of anti-oxidant activity of extracts of using
DPPH free radical scavenging method are shown in Table2.
RESULTS AND DISCUSSION:
The present study reveals the presence of
phytoconstituents such as flavonoids, steroids and carbohydrates. Both the
extract MEUCA and MERCA exhibited antibacterial and antifungal activity against
the tested microorganism.
The result of the present study also indicated that the
MEUCA was capable of scavenging free DPPH radical, thus providing to have more
antioxidant activity than the MERCA. The degree of discoloration indicates the
scavenging potential of the extracts.
The isolation of flavonol compound from this plant is
important to elucidate the exact mechanism involved in scavenging activity and
encompassing the In-Vivo behaviour of the extract and the present study
would be useful to predict the sensitive organism towards the drug extract of C.auranticum
fruit biomass, which in future can be developed as a potential antimicrobial
agent with reduced toxicity and untoward effect when compared with synthetic
chemotherapeutic agents.
Both the extract taken for study
are bio-waste of Citrus auranticum fruit that’s why they are not showing
that much antimicrobial and antioxidant activity to that of standard use.
ACKNOWLEDGEMENTS:
Authors are thankful to the Head of
Department of Chemistry and Department of Pharmacy, Rashtrasant Tukadoji
Maharaj Nagpur University, Nagpur, India for providing laboratory facilities.
REFERENCES:
1. Varies`s PS. Indian medicinal plants. Arya
Vaidya Sala, Kottakkal. 2006; Vol. 2 pp 97-100.
2. Khandelwal K. Practical Pharmacognosy,
Nirali publication. Aug 2000; 2nd Ed. p. 9-38
3. Trease GE. Evans MC. Textbook of
pharmacognosy. 15
th ed; 343.
4. Farnsworth NR. Biological and Phytochemical
Screening of Plants. Journal of Pharmaceutical Sciences. 1966; 55 (3):
p.225-286
5. Mazumder A. Saha BP. Basu SP and Mazumder R.
Antibacterial activity of methanolic extract of leaves of Lagerstroemia
parviflora. Indian.J.Nat.Prod. 2003; 19(3): pp 20-23.
6. Shanmungam S and Manavalan, R. Antimicrobial
activity of the extracts of Artocarpus heterophyllus. Hamdard medicus.
2007; 50: pp 24-25.
7. Cakir A. Isolation and characterization of
antioxidant phenolic compounds from the aerial parts of Hypericum
hyssopifolium L. by activity guided fractionation. Journal of
Ethnopharmacolog. 2003; 87 : 73-83.
8. Mazumdar UK. Studies on in vitro antioxidant
activities of methanol extract of Mucuna pruriens (Fabaceae) seeds. European
Bulletin of Drug Research.2005; 13: 31-39.