Hemolytic and Anti Microbial Effect in the Leaves of Acanthus ilicifolius
Acanthus ilicifolius is a very potential plant and it has more phyto chemical compounds. In present study, anti microbial and hemolytic effects of Acanthus ilicifolius leaves extracts of chloroform and aqueous in isolated protein. The antimicrobial activity of crude extract against bacterial and fungal pathogens showed the clear inhibition zone against Vibrio cholerae and Aspergillus niger in chloroform extract and aqueous extracts showed clear inhibition zone for Pseudomonas sp. and Candida albicans. Both the extracts exhibited hemolytic activity which was estimated as 10.80 ht mg-1 for chloroform extract and 9.5 ht mg-1 for aqueous extract. The partial purification of protein is done by using DEAE cellulose. On SDS-PAGE the crude protein yielded three well defined bands at 100.5, 52 and 21.4 kDa in both the extracts.
Antibiotics used are no longer desirable because of concerns about bacterial
resistance (Wallace, 2004). Increased awareness of the
potential problems associated with the use of antibiotics stimulates research
efforts to identify alternatives to their use. Novel approaches in the development
of new antimicrobial have been carried out, such as compounds to treat diseases
or to improve animal growth include dietary use of probiotics, prebiotics (Higgins
et al., 2007), organic acids (Immerseel et
al., 2006), medicinal herbs (Arab et al.,
2006; Du and Hu, 2004)
Hemoglobin S differs from HbA in the substitution of valine for glutamic acid in the sixth position of the β-chain amino acid sequence. Hemoglobin HbSS, in whom all the hemoglobin type is HbS, always manifest features of sickle cell disease, which may be fatal in childhood. The hemolytic activity of the extracts was also performed to rule out the possible cytotoxic mechanism and to check the safety of the phytocompound thus making it suitable for the preparation of natural drugs.
Acanthus ilicifolius is a commonly available as back mangrove plant
in almost all the coastal states of India. It is a folklore medicinal plant
used mainly against rheumatism, paralysis, asthma and snake-bites, skin disease,
ulcer. A decoction of the plant with sugar candy and cumin is used in dyspepsia
with acid eructations (Kathiresan and Ramanathan, 1997;
Ramanathan, 2000) It is also considered to be a diuretic
and is used as a cure for dropsy and bilious swellings. In Goa, the leaves are
employed as an emollient fomentation in rheumatism and neuralgia (Ananda
and Sridhar, 2002). The leaves are bruised and soaked in water for external
application and are also used as an expectorant. The analgesic, anti-inflammatory
(Kanchanapoom et al., 2001) and leishmanicidal
(Kapil et al., 1994) properties of A. ilicifolius
have been documented, whilst have reported the antioxidant and hepatoprotective
properties of the plant (Babu et al., 2001).
Hence, the present study was undertaken to evaluate the protective effects of
Acanthus ilicifolius extract and its comparison with active principle
hemolytic and anti microbial effects.
MATERIALS AND METHODS
Plant material: Chemical reagents were purchased to Sigma Chemicals Co. (St. Louis, MO). The fresh leaves of Acanthus ilicifolius was collected during September 2009 in Parangipettai, Chidambaram, Tamil Nadu, India. The leaf was identified and authenticated by Botanist at Annamalai University, Annamalai Nagar, Tamilnadu, India. The specimen was deposited in the departmental herbarium unit.
Extraction of crude toxin: Aqueous extraction: The aqueous extract of sponge was prepared by squeezing the sand-free specimens in triple distilled water. The resultant solution was filtered and dialyzed by using Sigma dialysis membrane-500 (Av Flat width-24.26 mm, Av. Diameter -14.3 mm and capacity approx -1.61 mL cm-1) against D-glucose to remove the excess water. The supernatant so obtained was lyophilized (Labcono Freeze Dry System) and stored at 4°C in a refrigerator for further use as crude aqueous extract.
Chloroform extraction: Crude toxin was extracted following the method
of Bakus (1981) with certain modifications. The sponge
was dried in air for 2 days and after that 10 g sponge tissue was shocked with
200 mL of chloroform, covered and kept standing for 5 h. The solvent was then
removed after squeezing the sponge and filtered through Whatman No. 1 filter
paper. The solvent was evaporated at low pressure by using a Buchi Rotavapor
R-200 at 45°C in refrigerator for further use as crude chloroform extracts.
Antimicrobial activity: Petri dishes with nutrient agar and PDA agar were inoculated with six different species of bacteria and fungus. Sponge extracts were sterilized by passing each through a 0.22 :m Millipore GV filter (Millipore, U.S.A). Round paper discs w with a radius of 0.8 cm were dipped into each sponge extract and placed in the center on inoculated Petri dishes. The bacterial and fungal colonies were allowed to grow overnight at 37 and 20°C respectively and then the inhibition zone around the disc was measured.
Protein estimation: Protein estimation was done as described by Lowry
and Lopez (1946) using Bovine serum Albumin at the rate of 1 mg mL-1
as the standard. Different concentrations of the standard ranging from 0.1 to
1 mg mL-1 were taken and made up to 1 mg mL-1. Then 5
mL of alkaline copper reagent was added, mixed well and allowed to stand for
10 min at room temperature. Then 0.5 mL of diluted Folins phenol reagent was
added and mixed well. The mixture was incubated for 30 min at room temperature.
The absorbance at 720 nm was read spectrophotometrically. The protein concentrations
of S. fibulatus extracts were estimated.
Partial purification of crude protein: Partial purification of the crude
extract was carried out using DEAE Cellulose Anion Exchange chromatography according
to the procedure of Stempein et al. (1970) method.
Gas chromatography: Gas chromatography of the crude extract was done as described. Identification of fatty acid was carried out on the basis of retention times of the standard mixtures of fatty acids.
Hemolytic assay: The micro hemolytic test was performed in 96 well V bottom micro titer plates. Different rows were selected for chick blood. Serial two fold dilutions of the crude toxin were made in 100 mL of normal saline. This process was repeated up to the last well. Then 100:l of RBC was added to all the wells. Appropriate controls were included in the test. To the 1% RBC suspension 100:l was added normal saline, which served as negative control. The plate was gently shaken and then allowed to stand for two hours at room temperature and the results were recorded. Uniform red colour suspension in the wells was considered as positive hemolysis and a button formation in the bottom of these wells was considered as lack of hemolysis. Reciprocal of the highest dilution of the crude toxin showing pattern was taken as 1 Hemolytic Unit (HU).
SDS-PAGE: One dimension sodium dodecyl sulphate (SDS) Polyacrylamide
gel electrophoresis (PAGE) was carried out following the modified method of
Laemmli (1970) SDS-PAGE was run on vertical slab gel
system. Proteins were electrophorised on 12% separating gel (0.75 mm thickness)
overlaid with 5% stacking gel. A 10% (w/v) stock solution was prepared in de
ionized water and stored in room temperature.
RESULTS AND DISCUSSION
The present study to investigated bioactivities of protein isolated from A.ilicifolius
leaves extracts. Chloroform extract of mangrove plant yielded a total amount
of 5.5 g crude extract from 500 g of A.ilicifolius. Similarly aqueous
extract pf total amount of 4.85 g of crude extract. Table 1
The crude aqueous and chloroform extract was determine the protein level of
in our sample in our result suggested that chloroform extract 1.520 mg mL-1
and followed by aqueous extract 1.234 mg mL-1 (Table
2). The crude of aqueous and chloroform extracts at different concentration
of 5, 10 and 15 mg mL-1 were tested against 6 species of bacteria
viz Pseudomonas sp., Streptococcus aureus, Vibrio parahaemolyticus,
Vibrio cholerae, E.coli and Vibrio parahaemlyticus and
3 species of fungus, A. flavus, A. niger and Candida albicans.
The results showed that the crude aqueous extract inhibit the growth of
V. cholerae whereas in the chloroform extract a clear inhibition zone
were observed only against Pseudomonas sp. The inhibition zone was measured
and it was found to be 2.7 cm for Pseudomonas sp. and 1.8 cm for Vibrio
cholerae (Table 4). The results showed that the crude
aqueous and chloroform extracts against fungal stains. In the aqueous extract
inhibit the growth of A.niger whereas in the chloroform extract a clear
inhibition zone was observed only against C. albicans.
|| Preparation of crude extract
||Hemolytic activity of Acanthus ilicifolius
||Anti microbial activity of Acanthus ilicifolius
The recent work was mention that maximum zone inhibition showed in Trichosporon
begelli and Candida albicans and minimum zone inhibition was showed
in Aspergillus niger and Penicillium chrysogenum in Spinifex
littoreus grass (Thirunavukkarasu et al., 2010).
The inhibition zone was measured and it was found to be 3.1 cm for A. niger
and 2.9 cm for Candida albicans. Burholder and Bedford
(1978) isolated two bromo compounds from Verongi fistularies and
V. vauliformis that inhibited the growth gram positive and gram negative
bacteria. The crude chloroform extract induced pronounced hemolysis on chicken
blood. The hemolytic titer in case of chloroform extract found to be 16 and
its specific hemolytic activity was estimated to be 10.8 HT mg-1
of protein (Table 3). The hemolytic titer of aqueous extract
of marine Acanthus ilicifolius was found to be 16 and its Hemolytic Activity
was found to be 9.5 HT mg-1 of protein. SDS-PAGE on 12% gel, the
crude protein toxins yielded 5 bands in aqueous extract and 8 bands in chloroform
extract of A. ilicifolius. Ranging from 7.8 to 116 kDa with three well
defined bands at 100.5, 52 and 21.4 kDa defined bands at in both extracts. Proteins
have recently been found in ginseng. Pananotin, from the roots of Panax notoginseng
was found to be toxic to Coprinus comatus, Physalospora piricola and
the phytopathogens, Botrytis cinerea and Fusarium oxysporum Other
proteins isolated from the Panax family (panaxagin and quinqueginsin)
also exhibit antifungal activity (Lam and Ng, 2002).
The chloroform and aqueous extracts of A. ilicifolius have great potential asantimicrobial proteins against microorganisms. Thus, they can be used in the treatment of infectious diseases caused by resistant microbes. And also it has great effect of hemolytic activity in chicken blood. Plant extracts against resistant bacteria leads to new choices for the treatment of infectious diseases.
The authors are grateful thanks to The Dean, Centre of Advanced study in Marine Biology, Faculty of Marine Sciences, Annamalai University and EASMA Institute of technology, Tamilnadu for providing all support during the study period.
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