![International Journal of Pharmaceutical Science Invention
ISSN (Online): 2319 – 6718, ISSN (Print): 2319 – 670X
www.ijpsi.org Volume 6 Issue 2 ‖ February 2017 ‖ PP. 31-36
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Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L
and Screening for Antioxidant and Antibacterial Activity.
N. Sharief Mohammada&B
, Mathewos Genetoa
, Dejene Deresh Abateneha
Mohammad Salahuddina
Mohammad Dilshad Manzara
Uma Maheswara Rao Vb
a
Department of Biomedical Sciences, Mizan Tepi University, Mizan, Ethiopia
b
Department of Microbiology, Acharya Nagarjuna University, Guntur, India
Abstract: The root extracts of Acanthus ilicifolius L finds a prominent place in folk medicine. In this study, we
extracted alkaloid, flavonoid, tannin and total phenols in benzene, ethyl acetate, acetone, methanol and
ethanol, their antibacterial activity and antioxidant activity was evaluated. The antioxidant activity is executed
by FRAP assay and agar well diffusion method is done to study the antibacterial activity against Enterobacter
aerogenes, Enterobacter cloacae, Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Streptococcus
pyogenes. The antibacterial activity of all the extracts was compared with standard antibiotic gentamicin.
The Minimum Inhibitory Concentration [MIC] was determined by serial dilution method. Alkaloids are rich in
acetone and Flavonoids are high in methanol extracts. The acetone extract showed higher antioxidant activity,
while benzene extract was identified to contain lower antioxidant activity. The extent of inhibition by the root
extracts diverge between the solvents used, among them ethanol extracts exhibited higher level of inhibition
against the gram positive test cultures compared to gram negative test cultures employed. Whereas, the acetone
extracts efficacy is more on gram negative test cultures than the gram positive cultures. The MIC was found to
be between 1mg/100µl to 5mg/100µl. This study gives the source for purification and characterization of
bioactive principles that possess antioxidant and antibacterial action from the root of Acanthus ilicifolius.
Keywords: Acanthus ilicifolius, Root, Antibacterial activity, Antioxidant activity
I. Introduction
Plants have been a rich source of medicines because they produce wide array of bioactive molecules,
most of which probably evolved as chemical defense against predation or infection. Mangroves are diversified
group of plants that grow in estuarine environment, have been a source of interest for their novel natural
products like alkaloids, flavanoids, glycosides, saponins, tannins, etc., are known to exhibit antiviral,
antibacterial and antifungal activities [1] Systematic study of mangrove species has revealed that crude extracts
of different plant parts in different solvents exerted potential antibacterial, antifungal, antiviral and antioxidant
activities [2]. Therefore, the present study has designed to extract the secondary metabolites and evaluate their
antibacterial and antioxidant activity from the roots of Acanthus ilicifolius.
II. Materials and Methods
Collection of Plant material
Acanthus ilicifolius belongs to the family Acanthaceae also be called as Holly Mangrove, Holly-
leaved Acanthus. The plant is used in traditional systems of medicine, including Traditional Indian Medicine
(TIM) and Traditional Chinese Medicine (TCM). The plant part were collected from Corangi Reserved Forest,
Kakinada, East Godavari, Andhra Pradesh, India. Geographic location - between 16o
39’ N longitude - 17o
N
longitude and 82o
14’ E latitude - 82o
23’E latitude. All the roots were surface sterilized with 1% mercuric
chloride solution and thoroughly washed with filter sterilized distilled water [3]. The washed root, were then
chopped to small pieces and shade dried until they were suitable for extraction in the selected solvents.
ISOLATION
Plant extracts in benzene, ethyl acetate, acetone methanol and ethanol were prepared according to the
standard protocols [4]. The chopped root material (1000g) was initially soaked in 1000ml of the respective
solvent at room temperature for 24h. Subsequently, the soaked material was refluxed for 6h below the boiling
point of the respective solvent. Infusions were filtered through Whatman No.1 filter paper and the residual
material was re-extracted with fresh solvent. After 24h the process was repeated. Pooled extracts were
individually concentrated by removing the solvent under reduced temperatures using vacuum rotator evaporator.
These extracts were further concentrated by solvent evaporation using thin film method.](https://image.slidesharecdn.com/h06023136-170318103903/85/Extraction-of-Secondary-Metabolites-from-Roots-of-Acanthus-Ilicifolius-L-and-Screening-for-Antioxidant-and-Antibacterial-Activity-1-320.jpg)
![Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for…
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Identification of Secondary Metabolites.
The crude extracts were subjected to the qualitative determination of secondary metabolites viz., alkaloids,
flavonoids, glycosides, tannins and total phenols by the standard protocols of phytochemistry [5]
Test for Alkaloids
To 5 ml of the crude extracts were stirred with 10 ml of 1% aqueous HCl on water bath and then filtered. To
2ml filtrate 4-6 drops drops of Dragendroff’s reagent was added. Formation of orange –red precipitate was
considered as positive to alkaloids.
To another 2 ml filtrate few drops of Mayer’s reagent was added and appearance of buff-coloured precipitate
was taken as existence of alkaloids.
Test for Flavonoids
About 5ml of the test solution was boiled with 10 ml of distilled water and then filtered. Then, 2 ml of lead
acetate solution was added to 2 ml of the filtrate. Appearance of buff coloured precipitates considered as
presence of flavonoids.
To 2 ml of the filterate, 5 ml of dilute ammonia solution was added followed by 4 – 6 drops of concentrated
sulphuric acid. Appearance of yellow color indicates the presence of flavonoids.
Test for tannins
About 5ml of each extract was stirred with about 10ml of distilled water and then filtered. Few drops of 1%
ferric chloride solution were added to 2ml of the filtrate. Occurence of a blue-black, green or blue-green
precipitate indicates the presence of tannins.
About 5 ml of each extract was added with 1 ml of 1% HCl solution. Formation of red precipitate indicates the
presence of tannins.
Tests for total phenols
Five ml of the extract was dissolved in 5 mL of distilled water. To this, few drops of neutral 5% ferric chloride
solution were added. Appearance of dark green color indicates the presence of phenolic compounds.
Five ml of the extract was dissolved in 5 mL of distilled water. To this, 3ml of 10% lead acetate was added.
Formation of bulky white precipitate indicate the presence of phenol compounds.
Determination of antioxidant activity by ABTS method
Total antioxidant capacity of each extract was measured using 2, 2´-azinobis [3-ethylbenzthiazoline]-6-
sulfonic acid (ABTS) assay. ABTS and potassium per-sulfate were separately dissolved in deionized distilled
water to a final concentration of 7mM and 2.45mM respectively. The two solutions were mixed and allowed to
stand in dark at room temperature for 16h before use in order to produce ABTS radical (ABTS•+). The resultant
intensely-coloured ABTS•+ radical cation was diluted with 0.01M PBS (phosphate buffered saline), pH 7.4, to
give an absorbance value of ~0.70 at 734 nm. The test compound was diluted 100X with the ABTS solution to a
total volume of 1ml. Absorbance was measured spectrophotometrically at time intervals of 3min after addition
of each extract. The assay was performed at least in triplicate. Controls were run using PBS in place of the
extract. The assay relies on the antioxidant capability of the samples to inhibit the oxidation of ABTS to
ABTS•+ radical cation. Percent inhibition was calculated using the following formula and it was compared with
ascorbic acid. [6]
% inhibition of oxidation of ABTS to ABTS•+ = Initial absorbance - Final absorbance X 100
Initial absorbance
Determination of antibacterial activity
The antibacterial activity of the crude extracts was screened with gram negative and gram positive
cultures viz., Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Bacillus subtilis,
Staphylococcus aureus and Streptococcus pyogenes. by agar well diffusion method [7]. About 20 ml of
melted Mueller Hinton agar was mixed with 1 ml of bacterial suspension homogeneously and allowed to
solidify in petri dishes (143 mm diameter). Wells (8mm diameter) were made using a sterile cork borer on the
solidified medium and are filled with 100 µl of the extract (5mg/100µl). The inoculated plates were incubated at
34-38o
C. The diameter of the inhibition zones were measured in mm. Each experiment was performed in
triplicate and the average value of inhibition and standard deviation was calculated. Gentamicin (30µg) discs
are used as positive control and DMSO with solvents (1:10 dilution) used as control. Results were expressed as
mean ± SD and the data were analyzed using one-way analysis of variance (ANOVA) to discover the
significant difference at the 5% (P<0.05)level.](https://image.slidesharecdn.com/h06023136-170318103903/85/Extraction-of-Secondary-Metabolites-from-Roots-of-Acanthus-Ilicifolius-L-and-Screening-for-Antioxidant-and-Antibacterial-Activity-2-320.jpg)
![Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for…
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Determination of MIC
Minimum Inhibitory Concentration [MIC] was determined by broth dilution assay method [8]. Plant extracts
were serially diluted in Mueller Hinton broth to get the concentrations of 1.0 to 5.0 mg/100µl. Each experiment
was repeated thrice and the mean values were tabulated.
III. Results
Plants are the important source for the development of new bio active principles, the knowledge of
secondary metabolites and their biological properties are desirable, not only for the discovery of novel bioactive
entities, but also for disclosing new sources of already known biologically active compounds. Acanthus
ilicifolius is a medicinal plant used against asthma, paralysis, snake bite, analgesic and anti inflammatory. The
reports of phytochemical studies are given in table 1. From this data it is very clear that tannins and total
phenols are there in all the solvents used for the extraction of secondary metabolites. However, flavonoids are
seen in all the solvents except benzene. Whereas, alkaloid is confined only to acetone, methanol and ethanol.
These secondary metabolites are incredible group of compounds plays an important role in many biological
activities.
The antibacterial activity of root extracts of Acanthus ilicifolius in benzene, ethylacetate, acetone,
methanol and ethanol was revealed by agar well diffusion method and the results are presented in table-2. All
the extracts have assorted degrees of antibacterial potential against cultures used. Among the tested extracts
benzene solubles of Acanthus ilicifolius exhibited highest potential of antibacterial activity (19.66mm) against
Enterobacter cloacae than gentamicin. The ethyl acetate soluble fraction is active against Escherichia coli,
Bacillus subtilis and Staphylococcus aureus with same zone size of 16.66 mm. However, metabolites of
Acanthus ilicifolius infused into acetone were found to be active on selective test cultures viz., Enterobacter
aerogene and Enterobacter cloaceae of gram negative and Staphylococcus aureus of gram positive bacteria.
The infusions of ethanol are susceptible to all the test cultures used except Enterobacter aerogenes and the
inhibitory effect against test cultures is in increasing order for Enterobacter cloacae (12.66), Escherichia coli
(13.33) Streptococcus pyogenes (17.66), Bacillus subtilis (18.33)and Staphylococcus aureus (18.66).
Whereas, the efficacy of methanol extracts are sensitive to Enterobacter aerogenes and Escherichia coli with
same zone size of 16.66 mm and zone size of 17.33 was observed with Bacillus subtilis and Staphylococcus
aureus. The maximum zone of inhibition exerted against gram negative test cultures is Enterobacter cloacae
(ethylacetate extract) 19.66 mm and gram positive is Staphylococcus aureus (ethanol extract) 18.66 mm. The
zone of inhibition exerted by gentamicin is same for all the gram positive test cultures employed. Whereas,
among gram negative Enterobacter aerogenes and Enterobacter cloacae are less sensitive then Escherichia
coli to gentamicin. The inhibitory effect of the root extracts of Acanthus ilicifolius in ethyl acetate and ethanol
against the test cultures is inferior than gentamicin. However the inhibitory outcome of acetone extracts were
superior than gentamicin against the two gram negative cultures used viz., Enterobacter cloacae and
Escherichia coli. Whereas, the infusions of methanol having higher inhibitory potential than gentamicin against
Enterobacter aerogenes and Enterobacter cloacae. We also studied the combination of different extracts and
the results are displayed in table-3. The effect of ethyl acetate and acetone extract mixture have mixed result it
has antagonistic effect on Bacillus subtilis and symbiotic effect on Escherichia coli. The mixture of ethyl
acetate and methanol have not net role on gram negative cultures used but have negative effect on the gram
positive cultures employed in terms of inhibition zone size. Ethyl acetate and ethanol mixture did not inhibit the
Enterobacter aerogenes, Enterobacter cloacae and Streptococcus pyogenes. But the zone of inhibition against
gram positive test organism viz., on Bacillus subtilis and Staphylococcus aureus is very high compare to that of
the individual extracts and the phenomenon is called symbiotic effect. The combination of acetone and methanol
extracts is the best as they control the growth of all the test organisms employed in our study. Acetone and
ethanol mixture has shows antagonistic effect with respect to inhibition. Methanol and ethanol mixture have
antagonistic effect on Enterobacter cloacae and Staphylococcus aureus.
Further, these extracts were analyzed to determine the minimum inhibitory concentration against both
Gram negative and Gram positive bacterial species. The MIC values ranged from 1.0-5.0 mg/100µl and varied
from extract to extract and the results are given in table-3. The MIC of root extracts in benzene is 2.0 mg/100µl
against Enterobacter cloacae. Ethyl acetate extracts were active at 3.0 mg/100µl with Escherichia coli and
2.0 mg/100µl with Bacillus subtilis and Staphylococcus aureus . Whereas, the MIC of acetone extract is 2.0
mg/100µl towards all the tested microorganisms. The methanol extracts of Acanthus ilicifolius root exhibiting
diversified range of MIC among the tested cultures between 1.0 - 4.0 mg/100µl. Whereas, the MIC of ethanol
extract is 5.0 mg/100µl against gram negative test cultures and 3.0 mg/100µl with gram positive
microorganisms used.
Several techniques have been used to determine the antioxidant activity, free radicals are known to
play a definite role in a wide variety of pathological symptoms. Antioxidants fight against free radicals and
protect us from various diseases. The non enzymatic antioxidant activity of plants is attributed to the secondary](https://image.slidesharecdn.com/h06023136-170318103903/85/Extraction-of-Secondary-Metabolites-from-Roots-of-Acanthus-Ilicifolius-L-and-Screening-for-Antioxidant-and-Antibacterial-Activity-3-320.jpg)
![Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for…
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metabolites. The antioxidant activity of the fractions was estimated spectrophotmetrically by ABTS method and
the data is given in Fig-1. The outcome of antioxidant activity by ABTS method is high for acetone extract
followed by ethanol, methanol ethyl acetate and benzene extracts.
IV. Discussion
In this study, we have qualitatively screened, specifically for the presence of alkaloids, flavonoids,
tannin and total phenols as well as the antioxidant and antibacterial potentials of Acanthus ilicifolius root
infusions in benzene, ethyl acetate, acetone, methanol and ethanol in order of increasing polarity. Alkaloids,
flavonoids, tannin and total phenols are associated with potent biological activities. Our results show that the
secondary metabolites present in the root extracts of Acanthus ilicifolius are diversified in the solvents used.
Acetone, methanol and ethanol fractions contain all the secondary metabolites checked. Our observation are in
accordance with the report of Asha et.al, they extracted flavnoids and phenolics from the root extracts of
Acanthus ilicifolius in ethanol [9 ]
. The efficacy of antibacterial action is high for ethanol extract and it
correlates with the presence of all the secondary metabolites studied. The present experimental data is in
accordance with that of Govindasamy et.al who studied the chemical constituents of different mangroves in
India [10]
. The secondary metabolites viz., alkaloids, flavonoids tannin and total phenols play an important
role in scavenging of free radical and are also known to be associated with other biological activities such as
antibacterial, antifungal, antidiuretic activities [11,12]
. The root extracts of in ethyl acetate, methanol and
ethanol are active against Escherichia coli Bacillus subtilis and Staphylococcus aureus these results are in
disagreement with reports of dev avijit et.al[13]
. They studied the stem extract of Acanthus ilicifolius in
methanol by maceration and continuous extraction method. The difference of the result may be due to the plant
part used or the protocol employed for the extraction.
The extracts in ethyl acetate were active against Escherichia coli, Bacillus subtilis and Staphylococcus
aureus with same zone size of 16.66 mm. This may be the compounds present in the extract may have the
similar effect on these organisms. The activity of ethanol extracts on all the bacterial cultures except
Enterobacter aerogenes confers their broad spectrum nature over the other extracts. The zone of inhibition
exhibited by the acetone extracts were higher towards gram negative organisms than gram positive organisms
and the ethanol extracts were more active on gram positive than gram negative cultures. This difference in the
rate of inhibition potential appear to be directly related to the qualitative diversity of the compounds that are
present in the extracts. This may be due to the permeability factor of cell membrane of the microorganism, or
this could be due to variation in the cell wall composition of Gram negative and Gram positive bacteria[14]
. The
Gram negative bacteria restrict the influx of many antibiotics. Multi drug efflux pumps at the trans-membrane
are also responsible for a higher intrinsic resistance in Gram negative bacteria. On correlating our results of
secondary metabolites and antibacterial activities it is inferred that alkaloids, flavonoids, glycosides tannins
and total phenol possess substantial antibacterial activity. Our results are in concurrence with the work of Bose
and Bose [15]
, with reference to the solvent system and type of microorganisms, i.e., gram positive cultures are
more sensitive than gram negative cultures used.
The more effectiveness of root extracts of Acanthus ilicifolius in methanol and ethanol than that of the
acetone, benzene and ethyl acetate in our study can be correlated with the medicinal preparation that use rum
and liquour to extract the active plant components. Benzene extracts did not inhibit Enterobacter aerogenes,
Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Streptococcus pyogenes. Similarly ethyl
acetate infusions also did not inhibit Enterobacter aerogenes, Enterobacter cloacae, and Streptococcus
pyogenes. Negative results did not mean the absence of bioactive principles in these extracts. The bioactive
principle may be insufficient to cross the membrane, or the microorganism may have mechanism to nullify the
effect of the bioactive principle present in these extracts.
In our study, the MIC value for all the positive extracts against the tested bacteria were between
1.0mg/100μl to 5mg/100μl. Gram negative test cultures showed higher MIC values than Gram positive text
cultures to the extracts in methanol. This difference may be explained by susceptibility testing condition,
physico chemical characters of the bioactive principle present in the extract and even strain to strain difference.
In comparison to some of the earlier reports on MIC values of pure compounds, our MIC may be higher [16]
.
But this can be substantiated by the argument that this value is for the crude extract. However, the purified form
of bioactive compound of the crude extract responsible for antibacterial activity may exhibit the inhibitory
effect at a lower concentration.
Fai-Chu Wong[17]
studied the MIC on S. aureus, M.luteus, E.coli and P.aeruginosa with ampicillin and
selected medicinal plant extracts and reported the MIC value of Amphicillin is between 0.02 - 1mg/1000µl and
that of the plant extracts are in the range of 6.3 – 50 mg/1000µl. Our results are far superior compare to that of
the plant extracts but inferior to that of the standard antibiotic.](https://image.slidesharecdn.com/h06023136-170318103903/85/Extraction-of-Secondary-Metabolites-from-Roots-of-Acanthus-Ilicifolius-L-and-Screening-for-Antioxidant-and-Antibacterial-Activity-4-320.jpg)
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References
[1]. Bandaranayake WM. (2002). Bioactivities, bioactive compounds and chemical constituents of mangrove plants. Wetlands Ecology
and Management. 10:421–52.
[2]. P. D. Abeysinghe, R. P. W., R. N. Pathirana, Evaluation of antibacterial activity of different mangrove plant extracts. Ruhuna
Journal of Science, 1 (2006) 104-112.
[3]. N. Sharief Md and UmaMaheswara Rao v.: Antibacterial activity of stem and root extracts of Avicennia officinalis L .
International Journal of Pharmaceutical Application. 2011; Vol2, Issue 4: 231-236.
[4]. P.Satya Veni, S.Sunitha and A. Srinivasulu (2013). Evaluation of antibacterial activity on selected bacteria and screening of
secondary metabolites of Avicennia Alba stem. International journal of Advanced Biotechnology and Research, 4: 511-517.
[5]. Rahmi Nurdiani, Muhamad Firduas, Asep Awaludin Prihanto. Phytochemical screening and antibacterial activity of methanol
extract of mangrove plant Rhizophora mucronata from Porong River Estuary. Journal of Basic Science And Technology 2012; 2:
27-29.
[6]. N.Sharief Md, A.Srinivasulu, P.Satya Veni and Uma Maheswara Rao V. International Journal of Biological & Medical Research.
2014; 5(1): 3869-3873.
[7]. Adeniyi BA, Odelola HA, Oso BA. African Journal of Medicine and Medical Sciences 1996, 25(3), 221–224.
[8]. Okoli, S. and Iroegbu, CU (2005). In vitro antibacterial activity of Synclisa scabrida whole root extracts, African journal of
biotechnology, 4(9): 946-52.
[9]. Asha KK, Suseela, Mathew, Lakshmi PT. Flavonoids and phenolics compounds in twomangrove species and their antioxidant
property. Indian Journal of Geo-Marine Sciences 2012; 3: 259-264.
[10]. Govindasamy Acoramoorthy, Fu-An Chen, Venugopalan Venkatesalu, Daih-Huang Kuo and Po-Chuen Shea: Evaluation of
antioxidant polyphenols from selected mangrove plants of India. Asian Journal of Chemistry 2008; 2: 1311-1322.
[11]. Saifullah Al Mamoon, Mohammad gulam Azam: Preliminary phytochemical screening and antidiarrhoeal activity of Derris
trifoliate Lour. International Journal of Pharmaceutical Sciences and Research 2012; 1: 97-100.
[12]. Raut, S. And S. Khan, 2012. Phytochemical fingerprinting of Acanthus ilicifolius Linn. Adv.Plant Sci., 25: 749-753.
[13]. Dev avijit, Rihan Sarkar Mohammad, Sariful Islam Howaldar Mohammad, Hamiduzzaman Mohammad, Monjur-Al- Hossain ASM.
Phytochemical screening and the evaluation of the antioxidant, cytotoxic and antimicrobial properties of Acanthus ilicifolius
(Acanthaceae). International Journal or Research Pharmacy. 2012; 3: 153-156.
[14]. Nikiado H. Antibiotic resistance caused by gram negative multidrug efflux pumps. Clinical Infectious Disease 1998; 27: 32-41.
[15]. Bose S, Bose A. Antimicrobial Activity of Acanthus ilicifolius (L.) Indian journal of Pharmaceutical sciences 2008; 6: 821-823.
[16]. Maria DC, Torrado T, Maria H, Sarragiotto, Benicio, Dias F: In Vitro Antibacterial activity of a 7-O- ß-D-glucopyranosyl-
nutanocoumarin from Chaptalia nutans (Asteraceae). Mem inst Oswaldo Cruz, Riode Janerio 2003; 98: 283-86.
[17]. Fai-Chu Wong, Anni-Li Yong, Hean-Chooi Ong, Tsun-Thai Chai : Evaluation of the antibacterial activities of selected medicinal
plants and determination of their phenolic consitutents. Science Asia 2013; 39: 591-595.](https://image.slidesharecdn.com/h06023136-170318103903/85/Extraction-of-Secondary-Metabolites-from-Roots-of-Acanthus-Ilicifolius-L-and-Screening-for-Antioxidant-and-Antibacterial-Activity-6-320.jpg)
Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for Antioxidant and Antibacterial Activity.
- 1. International Journal of Pharmaceutical Science Invention ISSN (Online): 2319 – 6718, ISSN (Print): 2319 – 670X www.ijpsi.org Volume 6 Issue 2 ‖ February 2017 ‖ PP. 31-36 www.ijpsi.org 31 | P a g e Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for Antioxidant and Antibacterial Activity. N. Sharief Mohammada&B , Mathewos Genetoa , Dejene Deresh Abateneha Mohammad Salahuddina Mohammad Dilshad Manzara Uma Maheswara Rao Vb a Department of Biomedical Sciences, Mizan Tepi University, Mizan, Ethiopia b Department of Microbiology, Acharya Nagarjuna University, Guntur, India Abstract: The root extracts of Acanthus ilicifolius L finds a prominent place in folk medicine. In this study, we extracted alkaloid, flavonoid, tannin and total phenols in benzene, ethyl acetate, acetone, methanol and ethanol, their antibacterial activity and antioxidant activity was evaluated. The antioxidant activity is executed by FRAP assay and agar well diffusion method is done to study the antibacterial activity against Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Streptococcus pyogenes. The antibacterial activity of all the extracts was compared with standard antibiotic gentamicin. The Minimum Inhibitory Concentration [MIC] was determined by serial dilution method. Alkaloids are rich in acetone and Flavonoids are high in methanol extracts. The acetone extract showed higher antioxidant activity, while benzene extract was identified to contain lower antioxidant activity. The extent of inhibition by the root extracts diverge between the solvents used, among them ethanol extracts exhibited higher level of inhibition against the gram positive test cultures compared to gram negative test cultures employed. Whereas, the acetone extracts efficacy is more on gram negative test cultures than the gram positive cultures. The MIC was found to be between 1mg/100µl to 5mg/100µl. This study gives the source for purification and characterization of bioactive principles that possess antioxidant and antibacterial action from the root of Acanthus ilicifolius. Keywords: Acanthus ilicifolius, Root, Antibacterial activity, Antioxidant activity I. Introduction Plants have been a rich source of medicines because they produce wide array of bioactive molecules, most of which probably evolved as chemical defense against predation or infection. Mangroves are diversified group of plants that grow in estuarine environment, have been a source of interest for their novel natural products like alkaloids, flavanoids, glycosides, saponins, tannins, etc., are known to exhibit antiviral, antibacterial and antifungal activities [1] Systematic study of mangrove species has revealed that crude extracts of different plant parts in different solvents exerted potential antibacterial, antifungal, antiviral and antioxidant activities [2]. Therefore, the present study has designed to extract the secondary metabolites and evaluate their antibacterial and antioxidant activity from the roots of Acanthus ilicifolius. II. Materials and Methods Collection of Plant material Acanthus ilicifolius belongs to the family Acanthaceae also be called as Holly Mangrove, Holly- leaved Acanthus. The plant is used in traditional systems of medicine, including Traditional Indian Medicine (TIM) and Traditional Chinese Medicine (TCM). The plant part were collected from Corangi Reserved Forest, Kakinada, East Godavari, Andhra Pradesh, India. Geographic location - between 16o 39’ N longitude - 17o N longitude and 82o 14’ E latitude - 82o 23’E latitude. All the roots were surface sterilized with 1% mercuric chloride solution and thoroughly washed with filter sterilized distilled water [3]. The washed root, were then chopped to small pieces and shade dried until they were suitable for extraction in the selected solvents. ISOLATION Plant extracts in benzene, ethyl acetate, acetone methanol and ethanol were prepared according to the standard protocols [4]. The chopped root material (1000g) was initially soaked in 1000ml of the respective solvent at room temperature for 24h. Subsequently, the soaked material was refluxed for 6h below the boiling point of the respective solvent. Infusions were filtered through Whatman No.1 filter paper and the residual material was re-extracted with fresh solvent. After 24h the process was repeated. Pooled extracts were individually concentrated by removing the solvent under reduced temperatures using vacuum rotator evaporator. These extracts were further concentrated by solvent evaporation using thin film method.
- 2. Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for… www.ijpsi.org 32 | P a g e Identification of Secondary Metabolites. The crude extracts were subjected to the qualitative determination of secondary metabolites viz., alkaloids, flavonoids, glycosides, tannins and total phenols by the standard protocols of phytochemistry [5] Test for Alkaloids To 5 ml of the crude extracts were stirred with 10 ml of 1% aqueous HCl on water bath and then filtered. To 2ml filtrate 4-6 drops drops of Dragendroff’s reagent was added. Formation of orange –red precipitate was considered as positive to alkaloids. To another 2 ml filtrate few drops of Mayer’s reagent was added and appearance of buff-coloured precipitate was taken as existence of alkaloids. Test for Flavonoids About 5ml of the test solution was boiled with 10 ml of distilled water and then filtered. Then, 2 ml of lead acetate solution was added to 2 ml of the filtrate. Appearance of buff coloured precipitates considered as presence of flavonoids. To 2 ml of the filterate, 5 ml of dilute ammonia solution was added followed by 4 – 6 drops of concentrated sulphuric acid. Appearance of yellow color indicates the presence of flavonoids. Test for tannins About 5ml of each extract was stirred with about 10ml of distilled water and then filtered. Few drops of 1% ferric chloride solution were added to 2ml of the filtrate. Occurence of a blue-black, green or blue-green precipitate indicates the presence of tannins. About 5 ml of each extract was added with 1 ml of 1% HCl solution. Formation of red precipitate indicates the presence of tannins. Tests for total phenols Five ml of the extract was dissolved in 5 mL of distilled water. To this, few drops of neutral 5% ferric chloride solution were added. Appearance of dark green color indicates the presence of phenolic compounds. Five ml of the extract was dissolved in 5 mL of distilled water. To this, 3ml of 10% lead acetate was added. Formation of bulky white precipitate indicate the presence of phenol compounds. Determination of antioxidant activity by ABTS method Total antioxidant capacity of each extract was measured using 2, 2´-azinobis [3-ethylbenzthiazoline]-6- sulfonic acid (ABTS) assay. ABTS and potassium per-sulfate were separately dissolved in deionized distilled water to a final concentration of 7mM and 2.45mM respectively. The two solutions were mixed and allowed to stand in dark at room temperature for 16h before use in order to produce ABTS radical (ABTS•+). The resultant intensely-coloured ABTS•+ radical cation was diluted with 0.01M PBS (phosphate buffered saline), pH 7.4, to give an absorbance value of ~0.70 at 734 nm. The test compound was diluted 100X with the ABTS solution to a total volume of 1ml. Absorbance was measured spectrophotometrically at time intervals of 3min after addition of each extract. The assay was performed at least in triplicate. Controls were run using PBS in place of the extract. The assay relies on the antioxidant capability of the samples to inhibit the oxidation of ABTS to ABTS•+ radical cation. Percent inhibition was calculated using the following formula and it was compared with ascorbic acid. [6] % inhibition of oxidation of ABTS to ABTS•+ = Initial absorbance - Final absorbance X 100 Initial absorbance Determination of antibacterial activity The antibacterial activity of the crude extracts was screened with gram negative and gram positive cultures viz., Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Streptococcus pyogenes. by agar well diffusion method [7]. About 20 ml of melted Mueller Hinton agar was mixed with 1 ml of bacterial suspension homogeneously and allowed to solidify in petri dishes (143 mm diameter). Wells (8mm diameter) were made using a sterile cork borer on the solidified medium and are filled with 100 µl of the extract (5mg/100µl). The inoculated plates were incubated at 34-38o C. The diameter of the inhibition zones were measured in mm. Each experiment was performed in triplicate and the average value of inhibition and standard deviation was calculated. Gentamicin (30µg) discs are used as positive control and DMSO with solvents (1:10 dilution) used as control. Results were expressed as mean ± SD and the data were analyzed using one-way analysis of variance (ANOVA) to discover the significant difference at the 5% (P<0.05)level.
- 3. Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for… www.ijpsi.org 33 | P a g e Determination of MIC Minimum Inhibitory Concentration [MIC] was determined by broth dilution assay method [8]. Plant extracts were serially diluted in Mueller Hinton broth to get the concentrations of 1.0 to 5.0 mg/100µl. Each experiment was repeated thrice and the mean values were tabulated. III. Results Plants are the important source for the development of new bio active principles, the knowledge of secondary metabolites and their biological properties are desirable, not only for the discovery of novel bioactive entities, but also for disclosing new sources of already known biologically active compounds. Acanthus ilicifolius is a medicinal plant used against asthma, paralysis, snake bite, analgesic and anti inflammatory. The reports of phytochemical studies are given in table 1. From this data it is very clear that tannins and total phenols are there in all the solvents used for the extraction of secondary metabolites. However, flavonoids are seen in all the solvents except benzene. Whereas, alkaloid is confined only to acetone, methanol and ethanol. These secondary metabolites are incredible group of compounds plays an important role in many biological activities. The antibacterial activity of root extracts of Acanthus ilicifolius in benzene, ethylacetate, acetone, methanol and ethanol was revealed by agar well diffusion method and the results are presented in table-2. All the extracts have assorted degrees of antibacterial potential against cultures used. Among the tested extracts benzene solubles of Acanthus ilicifolius exhibited highest potential of antibacterial activity (19.66mm) against Enterobacter cloacae than gentamicin. The ethyl acetate soluble fraction is active against Escherichia coli, Bacillus subtilis and Staphylococcus aureus with same zone size of 16.66 mm. However, metabolites of Acanthus ilicifolius infused into acetone were found to be active on selective test cultures viz., Enterobacter aerogene and Enterobacter cloaceae of gram negative and Staphylococcus aureus of gram positive bacteria. The infusions of ethanol are susceptible to all the test cultures used except Enterobacter aerogenes and the inhibitory effect against test cultures is in increasing order for Enterobacter cloacae (12.66), Escherichia coli (13.33) Streptococcus pyogenes (17.66), Bacillus subtilis (18.33)and Staphylococcus aureus (18.66). Whereas, the efficacy of methanol extracts are sensitive to Enterobacter aerogenes and Escherichia coli with same zone size of 16.66 mm and zone size of 17.33 was observed with Bacillus subtilis and Staphylococcus aureus. The maximum zone of inhibition exerted against gram negative test cultures is Enterobacter cloacae (ethylacetate extract) 19.66 mm and gram positive is Staphylococcus aureus (ethanol extract) 18.66 mm. The zone of inhibition exerted by gentamicin is same for all the gram positive test cultures employed. Whereas, among gram negative Enterobacter aerogenes and Enterobacter cloacae are less sensitive then Escherichia coli to gentamicin. The inhibitory effect of the root extracts of Acanthus ilicifolius in ethyl acetate and ethanol against the test cultures is inferior than gentamicin. However the inhibitory outcome of acetone extracts were superior than gentamicin against the two gram negative cultures used viz., Enterobacter cloacae and Escherichia coli. Whereas, the infusions of methanol having higher inhibitory potential than gentamicin against Enterobacter aerogenes and Enterobacter cloacae. We also studied the combination of different extracts and the results are displayed in table-3. The effect of ethyl acetate and acetone extract mixture have mixed result it has antagonistic effect on Bacillus subtilis and symbiotic effect on Escherichia coli. The mixture of ethyl acetate and methanol have not net role on gram negative cultures used but have negative effect on the gram positive cultures employed in terms of inhibition zone size. Ethyl acetate and ethanol mixture did not inhibit the Enterobacter aerogenes, Enterobacter cloacae and Streptococcus pyogenes. But the zone of inhibition against gram positive test organism viz., on Bacillus subtilis and Staphylococcus aureus is very high compare to that of the individual extracts and the phenomenon is called symbiotic effect. The combination of acetone and methanol extracts is the best as they control the growth of all the test organisms employed in our study. Acetone and ethanol mixture has shows antagonistic effect with respect to inhibition. Methanol and ethanol mixture have antagonistic effect on Enterobacter cloacae and Staphylococcus aureus. Further, these extracts were analyzed to determine the minimum inhibitory concentration against both Gram negative and Gram positive bacterial species. The MIC values ranged from 1.0-5.0 mg/100µl and varied from extract to extract and the results are given in table-3. The MIC of root extracts in benzene is 2.0 mg/100µl against Enterobacter cloacae. Ethyl acetate extracts were active at 3.0 mg/100µl with Escherichia coli and 2.0 mg/100µl with Bacillus subtilis and Staphylococcus aureus . Whereas, the MIC of acetone extract is 2.0 mg/100µl towards all the tested microorganisms. The methanol extracts of Acanthus ilicifolius root exhibiting diversified range of MIC among the tested cultures between 1.0 - 4.0 mg/100µl. Whereas, the MIC of ethanol extract is 5.0 mg/100µl against gram negative test cultures and 3.0 mg/100µl with gram positive microorganisms used. Several techniques have been used to determine the antioxidant activity, free radicals are known to play a definite role in a wide variety of pathological symptoms. Antioxidants fight against free radicals and protect us from various diseases. The non enzymatic antioxidant activity of plants is attributed to the secondary
- 4. Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for… www.ijpsi.org 34 | P a g e metabolites. The antioxidant activity of the fractions was estimated spectrophotmetrically by ABTS method and the data is given in Fig-1. The outcome of antioxidant activity by ABTS method is high for acetone extract followed by ethanol, methanol ethyl acetate and benzene extracts. IV. Discussion In this study, we have qualitatively screened, specifically for the presence of alkaloids, flavonoids, tannin and total phenols as well as the antioxidant and antibacterial potentials of Acanthus ilicifolius root infusions in benzene, ethyl acetate, acetone, methanol and ethanol in order of increasing polarity. Alkaloids, flavonoids, tannin and total phenols are associated with potent biological activities. Our results show that the secondary metabolites present in the root extracts of Acanthus ilicifolius are diversified in the solvents used. Acetone, methanol and ethanol fractions contain all the secondary metabolites checked. Our observation are in accordance with the report of Asha et.al, they extracted flavnoids and phenolics from the root extracts of Acanthus ilicifolius in ethanol [9 ] . The efficacy of antibacterial action is high for ethanol extract and it correlates with the presence of all the secondary metabolites studied. The present experimental data is in accordance with that of Govindasamy et.al who studied the chemical constituents of different mangroves in India [10] . The secondary metabolites viz., alkaloids, flavonoids tannin and total phenols play an important role in scavenging of free radical and are also known to be associated with other biological activities such as antibacterial, antifungal, antidiuretic activities [11,12] . The root extracts of in ethyl acetate, methanol and ethanol are active against Escherichia coli Bacillus subtilis and Staphylococcus aureus these results are in disagreement with reports of dev avijit et.al[13] . They studied the stem extract of Acanthus ilicifolius in methanol by maceration and continuous extraction method. The difference of the result may be due to the plant part used or the protocol employed for the extraction. The extracts in ethyl acetate were active against Escherichia coli, Bacillus subtilis and Staphylococcus aureus with same zone size of 16.66 mm. This may be the compounds present in the extract may have the similar effect on these organisms. The activity of ethanol extracts on all the bacterial cultures except Enterobacter aerogenes confers their broad spectrum nature over the other extracts. The zone of inhibition exhibited by the acetone extracts were higher towards gram negative organisms than gram positive organisms and the ethanol extracts were more active on gram positive than gram negative cultures. This difference in the rate of inhibition potential appear to be directly related to the qualitative diversity of the compounds that are present in the extracts. This may be due to the permeability factor of cell membrane of the microorganism, or this could be due to variation in the cell wall composition of Gram negative and Gram positive bacteria[14] . The Gram negative bacteria restrict the influx of many antibiotics. Multi drug efflux pumps at the trans-membrane are also responsible for a higher intrinsic resistance in Gram negative bacteria. On correlating our results of secondary metabolites and antibacterial activities it is inferred that alkaloids, flavonoids, glycosides tannins and total phenol possess substantial antibacterial activity. Our results are in concurrence with the work of Bose and Bose [15] , with reference to the solvent system and type of microorganisms, i.e., gram positive cultures are more sensitive than gram negative cultures used. The more effectiveness of root extracts of Acanthus ilicifolius in methanol and ethanol than that of the acetone, benzene and ethyl acetate in our study can be correlated with the medicinal preparation that use rum and liquour to extract the active plant components. Benzene extracts did not inhibit Enterobacter aerogenes, Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Streptococcus pyogenes. Similarly ethyl acetate infusions also did not inhibit Enterobacter aerogenes, Enterobacter cloacae, and Streptococcus pyogenes. Negative results did not mean the absence of bioactive principles in these extracts. The bioactive principle may be insufficient to cross the membrane, or the microorganism may have mechanism to nullify the effect of the bioactive principle present in these extracts. In our study, the MIC value for all the positive extracts against the tested bacteria were between 1.0mg/100μl to 5mg/100μl. Gram negative test cultures showed higher MIC values than Gram positive text cultures to the extracts in methanol. This difference may be explained by susceptibility testing condition, physico chemical characters of the bioactive principle present in the extract and even strain to strain difference. In comparison to some of the earlier reports on MIC values of pure compounds, our MIC may be higher [16] . But this can be substantiated by the argument that this value is for the crude extract. However, the purified form of bioactive compound of the crude extract responsible for antibacterial activity may exhibit the inhibitory effect at a lower concentration. Fai-Chu Wong[17] studied the MIC on S. aureus, M.luteus, E.coli and P.aeruginosa with ampicillin and selected medicinal plant extracts and reported the MIC value of Amphicillin is between 0.02 - 1mg/1000µl and that of the plant extracts are in the range of 6.3 – 50 mg/1000µl. Our results are far superior compare to that of the plant extracts but inferior to that of the standard antibiotic.
- 5. Extraction of Secondary Metabolites from Roots of Acanthus Ilicifolius L and Screening for… www.ijpsi.org 35 | P a g e Hence Acanthus ilicifolius is strongly recommended for considering a valuable source for isolation, identification and characterization of bioactive principle responsible for antibacterial activity on various bacteria and fungi. Also there is a need to study the different plant parts of Acanthus ilicifolius in other organic solvents apart from the solvents employed in our study. However, further work in this direction could lead to the discovery of powerful bioactive principles from the Acanthus ilicifolius Table-1 Secondary metabolites of root extracts of Acanthus ilicifolius + = Present - = Absent Table-2 Antibacterial activity of Acanthus ilicifolius root extracts Benzene Ethylacetate Acetone Methanol Ethanol Gentamicin Enterobacter aerogenes - - - 16.66±0.57 - 15.33±0.57 Enterobacter cloacae 19.66±0.57 - 17.33±0.57 - 12.66±1.52 15.33±0.57 Escherichia coli - 16.66±0.57 16.66±0.57 16.66±0.57 13.33±0.57 17.33±0.57 Bacillus subtilis - 16.66±1.15 - 17.33±0.57 18.33±1.15 18.66±0.57 Staphylococcus aureus - 16.66±1.52 14.33±0.57 17.33±0.57 18.66±0.57 18.66±0.57 Streptococcus pyogenes - - - - 17.66±1.52 18.66±0.57 Table-3 Antibacterial activity of Acanthus ilicifolius root extract Mixture Table-4 MIC of Acanthus ilicifolius root extracts (mg/100µl) Fig-1. Antioxidant activity of Acanthus ilicifolius by ABTS method. Alkaloid Flavonoid Tannin Total Phenols Benzene - - + + Ethyl acetate - + + + Acetone + + + + Methanol + + + + Ethanol + + + +
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