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Isolation, partial purification and evaluation of bioactive compounds from

leaves of Ageratum houstonianum

Article in EXCLI Journal · November 2011

DOI: 10.17877/DE290R-4911

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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

Original article:

ISOLATION, PARTIAL PURIFICATION AND EVALUATION OF

BIOACTIVE COMPOUNDS FROM LEAVES OF
AGERATUM HOUSTONIANUM
M. Zeeshan1*, S.M.D. Rizvi1, M.S. Khan1, A. Kumar2
1
Department of Biotechnology and Microbiology, Integral University, Lucknow-226 026,
India
2
Advanced Instrumentation Facility, University Science Instrumentation Centre, JNU, New
Delhi-1100 67, India
* corresponding author: e-mail: [email protected]

ABSTRACT
The present study deals with the isolation and partial purification of bioactive compounds
from the crude methanol extracts of the leaves of Ageratum houstonianum (Asteraceae). The
quantification and the identification of compounds in the crude extract and active bands iso-
lated by preparative TLC were accomplished using GC-MS analysis. The most important
compounds identified in the crude extract and active bands (AB-1 and AB-2) were 6-acetyl-7-
methoxy-2, 2-dimethylchromene, hexadecanoic acid and squalene, respectively. Crude extract
and active bands (AB-1 and AB-2) were investigated for their antibacterial activity against
Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Esche-
richia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and Pseudomonas aeruginosa.
The crude extract, AB-1 and AB-2 showed maximum zone of inhibition (10-13 mm) against
Staphylococcus epidermidis, however, the antibacterial potential of active bands was slightly
higher as compared to the crude extract. Dose-dependant increase in antioxidant potential was
noticed in crude extract as well as with both active bands measured by DPPH free radicals,
ion chelation and total antioxidants capacity. Our study reports various bioactive compounds
in the leaves of the A. houstonianum with significant antioxidant and antibacterial potential.

Keywords: Ageratum sp., antibacterial, antioxidants, bioactive compounds, GC-MS

INTRODUCTION large number of plants about 25,000 spe-

cies belonging to the family Asteraceae are
The increase in bacterial resistance
rich in secondary metabolites with biologi-
against the present drugs used in traditional
cal activity (Okunade, 2002). Various stud-
therapy caused the emergence of new anti-
ies have been carried out on some of
bacterial entities that can be explored from
a high number of natural sources such as Asteraceae plants, e.g. Achillea sp., Eupa-
torium sp., Tridax sp., Vernonia sp. and
plant materials to fight microbial diseases
Ageratum sp. The pharmacological proper-
(Eloff, 1998; Chariandy et al., 1999). The
ties, such as anti-inflammatory, antioxi-
functional properties – such as antibacteri-
dant, antispasmodic and anti-hemorrhoidal
al, antifungal and insecticidal – of essential
of the aerial parts of different species of
oils and plant extracts are widely used in
the genus Achillea have been reported. The
processed food preservation, pharmaceuti-
extracts of leaves of Eupatorium sp. pos-
cals, cosmetics, alternative medicine and
sess both antibacterial and antimalarial ac-
natural therapies (Bakkali et al., 2008). A

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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

tivity. Tridax sp. is commonly used in In- semivolatile compounds (Iordache et al.,
dian traditional medicine as anticoagulant, 2009). Therefore, the authors have at-
hair tonic, insect repellent, anti-diarrhea, tempted to isolate the bioactive com-
and anti-dysentery (Candan et al., 2003; pounds, evaluate the bioactive potential
Thakong, 1999; Saraf et al., 1991; Abuba- and characterize them by GC-MS analysis.
kar et al., 2011; Kasturi et al., 1973). Tra-
ditionally, Ageratum sp. is used as antidys- MATERIALS AND METHODS
enteric, in skin and wound diseases, in the
Plant material
treatment of leprosy and purulent ophthal- Plants of A. houstonianum, growing
mia. They are also used in treatment of wild in Pharmacy Herbal Garden, Integral
pneumonia by rubbing them on the chest of University, Lucknow, were collected in
the patient (Shirwaikar et al., 2003a; January, 2010. The authenticity of the
Begum and Nath, 2000). plant was confirmed by Dr. Tariq Husain,
Oxidative stress results due to the im- Senior Scientist, National Botanical Re-
balance of formation and neutralization of search Institute (NBRI), Lucknow, India
prooxidants, which is one of the major and voucher specimens are maintained at
cause of today's diseases such as cancer, the NBRI herbarium.
atherosclerosis, cardiovascular diseases,
ageing and inflammatory diseases (Braca et
Preparation of plant extracts
al., 2002; Maxwell, 1995). The defense Dried leaf powder (1 gm) was homog-
mechanism that includes enzymes such as enized in 10 ml methanol (HPLC grade,
superoxide dismutase (SOD) and catalase, Merck, India) and was extracted on a rota-
or compounds such as ascorbic acid, to- tory shaker in an Erlenmeyer flask at
copherol and glutathione are sometimes 40 rpm overnight (Daayf et al., 1997). The
fail to protect cell components from oxida- crude extracts were then filtered through
tive damage (Niki et al., 1994). Therefore, Whatman No. 1 filter paper and concen-
the supplementation of antioxidants are trated in vacuum at 40 °C using a rotary
vital to combat oxidative damage. In this evaporator. The concentrated extract was
direction much attention has been given in then dried aseptically with the help of dri-
search of ethnomedicines with strong anti- er.
oxidant properties (Bibhabasu et al., 2008).
The antioxidant potential of Vitex negundo
Isolation of the bioactive molecule
has been reported by employing various The methanol extract thus obtained was
established in vitro systems and the pres- redissolved in 1 ml methanol. For the TLC
ence of many polyphenolic compounds,
analysis, 25 µl of the extract was loaded on
terpenoids, glycosidic iridoids and alka-
the TLC (Silica gel 60) plates (Merck,
loids (Prakash et al., 2007).
Germany). The solvent ratio used for the
A large number of constituents has
separation of the compounds was Chloro-
been identified by GC-MS analysis of the
form: methanol (4:1, v:v). UV-trans-
essential oil of A. conyzoides and other
illumination of the plates at 365 nm re-
species also but pharmacological activities
vealed seven bands. These bands were
and chemical characterization of the most
eluted separately with a minimum amount
significant metabolites from A. houstonia-
of methanol. All seven bands were bioas-
num responsible for the medicinal proper-
sayed for antibacterial potential against
ties have not been done (Ekundayo et al.,
Staphylococcus epidermidis (NCIM 2493)
1998). In recent years the interest for the
(National Chemical Laboratory, Pune, In-
characterization of organic compounds
dia) by disc diffusion method on Mueller-
from plants has been developed. The GC–
Hinton (MH) agar (Bauer et al., 1966).
MS is an ideal technique for qualitative
Further preperative TLC was used to
and quantitative analysis of volatile and
pooled-up the amount of active bands but

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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

band AB-1 and AB-2 were selected for fur- sa (NCIM 5029) were used as test bacteria
ther studies. Active bands (AB-1 and AB- in the present study. For antibacterial test-
2) were scrapped off from the silica gel ing fresh inoculum was prepared for each
and eluted in methanol (HPLC grade, bacterium and incubated at 37 ± 2 °C for
Merck, India) followed by centrifugation at 24 h. The inoculum was adjusted with nu-
10,000 rpm. Clear supernatant was then trient broth to obtain turbidity comparable
dried aseptically and subjected to antibac- to that of MacFarland 0.5 standard
terial and antioxidant assays. GCMS anal- (108 CFU/ml) according to NCCLS
ysis was done to characterize these active (NCCLS, 1997). The cell suspension in
bands. each case was swabbed onto MH agar with
the help of a sterile cotton swab and incu-
GC-MS analysis bated at 37 ± 2 °C for 20 minutes. Thereaf-
For the identification of metabolites ter, the Whatman No. 1 filter paper discs
showing antibacterial and antioxidant po- impregnated with 10 μL of each extract
tentials, the samples were subjected to GC- either crude or active bands (AB-1 and
MS analysis. The sample (1 μl) was inject- AB-2) prepared in 50 % DMSO
ed into a RTX-5 column (60 m X 0.25 mm (40 mg/ml), was placed on the plates
i.d., film thickness 0.25 µm) of GC-MS (DMSO was used as a control) and incu-
(model GC-MS-QP-2010 plus, Shimadzu bated at 37 ± 2 °C for 18 h. All of the ex-
Make). Helium was used as carrier gas at a periments were performed in triplicate and
constant column flow 1.2 ml/min at the results (millimeters of the inhibition
173 kpa inlet pressure. Temperature pro- zone) were expressed as mean values.
gramming was maintained from 10 0°C to
200 °C with constant rise of 5 °C/min and Determination of free radical scavenging
then held isothermal at 200 °C for 6 min; activity
further the temperature was increased by The free radical scavenging activity of
10 °C/min up to 290 °C and again held iso- methanol crude extract and active bands
thermal at 290 °C for 10 min. The injector (AB-1 and AB-2) was measured by using
and ion source temperatures were 270 °C DPPH radical (Williams et al., 1995; Mil-
and 250 °C, respectively. The crude and iauskas et al., 2004). DPPH radicals have
active bands (AB-1) and (AB-2) (2 mg/ml) strong absorption maximum at 515 nm
dissolve in methanol (HPLC grade, Merck, which decreases due to the reduction by
India) were injected with a split ratio of antioxidants. The DPPH solution in meth-
1:10. Mass spectra were taken at 70 eV; a anol (6 × 10-5 M) was prepared freshly, and
scan interval of 0.5 s and fragments from 3 mL of this solution was mixed with
40 to 950 Dalton. The final confirmation of 100 μL of various concentration (0-
constituents was made by computer match- 0.5 mg/ml) of crude extract or TLC-eluted
ing of the mass spectra of peaks with the compounds. The samples were incubated
Wiley and National Institute Standard and for 20 min at 37 °C in a water bath, and
Technology (NIST) libraries mass spectral then the decrease in absorbance at 515 nm
database. was measured (Ae). A blank sample con-
taining 100 μL of methanol in the DPPH
Antibacterial assay solution was prepared and its absorbance
Staphylococcus epidermidis (NCIM was measured (Ab). All tests and analyses
2493), Staphylococcus aureus (NCIM were run in triplicates and the results ob-
2079), Bacillus subtilis (NCIM 2920), Ba- tained were averaged.
cillus cereus (NCIM 2156), Escherichia Radical scavenging activity was calcu-
coli (NCIM 2065), Enterobacter aerogenes lated using the following formula:
(NCIM 5139), Klebsiella pneumoniae % inhibition = [(Ab-Ae)/Ab] × 100
(NCIM 2957), and Pseudomonas aerugino-

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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

where Ab = absorbance of the blank compounds isolated by preparative TLC

sample, and Ae = absorbance of the metha- from the leaves of Ageratum sp. and their
nol extract. characterization by GC-MS analysis. The
methanol crude extract of leaves of Agera-
Total antioxidant activity tum houstonianum showed antibacterial
Spectrophotometric method was used activity against Staphylococcus epidermid-
for the determination of total antioxidant is, Staphylococcus aureus, Bacillus sub-
activity (Shirwaikar et al., 2003b). Various tilis, Bacillus cereus, and Enterobacter
concentrations (0–8 µg/ml) of the crude aerogenes whereas, no activity was noticed
extracts and active bands (AB-1 and AB-2) against Escherichia coli, Klebsiella pneu-
were prepared separately in methanol. The moniae, and Pseudomonas aeruginosa
reaction mixture contained 1 ml of the ex- (Table 1). The disc (400 µg) of crude ex-
tract and 2 ml of reagent solution (0.6 M tract exhibited maximum zone of inhibition
sulphuric acid, 28 mM sodium phosphate (10 mm) against S. epidermidis followed
and 4 mM ammonium molybdate). The by B. cereus (9 mm), E. aerogenes (9 mm),
mixture was incubated on water bath at B. subtilis (8 mm) and S. aureus (8 mm).
95 °C for 90 min and after cooling at room Based on these results methanolic crude
temperature, the absorbance of reaction extract of leaves was used to detect the bi-
mixture was measured at 695 nm against oactive compounds through TLC. The
blank. Ascorbic acid was used as the TLC results indicate the presence of seven
standard and total antioxidant capacity was bands under ultra violet light (365 nm) il-
expressed in terms of ascorbic acid equiva- lumination (Figure 1). Out of these, two
lents. All tests were run in triplicates and active bands (AB-1) and (AB-2) with Rf
averaged. value – 0.20 and 0.48, respectively were
selected to evaluate the bioactive potential
Ion chelating activity based on their good antibacterial activity
Ion chelating activity was evaluated by against S. epidermidis (data not shown).
the standard colorimetric method (Benzie Further, through preparative TLC the com-
and Szeto, 1999). The reaction mixture pounds of both active bands were pooled
contained 0.5 ml phenanthroline (0.05 %) separately and subjected to antibacterial,
in methanol, 1 ml ferric chloride (200 μM) antioxidant and G-MS analysis. The yields
and 1 ml different concentrations (0- of compounds were 25 µg/mg and 62.5
8 µg/ml) of the test compound. The reac- µg/mg of crude extract for AB-1 and AB-
tion mixture was further incubated at room 2, respectively. The GC-MS analysis of
temperature for 10 min and the absorbance crude and active band as shown in Figures
of the sample was read at 510 nm. Ascor- 2-4 indicates the presence of different
bic acid was used as the standard and ion components. The major components pre-
chelating activity was expressed as ascor- sent in the methanolic crude extract of
bic acid equivalents. All tests and analyses leaves of A. houstonianum were diazopro-
were run in triplicates and the results ob- gesterone (RT: 20.88), 6-acetyl-7-
tained were averaged. methoxy-2,2-dimethylchromene (RT:
22.30), 9,12-octadecadienoate (RT: 32.18),
Statistical analysis stigmast-5-en-3-ol (RT: 52.15) and 3,7-
Statistical analysis was carried out us- dimethyl-2,6-octadienyl acetate (RT:54.19)
ing the SPSS software (SPSS Inc., version (Table 2). Seven components were identi-
7.0). fied in active band (AB-1), out of which
hexadecanoic acid (RT: 27.94) and 3'-
RESULTS Acetyllycopsamine (RT: 29.40) were the
The present study tried to evaluate the major compounds (Table 3). Six compo-
bioactive potential of partially purified nents were detected in the active band

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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

(AB-2), however, squalene (RT: 31.83)

and phenol (RT: 31.96) were the major
components (Table 4).

Table 1: Antibacterial activity of crude extracts

and active bands (AB-1 and AB-2) isolated by
preparatory TLC

Zone of inhibition (mm)

Bacteria Crude AB-1** AB-2***

extract*
(400 µg/ (400 µg/ (400 µg/
disc) disc) disc) Figure 1: TLC separation of methanolic crude
extract of leaves of A. houstonianum using
S. epidermidis 10 13 13 chloroform:methanol (4:1) as mobile phase,
visualized under UV light 365 nm
S. aureus 8 9 10

B. subtilis 8 n.d. n.d. The results from Table 1 shows the an-
B. cereus 9 9 n.d. tibacterial potential of active bands AB-1
and AB-2 when tested against test bacteria.
E. coli n.d. n.d. n.d.
The disc prepared from active band (AB-1)
E aerogenes 9 11 8 showed maximum inhibition zone (13 mm)
K. pneumoniae n.d. n.d. n.d. against S. epidermidis followed by E. aer-
ogenes (11 mm), S. aureus (9 mm) and B.
P. aeruginosa n.d. n.d. n.d.
cereus (9 mm) while active band AB-2
showed inhibitory zone of 13, 10 and 8
n.d. - not detected at this concentration
* - Ageratum leaf crude extract in methanol mmm against S. epidermidis, S. aureus and
** - AB-1 is (active band with Rf value 0.20) E. aerogenes, respectively.
*** - AB-2 is (active band with Rf value 0.48).

Figure 2: GC-MS chromatogram of methanolic crude extract of A. houstonianum leaves

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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

Table 2: Bioactive compounds identified in the Table 3: Bioactive compounds identified in the
methanolic crude extract of the leaves of A. active band (AB-1) isolated from methanolic
houstonianum by GC-MS crude extract of the leaves of A. houstonianum
by GC-MS
Identified compounds RT (min) Chroma-
togram Identified compounds RT (min) Chroma-
% area togram
% area
2,6-Octadien-1-ol 07.984 0.74
6-Vinyl-7-methoxy-2,2- 21.354 1.50
Phenol (2,4,6- 13.755 0.41 dimethylchromene
Triisopropylphenol)
3-Eicosene 23.807 4.45
Ethanone 14.205 1.05
Hexadecanoic acid 27.948 28.52
Diazoprogesterone 20.885 32.94
2-Butenoic acid 29,.206 1.86
6-Acetyl-7-methoxy-2,2- 22.308 5.26
dimethylchromene 3’-Acetyllycopsamine 29.401 24.65
1,2-Benzenedicarboxylic acid 22.416 0.58 Phosphonic acid 29.687 4.49
3’-Acetyllycopsamine 24.769 0.48 3’-Acetyllycopsamine 30.032 34.53
Hexadecanoic acid 29.166 0.85

3’-Acetyllycopsamine 29.427 1.53

Table 4: Bioactive compounds identified in the
Benzene 29.719 0.34 active band (AB-2) isolated from methanolic
1-Hexadecyne 30.060 1.87
crude extract of the leaves of A. houstonianum
by GC-MS
9,12,15-Octadecatrienoic 30.700 0.52
acid Identified compounds RT (min) Chroma-
togram
Phytol 31.847 0.29 % area
Ethyl-9,12-octadecadienoate 31.977 0.74 1,2-Benzenedicarboxylic acid 30.903 1.23
Ethyl-9,12-octadecadienoate 32.180 4.70 Squalene 31.83 25.63
1H-Purin-6-amine 32.965 1.02 Phenol 31.963 58.64
Eicosane 33.085 2.41 Pentamethyl tetrahydro-5H- 32.161 5.75
chromene
1H-Purin-6-amine 36.142 0.61
Ethanone 32.334 6.94
1,2-Benzenedicarboxylic acid 37.063 1.22
6-Vinyl-7-methoxy-2,2- 33.375 1.80
Squalene 39.241 1.56 dimethylchromene
1-Eicosanol 41.049 2.06

2H-1-Benzopyran-6-ol 43.226 2.56

Stigmasterol 47.729 1.17

Stigmast-5-en-3-ol 52.159 5.46

1,6-Octadien-3-ol 54.008 2.38

3,7-Dimethyl-2,6-octadienyl 54.194 6.59

acetate

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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

Figure 3: GC-MS chromatogram of Active Band (AB-1) isolated from crude A. houstonianum leaves

Figure 4: GC-MS chromatogram of Active Band (AB-2) isolated from crude A. houstonianum leaves

For evaluation of antioxidant properties with the inhibition of 55.8 % at 0.5 mg/ml
of crude extract and TLC eluted compounds while the scavenging activity of active
of Ageratum sp. three assays have been bands AB-1 and AB-2 was lower as 42.3 %
used: DPPH radical scavenging, Total anti- and 39.05 % inhibition was noticed at
oxidant and Ion chelation assays and the 0.5 mg/ml, respectively. The results of total
results are shown in Figures 5, 6 and 7. antioxidant capacities of crude and partially
DPPH was reduced with the addition of purifed compounds based on the formation
crude and active bands in a concentration of phosphomolybdenum complex are
dependent manner (Figure 5). Crude extract shown in Figure 6. In this assay, crude ex-
was the most effective radical scavenger tract possessed more antioxidant potential
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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

with 9.4 µg equivalent of ascorbic acid at

8 µg/ml concentration than the partially pu-
rified compounds which showed only 1 and
2.5 µg equivalent of ascorbic acid at
8 µg/ml concentration. The ion chelation
activity was studied by interaction of crude
or partially purifed compounds with fer-
rous-o-phenanthroline complex (Figure 7).
Similar to this the crude extract has more
ion chelation capacity than the active bands.

Figure 7: Ion chelation activity of crude extract

and (AB-1 and AB-2) bands isolated through
TLC from leaves of A. houstonianum
*expressed in terms of ascorbic acid equivalent
(µg/ml). Values are mean ± SE of three
replicates

DISCUSSION
The present study was aimed to explore
the potential bioactive substances from
plant origin. Methanol is a polar solvent
Figure 5: Effects of crude extract and active that has ability to extracts most of the bio-
bands (AB-1 and AB-2) isolated through TLC
active compounds such as flavonoids, tan-
from leaves of A. houstonianum on the
scavenging of DPPH. Values are mean ± SE of nins and others (Niño et al., 2006). Recent-
three replicates ly, strong antibacterial and antifungal ac-
tivity of methanolic extracts of bark of Sar-
aca Indica has been reported (Sainath et al.,
2009).
The antibacterial results showed that
both crude extract and active bands (AB-1
and AB-2) were relatively more effective
against the Gram-positive bacteria as com-
pared to the Gram-negative bacteria. These
results are in agreement with the earlier studies
done with other plant species of Asteraceae
family (Boukamcha et al., 2004; Oueslati et
al., 2004). Similar pattern of antibacterial
sensitivity was also reported with the ex-
Figure 6: Total antioxidant activity of crude tract of plant Rhaponticum acaule DC
extract and active bands (AB-1 and AB-2) (Boussaada et al., 2008). The higher re-
isolated through TLC from leaves of A. sistance of the Gram-negative bacteria
houstonianum *expressed in terms of ascorbic
acid equivalent (µg/ml). Values are mean ± SE
could be due to the complexity of the cell
of three replicates wall of this group of microorganisms. In-
deed, the external membrane of Gram-
negative bacteria renders highly hydrophilic
surfaces whereas the negative charge of the
surface of the Gram-positive cell wall may
reduce their resistance to antibacterial com-
pounds (Smith-Palmer et al., 1998; Ultee et

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 EXCLI Journal 2012;11:78-88 – ISSN 1611-2156
Received: December 18, 2011, accepted: February 23, 2012, published: February 28, 2012

al., 1999). This relative difference in the cidal and fungicidal activities (Cowan,
potential may be due to presence of several 1999).
hydrophobic compounds in the crude ex- The presence of wide range of phyto-
tract, AB-1 and AB-2 of A. houstonianum chemical constituents as observed in the
leaves. partially purified bands indicates that fur-
Further, concentration dependent effect ther studies are required to pinpoint the role
of methanol crude extract was found to of various minor and major compounds in
have more antioxidant activity than active the antimicrobial and other biological activ-
bands (AB-1 and AB-2). The antioxidant ities. Thus, the present study reflects a hope
potential of the crude extract and active for the development of novel agents of bio-
bands (AB-1 and AB-2) may be attributed medical importance.
to differences in their chemical composition
such as phenolic compounds, polyphenyl ACKNOWLEDGEMENTS
compounds like squalene, hexadecanoic The authors are thankful to the Vice
acid and others (Kelly, 1999; Cowan, Chancellor, Integral University, Lucknow,
1999). India for providing necessary facilities for
Our GC-MS analysis of the crude ex- the research work. We also thank to Mr.
tract and AB-1 and AB-2 showed presence Ajai Kumar, Advanced Instrumentation Fa-
of certain metabolites (Tables 2-4). The an- cility, University Science Instrumentation
tibacterial activity of the crude extract Centre, JNU, New Delhi, for the GC-MS
might be due to the presence of 6-acetyl-7- analysis of the sample.
methoxy-2,2-dimethylchromene. The pre-
sent work also corroborates the earlier work
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