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International Journal of Biological & Medical Research
Int J Biol Med Res. 2011; 2(4): 1038 – 1042
Effect of Bioactive Compounds and its Pharmaceutical Activities of Sida cordifolia
(Linn.)
A R T I C L E I N F O A B S T R A C T
Keywords:
Original Article
Pathogenic bacteria
Sida cordifolia
Cytotoxicity
Antibacterial activity
1. Introduction
Plants are used medicinally in different countries and are a source of many potent and
powerful drugs. The general objective of this study shows to identify the bioactive compounds
from the Sida cordifolia plant and to study the antimicrobial, cytotoxic effect on HeLa cell lines.
The GCMS analysis results showed mainly four different compounds such as Vasicinol,
Ephedrine, Vasicinone and Hypaphorine in various criteria like retention time and peak
observation among the four phytochemical compounds based upon in vivo study. The agar disk
diffusion method was used to study the antibacterial activity of S. cordifolia extracts against 7
bacterial strains. The Minimum Inhibitory Concentration (MIC) of the plant extracts were
tested using two fold agar dilution method at concentrations ranging from 6 to 18 μm/ml. The
cytotoxicity results of S. cordifolia (L.) extract on HeLa cell lines clearly reflected the treated
cell lines are fortunately all the uncontrolled growth has been arrested there is declined level of
cancerous cells observed. The methanol extract was found to be an effective against all
phytopathogens with low MIC of 6μm/mm and the methanol extract exhibited a higher
inhibition activity against Escherichia coli, Bacillus subtilis, Enterobacter aerogenes,
Mycob acter ium sp., a nd M icro coccu s va rianc e, Pseudo monas aeru ginosa and
B. subtilis. Meanwhile, the results also indicate the presence of major phytochemical
compounds such as vasicinol, Ephedrine, vasicinone, Hypaphorine in the S. cordifolia extracts.
Hence, the isolation and purification of therapeutic potential compounds from S.
cordifolia could be used as an effective source against bacterial diseases in human and plants.
Sida cordifolia Linn. (Family: Malvaceae) commonly known as
berela (Bengali) is herb that is extensively used as a common herbal
drug in the Indian subcontinent. The water extract of the leaves was
reported to possess analgesic and anti-inflammatory activities in
animal models (Gunatilaka et al., 1980). It is used in Ayurvedic
medicine. (Pole et al 2006). It has been investigated as an anti-
inflammatory (Franzotti et al., 2000) for treating cancer (Jenny et al
2005) and for encouraging liver re-growth. (Silva et al 2006).
Medicinal plants represent a rich source of antimicrobial agents.
Plants are used medicinally in different countries and are a source
of many potent and powerful drugs. Due to its ephedrine content, it
possesses psychostimulant properties, affecting the central
nervous system and also the heart (Adam et al 2006). It is also used
as a fat-burning supplement. It is used in Ayurvedic medicine
(Sebastian, 2006), it has been investigated as an anti-inflammatory
(Franzotti et al., 2000) for treating cancer (Jenny, et al., 2005) and
for antibacterial growth Isman et al. (2003; Silva et al., 2006). It has
a depressive effect on the central nervous system (Franco et al.,
2005). Moreover, previous phytochemical studies on the roots had
shown the presence of ephedrine, vasicinol, asicinone and N-
methyl tryptophan (Franzotti, 2004). Recent analyses have
revealed that ephedrine and pseudoephedrine constitute the
major alkaloids from the aerial parts of the plant, which also show
traces of sitosterol and palmitic, stearic and hexacosanoic acids.
From seed oil sterculic, malvalic and coronaric acids are isolated
along with other fatty acids.
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International Journal of
BIOLOGICAL AND MEDICAL RESEARCH
www.biomedscidirect.com
Int J Biol Med Res
Volume 2, Issue 4, Oct 2011
Baby Joseph, A.U.Ajisha, Satheesna Kumari and S.Sujatha*
International Centre for Bioresources Management Malankara Catholic College, Mariagiri-629513.
* Corresponding Author : Sujatha
International Centre for Bioresources Management
Malankara Catholic College, Mariagiri-629513
E mail : sujatharbs@rediffmail.com
Copyright 2010 BioMedSciDirect Publications. All rights reserved.
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2. Materials and Methods
2.1.Collection and identification of Experimental plants
2.2.Compound study of crude extract using GCMS
1039
Baby Joseph et.al / Int J Biol Med Res. 2011; 2(4): 1038 – 1042
Very recently, Swathy et al., 2010 studied that 50% ethanolic
extract of Sida cordifolia has got potent antioxidant and
antiinflammatory activity and the activity was comparable with
the standard drug diphenyl. Since, there no more works has been
available this kind of medicinal plants. Hence this research works
having designed the following objectives. To identify the
responsible secondary metabolites for antibacterial activity of
best active plant by performing GCMS analysis. In addition to study
the cytotoxic effect of S. cordifolia on HeLa Cells.
The whole plants of Sida Cordifolia were collected from
Parassala situated in and around the college campus (Malankara
Catholic College) Trivandrum District, Kerala. The selected plant
was identified by the Herbarium of TBGRI (Tropical Botanical
Garden Research Institute, Palode and Trivandrum) and the flora
of the presidency of Madras.
The crude plant extracts were subjected to centrifugation at
about 10,000 rpm for about 30minutes to remove the particulates.
The clear supernatant was aspirated using a pipette and
transferred into a clean vial and labeled. Then the supernatants
were subjected to gas chromatography analysis using a Varian Cp
3,800 model gas chromatography equipped with two flame
ionization detectors and connected with two flame ionization
detectors and connected with two flame ionization detectors and
connected with Cp-ware (Polyethylene glycol) (60m x0.25nm) and
Cp-5 (100% dimethyl polysiloxane) capillary column.
The peak area calculations were done by star work station and
peak identification by comparison with authentic, wherever
available calculations of Kovats Retention index was done. The
Kovats index system has been widely used in the analysis of food
flavors, pesticides and essential oil analysis. Kovats retention
index, (1) is defined and calculated by following equation
(Douglas, 2000).
Mass spectrometry analysis was performed on a Shimadzu GC
17 A QP 5,000 MS coupled with a mass detector, fitted non-polar
DB-5 (Diphenyl siloxane) capillary column of length 25 m × 0.25
mm id GC MS operation conditions at initial temperature 60 0 C –
3000C. The injection volume was 0.1μl with helium gas as carrier
at the flow rate of 0.6ml per minute. Relative retention times
(RRts) of constituents were determined using c5-c30 straight
chain alkanes as standards. Individual constituents of the extract
were identified by WILEY and NIST database matching by
comparison of mass spectra with published data and by
comparison of Mass spectra with published data and by their
comparison of their RRts.
1= 100 N + 100 log'R (N+n) log'R(N).
Where
t'R (N) = adjusted retention time of n paraffin hydrocarbon of
carbon number eluting before solute A.
T'R (N+n) = adjusted retention time of n paraffin hydrocarbon of
carbon number (N+n) eluting after solute A.
T'R (A) = adjusted retention time of solute-A.
2.3.Statistical analysis
3.Results and Discussion
3.1.Bioactive compounds in S. cordifolia leaves by GCMS
analysis
Table 1. GCMS analysis depicted important phytochemical
compounds present in the Sida cordifolia leaves
Fig. 1. GCMS Analysis of Natural Callus Extract of Sida Cordifolia
From the figure 1 shows two peak higher activity shows the above
said compounds such as vasicinol and Ephedrine. Suddenly
declined level of secondary metabolic compound observed
vasicinone and Hypaphorine of these four compounds maximum
well processed bioactive compound is vasicinol. The peak could be
identified as Vasicine, when compared with the standard structure
and properties were identified with a help of this web address
(www.ncbi.nlm.nib).
Gcms Analysis
The results were analyzed for statistical significance using one-
way analysis of variance (ANOVA) followed by Dunnet's test.
Values with p<0.05 were considered significant.
GCMS analysis results showed mainly four different compounds
such as Vasicinol, Ephedrine, Vasicinone, Hypaphorine in various
criteria like retention time and peak observation among the four
phytochemical c ompounds based u pon in vivo type of
experimental method expressed maximum retention time 12.8
denoted as Hypaphorine followed by 11.29, 7.30, 5.62 revealed
biochemical compounds such as vasicinol, Ephedrine, vasicinone
respectively. Though according to area observation the highest
level noticed on 68.7 represent bioactive compound was
Ephedrine and minimum as well as similar area observed in
Hypaphorine, vasicinol. In case of in vitro treated plant possessed
max retention time expressed the same in vivo case Hypaphorine
and minimum vasicinol 3.73. Furthermore, the area of the peak
noticed 72.14 represented Ephedrine compounds also minimum
area of the peak obtained on Hypaphorine compounds (Table-1).
In vivo In vitro
Compounds Retention time Area AreaRetention time
Vasicinol
Ephedrine
Vasicinone
Hypaphorine
5.62
7.30
11.29
12.28
20.99
68.27
3.97
3.97
3.73
7.06
11.11
12.96
24.90
72.14
1.98
0.98
3.4. Lactose dehydrogenase cytotoxicity detection assay
Concentration
(µg/ ml)
Blank
3.3.Trypan blue cell viability assay
Table 3. LDH Cytotoxicity detection assay of S. cordifolia
5000.00 10000.00 20000.00
1040
When compared with control and treated with S. cordifolia
extracts on the HeLa cells this extract applied with cancer cells
visibly observed the following changes initially the cells are
scattered with abnormal proliferation and the cells were
elongated number of the cell divisions range was decreased when
compared with control. Meanwhile, the results of S. cordifolia
extracts cytotoxicity on HeLa cell lines are shown in Figure 2a and
b. It represent the cytotoxicity results of S. cordifolia (L.) extract
on HeLa cell lines clearly reflected the treated cell lines are
fortunately all the uncontrolled growth has been arrested there is
declined level of cancerous cells observed. But incase of control
cell lines explained clumped aggregated cells were spreaded
throughout the HeLa cell lines. The external morphological
changes observed using the thymidine fluorescence assay showed
slight to higher alterations in cellular components after 96hrs post
treatment. Though, a treated cell shows the more prominent
growth inhibition and shrinkage of the cells takes place.
Furthermore, the inhibitory effect of on HeLa cell lines was mainly
influenced with dose-dependent the difference was remarkable
starting from 150µgm/mL.
Lactose dehydrogenase Cytotoxicity detection was observed by
the absorbancy of cell line culture. The obserbency was higher in
control (0.78) the cell used at different levels such as 5000, 10000,
20000 cells/ml. In addition, this table revealed when the lowest
concentration of cells through the LDH cytotoxicity assay
possessed the more or less similar absorbance values. Though,
other two cell concentrations (5000.00 and 20000.00) were
remarkably stands for the irregular manner of absorbance value
hence, most probably that was very less abnormality (0.18) 20000
cells/ml was showed higher abnormality (0.39). However, among
the three different concentrations of cells treated maximum
average of cells noted on 20000.00 followed by 10000 and 5000
cells /1ml (Table 3).
Thus, the results of the previous study demonstrated the
compounds like (5-Hydroxymethyl-1-(1, 2,3, 9-tetrahydro-
pyrrolo [2, 1-b] quinazolin-1-yl)-heptan-1- one) has significant
analgesic and anti-inflammatory activities. However a more
extensive study is necessary to determine exact mechanism(s) of
action.
The acetic acid induced writhing test is normally used to
evaluate the peripheral analgesic effect of drugs. The response is
thought to be mediated by peritoneal mast cells acid sensing ion
channels (Voilley, 2004) and the anticancerous activity (Asha and
The vasicine compound was more in vitro plant extracts
compare with the natural extracts. Its molecular formula was
C11H12N2O. Another one peak observed in GCMS chromatogram
at 7.06 retention time in cs1 (in vitro extracts) but the same peak
was observed at 7.3 retention time in natural extracts. This peak
was identified as Ephedrine, when compared with the standard. Its
molecular formula was C10H15NO. Some other small peak were
also observed at a retention time of 11.1, 12.9, 17.8 and 19.06
compared with the standard it was conformed as vasicinol,
hypaphorine, vasicinone and phytosterols.
To determine the anticancer effect of S. cordifolia the extracts
were subjected to cytotoxicological studies.
Table-2. Antibacterial Activity of Natural and In Vitro Plant
Extract of S. cordifolia
C1 NA 1.00mg/l + Kinetin 1.00mg/l
C2 - NA 1.00mg/l + Kinetin 0.5mg/l
N3 in vivo plant extract of Sida cordifolia
Values are Mean ± SEM (n = 6);
Paw volume is expressed in change of height (in mm) of Hg bath (in
parentheses). *p<0.01 compared to control.
Two samples were used in this test. One was used as the control
(NIC). The plant extract was the test sample (NIT). The tumor cells
were viable in the natural sample. The average percentage of
viability was 96.33%. But in case of natural extract, most of the
cells were dead. The percentage of viability was 30.6% (Table. 3).
Fig. 2. Effect of S. cordifolia on HeLa cells Cytotoxicity
(Direct contact assay) (a) florescence HeLa cell control, (b)
HeLa cells treated (Probable apoptosis) with essential
3.2.Cytotoxicity
Absorbance
Average
0.77
0.72
0.84
0.78
0.16
0.20
0.19
0.18
0.24
0.25
0.25
0.25
0.44
0.39
0.35
0.39
Bacteria Zone of inhibition (µg/mm)
Mycobacterium species
Escherichia coil
Bacillus subtilis
Klebsiella pneumoniae
Micrococcus variance
Staphylococcus aureus
Pseudomonas aeruginosa
18
14
13
6
10
8
11
C1 C2 N3
10
5
8
7
9
9
14
15
12
16
10
6
11
13
Baby Joseph et.al / Int J Biol Med Res. 2011; 2(4): 1038 – 1042
1041
Bannerjee, 1985; Ramar et al., 2008) A large variety of
phytochemicals that have been reported from the natural product
research has been proven successfully as anticancerous agents
(Ranajit et al., 2008). The methanol extract of this medicinal plant
has been indicative effect of probable to regulate the apoptosis. In
this result shows the leaf of this experimental plant having highest
cytotoxic effect on HeLa cells. This is the first report that S.
cordifolia leaf possesses cytotoxic activity on HeLa cells (Figure
2b). In previous work, major constituents of its leaves were
identified anti-bacterial activity (Newman et al., 2000; Pole, 2006).
This is the similar kind of works have been identified as
anticancerous agent. In this work also identified major
constituents of leaf that have Cryptonine alkaloid, Dihydro
benzophenanthridine (cytotoxic, antitumor alkaloid), as well as
constituent that. In this present study proved that was the must be
capable of probable apoptosis on Human cervical cancer cell
(HeLa) lines. The cytotoxic activity may depend on following
bioactive compounds such as vasicinol, Ephedrine, vasicinone,
Hypaphorine. These may have cytotoxic effect because most of the
alkaloids have been cytotoxic effects (Mohammad et al., 2008;
Haque et al., 2000). The absence of normal apoptosis account for
uncontrolled cell multiplication and these neoplastic cells have
undergone alterations that tend to resist their susceptibility to
probable apoptosis. Hence, for aqueous essential oil with cytotoxic
activity to be formulated as a potent antitumor agent it should have
a direct and selective action on these resistant tumor cervical
cancer cells. Apart from this result clearly reveals that S. cordifolia
leaf consisted bioactive compounds having cytotoxic effect and its
profound cellular damage and hence scientifically substantiates
the folklore claims. Earlier phytochemical studies on the roots had
shown the presence of ephedrine, vasicinol, vasicinone and N-
methyl tryptophan (Khan et al., 1989; Kanth and Diwan, (1999). In
continuation of our studies on medicinal plants available in
Bangladesh for their chemical constituents and biological
activities we isolated (5-Hydroxymethyl -1-(1,2,1`3,9-tetrahydro-
pyrrolo [2, 1-b] quinazolin-1-yl)-heptan-1-one). Ekramul Islam et
al., (2004) and Khatoon et al., 2005 depicted the cytotoxicity and
antibacterial activities of crude extracts from the leaves of Sida
rhombifolia were investigated. This kind of similar experiment
were conducted and the results were showed few contradiction
that was ethyl acetate extract showed potent cytotoxicity with
LC50 values (5.41 ppm) comparable to the reference standard,
gallic acid. All the extracts showed weak antibacterial activity
against both Gram-positive and Gram-negative test organisms
[4] Ekramul Islam, M., Ekramul Haque, M. Mosaddik. M. A. 2002.
Cytotoxicity and antibacterial activity of Sida rhombifolia (Malvaceae)
g r o w n i n B a n g l a d e s h , 1 7
(8) :973 – 975.
[5] Franco I.W., R.C. Kassulke, and K.L. Harley. S. 2005. Host specificity and
aspects of the biology of Calligrapha pantherina (COL: Chrysomelidae),
a biological control agent of Sida acuta [Malvaceae] and S. rhombifola in
Australia. Entomoph.37: 409-417
[6] Franzotti EM, Santos CVF, Rodrigues HMSL, Mourao RHV, Andrade MR,
Antoniolli AR. 2000. Anti-inflammatory, analgesic activity and acute
toxicity of Sida cordifolia L. (Malva-branca). J. Ethnopharmacol.
72:273–38.
[7] Franzotti, EM, Santos, CV, Rodrigues, HM, Mourão, RH, Andrade, M
Antoniolli, A. 2000. "Anti-inflammatory, analgesic activity and acute
toxic ity of Si da cor difoli a L . (Mal va- bran ca) .” Jour nal of
Ethnopharmacology 72 (1-2): 273–7.
[8] Gunatilaka AAL, Sotheeswaran S, Balasubramaniam S, Chandrasekara
AI, Badrasriyani H T. 1980. Studies on medicinal plants of Sri Lanka.
Planta Medica. 39:66-72.
[9] Haque, N., Choudhury, S.A.R., Nutan, M.T.H., Rahman, G.D.S. and Rashid,
M.A. 2000. Evaluation of antitumor activity of some medicinal plants of
Bangladesh by potato disk bioaasay, Fitoterapia 71: 544 - 549.
[10] Islam , E. Haque, E. Mossadik, M.A. (2003). Cytotoxicity a nd
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[11] Jenny, M; Schwaiger, W; Bernhard, D; Wrulich, Oa; Cosaceanu, D; Fuchs,
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constituents of Sida cordifolia Linn. J. Bangl. Acad. Sci. 13: 55-60.
[13] Kanth V.R.and Diwan, P.V. (1999). Analgesic, anti-inflammatory and
hypoglycaemic activities of Sida cordifolia. Phytother Res. 13: 75-7.
[14] Khatoon, S.; Srivastava,M.; Rawat, A.K.S.; Mehrotra, S. 2005. HPTLC
method for chemical standardization of Sida species and estimation of
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[15] Mohammad S. Rahman, Bilkis B., Rasheduzzaman C., Khondaker M.
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[16] Newman, D.J., G.M. Cragg and K.M. Snader, 2000. The influence of
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[17] Pole, S (2006). Ayurvedic Medicine. Elsevier Health Sciences. Pp. 137.
[18] Ramar, P, S., Peter N.P., and Ponnampalam G., 2008. A compilation of
Bioactive Compounds from Ayurveda, Bioinformation. 3 (3): 100–110.
[19] Ranajit K. S., Matior Rahman, AKM., Mesbahuddin, A., Sitesh, C. B.,
Achinto, and Samar, K G. (2006). Bioactive Alkaloid from Sida cordifolia
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of pharmacology and therapeutics. 5 (2): pp. 175-178.
[20] Ranajit, K. S., Matior, R. M. A., Sitesh, C.B. Achinto, S and Samar, K.G.2008.
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[22] Silva, Rl; Melo, Gb; Melo, Va; Antoniolli, Ar; Michellone, P., Zucoloto, S.,
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