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Ethnobotanical Leaflets 13: 1485-1503, 2009. Studies
on Phytochemical constituents, Functional Group Identification and
Antimicrobial Activity of� Solanum
nigrum (Solanaceae) *D.venkatesan1;
C.M. Karrunakaran2; S. Selva kumar3 *1Department of Industrial
Biotechnology, Tamil 2Dean-Research,
3Lecturer, *Corresponding
author: venkat_micro@sify.com Issued� 01
December, 2009 Abstract Phytochemical
constituents, functional group identification and antimicrobial activity of
ethanolic extract of Solanum nigrum were
investigated. The phytochemical screening of the crude extract revealed the
presence of alkaloids, reducing sugars, tannis, flavonoids, Phlobatannis, and
steriods. The extract were subjected for identification of functional groups
using infra red spectrophotometer, presence of�� C=O,��
C-H,� C=C and C-O were
identified, these bonding structures are responsible for presence of alkyl
groups, methyl groups, alcohols, ethers, esters, carboxylic acid and
anhydrides. And the crude ethanolic extract of Solanum nigrum were assayed for in vitro antimicrobial activity
against gram positive and gram negative bacteria and they were also subjected
for antifungal activity and the zone of inhibition were compared with control
drug Penicillin and Nystatin. Staphylococcus
aureus as the most sensitive strains exhibited maximum zone of inhibition
about 25.7mm among gram positive organism tested. Escherichia coli exhibits 30.1mm zone than that of standard
penicillin positive control. Trichophyton
mentagrophytes exhibits maximum zone of inhibition of about 26.1mm among
the tested fungal species. Key words ����������� Solanum nigrum,
Staphylococcus aureus, Escherichia coli, Trichophyton
mentagrophytes, Infra red spectrophotometer. Introduction Natural
products drug discovery will be more holistic, personalized and involve wise
use of ancient and modern therapeutic skills in a complementary manner so
that maximum benefits can be occurred to the patients and community
(Patwardhan and Hopper, 1992). The antiseptic qualities of aromatic and
medicinal plants and their extracts were recognized in the laboratory data
back to the early (Dorman and Dean, 2000). As result of ecological and
fitgeographical diversification, endemism of species is high (Tan, 1992;
Dagic et al., 2002). �Ayurvedic remaining one of the most ancient
and yet living traditions practiced widely in developing countries as well as
developed countries and has a sound philosophical and experimental basis
(Dahanukar and Thathe, 2000; Chopra and Doiphode, 2002). In
recent years investigation found several plants of the ethnomedicine posse�s
really interesting biological activities which could be of interest for all
parts of the world (El- Fiky et al., 1995; Awadh Ali et al., 2001; Mothana
and Lindequist, 2005; Mothana et al., 2006; Al-Fatimi et al., 2007). Approximately
20% of the plants found in the world have submitted to pharmacological
activities (Suffrendini et al., 2004). And the plants have organic chemicals
(Evans et al., 1986). The plants posses chemotherapeutic, bacteriostatic and
antimicrobial agents (Purohit and Mathur, 1999) �The source of plants is models for the
synthesis of new drugs with better therapeutic, chemical (or) physical
properties than the original compound (Baerheim svendsen and Scheffer, 1992).
In recent year�s plant material have been investigated as a source of
medicinal agents (Krishnaraju et al., 2005). And the plants materials used
for treatment bacterial infections (Balandrin et al., 1985).� However world health organization also has
recognized the importance of traditional medicine and has been active in
crating strategies, guidelines and standards for botanical medicines (WHO,
2002). �Plant derived drugs remains important
resource especially in developing countries, to combat serious disease.
Approximately 62 � 80% of the world�s population still relies on traditional
medicines for the treatment of common illness (Zhang, 2004). In fact, plants
produce a diverse range of bioactive molecules making them a rich source of
different types of medicines. Higher plants, as sources of medicinal
compounds, have continued to play a dominant role in the maintenance of human
health since ancient times (Farombi, 2003). Over 50% of all modern clinical
drugs are of natural product origin (Stuffness and Douros, 1982). And natural
products play on important role in drug development programmes in the
pharmaceutical industry (Baker et al., 1995). There
are a few reports on the use of plants in traditional healing by either
tribal people or indigenous community (Sandhy et al., 2006; Ayyanar and
Ignacimuthu, 2005; Rajan et al., 2002; Natarajan et al., 1999; Ignacimuthu et
al., 1998). The
activities have been have been selected because of their great medicinal
revelence with the recent years, infections have increased to a great extent
and resistant against antibiotics, becomes an ever increasing therapeutic
problem (Austin et al., 1999). Because natural products of higher plants may
give a new source of antimicrobial agents. There may research group that are
now engaged in medicinal plants research (Samy et al., 1998; Hamil et al.,
2003; Motsei et al., 2003). The
development drug resistance in human pathogens against commonly used
antibiotics has necessitate a search for new antimicrobial substance from
other sources including plant screening of medicinal plants for antimicrobial
activities and photochemical is important for finding potential new compounds
for therapeutic uses. In
present study focus phytochemical constituent, functional group
identification and in vitro evaluation of antimicrobial activity of the
separated fractions of ethanolic extracts from the aerial parts of Solanum nigrum are bioassay against pathogenic
microrganism.� Materials and Methods Collection
of Plant materials and Identification Plant material of Solanum nigrum was purchased from a local Ayurvedic medicinal
shop "Chennai," and the material was identified and authenticated
by the Chief Botanist, Tamil Nadu Aromatic Medicinal Plants Corporation
Limited (TAMPCOL), Arignar Anna Siddha Medical College and Hospital Campus,
Chennai, Tamil Nadu, India. Ethanolic
extraction The
plant materials were dried in shaded and powdered by mechanical grinder. The
powder of Solanum nigrum plant material were initially defated with
petroleum benzene ( 60 - 80C) followed by 1000 ml of ethanol by using a
Soxhlet extractor for 72 hours at a temperature not exceeding the boiling
point of the solvent. The extract was filtered using whattman filter paper
(No 1) and then concentrated in vaccum and dried at 45C for ethanol
elimination and the extract were kept in a sterile bottle under refrigeration
condition of about 2-8C. Test
for Phytochemical analysis The extracts were analyzed for the presence of
alkaloids, terpenoids, reducing sugars, Saponins, tannis, Carbonyls,
Flavonoids, Phlobatannis and steriods (Adetuyi and Popoola 2001; Trease and Evans, 1989; Sofowora,
1982). Test
for Alkaloids Weigh about 0.2 gm of plant extract in separate
test tube and warmed with 2% Sulphuric acid for 2 minutes. And it was
filtered in separate test tube and few drops of Dragencloffs reagent were
added and observed for the presence of orange red precipitates for the
presence of alkaloids. Test
for Cardiac glycoside Keller-Killani
Test Weigh about 0.5 gm of plant extract in a separate
test tube with 2 ml of glacial acetic acid containing a drop of ferric
chloride solution. This was under layered with 1 ml of concentrated tetra oxo
sulphate (VI) acid. And observe for brown ring formation at the interface
(Finar, 1983). Test
for Terpenoids Weigh about 0.5 g plant extract in separate test
tubes with 2 ml of chloroform. And add concentrated Sulphuric acid carefully
to form a layer. And observe for presence of reddish brown color interface
was formed to show positive results for the presence of terpenoids. Test
for reducing sugars Take a test tube and add 2 ml of crude plant
extract and add 5 ml of Distill water and filtered. The filtrate was boiled
with 3-4 drops of Fehling�s solution A and B for 2 minutes. Observe for
orange red precipitate indicates the presence of reducing sugars. Test
for Saponins Weigh about 0.2 gm of plant extract in the test
tube and add 5 ml of distill water and then heat to boil. Observe for
frothing (appearance of creamy mass of small bubbles) shows the presence of
Saponin Test
for Tannis To small quantity of plant extract was mixed with
water and heated on water bath. The mixture was filtered and ferric chloride
was added to the filtrate. And observe for dark green solutions indicates the
presence of tannis�� Test
for Carbonyl Take 2 ml of�
plant extract in separate test tubes and add few drops 2,4, di nitro
phenyl hydrazine solution and shaken. And observe for the presence of yellow
crystals immediately for the presence of an aldehyde. Test
for Flavonoids Weigh about 0.2 gm plant extract in separate test
tubes and dissolved diluted Sodium hydroxide and add diluted Hydrochloride.
And observe for yellow solutions that turns colorless indicates the presence
of flavonoids Test
for Phlobatanin Weigh about 0.5 gm of plant extract in a test
tubes and dissolved with distill water and filtered. The filtrate was boiled
with 2% Hydrochloric acid solution. Observe for red precipitate shows the
presence of Phlobatanin Test
for Steroids To the plant extract add 2 ml of acetic anhydride
and add 0.5 gm of ethanolic extract of each sample with 2 ml of Sulphuric
acid .Observe for the color change from violet to blue or green in samples
indicating the presence of steriods Identification
of functional group Instrument used������� : FTIR (Fourier Transformer Infra
red� ��������������������������������������������
Spectrophotometer) Make������������������������� : Shimadzu Sample
preparation for Infrared Spectrophotometer A drop of ethanolic extract of Solanum nigrum was placed on the
Sodium chloride cell to obtain the thin layer. Then the cell was placed in
the FTIR compartment and scanned accordance with ASTM (American standard
testing material) 1252-98 through IR region for about 25 scans and
chromatogram was obtained. Antibacterial
activity Bacterial
strains and Growth conditions The
following cultures of gram positive organism were used Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus
faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus
anthracis, Bacillus subtilis and gram negative organism of Escherichia coli,
Klebsiella pneumoniae, Shigella flexneri, Shigella sonnei,� Shigella boydii, Salmonella typhi,
Salmonella paratyphi, Vibro cholera and Pseudomonas aeruginosa. The
fungal cultures used were Microsprum
gypsum, Trichophyton mentagrophytes, Epidermophyton floccosum and Aspergillus
Preparation
of plant extract, antibiotic and Inoculum preparation The
dried plant extract were weighed and dissolved in sterile distilled water to
prepare appropriate dilution to get required concentration of 5.0 mg/ml and
antibiotic at 50�g/disc. Inoculum of bacterial cultures were prepared in
nutrient broth medium and kept incubation at 35C for 8 hours and fungal
cultures at 25C for 24 hours. Procedure
for performing the Disc Diffusion test (Bayer et al., 1986) The
required amount of Mueller-Hinton plates (Hi media) is prepared as per
manufacturer instructions. A sterile cotton swab was dipped into the turbid
culture suspension. The dried surface of Muller-Hinton agar plate was
inoculated by streaking two more times rotating the plate approximately 60each
time. The lid may be left aside for 3-5 minutes and allow to dry for the
excess surface moisture content. The
ready prepared sterile Himedia discs (0.7mm) were poured with different
concentration of above prepared antibiotic and plant extract solutions, the
discs were placed on the medium and the plate were incubated at 5C for
1 hour to permit good diffusion and then transferred to an incubator at 37C
for 24 hours for bacterial cultures and for fungal cultures the plates should
be incubated at 25C for 72 hours. The negative control was included
without adding the cultures to know the sterile conditions. The antibacterial
activity was recorded by measuring the width of the clear inhibition zone
around the disc. Results and Discussion Ayurveda
remains one of the most ancient and yet living traditions practiced widely in
The ethanolic extract of Solanum nigrum were subjected for phytochemical analysis, the
results shows that positive for tests of alkaloids, reducing sugars, tannis,
flavonoids, phlobatannis, and steriods but shows negative results for cardiac
glycosides, terpenoids, saponins and carbonyl were discussed in the table -1 Table 1.� Phytochemical constituents of Solanum nigrum.
Key� + = Positive ; - = Negative The
Infra red spectroscopic (IR) analysis of ethanolic crude extract of Solanum nigrum, reveals that the
presence of different functional group ranging from 4000 to 400 wave number,
C-Hstrl, alkyl group, (2926.6cm-1), C=O stretching for carbonyls
(2162.1 cm-1), C-O bending for alcohols, ethers, esters,
carboxylic acid and anhydrides (1310.6 �1059.6cm-1), C-H bending
alkyl (1453.4 � 1376.2 cm-1), C=C stretching (1660.0- 1667.0) and
C-H bending for methyl group (864.1 � 668.4 cm-1) The
spectroscopic analysis of the plant extract of Solanum nigrum, figure -1 shows peaks range from 2000 � 400 wave
number were discussed in the table -2 the extract revealed the presence of
C=O, C-H, C=C and C-O bond stretching. Generally O-H stretching will be
present in all phenolic compounds and C-N is commonly found in all types of
Alkaloids. Table 2.� IR Spectroscopic analysis of ethanolic
extract of Solanum nigrum, which
showing characteristic absorption peaks at IR range.
Figure 1.� IR Spectroscopic analysis of ethanolic
extract of Solanum nigrum.
The
in vitro antimicrobial activity of ethanolic extract of Solanum nigrum was assessed qualitatively and quantitatively by
the presence or absence of inhibition zones, Zone diameter values, the
ethanolic extract of Solanum nigrum
showed great in vitro antimicrobial activities against 16 types of bacteria
and 4 fungus species tested. In this study the antimicrobial activities of
ethanolic extract were compared with standard antibiotics such as Penicillin
and Nystatin used as positive control. Results
from the antimicrobial disc diffusion assay were furnished in table-3, the
data indicates that gram positive Staphylococcus
aureus was the most sensitive strain tested to the ethanolic extract of Solanum nigrum with greatest
inhibition zone of about 25.7mm. Staphylococcus
epidermidis, Bacillus anthracis
and Bacillus subtilis were also
found more sensitive with zone of inhibition 22.3mm, 20.1mm and 22.1mm. Streptococcus pyogenes show less
activity when compared with control standard standard penicillin with zone of
inhibition about 10.2mm. The
ethanolic extract of Solanum nigrum
also shows excellent activity against Escherichia
coli, tested gram negative organism with 30.1mm zone of inhibition to
that of standard penicillin positive control. Klebsiella pneumoniae, Shigella flexneri, Salmonella typhi and Pseudomonas aeruginosa exhibited
maximum significant zones to the tested ethanolic crude extract of Solanum nigrum with inhibited zones of
about 28.1mm, 26.0mm, 18.2mm, and 21.5mm. Shigella
boydii, Salmonella paratyphi
and Vibro cholera exhibited better
significant zones of about 10.0mm, 09.1mm and 12.5mm. The
ethanolic extract of Solanum nigrum
also exhibited the highest antifungal activity against Microsporum gypsum, Trichophyton
mentagrophytes, Epidermophyton
floccosum and Aspergillus Table 3.� Antimicrobial activity expressed as minimum
inhibitory concentration (MIC) of ethanolic extract of Solanum nigrum by disc diffusion method.
Note Values
in the means of triplicate Ethanolic
extract concentration 5.0 mg/ml Penicillin
50�g/disc Nystatin
50�g/disc Conclusion ����������� This
research work states that the presence of presence of alkaloids, reducing
sugars, tannis, flavonoids, Phlobatannis, and steriods are responsible for the antimicrobial activity, the ethanolic
extract of Solanum nigrum have of
C=O, C-H, C=C and C-O bond stretching compounds exhibits maximum zone of
inhibition against the tested organism when compared with control drug
penicillin and Nystatin .Hence present study suggests that pathogenic
microorganism become resistant to the existing drugs.� And this study shows that the plant is
promising development of phytomedicine for antimicrobial properties. In this
endeavor, traditional herbal medicines must perforce be granted the benefits
of modern science and technology to serves further global needs. The drugs
derived from herbs may have the possibility of using in medicine because of
its antibacterial activity. �Acknowledgements We
wish to acknowledge and thankful to Dr. C.M. Karrunakaran, Ph D.,
Dean-Research, Bharath University for their valuable and consistent support
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