Vijisaral Elezabeth D and Subramanian A, Identification of Phytochemical Constituents and Antimicrobial Activity of Indigofera

Suffruticosa Leaves, Int.J.Curr.Biotechnol., 2013, 1(7):6-10.

International Journal of Current

Biotechnology
ISSN: 2321 - 8371
Journal Homepage : http://ijcb.mainspringer.com

Identification of Phytochemical Constituents and Antimicrobial Activity of

Indigofera Suffruticosa Leaves
D.Vijisaral Elezabeth and Arumugam Subramanian*
PG & Research Department of Chemistry, Nehru Memorial College, Puthanampatti - 621 007, Tiruchirappalli,
Tamilnadu, India.
.
A R T I C L E I N F O A B S T R A C T

Article History:
Indigofera suffruticosa (rPi kePyp) is an Indian herb used for various ailments by
Received 10 September 2013
traditional healers. The present study is carried out to identify the phytochemical
Received in revised form 13 September 2013
constituents of Indigofera suffruticosa plant leaves. Secondary metabolites like
Accepted 15 September 2013 total phenolic and flavonoid content were analyzed by UV spectroscopy and anti-
Available online 28 September 2013
microbial activity of ethanol extracts of the leaves were detected. Phytochemical
studies revealed the presence of reducing sugar, tannin, flavonoid, phenol, alka-
loid, glycoside, and steroid. Susceptibility testing by disc diffusion assay revealed
significant antimicrobial activity of ethanol extracts of leaves against bacteria
Key words: such as Staphylococcus aureus (22mm), Escherichia coli (14mm), Pseudomonas
Antimicrobial activity, Indigofera aeruginosa (16mm) and fungi against Klebsiella pneumonia (20mm), Aspergillus
suffruticosa, Medicinal plants, Alkaloid. niger (22mm) and Candida albicans (38mm). These findings provide supportive
evidence for the use of Indigofera suffruticosa in traditional medicines.
Introduction activities, high safety margins and lesser costs. Herbal
molecules are safe and would overcome the resistance
India has a rich culture of medicinal herbs and spices, produced by the pathogens as they exist in a combined
which includes about more than 2000 species and has form or in a pooled form of more than one molecule in
a vast geographical area with high potential abilities the protoplasm of the plant cell. Traditional use of
for Ayurvedic, Unani, Siddha traditional medicines but medicine is recognized as a way to learn about potential
only very few have been studied chemically and future medicines. Researchers have identified number
pharmacologically for their potential medicinal value. of compounds used in mainstream medicine which were
Human beings have used plants for the treatment of derived from “ethnomedical” plant sources. Plants are
diverse ailments for thousands of years (Sofowara, used medicinally in different countries and are a
1993). According to the World Health Organization, source of many potent and powerful drugs. Indigo
most populations still rely on traditional medicines obtained from species of Indigofera suffruticosa was
for their psychological and physical health used in the Old World, the use of wild indigo by pre-
requirements, since they cannot afford the products of Columbian natives of Mexico to dye cloth and paint in
Western pharmaceutical industries, together with their various shades of blue was passed down to the Spanish
side effects and lack of healthcare facilities. Rural areas colonists (Haude, 1997). Indigofera suffruticosa species
of many developing countries still rely on traditional became important commercial crops in various
medicine for their primary health care needs and have tropical and subtropical areas. The blue dye was
found a place in day-to-day life. These medicines are produced by fermentation of the leaves, usually with
relatively safer and cheaper than synthetic or modern caustic soda or sodium hydrosulfite, and the exudates
medicine. People living in rural areas from their processed into dry cake. The blue color developed as
personal experience know that these traditional the cake was exposed to the air (Simon et al., 1984).
remedies are valuable source of natural products to Indigo was used as a bluing to counter the yellowing in
maintain human health, but they may not understand clothes from washing with soap (Velez and van
the science behind these medicines, but knew that some Overbeek, 1950). In the last few decades, natural indigo
medicinal plants are highly effective only when used has been almost wholly replaced by synthetic dyes.
at therapeutic doses. Herbal medicines are in great Poultices and extracts of wild Indigofera suffruticosa
demand in both developed and developing countries leaves, alone or in combination with other ingredients,
as a source of primary health care owing to their are used in herbal medicine to treat fever, headaches,
attributes having wide biological and medicinal hemorrhages, convulsions, acute cough, skin parasites,
and boils (HealthLink, 2001).
*Corresponding author.
Email address: nmcaaru@gmail.com

Volume 1; Issue 7; Sep, 2013 Int.J.Curr.Biotechnol. 6

Materials and Methods Test for Alkaloid
About 0.2 g of plant extract was weighed in separate
Collection of Plant Material test tube and warmed with 2% Sulphuric acid for 2
The leaves of Indigofera suffruticosa were collected from minutes. And it was filtered in separate test tube and
Vaithiynathpuram Village in Perambalur District of few drops of Dragencloffs reagent were added and
Tamilnadu, India during January to December, 2012 and observed for the presence of orange red precipitate for
authenticated by the Director of the Rapinat Herbarium the presence of alkaloid.
and Centre for Molecular Systematics, St.Joseph’s
college campus, Trichirappalli, Tamilnadu, India. Fresh Test for Glycoside : Legal’s Test
leaves were cleaned with running tap water and dried About 0.5 g of plant extract was weighed in a separate
under the shade (sunlight). Then the dried plant leaves test tube with pyridine and sodium nitroprusside
were ground to fine powder mechanically and preserved reagent was added and made alkaline with NaOH
in airtight containers for further analysis. solution and observed for the presence of pink to red
color solution which indicates the presence of
Preparation of Plant Extract glycoside.
The powdered leaves were extracted in a Soxhlet
apparatus with ethanol and water successively. The Test for Terpenoids
resultant extracts were evaporated under reduced About 0.5 g of plant extract in separate test tube was
pressure in a rotary evaporator to obtain the crude taken with 2 ml of chloroform; 5 ml of concentrated
extract of Indigofera Suffruticosa. sulphuric acid was carefully added to form a layer and
observed for presence of reddish brown color interface
Test for Phytochemical Analysis to show positive results for the presence of terpenoid.
The extracts were analyzed for the presence of alkaloid,
terpenoid, reducing sugars, tannin, flavonoid Test for Reducing Sugars
carbohydrate, phenol, anthocyanin, protein and steroid Two ml of crude plant extract and add 5ml distilled
(Sofwora, 1993; Harborne, 1973). water were stirred and filtered. The filtrate was boiled
with 3-4 drops of Fehlings solution A and B for 2 minutes

Figure: 1 Antimicrobial Activity of Leaves of Indigofera Suffruticosa

7 Int.J.Curr.Biotechnol. Volume 1; Issue 7; Sep, 2013
 Table 1: Results of Phytochemical Analysis Ethanolic Table 3: Antimicrobial Efficacy of Ethanol Extract of
and Aqueous Extract of Indigofera Suffruticosa Indigofera suffruticosa Leaves.
N a m e o f th e Zone of inhibition in mm
P h y t o c h e m ic a l E th a n o l A q u eo u s
C o n s t i t u e n ts e xtrac t e x t ra c t
S. No Name of the
Microorganism Ethanol extracts Solvent
of leaves of control Standard
A l k a l o id + +
Indigofera
G ly c o s id e + + suffruticosa
1 Staphylococcus 20 - 35
R ed u ci n g su g a r + + aureus
(NCIM2079)
T a n n in + +
2 E.coli 14 - 38
F la v o n o i d + + (NCIM2065)
3 Pseudomonas 16 - 35
S te r o i d + + aeruoginosa
(NCIM2036)
A n t h o c y a n in - -
4 Klebsiella 20 - 30
P h en o l + + pneumonia
(NCIM2098)
A m in o a c id - - 5 Aspergillus 22 - 35
niger
P r o t e in - -
(NCIM105)
+: Indicates the presence and -: Indicates the absence 6 Candida 38 - 25
of phytoconstituents albicans
Table 2: Determination of Secondary Metabolites in (NCIM3102)
two Different Solvent Extracts of Indigofera
suffruticosa Leaves Standard - Ciprofloxacin 5µg / disc for bacteria;
Ethan ol Aqueous Nystatin 100 µg / disc for fungi. Solvent-DMSO
Secondary metab olites extract extract
Test for Phenol
(%) (%) Ferric Chloride Test
About 0.2 g of plant extract was weighed and treated
Total Phenoli c content 0.54 0.42 with 5% ferric chloride and observed for the formation
Total flavonoid content 0.22 0.14 of deep blue color which indicates the presence of
phenol.

and observed for orange red precipitate which Test for Amino acid
indicates the presence of reducing sugars. Ninhydrin Test
About 0.2g of plant extract was weighed and treated
Test for Tannin with Ninhydrin solution and observed for a
Small quantity of plant extract was mixed with 5ml of characteristic purple color which indicates the
distilled water and heated on water bath. The mixture presence of amino acid.
was filtered and ferric chloride was added to the
filtrate and observed for dark green solutions that Test for Protein
indicate the presence of tannin. Million’s Test
Small quantity of plant leaf extract was treated with
Test for Flavonoid few drops of Million’s reagent and observed for the
About 0.2 g of plant extract was weighed in separate formation of white precipitate which indicates
test tubes and dissolved in diluted sodium hydroxide presence of protein.
and diluted Hydrochloride added and observed for
yellow solutions that turn colorless. This indicates the Estimation of Secondary Metabolites
presence of flavonoid. Determination of Total Phenolic Content
The total phenolic content of Indigofera suffruticosa
Test for Steroid leaves was determined in two different solvent extracts
Two ml of acetic anhydride was added to 0.5 g extract spectrophotometrically according to the Folin-
with 2ml of Sulphuric acid and observed for the color Ciocalteu method (Singleton et al., 1999) using Gallic
change from violet to blue or green in samples acid as a standard (the concentration range; 0.25 to
indicating the presence of steroid. 0.5mg mL-1). The total phenolic content was expressed
as milligram per gram dry extract.
Test for Anthocyanin
Sodium Hydroxide Test Determination of total Flavonoid Content
About 0.2 gm of plant extract was weighed in separate The total flavonoid content was determined according
test tube, 1ml of 2N Sodium hydroxide was added, and to the aluminum chloride colorimetric method (Lin and
heated for 5 minutes at 100 ± 2°C and observed for the Tang, 2007). Rutin was chosen as a standard (the
formation of bluish green color which indicates the concentration range; 0.005 to 0.1mg mL-1) and the total
presence of anthocyanin.

Volume 1; Issue 7; Sep, 2013 Int.J.Curr.Biotechnol. 8

flavonoid content was expressed as milligram per gram Albicans and minimum activity was seen against
of dry extracts. Klebsiella pneumonia and Aspergillus niger.
Antimicrobial Assay Discussion
The following organisms were employed for this study
as test organisms; bacteria such as Staphylococcus The phytochemical analysis estimated the secondary
aureus, E.coli, Pseudomonas aeruginosa and Klebsiella metabolites such as flavonoid and phenol and revealed
pneumonia, fungi such as Aspergillus niger and Candida the antimicrobial activity of Indigofera suffruticosa
albicans. The test microbial pathogen cultures were leaves. The presence of notable phytochemicals;
obtained from National Chemical Laboratory (NCL) alkaloid, glycoside, terpenoid, steroid, flavonoid,
Pune, maintained by periodical sub culturing on reducing sugar, tannin and phenol in the plant leaf
Nutrient agar and Sabouraud dextrose agar medium. might have been responsible for its uses in traditional
health care. The phenolic compounds are one of the
Antimicrobial and antifungal activity of ethanol extracts largest and most ubiquitous groups of plant
were tested using the disc diffusion method described metabolites. They possess biological properties such
by Indian pharmacopoeia, (1996, Vol II A-105). All the asantiapoptosis, antiaging, anticarcinogen,
above mentioned bacteria were inoculated into nutrient antiinflammation, antiatherosclerosis, cardio vascular
agar medium and fungi inoculated to Sabouraud protection and improvement of endothelial function,
dextrose agar medium. The well of 8mm diameter was as well as inhibition of angiogenesis and cell
punctured in the culture medium using sterile cork proliferation activities (Han et al., 2007). Non toxic
borer. Ethanol extracts were administered to fullness glycoside can also be hydrolyzed to release phenolic
in each well. Culture plates were incubated at 37° ± 2° compounds that are toxic to microbial pathogens. These
for 24 h for bacteria and incubated at 37° ± 2° for 4 days bioactive compounds exert antimicrobial activity
for fungi. Bioactivity was determined by measuring through different mechanisms. Natural antioxidants
diameter of inhibition zones in mm. Solvents used for mainly come from plants in the form of phenolic
extraction served as control. compounds such as flavonoid, phenolic acids,
tocopherols etc. (Ali et al., 2008). Tannins bind to
Results proline rich protein and interfere with protein synthesis.
Phytochemical Analysis There is no report on the total phenolic and flavonoid
Ethanolic extracts of the leaves of Indigofera suffruticosa present in Indigofera suffruticosa leaves extract in
showed the presence of alkaloid, glycoside, reducing previous studies. Derived polyphenols from plants are
sugar, tannin, flavonoid, steroid and phenol. Amino of great importance because of their potential
acid, anthocyanin and protein were not found in antioxidant and antimicrobial properties. Phenolic
ethanolic extract of Indigofera Suffruticosa. Similarly, compounds exhibit a considerable free radical
the aqueous extract of the plant revealed the presence scavenging (antioxidant) activity (Wojydylo et al., 2007).
of alkaloid, glycoside, reducing sugar, tannin, Thus the phenolic compound present in the plant
flavonoid, steroid and phenol and except anthocyanin, Indigofera suffruticosa might contribute various
amino acid and protein. Table 1 showed the results of therapeutic uses of the extract’s traditional system of
phytochemical analysis of the ethanolic and aqueous medicine. Antimicrobial assay of ethanolic extracts
extracts of Indigofera Suffruticosa. were carried out against bacteria and fungi. Among the
extracts, the leaf extract of Indigofera suffruticosa were
Estimation of Secondary Metabolites effective against bacteria and fungi. The plants are vital
Total Phenolic Content source of innumerable number of antimicrobial
Total phenolic content of two different solvent extracts compounds. Several phytochemical constituents like
of leaves of Indigofera suffruticosa was determined by flavonoids (Tsuchiya et al., 1996), phenolics and
spectrophotometricaly according to the Folin-Ciocalteu polyphenols (Mason and Wassermann, 1987), tannins
method and the results are given in Table 2. It is clear (Ya et al., 1988), etc. are effective antimicrobial
from the table that among all the extracts, the aqueous substances against a wide range of microorganisms.
extract showed high amount of phenolic content 0.54% One of the largest groups of chemical produced by plant
compared to ethanol extracts. The ethanolic extract is the alkaloids and their amazing effects on humans
showed the least phenolic content 0.42%. have led to the development of powerful painkiller
medications (Raffauf, 1996). Alkaloids of
Total flavonoid content Heliotropium indicum and Chorizanthe procumbens are
The total flavonoid content was determined according used for the treatment healing, antitumor and analgesic,
to the aluminum chloride colorimetric method in two hence different formulations could be prepared for
different solvent extracts of leaves of Indigofera clinical trials (Boomination and Ramamurthy, 2009).
suffruticosa and the results are given in the Table 2. The
ethanol extract showed high amount of flavonoid Acknowledgement
content 0.22% when compared to aqueous extract. The The authors would wish to acknowledge the
aqueous extract showed the least flavonoid content management and Principal of Nehru Memorial College
0.14%. for providing research facilities and encouragements.
Antimicrobial assay Reference
The results of antimicrobial assay of the ethanolic Ali, S.S., Kasoju, N., Luthra, A., Singh, A., Sharanabasava,
extracts of the leaves of Indigofera suffruticosa against H.,Sahuand, A., Bora, U. (2008). Indian medicinal herbs
tested strains were shown in Table 3. The antimicrobial as sourceof antioxidants. Food Res. Int., 41: 1-15.
analysis reveals maximum activity against the Boominathan, M., Ramamurthy, V., (2009).
Staphylococcus aureus and minimum activity was noted Antimicrobial activity of Heliotropium Indicum and
against the E.coli and Pseudomonas aeruginosa. Fungal Coldenia procumdens., J. Ecobiol., 24: 11-15.
pathogens activity was maximum towards Candida

9 Int.J.Curr.Biotechnol. Volume 1; Issue 7; Sep, 2013

Han, X., Shen, T., Lou, H. (2007). Dietry polyphenols and
theirbiological significance. Int. J. Mol. Sci., : 950-988.
Harborne J.B., (1973). Phytochemical Methods: A Guide
to Modern Technique of Plant Analysis.
Haude, M.E., (1997). Indentification and classification
of colorants used during Mexico’s early colonial period.
Book and Paper Group Annual Vol.16. The American
Institute of Conservation. http://aic.stanford.edu/
conspec/bpg/annual/v16//bp16-05.html. 26 p.
HealthLink., (2001). Monograph: Indigo naturalis. http:/
/www.healthlink.com.au/nat_lib/htm-data/htm-herb/
bhp1016.htm. 3 p.
Lin, J.Y., and C.Y., Tang. (2007). Determination of total
phenolic and flavonoid contents in selected fruits and
vegetables, as well as their stimulatory effects on mouse
splenocyte proliferation. Food Chem., 101: 140-147.
Mason, T.L., and Wassermn, BP., (1987). Inactivation of
red beet betaglucan synthase by inactive and oxidized
phenolic compounds. Phytochemistry. 26: 2197-2202.
Raffauf, R.F., (1996). A Guide to Their Discovery and
Distribution, Hawkworth Press Inc, New York, P.35.
Sofowora, A., (1993). Medicinal Plants and Traditional
Medicines in Africa, Chichester John Wiley & Sons, New
York, 97-145.
Simon, J.E., A.F. Chadwick, and L.E. Craker. (1984). Herbs.
An indexed bibliography. (1971-1980). Scientific
literature on delected herbs, and aromatic and
medicinal plants of the temperature Zone. Archon
Books, Hamden, CT. 770 p.
Singleton, V.L., R. Orthofer and R.M., Lamuela-Raventos,
(1999). Analysis of total phenols and other oxidation
substrates and antioxidants by means of folin-ciocalteu
reagent. Methods Enzymol., 299: 152-178.
Tsuchiya, H., Sato, M., Miyazaki, T., Fujiwara, S.,
Tanigaki, S., Ohyma, M., Tanaka, T., and Ilinuma, M.,
(1996). Comparative study on the antibacterial of
phytochemical flavanones against methicillin resistant
Staphylococcus aureus. J. Ethnopharamacol., 50: 27-34.
Velez, I., and J. van Overbeek. (1950). Plantas
indeseables en los cultivos tropicales. Editorial
Univeresitaria, Rio Piedras, PR. 497 p.
Wojdylo, A., J. Oszminaski and R. Czemerys, (2007).
Antioxidant activity and phenolic compounds in 32
selected herbs. Food Chem., 105: 940-949
Ya, C., Gaffiney, S.H., Lilley, T.H. and Haslam, E. (1988).
Carbohydrate-polyphenol complexation. P. 553. In:
Hemingway, R.W. and Karchesy, J.J. (ed.), Chemistry and
significance of condensed tannins. Plenum Press, New
York.

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