INTRODUCTION:
Nature has bestowed on us a very rich
botanical wealth and a large number of diverse types of plants in different
parts of the country1. Plants have been one of the major sources of
medicine since the beginning of human civilization2. Legend says,
medicine and health starts from kitchen. Food has been the main source of medicine
right from the ancient days. That’s why siddha and homeopathy treatments
involve herbs and spices for treating diseases. Before the birth of allopathic
medicines herbal medicines were used to fuse even broken bones, treat big
ailments and even cure infections. Even today tribal people believe in herbal
medicine and they have cure for almost all diseases except for cold which they
get from non-tribal people. In rural India, 70% of the population is dependent
on the traditional system of medicine, the Ayurveda3.
Ethno-medicinal studies have offered immense scope and opportunity for the
development of new drugs.
Public, academic and
government demand in traditional medicines is increasing exponentially due to
growing incidence of the adverse drug reactions and economic burden of the
modern system of medicine4. Medicinal plants are still major parts
of traditional medicinal systems in developing countries. Many infectious
diseases are known to be treated with herbal remedies throughout the history of
mankind. Even today, plant materials continue to play a major role in primary
health care as therapeutic remedies in many developing countries5.Medicinal
plants, which form the backbone of traditional medicine, have in the last few
decades been the subject of very intense pharmacological studies6.
As each day proceeds the
number of diseases caused by microbes increases. So the demand of discovering
new antimicrobial compounds keep on increasing. So new compounds are being
tested to study its spectrum and see if they can kill the pathogens without
affecting the natural flora that are required for the body. Each plant possess
many novel biologically active compounds as very few plant species have been
thoroughly investigated for their medicinal properties7. These
compounds possess different activities within themselves. One of such is
antimicrobial activity which helps both the plant and humans when extracted.
These plant based systems continue to play an essential role in health care and
it has been estimated by the World Health Organization that approximately 80%
of the world’s inhabitants rely mainly on traditional medicines for their
primary health care. Plant products also play an important role in the health
care system of the remaining 20% of the population, mainly residing in
developed countries8,9. Green plants are the symbol of a reservoir
of resourceful chemotherapeutics that provide important source of natural
antimicrobials10.
Cardiospermum halicacabum is a woody perennial vine, commonly known as Balloon
vine or Love in a puff. Climbing plant which can be easily spotted on road
sides and considered as weed. It is well known for its medicinal properties in
homeopathic medicine and used to treat various diseases. It is one among
Kerala’s “Dasapushpam” which means ten sacred flowers of Kerala.
Solanum nigrum is from the family solanaceae, It is mostly found as
a garden weed. It is a short lived perennial shrub. In traditional medicine it
is used to treat skin disease, rheumatism and gout. Its juice is given for the
chronic enlargement of liver. It can also cure ear and eye disease. It is
widely known as the anti-ulcer plant. It is used to treat mouth cancer in
homeopathy medicine. It is also used to treat various liver related ailments
including jaundice. It is also used against asthma and whooping cough. It is
anti-tumorigenic, antioxidant, Anti-inflammatory, hepatoprotective, diuretic
and antipyretic.
Fig 1.Cardiospermumhali cacabum
Fig 2. Solanum nigrum
MATERIALS AND METHODS:
Sample collection:
The selected herb samples Cardiospermum halicacabum and Solanum nigrum
were collected from Vellore (Fig. 1 and 2). They were initially washed in
running water to remove the dirt and extraneous matter present in it. They were
transferred to the laboratory in sterile polythene cover. Then they were washed
in distilled water twice to cleanse it further. The leaves were air-dried in a
shady place for a week and ground into powder and stored for further use.
Sample extraction:
Extraction 1(Shaker): 10gm of the powder was extracted with 100ml of two
different solvents such as methanol and water. It was kept in a shaker at
120rpm for 48 hours. It was then filtered and stored.
Extraction 2(Using low boiling point): 15 gm of the powder was extracted with
100ml of different solvents such as methanol and water. It was boiled. Then it
was filtered and stored.
Preliminary phytochemical
analysis were done to detect the presence of various chemicals like
carbohydrates, proteins, amino acids, flavonoids, terpenoids, saponins,
phytosterols, phenols and tannin.
Phytochemical analysis:
The following test were
carried out using standard procedures to identify the various constituents
present11.
Detection of carbohydrates
(Fehling’s test):
1 g of extract dissolved in
100 ml distilled water and filtered and it was subjected to Phytochemical test.
1mL of the filtrate were
treated with Fehling’s A and B solutions and then heated on a water bath for
five minutes. Brick red coloured precipitate was formed which indicates the
presence of carbohydrates.
Detection of proteins and
amino acids:
a) Ninhydrin test:
To the 1 mL of filtrate
few drops of 0.25% ninhydrin reagent was added and boiled for a few minutes.
Formation of blue color indicates the presence of amino acids.
b) Biuret test:
1 mL of filtrate were
treated with 1 mL of 10% sodium hydroxide solution and heated. To this a drop
of 0.7% copper sulphate solution was added to it. Formation of purplish violet
color indicates the presence of proteins.
Detection of flavonoids
(Lead acetate test):
To the 1 mL of filtrate, a few
drops of 10% lead acetate solution were added. Yellow colour precipitate was
obtained indicating the presence of flavonoids.
Detection of terpenoids
(Salkowski’s test):
To the 2 mL of extract was
mixed with 2 mL of chloroform, and concentrated sulphuric acid was carefully
added to form a layer. A reddish brown coloration at the inter face was formed
indicating the presence of terpenoids.
Detection of saponins
(Foam test):
To the 2 mL of extract was
diluted with 10mL of distilled water and it was agitated for few minutes. Foam
was produced indicating the presence of saponins.
Detection of phytosterols
(Salkowski’s test):
50 mg of the extract was
dissolved in 5mL of chloroform separately and then subjected to Salkowski test.
The prepared filtrates were treated with a few drops of concentrated sulphuric
acid, shaken and allowed to stand. Golden yellow colour was produced indicating
the presence of sterols.
Detection of phenols
(Ferric chloride test):
1 mL extract was treated
with a few drops of ferric chloride solution. Formation of bluish black colour
indicates the presence of phenols.
Detection of tannins
(Gelatin Test):
To the 1 mL of extract, 1%
gelatin solution containing sodium chloride was added. Formation of white
precipitate indicates the presence of tannins.
Antimicrobial activity:
The antimicrobial property of
the two spices were tested using the disc plating method. The inoculum prepared
were used for surface inoculation spread plate technique on a petri dish
containing 10ml of nutrient agar or potato dextrose agar. 0.3ml of inoculum was
spread on each petri dish equally with the help of L-rod. Bacterial strains
were inoculated on nutrient agar plates and fungus and yeast were inoculated on
potato dextrose agar plates. All the experiments were repeated to avoid
false-positive and false-negative results. All the plates were incubated at
37°C for overnight. The area of zone of inhibition were observed after 24
hours.
RESULTS:
Preliminary phytochemical analysis:
The methanolic and aqueous
extracts of Cardiospermum halicacabum and
Solanum nigrum were tested and the
presence of carbohydrates, amino acids, proteins, flavonoids, terpenoid,
saponin, phytosterol, phenol and tannin were reported in Table 1. In the
aqueous and methanolic extract the terpenoid and phenol was absent in S. nigrum. Various tests has been
conducted to find out the phytochemical constituents and the results are
tabulated below.
Table 1: Showing the presence or absence of compounds
in C. halicacabum and S. nigrum
|
S.
No.
|
Test
|
Cardiospermum halicacabum
|
Solanum nigrum
|
|
Aqueous
|
Methanol
|
Aqueous
|
Methanol
|
|
1.
|
Carbohydrates
|
+
|
+
|
+
|
+
|
|
2.
|
Amino acids
|
+
|
+
|
+
|
+
|
|
3.
|
Proteins
|
+
|
+
|
+
|
+
|
|
4.
|
Flavonoid
|
+
|
+
|
+
|
+
|
|
5.
|
Terpenoid
|
+
|
+
|
-
|
-
|
|
6.
|
Saponin
|
+
|
+
|
+
|
+
|
|
7.
|
Phytosterol
|
+
|
+
|
+
|
+
|
|
8.
|
Phenol
|
+
|
+
|
-
|
-
|
|
9.
|
Tannin
|
+
|
+
|
+
|
+
|
Anti-microbial activity:
Extraction method 1:
The two phytochemical samples
of both solvent using extract 1 method
(Table 2) showed good anti-microbial activity against E. coli, S. aureus, B. subtillis, P. putida (Fig.
3)and A. niger no anti-microbial
activity was seen against S. cerevisiae (Fig 5).The inhibition of E. coli by
aqueous and methanol extract showed high zone of inhibition in C. halicacabum. The high inhibition of S. aureus in aqueous extract showed
almost same zone while in methanol extract C.
halicacabum showed high zone of inhibition. The aqueous and methanol
extract showed equal zone of inhibition for B.
subtilis. C. halicacabum showed high zone against P. putida in both the extract (Fig 3 (iv) ).
Extraction method 2:
Similar results were also
seen for extract 2 (Table 3) over two samples and two solvents except at
aqueous extract of C. halicacabum no
activity was observed for B. subtilis and
in the S. nigrum of aqueous extract
no activity was observed at the S. aureus.
The aqueous extract showed same zone of inhibition for both the herbs
against E.coli where as in methanolic
extract S.nigrum showed high zone of
inhibition. Methanolic extract showed inhibition against S. aureus in both samples. Methanolic extract against B.subtilis showed high zone of
inhibition for C. halicacabum.
Inhibition of P. putida in aqueous
extract showed same zone of extraction for both while in methanol extract C. halicacabum showed high zone of
inhibition (Fig 4).
Fig 5 Resistance of S.cerevisiae
by extraction 1 and 2
Shows the resistance of Saccharomyces cerevisiae using the
extraction method one and two. (a- Cardiospermumhalicacabum,
b- Solanumnigrum) Left – aqueous
extract, right- methanol extract, top – Extraction method one, bottom –
extraction method two.
DISCUSSION:
Phytochemicals are bioactive
chemicals of the plant origin. They are regarded as the secondary metabolites
as their need is less them. The quantity and quality of phytochemicals present
in different parts of the plant differ12. Successful determination
of biologically active compounds from plant material is largely dependent on
the type of solvents used in the extraction method13.
In the present study aqueous
and methanol extract of C. halicacabum showed
presence of carbohydrates, amino acids, proteins, flavonoid, terpenoid,
saponin, phytosterol, phenol and tanin.Similar results was reported by Suresh1
in C. halicacabum stem and the
phytochemical analysis bymethanolic extract analysed for the compounds such as
alkaloids, cardiac glycosides, flavonoids, glycosides, saponins, steroids and
tannins of which glycosides was absent. In a study by Deepan14aqueous
and alcoholic extract of C. halicacabum
showed the presence of six compounds alkaloids, carbohydrates, saponins,
proteins and free amino acids, lignin and polysterol and absence of other five compounds
oils and fats, tannins and phenolic compounds, gums and mucilage, flavonoids
and glycosides.
Phytochemical and
antibacterial activities of C. halicacabum
leaf extract by Gopal15 showed the presence of triterpenoids,
phenolics, ferric chloride gelatinxantho protein, and carbohydrate; and absence
of alkaloids, lead acetate, anthracene, steroids etc.
The next plant S. nigrum showed the presence of all
phytochemicals as that of C. halicacabum
except for terpenoids and phenol in both the extract of aqueous and methanol.
Similarly the leaves of S. nigrum
showed absence of terpenoids and volatile oils whereas large quantity of
alkaloids, saponins, glycosides and flavonoids; medium quantity of tannins and
coumarins16.
Phytochemical investigation
of S. nigrum using hexane and benzene
extracts showed the presence of saponins, phytosterols, tannins and fixed oils
and fats. The ethanolic and the aqueous extract showed carbohydrates,
flavonoids, coumarins and phytosterol17.
While comparing the
phytochemicals extracted from C. halicacabum
and S. nigrum the later one
showed more compounds in the selected extraction method and solvents.
Plants have been playing a
very important role in human medicine. Several medicinal plants have been tried
against pathogenic microorganisms18.
S. nigrum extract observed by method I showed activity against all the bacterial
and fungal strain but no activity was seen for the yeast S. cerevisiae. The extract from method II showed the absence of S. cerevisiae in both the aqueous and
methanol while S. aureus was absent
in aqueous extract.
The antibacterial property of
C. halicacabum showed a maximum
activity against S. aureus gram
positive bacteria and the least activity was observed against Proteus vulgar is a gram negative
bacteria. The moderate activity was seen against B. subtilis, E.coli and Klebsilla
pneumonia1.
The ethanolic extract of S. surattnese Burm. F. leaf showed
varying level of antibacterial activity in which the inhibitory effect of
extracts was directly proportional to increasing concentration of extract.
Maximum zone of inhibition was obtained at 500 ug concentration of all bacteria
screened (S. aureus, Streptococcus sp.,
Bacillus subtilis, E. coli, P. aeroginosa, Salmonella tyhpi and Vibrio cholerae)
except Shigella dysenteriae where no
inhibitory effect was seen between any concentration19.
The study by Abbas20
in S. nigrum and S. xanthocarpum, the fruit extract of S. xanthocarpum has more
antimicrobial activity against gram negative bacteria (Salmonella typhi, Pasteurella maltocida, E. coli, Klebsiella pneumonia,
Vibrio cholerae) as well as antifungal activity (Aspergillus niger, A. flavus, A. fumigatus), whereas S. nigrum was more potent against gram
positive bacteria (Micrococcus varians,
M. luteus, Staphylococcus aureus) this is because S. xanthocarpum contains carpesterol and other steroidal glycoside
which are absent or present in little quantity in S. nigrum.
In a study by Skrinjar21
out of 27 essential oils (EOs)thirteen were active against at least for
one bacterial strain. Among all Armoracia
rusticana, inhibited both gram- positive and negative strains and Allium sativam was more active against
gram positive bacteria than gram negative bacteria. S. aureus was the most susceptible bacterium followed by E. coli, L. monocytogenes, S. enteritidis and
P. aeruginosa. EOs from horseradish,
garlic, oregano and thyme etc. were used to fight against food borne bacterial
pathogens.
CONCLUSION:
In the present study, the
methanol extracts showed higher activity than the aqueous extracts in both the
extraction types. Extraction using shaker showed more inhibition than the other
when compared to each other. They are effective against gram positive, gram
negative bacteria but less effective against fungus which can be taken as a
proof for their wide spectrum.
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