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*Author for correspondence; E-mail: shaziz2408@yahoo.com; Tel: 088-01713005011;
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Int. J. Chem. Sci.: 12(4), 2014, 1328-1336
ISSN 0972-768X
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PHYTOCHEMICAL AND ELEMENTAL SCREENING ON
LEAVES AND FLOWERS OF CATHARANTHUS ROSEUS : AN
IMPORTANT MEDICINAL PLANT OF BANGLADESH
SHAHIN AZIZ*, KOUSHIK SAHAa, NASIM SULTANAb,
SHAMIM AHMEDb and ABDULLAH AL-MANSURb
Chemical Research Division, BCSIR, DHAKA, BANGLADESH
aJahangirnagar University, SAVAR, BANGLADESH
bBangladesh Council of Scientific and Industrial Research (BCSIR) Laboratories,
DHAKA, BANGLADESH
ABSTRACT
Qualitative analysis of Catharanthus roseus plant confirms the presence of various
phytochemicals like alkaloids, flavonoids, terpenoids, saponins, steroids, carbohydrates, anthaquinone
glycosides etc in different extracts of its leaves and flowers. Some minerals have also been identified in
the leaves and flowers of the plant by ICP-MS techniques.
Key words: Phytochemical screening, Catharanthus roseus, Plant species.
INTRODUCTION
Bangladesh is a rich storehouse of medicinal plants. All secondary metabolites in
natural products can be termed bioactive molecules, as every diverse molecule possesses one
or multiple kinds of pharmacological activities. The beauty of natural products is the
uniqueness in their chemical and structural diversity that in relation to their various
biological actions. Herbal medicine refers to use different parts of plants for medicinal
purpose. Herbal drugs played an important role in drug discovery and were the basis of most
early medicines. Catharanthus roseus commonly called Madagascar periwinkle is an
evergreen shrub or herbaceous plant, which exhibits the anticancer activity due to the
presence of vincristine and vinblastine1. The different parts of the medicinal plant,
Catharanthus roseus is used for medicinal purposes for thousands of years in India, or
subcontinetal. It is one of the chief herbs for treating dermatitis, abscesses, eczema, psoriasis,
Int. J. Chem. Sci.: 12(4), 2014 1329
sores, corns, ringworm, scabies, epilapsy, malaria, heart tonics and tumor. Mainly leaves and
flowering tops of the plant are used for the extraction of oil. This oil has been found to have
anti-bacterial and anti-yeast action. Researchers have been found that it can kill some
intestinal parasites and have mild antibiotic effects. It was also observed that the its leaves
are used extensively in folk medicine for decreasing sugar level of blood and showed
significant anti-hyperglycemic effect1-10. The literature review reveals that scientists are
much more interested to find out elements and organic compounds present in different parts
of the plants. Some phytochemical works have been done previously on this medicinal plant
in some other countries like India, Pakistan, Thailand etc. But in Bangladesh, no systematic
phytochemical screening was done on extracts from different parts of this plant so far. After
all, Bangladesh is not only geographically but zeologically different from other countries. It
is well known that physiological changes do occur in plants due to changes in geographical
sites, climatic and environmental conditions, which result in the production of nonidentical
plant metabolites in this plant grown in different geographical regions. By considering theses
facts and the medicinal values of the plant, phytochemical screening on different extracts
from leaves and flowers solvent extract of Catharanthus roseus was carried out along with
the identification of metals in dried leaves and flowers using ICP-MS techniques.
EXPERIMENTAL
Materials and methods
Collection of plant material
Fresh leaves and flowers of C. roseus were collected from the gardens of Botany
Department of Dhaka University, Bangladesh in June, 2013 and identified by the taxonomist
of Bangladesh National Herbarium, Dhaka, where a voucher specimen (No. = 39512) has
been deposited.
Preparation of the solvent extracts (Cold extraction)
Freshly collected leaves and flowers of C. roseus were dried in an oven at 38oC and
powdered by using a grinding machine. The powder of leaves (510 g) was extracted with
methanol at room temperature for 5 days. The filtrate was dried into a gummy mass using
rotary evaporator under reduced pressure. The methanol extract (40.0 g) was then triturated
by n-hexane (100 mL x 3), then by ethyl acetate (100 mL x 3) and finally by n-butanol (100
mL x 3). Then these extracts were dried by using a rotary evaporator to get n-hexane extract
(11.0 g), ethyl acetate extract (9.0 g) and n-butanol extract (8.5 g). The residual methanol
soluble part (11.5 g) was finally denoted as methanol extract. Powder of the flower (200 g)
was extracted successively with different solvents at room temperature. At first, it was
S. Aziz et al.: Phytochemical and Elemental Screening on….
1330
extracted with n-hexane for 5 days and the extract was dried to get a gummy mass (7.15 g)
using rotary evaporator. Then the residual part of the flower was extracted with
dichloromethane for 5 days and the extract was dried to gummy mass (5.80 g). Again the
residual part was extracted with methanol and the filtrate was dried under reduced pressure
to gummy mass (22.69 g). All solvents were of analytical grade and obtained from
commercial sources (Sigma-Aldrich, St. Louis, MO, USA).
Reagent preparation for the detection of different class of compounds
(a) 1% Picric acid: 1 mL of picric acid dissolved in 99 mL distilled water.
(b) Dragendroff’s reagent: It is used for the detection of alkaloids. 0.17 g Bismuth
nitrate was dissolved in 2 mL acetic acid solution and 8 mL of distilled water
was added. (Solution A). 4 g of potassium iodide was dissolved in 10 mL acetic
acid solution and 20 mL of distilled water was added (Solution B). Solution A
& B were mixed and made up to 100 mL with distilled water.
(c) Mayer’s reagent: It is used for the detection of alkaloids. Solution (A) was made
by dissolving 0.68 g mercuric chloride in 30 mL of distilled water. Solution (B)
was made by dissolving 2.5 g of potassium iodide in 10 mL of distilled water.
Solution A & B were mixed and the volume was adjusted to 100 mL with
distilled water.
(d) Molisch’s reagent: 10 g of α-naphthol was dissolved in 100 mL of 95% alcohol.
It is used for the detection of carbohydrates.
(e) Fehling’s solution: It is used for the detection of reducing sugar. 3.4650 g Copper
sulphate was dissolved in distilled water and the volume was made up to 50 mL
(Solution A). 17.30 g of potassium sodium tartarate and 5 g of sodium hydroxide
was dissolved in distilled water and volume was made up to 50 mL (Solution B)
with water. Two solutions were mixed in equal volume prior to use.
Test for qualitative estimation of bioactive compounds from different solvent
extracts of leaf and flower of Catharanthus roseus
The extracts of leaves and flowers of Catharanthus roseus were subjected to
qualitative analysis to detect the presence of different classes of chemical constituents in the
plant.
(i) Test for alkaloids
n-Hexane, ethyl acetate, n-butanol and methnol extracts of leaf & n-hexane,
dichloromethane and methanol extracts of flower of Catharanthus roseus were warmed
Int. J. Chem. Sci.: 12(4), 2014 1331
separately with 2% H2SO4 for two min. It was filtered and few drops of following reagents
were added, which indicated the presence of alkaloids.
(a) Dragendroff’s reagent: A red precipitation indicated the positive test.
(b) Mayer’s reagent: A creamy white colored indicated the positive test.
(c) Picric acid (1%): A yellow precipitation indicated the positive test.
(ii) Test for flavonoids
A small quantity of the extract was heated with 10 mL of ethyl acetate in boiling
water for 3 min. The mixture was filtered and the filtrates were used for the following test.
(a) The filtrate was shaken with 1 mL of dilute ammonia solution (1%). The layers
were allowed to separate. A yellow coloration was observed in ammonia layer
indicating the presence of flavonoid.
(b) The filtrate was shaken with 1 mL of 1% ammonium chloride solution, where
light yellow color was observed. It indicated the presence of flavonoid.
(iii) Test for carbohydrates
The extracts were shaken vigorously with water and then filtered. A few drops of
Molisch’s reagent was added to the aqueous filtrate, followed by vigorous shaking again.
Concentrated H2SO4 (1 mL) was carefully added to form a layer below the aqueous solution.
A brown ring at the interface indicated the positive test.
(iv) Test for saponins
A small quantity of different extracts was diluted with 4 mL of distilled water. The
mixture was shaken vigorously and then observed on standing for stable brake, which
indicated the positive test.
(v) Test for steroids
2 mL of acetic anhydride and 2 mL H2SO4 were added to the extracts. The color
changed from violet to blue or green, which indicated the presence of steroids.
(vi) Test for anthraquinone glycosides (Borntragers’s test)
Dil. H2SO4 was added to the extracts and boiled. Then it was filtered and cooled. To
the cold filtrate, 3 mL of benzene was added and mixed. The benzene layer was separated
S. Aziz et al.: Phytochemical and Elemental Screening on….
1332
and ammonia (2 mL) solution was added to it. A rose pink to red color in ammonical layer
was observed, which indicated positive test.
(vii) Test for cardiac glycosides (Legal’s test)
To each extract, 1 mL of pyridine and 1 mL of sodium nitroprusside solution were
added and observed. A deep red color was observed indicating the positive test.
(viii) Test for terpenoids (Salkowski test)
Each extract was mixed with 2 mL of chloroform and then concentrated H2SO4
(3 mL) was carefully added to form a layer. A reddish brown coloration at the interface
indicated positive result for the presence of terpeniods.
(ix) Test for gum and mucilages
Each extract was dissolved in 10 mL of distilled water and 25 mL of absolute
alcohol was added to it with constant stirring. White or cloudy precipitate indicated the
presence of gum and mucilages.
(x) Test for proteins and amino acids
Each extract was dissolved in 10 mL of distilled water and the filtrate was subjected
to test the presence of proteins and amino acids.
(a) Biuret test: 2 mL filtrate was treated with one drop of 2% copper sulphate
solution and then 1 mL of ethanol (95%) was added to it followed by excess
potassium hydroxide pellets. Pink color in the ethanolic layer indicated the
presence of proteins.
(b) Ninhydrin test: Two drops of ninhydrin solution (10 mg of ninhydrin in
200 mL of acetone) were added to 2 mL of aqueous filtrate. A characteristic
purple color indicated the presence of amino acids.
Determination of elements
Major minerals/elements serve as structural components of tissues and function in
cellular and basal metabolism, water and acid-base balance, clotting of blood and formation
of bones and teeth etc.11-15 Inductively Coupled Plasma-mass Spectrometry (ICP-MS)
(Franklin, USA) was used for the determination of elements in the dry powder of leaves and
flowers of Catharanthus roseus. Definite amount (10 g) of leaf and flower samples were
taken and kept in furnace at 400oC for 8 hrs. Then there was a formation of ash. The ash
Int. J. Chem. Sci.: 12(4), 2014 1333
samples were transferred into a mixture of HNO3 and HClO4 (2:1), which was prepared
earlier in a Kjeldahl flask and left overnight. Before starting digestion, ice bath was used for
cooling Kjeldahl flask. Then it was placed on heating mantle set at low temperature. Once
boiling was initiated, red orange fumes of NO2 were driven off. Gentle heating was
continued until HNO3 and H2O was driven off. At this point, effervescent reaction occurred
between organic material and HClO4. Then the flask was put on heating mantle, which was
at room temperature and digestion was allowed to proceed with occasional heating from
mantle. It is important that the reaction between organic material and HClO4 should not to
go too fast, because then charring would occur. If charring occurred, immediately the flask is
placed in ice bath to stop digestion. Then I mL of HNO3 will added and resume gentle
boiling. After reaction of test portion with HClO4 was completed (identified by cessation of
effervescent due to reaction between organic material and HClO4). High heat was applied
for Ca for 2 min. Then the flask was removed from heating mantle and left to cool. Each
digest sample was transferred to 50 mL volumetric flask and diluted with H2O. Then each
digest sample from leaves and flowers of Catharanthus roseus was ready for elemental
analysis in ICP-MS spectrometry.
RESULTS AND DISCUSSION
The results of the qualitative analysis of different extracts from leaves and flowers of
Catharanthus roseus are presented in Table 1. The medicinal value of these plants lies in
some chemical substances that have a definite physiological action on human body. The
most important of these bioactive constituents of the plants are alkaloids, terpenoids,
flavonoids, steroids, cardiac glycosides and protein compounds. Results showed that the
polar compounds like alkaloids, carbohydrates, amino acids and glycosides are present in the
polar fractions of extracts from leaves and flowers. But flavonoids, terpenoids and steroids
are present in all the extracts; may be because of their polarity difference due to the
availability of various substituents in their structure.
Table 1: Results of screening various extracts from leaves and flowers of Catharanthus
roseus
Test for LH LE LBn LM FH FD FM
Alkaloids
(a) Dragendroff;s test
(b) Mayers test
(c) Picric acid test
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Cont…
S. Aziz et al.: Phytochemical and Elemental Screening on….
1334
Test for LH LE LBn LM FH FD FM
Flavonoids Positive Positive Positive Positive Positive Positive Positive
Carbohydrates Negative Positive Positive Positive Negative Negative Positive
Saponins Negative Negative Positive Positive Negative Negative Positive
Steroids Positive Positive Positive Positive Positive Positive Positive
Anthraquinone glycosides Negative Positive Positive Positive Negative Negative Positive
Cardiac glycosides Negative Positive Positive Positive Negative Negative Positive
Gum and mucilages Positive Positive Negative Negative Positive Positive Positive
Terpenoids Positive Positive Positive Positive Positive Positive Positive
Proteins and amino acids Negative Positive Positive Positive Negative Negative Positive
LH : Leaf n-hexane extract, LE : Leaf ethyl acetate extract, LBu-leaf butanol extract,
LM : Leaf methanol extract FH : Flower nahexane extract, F : Flower dichloromethane extract,
FM : Flower methanol extract
There was a tremendous legacy of folklore uses of different parts of Catharanthus
roseus in medicine. At present for the prevention of several diseases, there is an increasing
interest for the importance of dietary minerals. The trace elements, together with other
essential nutrients, are necessary for growth, normal physiological functioning and
maintaining life. They must be supplied by food, since the body can not synthesize them. So
it is necessary to find out, which elements are present in the selected plant. The results of
elemental detection in the leaves and flowers of Catharanthus roseus are presented in
Table 2. Results indicated the presence of Na, K. Fe, Ni, S and Cl2 in both; the leaves and
flowers.
Table 2: Minerals/elements detection (calculated on dry matter basis) by ICP-MS
techniques for leaves and flowers of Catharanthus roseus
Component as element Leaf Flower
Magnesium (Mg) Absent Absent
Calcium (Ca) Absent Absent
Sulphur (S) Present Present
Iron (Fe) Present Present
Sodium (Na) Present Present
Cont…
Int. J. Chem. Sci.: 12(4), 2014 1335
Component as element Leaf Flower
Chorine (Cl) Present Present
Potassium (K) Present Present
Manganese (Mn) Absent Absent
Chromium (Cr) Absent Absent
Lead (Pb) Absent Absent
Nickel (Ni) Present Present
Cadmium (Cd) Absent Absent
CONCLUSIONS
The plant studied here can be seen as a potential source of useful drugs. It also
justifies the folklore medicinal uses and claims about the therapeutic values of this plant as
curative agent. We therefore, suggest further the isolation, purification, and characterization
of the bioactive compounds of the leaf and flower part of Catharanthus roseus with a view
to obtain some useful chemotherapeutic agents.
ACKNOWLEDGEMENT
We are thankful to the former Director, BCSIR Laboratories, Dhaka, Mr. Abu Anis
Jahangir for providing necessary facilities to carry out this research work.
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Accepted : 23.07.2014