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Veni SN., et.al., A, Int. J. Pharmacol. Pharm. Sci. (2015) 2:3; 22-31 22
International Journal of Pharmacology and Pharmaceutical Sciences 2014; Vol: 2, Issue: 3, 22-31.
Review Article
RESEARCH ARTICLE ISSN: 2394-613X
Phytochemical profile of Codiaeum variegatum (L.) Bl.
Sangha R Bijekar1*, M.C.Gayatri2
1Department Molecular Biology, Bangalore University, Bangalore-India
2Department of Botany, Bangalore University, Bangalore - India
* Corresponding Author
Abstract
The present study investigates the qualitative and quantitative analysis of the major bioactive constituents of different parts like
root, stem and leaf of medicinally important plant Codiaeum variegatum using seven different solvents. Qualitative study of
alkaloids, carbohydrates, glycosides, steroids, flavonoids, coumarins, saponins, fatty acids, tannins, protein and amino acids, gum
and mucilage, terpenoids, anthroquinones and phenols showed different types of results in different solvents. Quantitative
estimation revealed that phytochemicals are in between the following range alkaloids in between the range of (4.66 -10.2%),
flavonoids (33.1-37.63%), saponins (11.36-13.76%), phenolics (35.43-39.76%), tannins (10.5-18.5%), terpenoids (27.56-
30.3%).
Key Words: Codiaeum variegatum, Euphorbiaceae, phytochemicals screening.
INTRODUCTION
Codiaeum variegatum (L.) Bl. ("garden croton" or "variegated croton"; syn. Croton variegatum L. is a species of plant in the genus
Codiaeum, which is a member of the family Euphorbiaceae. It is native to southern India, Sri Lanka, Indonesia, Malaysia, and the
western Pacific Ocean islands, growing in open forests and scrub (Huxley A, 1992) It is an evergreen shrub growing to 3 m tall and
has large, thick, leathery, shiny evergreen leaves, alternately arranged, 5–30 cm long and 0.5–8 cm broad. The inflorescences are
long racemes 8–30 cm long, with male and female flowers on separate inflorescences; the male flowers are white with five small
petals and 20–30 stamens, the female flowers yellowish, with no petals. The fruit is a capsule 9 mm diameter, containing three 6
mm seeds. The stems contain milky sap that bleeds from cut stems Croton ‘Gold Dust’ is a compact plant with elliptical to oval
leaves which are medium green and liberally dusted with paint-like specks of yellow. Mature crotons are "V" shaped. Crotons grow
best in well-drained soil and need to be shaded from the hottest sun of the day. Too much sun bleaches the color out of the leaves
and too little will make them more green. The leaves extracts of crotons are reported to have many medicinal properties including
purgative, sedative, antifungal, antiamoebic and anticancerous activities (Deshmukh & Borle, 1975; Kupchan et al., 1976). Anti-
inflammatory, Antifungal, antiamoebic and anticancerous activities (Olusola et al., 2007) Used to treat irregular menstruation
(Bourdya et al. 1992), wound healing (Sangeetha et al. 2011). A plant’s medicinal value is due to the presence of some chemical
substance that produces a physiological action on the Human body. These chemicals are of two types, primary and secondary
metabolites. Primary metabolites are those which are required for growth and development of plant and secondary metabolite are
the byproducts of metabolic pathway and play an important role in defense system. Secondary metabolites are such as alkaloids,
carbohydrates, glycosides, steroids, flavonoids, coumarins, saponins, fatty acids, tannins, protein and amino acids, gum and
mucilage, terpenoids, anthroquinones and Phenols. Study of these phytochemicals is crucial because these chemicals can be
exploited to synthesize future drugs.
MATERIAL AND METHODS
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Pharmaceutical Sciences
Veni SN., et.al., A, Int. J. Pharmacol. Pharm. Sci. (2015) 2:3; 22-31 23
Collection of Plant material
Codiaeum variegatum (L.) Bl. was collected from Lal Bagh Botanical garden, Bangalore and now plant is being maintained in the
Department of Molecular Biology, Bangalore University, Bangalore.
2.2 Preparation of plant extract
Leaf, stem, root and flowers were collected from Codiaeum variegatum. They were dried for one week at room temperature (in
shade). Dried plant parts were grinded in a blender to fine particles. Crude plant extract was prepared by Soxhlet extraction method.
Seven different solvents were used namely methanol, ethanol, water, chloroform, petroleum ether, hexane and acetone. 20gm of
dried fine grinded powder was uniformly packed into thimble and phytochemicals were extracted with 250mL of seven mentioned
solvents separately. The extraction was carried out for 24 hours. Later extract was concentrated by keeping it on hot plate at 30 to
40°C and stored at 4°C for further research.
Phytochemical profiling
Qualitative study
Test for alkaloids (Gibbs 1974)
The test solution of the extracts was dissolved in chloroform and the solution was extracted with Dil. HCL or H2SO4 and acid layer
taken and tested for presence of alkaloids.
Dragendroff’s test
To 2mL of acid layer of test solution, add 2mL of Dragendroff’s reagent (potassium bismuth iodide solution) and 2mL of Dil. HCL.
An orange-red precipitate indicates the presence of alkaloids.
Mayer’s test
To the 1 mL of acid layer of test solution, add 1mL of Mayer’s reagent (potassium mercuric iodide solution). Whitish or cream
colored precipitate indicates the presence of alkaloids.
Wagner’s test
To the 1mL of acid layer of test solution, add 2mL of Wagner’s reagent (iodine in potassium iodide). Reddish brown colored
precipitate indicates the presence of alkaloids.
Anthraquinone (Borntrager’s test)
About 0.5 g of the test solution was taken into a dry test tube and 5 mL of chloroform was added and shaken for 5 min. The extract
was filtered and the filtrate shaken with equal volume of 10% ammonia solution. A pink violet or red color in the ammoniacal layer
(lower layer) indicates the presence of anthroquinone (Aiyelaagbe et al. 2009)
Test for carbohydrates
Molisch’s test
To 2mL of the test solution, add 1mL of α-napthol solution, add concentrated sulphuric acid (H2SO4) through the sides of the test
tube. Purple or reddish violet color at the junction of the two liquids reveals the presence of carbohydrates.
Barford’s test
Veni SN., et.al., A, Int. J. Pharmacol. Pharm. Sci. (2015) 2:3; 22-31 24
To 0.5mL of test solution, add 3mL of Barfoed's solution, Place the tubes in a boiling water bath A rusty or brownish-red color will
indicate monosaccharides.
Benedicts test
To 0.5mL of test solution, add 3 ml of Benedict's reagent. Shake each test tube to assure thorough mixing. Place the tubes in a
boiling water bath for 3 minutes. Red or green or yellow ppt obtained shows presence of reducing sugar
Cardiac glycosides (Keller Killiani’s test)
About 100 mg of test solution was dissolved in 1 mL of glacial acetic acid containing one drop of ferric chloride solution. This was
then underlayer with 1 mL of concentrated Sulphuric acid. A brown ring obtained at the interface indicated the presence of Cardiac
glycosides (Aiyelaagbe et al, 2009)
Coumarins test
In a test tube, 1 g of each of the extracts were placed and covered with filter paper moistened with dilute sodium hydroxide (NaOH),
then heated on water bath for a few minutes. The filter paper was examined under UV light, yellow fluorescence indicated the
presence of coumarins (El-Tawil et al., 1983)
Fatty acids test:
5mL of test solution was mixed with 5mL of ether. This extract was allowed to evaporate on filter paper and dried the filter paper.
The transparency on filter paper indicates the presence of fatty acids (Chandrashekar and Rao, 2013)
Test for Flavonoids
Shinoda’s test
About 1 g of each of the extracts was dissolved with 5 ml of ethanol (98 %). To this a small piece of magnesium foil metal was
added, this was followed by drop wise addition of concentrated hydrochloric acid. Intense cherry red colour indicated the presence
of flavonones. Orange red colour indicated the presence of flavonols (Brain et al., 1975).
Lead acetate test
Few drops of lead acetate solution were added to each of the extracts in test tubes. Formation of yellow coloured precipitate indicated
the presence of flavonoids (Tiwari et al., 2011).
Alkaline reagent test
About 1 ml test solution was treated with few drops of sodium hydroxide solution and observed for intense yellow coloration which
disappeared on the addition of dilute HCl. (Veena et al. 2013)
Detection of Gum and Mucilage
The plant extract was dissolved in 10mL of distilled water and to this; 25 mL of absolute alcohol was added with constant stirring.
White or cloudy precipitate indicated the presence of gums and mucilages (Sai Koteswar Sarma et al. 2011).
Ninhydrin test
Veni SN., et.al., A, Int. J. Pharmacol. Pharm. Sci. (2015) 2:3; 22-31 25
Ninhydrin test: Add two drops of freshly prepared 0.2% ninhydrin reagent (0.1% solution in n-butanol) to the small quantity of
extract solution and heat. Development of blue color reveals the presence of proteins, peptides, or amino acids. (Saxena Mamta et
al. 2012)
Test for Phenols
Ferric chloride test: To 10mL of alcoholic solution of extract, 2mL of distilled water followed by drops of 10% aqueous Ferric
chloride (FeCl3) solution were added. Formation of blue colour indicates the presence of phenols (Chandrashekar and Rao, 2013)
Test for Saponins
About 0.5 g of the plant extract was shaken with water in a test tube. Frothing, which persist on warming was taking as a preliminary
evidence for the presence of saponin. Few drops of olive oil was added to 0.5 g of the extract and vigorously shaken. Formation of
soluble emulsion in the extract indicates the presence of Saponin (Odebiyi and Sofowora, 1978)
Test for Steroids
Liebermann Burchard Test:To 1mL of extract, 1mL of glacial acetic acid and 1mL of acetic anhydride and two drops of concentrated
sulphuric acid were added. The solution becomes red, then blue and finally bluish green, indicates the presence of steroids (Seema
Firdouse and Parwez Alam 2011)
Tannins test
The extract of the sample was treated with 15% ferric chloride test solution. The resultant colour was noted. A blue colour indicated
the presence of hydrolyzable tannin. Or into 10 mL of freshly prepared potassium hydroxide (KOH) in a beaker, 0.5 g of the extract
was added and shaken to dissolve. A dirty precipitate observed indicates the presence of tannin (Odebiyi and Sofowora, 1978;
Sofowora, 1982).
Test for terpenoids
Salkowski test: To 0.5 mL of each the extract was added 2mL of chloroform. Three mL of concentrated sulphuric acid (H2SO4)
was carefully added to form a layer. A reddish brown colouration of the interface indicates the presence of terpenoids. (Sofowora,
1982)
Phytochemical quantitative test
The extracts were subjected to quantitative phytochemical tests for plant secondary metabolites such as alkaloids, flavonoids,
saponins, phenolics, tannins and terpenoids.
Alkaloid determination using Harborne (1973) method
In 250mL conical flask, 5g of the dried fine powdered sample is taken and 200 ml of 10% acetic acid in ethanol was added and
covered and allowed to stand for 4h. This was filtered and the extract was concentrated on a water bath to one-quarter of the original
volume. Concentrated ammonium hydroxide was added dropwise to the extract until the precipitation was complete. The whole
solution was allowed to settle and the precipitated was collected and washed with dilute ammonium hydroxide and then filtered.
The residue is the alkaloid, which was dried and weighed.
Flavonoid determination by the method of Bohm and Kocipai- Abyazan (1994)
10 g of the plant sample was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The whole solution
was filtered through whatman filter paper No 42 (125 mm). The filtrate was evaporated into dryness over a water bath and weighed.
Veni SN., et.al., A, Int. J. Pharmacol. Pharm. Sci. (2015) 2:3; 22-31 26
Saponin determination (Obadoni and Ochuko, 2001)
In conical flask, 20 g of dries fine particles plant sample was takem and 100 mL of 20% aqueous ethanol was added. This mixture
was heated (55˚C) on water bath for 4 h with continuous stirring. Later the mixture was filtered and the residue reextracted with
another 200 ml of 20% ethanol. This extracts was further reduced to 40 mL over hot water bath (90˚C). The concentrated extract
was transferred into a 250 ml separating funnel and 20 ml of diethyl ether was added and shaken vigorously. Ether layer was
discarded and aqueous layer was collected. This step of purification was repeated. 60 ml of n-butanol was added. The combined n-
butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a waterbath.
After evaporation the samples were dried in the oven and weighed.
Determination of tannin content of the sample (Van Buren, J. P. and Robinson, W. B. 1981):
In 250mL conical flask, 5g of powdered plant sample was taken and 50mL of distilled water was added and shook it vigorously for
an hour. Later this solution was filtered into a volumetric flask and 5mL of this filtrate is pipetted out into a test tube. The sample
were incubated for 1.5 hours at 20 – 300 C and the sample was then filled with distilled water up to mark of 50 mL of the volumetric
flask. Tannic acid was used as standard; 0.1g of tannic acid was dissolved in 100 mL of water to form tannic acid solution. Distilled
water was used as blank. The absorbance of the samples was measured at 760 nm. The values generated were used to calculate the
tannin content.
Determination of total phenols (Edeoga et al., 1983 and Jing-Chung et al., 2007):
The sample was boiled with 50 ml of ether for the extraction of the phenolic component for 15 min. 5 ml of the extract was taken
into a 50 ml flask, then 10 ml of distilled water was added. 2 ml of ammonium hydroxide solution and 5 ml of concentrated amyl
alcohol were also added and left to react for 30 min for colour development. This was measured at 505 nm. The standard curve was
prepared using 0, 50, 100, 150, 200, 250 mg/l solutions of gallic acid in methanol: water (50:50 v/v)
RESULTS AND DISCUSSION
Codiaeum variegatum is an ornamental plant and also known for its medicinal properties. The medicinal properties of plants are
due to the phytochemicals. These phytochemicals are the secondary metabolites which are produced in high quantity under stressed
conditions, which allows the plant to protect itself from adverse environmental factors. Dietary intake of these phytochemicals may
promote health benefits, protecting against chronic degenerative disorders, such as cancer, cardiovascular and neurodegenerative
diseases. Majority of foods, such as whole grains, beans, fruits, vegetables and herbs contain phytochemicals. These phytochemicals,
either alone or in combination, have tremendous therapeutic potential in curing various ailments (Bhatt et al., 1982). The results of
qualitative test of phytochemicals are presented in table 1. Different phytochemicals viz. alkaloids, anthroquinone, carbohydrates,
cardiac glycosides, coumarins, fatty acids, flavonoid, gum and mucilage, protein and amino Acids, phenols, saponin, steroids,
tannins and terpenoids were found to be present in different solvents depending upon their solubility. This result agrees with the
findings of Sangha R.B. et al., (2014) on qualitative phytochemical work carried on Baliospermum montanum (member of
Euphorbiaeae family).
Table 1- Qualitative analysis of different parts like root, stem, leaf, flower and latex of Codiaeum variegatum using
different Solvents-
Plant name
W
Ac
Et
Mt
Ch
Hx
Eth
Tests
Phytochemicals
Cv(leaf)
-
-
+
-
-
-
+
Dragendorff’s
Cv (stem)
-
-
+
-
-
-
+
Cv (roots)
-
-
+
-
-
-
+
Veni SN., et.al., A, Int. J. Pharmacol. Pharm. Sci. (2015) 2:3; 22-31 27
Cv(leaf)
-
-
+
-
-
-
+
Mayer’s test
Alkaloids
Cv (stem)
-
-
+
-
-
-
+
Cv (roots)
-
-
+
-
-
-
+
Cv(leaf)
-
-
+
-
-
-
+
Wagner’s test
Cv (stem)
-
-
+
-
-
-
+
Cv (roots)
-
-
+
-
-
-
+
CV (leaf)
-
-
+
-
+
+
+
Borntreger’s test
Anthroquinones
CV (stem)
-
-
+
-
+
+
+
CV (roots)
-
-
+
-
+
+
+
Cv(leaf)
+
-
+
+
-
-
+
Molisch’s test
Carbohydrates
Cv (stem)
+
+
+
+
-
-
-
Cv (roots)
-
+
+
+
-
-
-
Cv(leaf)
+
-
+
+
-
-
+
Barford’s test
Cv (stem)
+
+
+
+
-
-
-
Cv (roots)
-
+
+
+
-
-
-
Cv(leaf)
+
-
+
+
-
-
+
Benedicts test
Cv (stem)
+
+
+
+
-
-
-
Cv (roots)
-
+
+
+
-
-
-
Cv(leaf)
+
+
+
+
+
-
-
Keller- Killiani
test
Cardiac Glycosides
Cv (stem)
+
+
-
+
-
-
-
Cv (roots)
+
+
-
+
-
-
Cv(leaf)
-
-
-
-
-
+
-
Using NaOH
Coumarins
Cv (stem)
-
-
-
-
-
-
+
Cv (roots)
-
-
-
-
-
-
-
Cv(leaf)
-
-
-
-
-
-
-
Paper test
Fatty acids test
Cv (stem)
-
-
-
-
-
-
-
Cv (roots)
-
-
-
-
-
-
-
Cv(leaf)
-
+
+
+
+
-
-
Shinoda test
Flavonoids
Cv (stem)
-
+
+
+
+
-
-
Cv (roots)
-
+
+
+
+
-
Cv(leaf)
+
+
+
+
+
+
-
Alkaline reagent
test
Cv (stem)
-
+
+
+
+
-
+
Cv (roots)
-
+
+
+
+
-
Cv(leaf)
+
+
+
+
+
-
-
Lead acetate test
Cv (stem)
-
-
+
+
+
-
+
Cv (roots)
-
-
+
+
+
-
-
Veni SN., et.al., A, Int. J. Pharmacol. Pharm. Sci. (2015) 2:3; 22-31 28
Cv(leaf)
-
-
-
-
-
-
-
Gum and Mucilage
Cv (stem)
-
-
-
-
-
-
-
Cv (roots)
-
-
-
-
-
-
-
Cv(leaf)
+
+
-
-
-
-
-
Ninhydrin test
Protein and Amino
Acids
Cv (stem)
+
-
-
+
-
-
-
Cv (roots)
+
-
-
+
-
-
-
Cv(leaf)
-
+
+
+
-
-
-
Ferric chloride
test
Phenols
Cv (stem)
-
+
+
+
-
-
-
Cv (roots)
-
+
+
+
-
-
-
Cv(leaf)
+
-
+
+
-
-
+
Frothing test
Saponins
Cv (stem)
+
-
+
+
-
-
-
Cv (roots)
+
-
+
+
-
-
-
Cv(leaf)
-
-
-
-
-
-
+
Liebermann
Burchard reaction
Steroids
Cv (stem)
-
-
-
-
+
+
+
Cv (roots)
-
-
+
-
-
-
Cv(leaf)
+
-
+
+
-
-
-
Using FeCl3
Tannins test
Cv (stem)
+
-
+
+
-
-
-
Cv (roots)
+
-
+
+
-
-
-
Cv(leaf)
-
+
-
+
+
-
-
Salkowski test
Terpenoids
Cv (stem)
-
-
-
-
-
-
-
Cv (roots)
-
-
-
-
-
-
-
CV= Codiaeum variegatum, W=water, Ac=Acetone, Et= Ethanol, Mt= Methanol, Ch= Chloroform, Hx=Hexane, Eth= ether, + =
present, - = absent
Fig:1- Quantitative estimation (%) Secondary metabolites of different parts of Codiaeum variegatum, n=3, Data is
presented as Mean± SD.
0
5
10
15
20
25
30
35
40
45
Quantitative estimation
Phytochemicals
leaf
stem
root
Veni SN., et.al., A, Int. J. Pharmacol. Pharm. Sci. (2015) 2:3; 22-31 29
Quantitative estimation revealed that alkaloid is in between the range of (4.66 -10.2%), flavonoids (33.1-37.63%), saponins (11.36-
13.76%), phenolics (35.43-39.76%), tannins (10.5-15.32%), terpenoids (27.56-30.3%). It has also found that the plant is rich in
flavonoids, phenols and terpenoids. The estimated quantitative data is presented in Fig 1.
The Codiaeum variegatum is known to have medicinal properties like anticancerous and anti-inflammatory properties. This study
revealed the presence of medicinal active constituents of Codiaeum variegatum. The phytochemicals like flavonoids, phenols and
terpenoids which are rich in Codiaeum variegatum may be contributing in providing the medicinal properties to the plant. The
inflammation is mainly caused by cyclooxygenase and lipooxygenase pathway. Phenolic compounds were shown to inhibit the
cyclooxygenase and lipooxygenase pathway (Ferrandiz et al. 1991; Ferrandiz et al. 1991; Laughton et al. 1991). Flavonoids are
found to inhibit Ornithine decarboxylase enzyme, it is rate-limiting enzyme in polyamine biosynthesis, which has been correlated
with the rate of DNA synthesis and cell proliferation in several tissues, hence inhibiting cell proliferation (Tanaka et al., 1997a;
Tanaka et al., 1997b; Makita et al., 1996). Flavonoids inhibit growth of microorganism by depolarizing membrane, inhibiting DNA,
RNA and protein synthesis (Dzoyem et al., 2013). Study of flavonoids, phenols and terpenoids from this plant may help us to exploit
more of the medicinal properties of Codiaeum variegatum.
CONCLUSION
The Codiaeum variegatum was screened for phytochemical constituents and found to be good source of medicinally active elements
which can be further exploit to isolate and synthesize modern medicines. This work justifies the need to isolate and characterize the
medicinally active compounds.
ACKNOWLEDGEMENT
UGC BSR Faculty fellow grant by UGC, New Delhi sanctioned to Dr. M.C.Gayatri is gratefully acknowledged.
Conflict of interest: ‘No conflict to be disclose’’.
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