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Phytochemical investigation and antioxidant screening of crude leaves extract

from Epipremnum aureum

Article · August 2015

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International Journal of Pharmacognosy and Phytochemical Research 2015; 7(4); Galley Proof

ISSN: 0975-4873
Research Article

Phytochemical Investigation and Antioxidant Screening of Crude

Leaves Extract from Epipremnum aureum
Sreemoy kanti Das*, Pinaki Sengupta, Mohd. Shahimi Mustapha, Md. Kifayatudullah, Md.
Gousuddin
Faculty of Pharmacy, Lincoln University College, Selangor, Malaysia

Available Online

ABSTRACT
Objective: Epipremnum aureum belonging to the family Araceae is commonly known as money plant having indoor air
pollution removing capacity. The aim of the present study was to investigate the presence of various phytochemical
constituents which is responsible for the medical activities of the plant. Methods: The leaves were successively extracted
in three different solvents viz. ethanol, acetone and chloroform. The phytochemical analysis of plant extract was performed
using thin layer chromatography and preliminary screening methods. Different concentration of the crude plant extract was
evaluated for antioxidant activity using DPPH scavenging activity and reducing power activity. Results: Preliminary
qualitative chemical test for different extract shows the presence of steroids, terpinoids, alkaloids, saponins, tannins and
flavonoids. All the three extracts were proven effective against free radicals. Ethanol extract was found to possess highest
antioxidant activity compared to acetone and chloroform. Conclusion: Thus the positive results suggest that Epipremnum
aureum extracts should be further studied to determine the bioactive chemical compounds as well as to understand the
possible mechanism of action and evaluate their toxicity looking towards pharmaceutical actions.

Keywords: Epipremnum aureum, DPPH, Reducing power, Phytochemical, Thin Layer Chromatography

INTRODUCTION phytochemical constituents. Many active

Plants, the sources of bioactive constituents have been phytoconstituents such as Flavonoids exhibits potential
used traditionally to cure various ailments in Ayurveda, antidiabetic and neuroprotective activity. Alkaloids are
Unani & Siddhi. Though, during last few years, synthetic used as anaesthetic agents5. Terpenoids exhibit various
drugs occupy the position for curing various diseases, but, important pharmacological activities such as anti-
due to their side effects, scientists are now focusing to inflammatory, anticancer, antihyperlipedic, anti-viral and
explore the potentiality of traditional medicines 1. Araceae anti-bacterial activities. The effective identification of
is a large family comprising of many therapeutically active phytoconstituents plays an important role in determining
medicinal plants. Epipremnum aureum is known by many the therapeutic activity. In this case the standardized thin-
names but most common is “Money Plant”. It is a large layer chromatographic procedures can be used effectively
root-climber belongs to the botanical family of Araceae for the screening analysis as well as quality evaluation of
and a common house plant with several cultivars and the plant or its derived herbal products. Though the
capable of removing indoor air pollutants such as xylene, traditional system of medicine has a long history of use but
formaldehyde and benzene2. Epipremnum aureum has they lack scientific evidence particularly in modern
shiny heart-shaped leaves and long slender stems, which scientific knowledge. Large number of medicinal plants
can grow up to 3m in length. However, the stems can be has been investigated for their antioxidant properties.
wound round sticks or attached to supports to keep the Natural antioxidants in the form of raw extract or their
plant from taking up too much space. The plant generally chemical constituents are very effective to prevent the
stands at a height of between 5m and 9m and has a total destructive processes caused by oxidative stress. If excess
spread of 1.5m to 2.5m. Epipremnum aureum produces ROS are not eliminated by antioxidant system, reactive
small green flowers in summer. This plant is widely known oxygen species (ROS) will exert oxidative damaging
in Malaysia and Singapore and has a reputation as a effects by reacting with mostly every molecules found in
traditional anticancer preparation as well as a remedy for living cells including lipid, amino acids and DNA. They
skin diseases3. A decoction of the fresh leaves with meat play important roles in aging and in the pathogenesis of
or eggs or as tea was reported to be a common practice age related disorders such as cancer, hypertension,
among the locals. Aerial roots and leaves of Epipremnum atherogenesis, neurodegenerative disorders such as
aureum show great potential for antimicrobial activity4. Alzheimer’s disease and Parkinson’s disease. Thus the
The medicinal plants are useful for healing as well as for present study aims to analyse different phytoconstituents
curing of human diseases because of the presence of

*Author for Correspondence

 Das at al. / Phytochemical Investigation and…

Table 1: Data showing the extractive value of Epipremnum aureum

Plant Name Part Used Method of Percentage yield %
extraction Ethanol Acetone Chloroform
Epipremnum aureum Arial Parts Hot Percolation 2.8g 2.1g 1.6g
using Soxhlet
Extraction

Table 2: Data showing the preliminary phytochemical screening of the various extracts of Epipremnum aureum
Sl No. Constituents Ethanol extract Acetone Extract Chloroform extract
1 Alkaloids + + +
2 Flavonoids + + +
3 Glycosides + + +
4 Sterols + - -
5 Terpinoids + + -
6 Tannins + - -
7 Fixed oils and Fats - + +
8 Phytosterols + - -
9 Quinones - - -
10 Coumarin - - -

Table 3: Flavonoid content of Epipremnun aureum Acetone Extract

plant extract The marc left after chloroform extract was dried and then
Extract Flavonoid content extracted with acetone (55-56°C) until extraction was
Ethanol 1.81 completed. After completion of extraction, the solvent was
removed by distillation. Dark brownish green colour
Acetone 1.65 residue was obtained. The residue was then stored in
Chloroform 1.47 dessicator.
Ethanol Extract
The marc left after chloroform extract was dried and then
present in the leaf extract of Epipremnum aureum and to extracted with ethanol 95% (75-78°C) until extraction was
evaluate its antioxidant potential. completed. After completion of extraction, the solvent was
removed by distillation. Dark brown colour residue was
MATERIALS AND METHODS obtained. The residue was then stored in dessicator.
Collection and authentication of Plant Phytochemical analysis of plant extract
The fresh whole plant of Epipremnum aureum was Test for Glycosides
collected from Kepong district, Malaysia. The plant was Keller Killiani Test – Test solution was treated with few
identified by Miss Tan Ai Lee, Research officer, Natural drops of glacial acetic acid and Ferric chloride solution and
products, Forest Research Institute Malaysia. The voucher mixed. Concentrated sulphuric acid was added, and
specimen (No. SBID: 001/15) was prepared and deposited observed for the formation of two layers. Lower reddish
in the Faculty of Pharmacy, Lincoln University College, brown layer and upper acetic acid layer which turns bluish
Malaysia for imminent reference. green would indicate a positive test for glycosides.
Preparation of crude drug for extraction Detection of alkaloids
The authenticated leaves were washed with fresh water and Extracts were dissolved individually in dilute
dried under shade of sunlight for 5 days. The dried plant Hydrochloric acid and filtered. a) Mayer’s Test: Filtrates
leaves were coarsely powdered with the help of were treated with Mayer’s reagent (Potassium Mercuric
mechanical grinder. The powder was stored in an airtight Iodide). Formation of a yellow coloured precipitate
container for further use. indicates the presence of alkaloids. b) Wagner’s Test:
Method of extraction Filtrates were treated with Wagner’s reagent (Iodine in
The crude Continuous hot percolation process by using Potassium Iodide). Formation of brown/reddish precipitate
Soxhlet apparatus was used for the extraction of the crude indicates the presence of alkaloids.
leaves of Epipremnum aureum. The extraction was carried Detection of phenols
out as per the polarity of the solvents with chloroform, Ferric Chloride Test: Extracts were treated with 3-4 drops
acetone and finally with ethanol6. of ferric chloride solution. Formation of bluish black
Chloroform Extract colour indicates the presence of phenols.
The shade dried coarsely powdered leaves of Epipremnum Detection of tannins
aureum (500g) was extracted with chloroform (58-62°C) Gelatin Test: To the extract, 1% gelatin solution containing
until the extraction was completed. After completion sodium chloride was added. Formation of white precipitate
extraction, the solvent was removed by distillation. Dark indicates the presence of tannins.
greenish yellow colour residue was obtained. The residue Detection of flavonoids
was then stored in dessicator. a) Alkaline Reagent Test: Extracts were treated with few

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 Das at al. / Phytochemical Investigation and…

Table 4: Preliminary detection of various phenolic compounds against reference compounds

Extract Compound Rf value of Sample Reference compound (Rf Value)
Caffeic acid 0.99, 0.75, 0.90 Caffeic acid (0.90)
Arial parts of
Rosmarinic acid 0.83, 0.78, 0.78 Rosmarinic acid (0.83)
Epipremnum
Ferulic acid (0.95)
aureum
Ferulic acid 0.91, 0.95, 0.96

Table 5: Antioxidant activities of plant extracts on DPPH free radical scavenging on UV visible Spectrophotometer at
517nm of wavelength
Sample Concentration Mean absorbance %age DPPH free radical
mg/l scavenging
Ascorbic acid 0.25 0.053±0.024 82%
0.75 0.032±0.018 89%
1.0 0.024±0.012 92%
Ethanol extract 0.25 0.179±0.071 42%
0.75 0.152±0.011*** 51%
1.0 0.122±0.045*** 60%
Acetone extract 0.25 0.216±0.027 30%
0.75 0.210±0.024 34%
1.0 0.179±0.017*** 42%
Chloroform extract 0.25 0.213±0.021 31%
0.75 0.188±0.011 39%
1.0 0.157±0.018*** 49%
Data are expressed as Mean ±SD. Values are considered as significant at ***p<0.001, when compared to control N= 3

drops of sodium hydroxide solution. Formation of intense The TLC plates were prepared by using silica gel G coated
yellow colour, which becomes colourless on addition of over glass plates of 10X10 cm dimension. Silica gel plate
dilute acid, indicates the presence of flavonoids. b) Lead was impregnated by dipping into 4 % solution of sodium
acetate Test: Extracts were treated with few drops of lead acetate in methanol –water 3:2 for 5s followed by drying
acetate solution. Formation of yellow colour precipitate at room temperature. The plates were activated by heating
indicates the presence of flavonoids. the plates at 100°C to 110°C which is necessary for linear
Detection of phytosterols movement of solutes over stationary phase. Glass
a) Salkowski’s Test: Extracts were treated with chloroform capillaries were used to spot the sample extract at distance
and filtered. The filtrates were treated with few drops of of 1 cm at 3 track. In this case Chloroform – ethyl acetate
Conc. Sulphuric acid, shaken and allowed to stand. – formic acid, 5:4:1 and ethyl acetate – methanol – water,
Appearance of golden yellow colour indicates the presence 77:13:10 after pre-saturation with mobile phase for 20 min
of triterpenes. for development were used8. Solutions of standard
Test for Terpenoids substances caffeic acid, rosmarinic acid and ferulic acid,
2 ml of the organic extract was dissolved in 2 ml of were prepared by dissolving of 10 mg in 1 ml of distilled
chloroform and evaporated to dryness. 2 ml of water. Substances were identified using UV detection at
concentrated sulphuric acid was then added and heated for 254 nm. Visualization was carried out by spraying with
about 2 min. Development of a greyish colour indicates the solution of iron III chloride (2% methanol) and aluminium
presence of terpenoids. chloride (1% in ethanol) a blue or light blue spot indicates
Tests for steroids the presence of caffeic acid, Rosmarinic acid and ferulic
A red colour produced in the lower chloroform layer when acid9.
2 ml of organic extract was dissolved in 2 ml of chloroform The Rf value of the sample was calculated and compared
and 2 ml concentrated sulphuric acid was added in it, with the Rf value of the standard compounds. The
indicates the presence of steroids. ii. Development of a identified compounds with the respective Rf values were
greenish colour when 2 ml of the organic extract was depicted in table no.3.
dissolved in 2 ml of chloroform and treated with sulphuric Determination of total flavonoids by colorimetric method
and acetic acid indicates the presence of steroids. All the three crude extracts were used to determine the total
Test for Quinones flavonoids contents. The total flavonoids contents of
Dilute NaOH was added to the 1 ml of crude extract. Blue different crude extracts were estimated by aluminium
green or red coloration indicates the presence of quinones. chloride colorimetric method10. Sodium nitrate (2.5 g) was
Test for Coumarin taken in a volumetric flash (50 mL) and added water upto
10 % NaOH was added to the extract and chloroform was the mark that was 5% sodium nitrate. Sodium hydroxide
added for observation of yellow color, which shows the (2.5 g) was taken in another volumetric flash (50 mL) and
presence of Coumarin7 added water upto the mark that was 4% sodium hydroxide.
Qualitative analysis of phenolic acids by Thin Layer Then 10% aluminium chloride solution was prepared the
Chromatography same procedure. The different crude extracts (0.25 mg)

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 Das at al. / Phytochemical Investigation and…

100
ascorbic acid
80 ethanol extract
chlorofom extract
% Inhibition

60 acetone extract

40

20

0
0.0 0.5 1.0 1.5
Concentration (mg/ml)

Figure 1: DPPH radical scavenging activities of the various extracts of Epipremnum aureum and ascorbic acid. Result
represents means of triplicates of different concentrations analyzed.
1.0
Ascorbic acid
0.8 Ethanol extract
chloroform extract
absorbance

0.6 acetone extract

0.4

0.2

0.0
1
25

75
0.

0.

Concentration (mg/ml)

Figure 2: Reducing power activities of the various extract of Epipremnum aureum comparison with ascorbic acid.

were taken in a test tube and added water (1.25 mL) and 100 μl of the sample at various concentrations (0.25, 0.75
sodium nitrate (0.75 µL) then mixed together. All the test and 1 mg/ml). The reaction mixture was shaken well and
tubes were kept in the dark place for 6 min. Then 10% incubated in the dark for 15 min at room temperature. Then
aluminium chloride (0.150 µL) was added into the test tube the absorbance was taken at 517 nm. The control was
and wait for 5 min in the dark for complete reaction. prepared as above without any sample. The scavenging
Finally, 5% sodium hydroxide (0.5 mL) and water (0.275 activity was estimated based on the percentage of DPPH
mL) were added to the test tube. The absorbance was radical scavenged. Lower absorbance of the reaction
measured of all samples at a fixed wavelength 510 nm mixture indicates higher free radical scavenging activity.
using UV spectrophotometer. Quercetin standard was used The percentage of inhibition was calculated by the
for the calibration curve. The estimation of total flavonoids following:
contents in the crude extracts was carried out in triplicate Scavenging (%) = [A1 – A2 / A1] X 100
and the results were averaged. The total flavonoid was A1 = Absorbance of Control
calculated by the following formula: A2 = Absorbance of sample
X= (A. mo)/(Ao. m) Reducing power
Where “X” is the flavonoid content, mg/g plant extract, The reducing power was based on Fe (III) to Fe (II)
“A” is the absorption of plant crude extract solution, “Ao” transformation in the presence of the solvent fractions13.
is the absorption of standard quercetin solution, “m” is the The Fe (II) can be monitored by measuring the formation
weight of crude drug extract in mg and “mo” is the weight of Perl’s Prussian blue at 700 nm. Various concentrations
of quercetin in the solution in mg. of the sample (2 ml) were mixed with 2 ml of phosphate
Antioxidant evaluation buffer (0.2 M, pH 6.6) and 2 ml of potassium ferricyanide
DPPH scavenging activity (1%). The mixture was incubated at 50°C for 20 min
The free radical scavenging activity of the fractions was followed by addition of 2 ml of trichloroacetic acid (100
measured in vitro by 2, 20 - diphenyl-1-picrylhydrazyl mg/l). The mixture was centrifuged at 3000 rpm for 10 min
(DPPH) assay11. The stock solution was prepared by to collect the upper layer of the solution. A volume of 2 ml
dissolving 24 mg DPPH with 100 ml methanol and stored from each of the mixture earlier mentioned was mixed with
at 20°C until required. The working solution was obtained 2 ml of distilled water and 0.4 ml of 0.1% (w/v) fresh ferric
by diluting DPPH solution with methanol and the chloride. After 10 min reaction, the absorbance was
absorbance obtained was 0.3098±0.02 at 517 nm using the measured at 700 nm. Higher absorbance of the reaction
spectrophotometer which was taken as absorbance of mixture indicates a higher reducing power.
control12. A 3 ml aliquot of this solution was mixed with

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 Das at al. / Phytochemical Investigation and…

RESULTS AND DISCUSSION extract may be attributed due to the presence of phenolic
The phytoconstituents were extracted by using different acids which were confirmed by TLC profiling. In
solvents of increasing polarity like chloroform, acetone comparison to the standard ascorbic acid the ethanolic
and finally with ethanol. The extractives value was given extract showed more than 60% of free radical scavenging
in Table 1. efficacy; whereas acetone soluble fraction shows least
The results confirm the presence of constituents which are antioxidant efficacy of about 30% free radical scavenging
known to exhibit medicinal as well as physiological activity. The ethanolic extract showed the highest
activities. The qualitative phytochemical screening percentage of inhibition at a concentration of 1mg/l
revealed the ethanolic extract richness in tannins, saponins, whereas the lowest inhibition percentage of 42% as shown
terpinoids, flavonoids, phytosterols, glycosides and in (figure 1) was observed at a concentration of 0.025 mg/l.
alkaloids. It was found that the chloroform extract contains The acetone and chloroform extract also followed the
fixed oils and fats which was absent in ethanolic extract. concentration dependent activity as shown in (table 4).
Moreover quinones and coumarin were undetected in any Reducing power is associated with antioxidant activit
of the extracts. The phytochemical characteristics of the y and may serve as a significant reflection of the ant
aerial plant extract of Epipremnum aureum investigated ioxidant activity. Compounds with reducing power ind
are summarized in table 2. icate that they are electron donors and can reduce the
Typical phenolics that possess antioxidant activity have oxidized intermediates of lipid peroxidation processes,
been characterized as phenolic acids and flavonoids. so that they can act as primary and secondary antioxi
Phenolic acids have repeatedly been implicated as natural dants. It was observed that the reducing ability of the
antioxidants in fruits, vegetables, and other plants. extract was dose dependent and all the three extract
Flavonoids are hydroxylated phenolics and are potent showed an increase in absorbance with the increase in
water-soluble antioxidants which help in radical dose. The ethanolic extract showed an absorbance of 0.603
scavenging and prevention of oxidative cell damage. which was higher acetone and chloroform extract. The
Moreover they are found to be effective in scavenging free results of reducing power activity reveal that ethanolic
radicals as a result of their redox properties that allow them extract has higher antioxidant potential as compared to
to act as reducing agents. In the present study among the acetone and chloroform extract. In case of both DPPH
three crude extracts from epipremnum aureum, ethanol scavenging activity and reducing power activity the three
extract was found be containing highest amount of extracts were found to dose dependent which was directly
flavonoids content compounds followed by acetone and proportional to concentration. The results strongly suggest
chloroform extract. that phenolic compounds are important components of this
The result of total flavonoid contents of the three crude plant, and some of its pharmacological effects could be
extracts of Epipremnum aureum is given in table 3. Among attribute to the presence of these valuable constituents.
the three crude extracts, ethanol extract contained the
highest (1.81 mg/g) amount of flavonoids content CONCLUSION
compounds followed by acetone (1.65 mg/g), chloroform The present study clearly indicates that the plant
(1.47 mg/g). The variation may be due environmental Epipremnum aureum is a rich source of active
conditions, which can modify the constituents of the plant. phytoconstituents responsible for pharmacological
The qualitative determination of constitutes by TLC activities. The results of the present study suggest that
analysis showed the presence of three different phenolic tested plant extracts have moderate to potent antioxidant
compounds caffeic acid, ferulic acid and rosmarinic acid activity and or free radical scavenging activity.
in the investigated extracts. The phenolic compounds may Nevertheless, the great antioxidant potential will be of
contribute directly to antioxidative effect13. The Rf values immense benefit from the consumption of these medicinal
of the obtained spots were compared with the Rf values of plant extracts. The qualitative TLC analysis of the three
the standard compounds. The Rf values of the sample different plant extract reveals the presence of phenolic
extract was found to be (0.90, 0.83, 0.95) which proves the compounds which contributes the antioxidant potential of
presence of caffeic acid, rosmarinic acid and ferulic acid the extracts. Hence, it is proved that Epipremnum aureum
respectively which are shown in table 4. In the present contains effective phytoconstituents which needs to be
study the presence of phenolic compounds in the plant explored on the basis of pharmacological importance.
extract also supports the antioxidant property of
Epipremnum aureum. ACKNOWLEDGEMENT
Antioxidant activity is evaluated by different methods but The authors are grateful to Prof. Dr. Shahimi Mustapha,
the most widely used methods are those that generate free Dean, Faculty of Pharmacy, Lincoln University College,
radical species which are then neutralized by antioxidant Malaysia for providing the physical facilities during the
compounds. In the present study the antioxidant activity work. The authors are also thankful to Dr. Arindam Das,
test was found to be positive for each extract which can be Director of Post Graduates studies, Lincoln University
attributed to the presence of phenols and flavonoids as College, for this continuous support during the study.
shown in the phytochemical screening test. The
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