article_wjpps_1408968504

www.wjpps.com Vol 3, Issue 9, 2014. 136
Aqna Malik World Journal of Pharmacy and Pharmaceutical Sciences
REVIEW OF STUDY ON PHYSIOCHEMICAL, ANTIOXIDANT,
PROPERTIES OF TAMARIXDIOICA
*Dr Aqna Malik
Doctor of Pharmacy, M.Phil. Pharmacology Reg. No. 10-M.PH/LCWU-17981
Lecturer in Hashmat Medical & Dental College, Tanda Chowk, JPJ, Gujrat, Pakistan.
ABSTRACT
TamarixDioica Leaves collected from marshy areas near river banks. It
has been used traditionally as anti-Infective (Said,1969), aphrodisiac
(Katewa et al., 2006), Cough and Flu (UK Saad et al.,2009) and
Astringent. In my study Phytochemical constituents, antioxidant
activity of various extracts of TamarixDioicia was carried out.
Phytochemicals were extracted from different extracts of
TamarixDioica using various solvents such as, chloroform, ethanol,
ethyl acetate, methanol. Screening of phytochemicals showed positive
results for the presence of flavonoids, alkaloids, phenols, steroids,
glycosides, carbohydrates, terpenoids and tannins. Phytochemicals
were extracted best in methanol. Separation of compounds was also
done by TLC method. Free radical scavenging activity was determined
using FeCL3 method, is a stable antioxidant. Both Methanolic and Ethanolic extracts was
found to have maximum antioxidant activity. These activities may be due to the presence of
flavonoids, alkaloids, phenols, steroids, glycosides and tannins in Tamarixdioicia.
KEY WORDS: Phytochemicals, Tamarixdioicia, solvents, antioxidant activity,Thin Layer
Chromatography.
1. INTRODUCTION
Properties of medicinal plant species contributed a lot in progression of traditional herbal
therapies. Due to insufficiency of written documents and comparatively low income in those
times value of these traditional knowledge systems declined with passage of time(Myers N,
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Article Received on
19 May 2014,
Revised on 24 June
2014,
Accepted on 04 August 2014
*Correspondence for Author
Dr. Aqna Malik
Doctor of Pharmacy, M.Phil.
Pharmacology Reg. No. 10-
M.PH/LCWU-17981
Lecturer in Hashmat Medical
& Dental College, Tanda
Chowk, JPJ, Gujrat, Pakistan

1991) (Lacuna-Richman C, 2002). Now a day’s gradually increasing interest of people in
herbal medicines gave medicinal plants a new boom.
1.1. Hepatoprotective herbs
Hepatitis is categorized as life threatening diseases worldwide. Medicines developed
according to allopathic medicines for hepatitis have shown limits in efficacy, cost especially
for the developing world. So search for phytoconstituents for treatment of liver diseases is
need of hour to gain benefits of easy approach will be free from painstaking pharmaceutical
synthesis
Following limiting factors involved in Practical application of Natural remedies ( herbs)
(i) Lack of standardization of the herbal drugs.
(ii) Lack of identification of active ingredients
(iii) Lack of randomized controlled clinical trials (RCTs),
(iv) Lack of toxicological evaluation (Radha et al., 2005).
Phytochemicals being Bio-active non-nutrient plant compounds present in fruits, vegetables,
grains, and other plant foods play important role in decreasing the risk of pathological
manifestations from major chronic diseases. A lot of phytochemicals have been discovered,
these may also known as secondary metabolites. Their occurrence and distribution vary
according to plant species. CCl4 and other toxic materials which damage liver cells through
free radicals production ending in numerous physiological and pathological events e.g.
inflammation, aging, mutagenicity and carcinogenicity. Very reactive chemical species free
radicals (capable of independent existence) with one or moreunpaired electrons are formed
due to Low oxygen partial pressure increasing the reductive metabolism of toxicant and thus
promote covalent binding with cellular constituents leading to cellular damage. Antioxidants
have free-radical scavenger ability to guard living organisms from damage caused by
Uncontrolled reactive oxygen species which results in loss of calcium homeostasis and,
ultimately, apoptosis and cell death. Near than 160 phytoconstituents possess liver protective
activity.
1.2. Thin Layer Chromatography
i.TLC Importance
TLC has its established place in detection of chemicals from more than 100 years. (M. W.
Beyerinck, 1889). TLC has proven Highly efficient in micro-analytical separation technique.
Also TLC has extraordinary sample throughput in a less time period.

The methanol, chloroform, ethanol and ethyl-acetate extracts of TamarixDioica were
subjected to TLC analysis, to find presence of different chemical ingredients in this medicinal
shrub to support the chemical tests. Pre-coated TLC plates were used.
2. MATERIALS AND METHODS
My Research work was aimed at finding phytoconstituents which possess hepatoprotective
activity from a well-known medicinal plant TamarixDioica using mice as hepatotoxicity
model.
2.1.Phytochemical Analysis
Different extracts of TamarixDioicawere tested forvaious phytochemicals by qualitative
chemical tests to prove presence of hepatoprotectivephytoconstituents in TamarixDioica
leaves Tests performed for pre-liminary phytochemical screening are as follows.
2.1.1. Test for Alkaloids
i.Wagner's test
Both methanolic and aqeous extracts of TamarixDioica gave a reddish brown precipitate
with Wagner's reagent (Solution of iodine in potassium iodide).
ii. Hager's test
Both methanolic and aqeous extracts of TamarixDioica gave yellow color precipitate with
Hager's reagent [saturated solution of Picric acid].
2.1.2.Test for Glycosides
The extracts were tested for free sugars
i.Bromine water test
Test solution of Bothmethanolic and aqeous extracts of TamarixDioica when treated with
bromine water gave yellow precipitate.
2.1.3.Test for Saponin Glycosides
i.Froth Test
1ml solution of both methanolic and aqeous extracts of TamarixDioica produced a stable
froth when placed in water in a semi-micro tube and shaken well.

2.1.4.Test for Tannins & Phenolic Compounds
I .chloride test
Test solution of methanolic extract of TamarixDioica gave visible blue green color with
ferric chloride.
Alkaline reagent test
Test solution methanolic extract of TamarixDioica when treated with sodium hydroxide
solution gave yellow to red precipitate within short time.
2.1.5 .Test for Flavonoids
Alkaline reagent test
Test solution of aqeous extracts of TamarixDioica , when added with few drops of sodium
hydroxide solution, an intense yellow color appeared which turned to Colorless on addition
of few drops of dil. Acid indicating presence of Flavonoids.
2.1.6 .Test for Proteins & Amino Acids
Ninhydrin test
Aqeous extract of TamarixDioica when boiled with 0.2% solution of Ninhydrin (Indane 1, 2,
3 trione hydrate), strong Violet color appeard.
2.1.7. Test for Sterols &Triterpenoids
Libermann- Buchard test
Aqeous extracts of TamarixDioica when treated with few drops of acetic anhydride, boiled
and cool, con. Further sulfuric acid is added from the sides of the test tube,A brown ring at
the junction of two layers visible and the upper layer turned green showing the presence of
Steroids and formation of deep red color indicated the presence of triterpenoids.
2.1.8. Test for Fats & Fixed Oils
i.Stain test
Small quantity of aqeous extracts of TamarixDioica was pressed between two filter papers,
the stain on I filter paper indicates the presence of fixed oils.
ii.Saponification test
Few drops of 0.5N of alcoholic potassium hydroxide added to a small quantity of methanolic
extract of TamarixDioica along with a drop of Phenolphthalein separately and heat on a water

bath for 1-2 hrs. The formation of soap or partial neutralization of alkali indicates the
presence of Fixed oils and Fats.
2.1.9 Anti- Oxidant Activity
I have used FeCl3 method to check free radical scavenging activity (Lim et al.,2007). Diluted
extracts of leaves of TamarixDioica in a quantity of 1ml were mixed with of potassium
phosphate buffer in a quantity of 2.5 ml which was prepared in 0.2 M concentration and at
6.6 pH 6.6 and potassium ferricyanide taken in 2.5ml quantity. The whole mixture was
incubated at 500C° for 20 min .10% tricoloracetic acid in qty of 2.5 ml was added to the
mixture to stop the reaction. Before the addition of 0.5 ml of FeCl3 ultra-pure water was
added in a same quantity in 2.5 ml of the mixture. Same procedure was repeated 3 times and
allowed to stand for 30 min and finally absorbance was recorded at 700 nm.
2.2.Thin Layer Chromatography
2.2.1. Procedure performed for developing TLC plates
i.EXTRACT PREPARATION
In respected solvents of methanol, chloroform, ethanol and ethyl-acetate in ratio of (200mg:
20ml) TamarixDioica (Powdered form) was dissolved in each, shaken for few minutes ,
placed on Hot plate for very short time followed by filtration & further heating to obtain
minimal amount of sample to be applied on TLC plates.
ii.SOLVENT APPLICATION
Multiple times spotted sample (with the help of a projected capillary tube). After observing
visible spotted point can be seen on line drawn 1.5cm above from bottom of plate TLC plate
was placed at 45 degree angles in development chamber in a way that bottom of the plate was
dipped in solvent up to nearly 1 cm. Solvent front was marked &plate was dried finally.
iii.DEVELOPMENT OF CHROMATOGRAM
Chromatogram clearly showed eluted coloured substances while Iodine Vapours were used as
visualizing agent to detect colourless components by defining migrating behavior of
separated substances in terms of Rf values qualitative evaluation of plates can be performed. I
haveaccessed Spots on plates both before and after iodine spray under UV light at 366nm and
254nm.

iv. SOLVENT SYSTEM PREPARATION
Solvent system selected for the TLC of ethanol extract was Chloroform: Methanol in ratio of
(9.9:0.1). Similarly for the TLC of methanol extract was Ethylacetate: Formic acid: Acetic
acid: Water of ratio of ratio of (100:11:11:27).For the TLC of Chloroform extract was
Chloroform: Ethyl acetate in ratio of (4:6). Solvent system for the TLC of Ethyl acetate
extract was Toluene: Ethyl acetate in ratio of (8:2).
3. RESULTS AND DISCUSSION
3.1 Results of Phytochemical Analysis
Present study proved that Phytochemicals e.g.Saponin Glycosides were detected in froth Test
where 1ml solution of both methanolic and aqueous extracts of leaves of TamarixDioica
produced a stable froth when placed in water in a semi-micro tube and shaken well. Tannins
& Phenolic compounds were also detected by ferric chloride test where test solution of
methanolic extract of TamarixDioica gave visible blue green color with ferric chloride. In
Alkaline reagent test for flavonoids test solution of aqueous extracts of TamarixDioica,
produced an intense yellow colorwhen added with few drops of sodium hydroxide solution,
which turned to Colorless on addition of few drops of dil.acid indicating presence of
Flavonoids.
Proteins & Amino Acids were also detected when aqueous extract of TamarixDioica was
boiled with 0.2% solution of Ninhydrin (Indane 1, 2, 3 trione hydrate) and strong Violet color
appeared.
Libermann-Buchardtest proved presence of Sterols&Triterpenoids where aqueous extracts of
TamarixDioica when treated with few drops of acetic anhydride, boiled and cool. Further
concentrated, sulphuric acid is added from the sides of the test tube, a ring of brown color
appeared at the junction of two layers visible and the upper layer turned green showing the
presence of Steroids and formation of deep red color indicated the presence of triterpenoids.
Presence of fats &fixed Oils was confirmed with stain test and saponification test.

Table 3.1. Phytochemical tests for both methanolic and aqueous extracts of Leaves of
TamarixDioica
Phytochemicals Tests
Methanolic
extract
Aqueous
Extract
Alkaloids Wagner's test - -
Hager's test - -
Glycosides Bromine water test - -
Saponin Glycosides Froth Test + +
Tannins&Phenolic Compounds Ferric chloride test + -
Alkaline reagent test False positive -
Flavonoids Alkaline reagent test - +
Sterols &Triterpenoids
Libermann- Buchard
test
- +
Proteins & Amino Acids Ninhydrin test - +
Fats & Fixed Oils Stain test + -
Saponification test + -
1 + sign of positive test
2 - Sign of negative test
Above Results confirmed presence of Saponin Glycosides, Tannins&Phenolic Compounds,
Flavonoids, Sterols &Triterpenoids, Proteins & Amino acids, Fats& Fixed Oils in methanolic
and aqueous extracts of leaves of TamarixDioica.
3.2 Results of Antioxidant Activity
Free radical scavenging activity was checked by method described as follows (Lim and
Murtijaya, 2007) i.e.FeCl3 method, Diluted methanolic and ethanolic extracts of leaves of
TamarixDioica (1 ml) gave absorbance at 700nm. But aqeous extract of leaves of
TamarixDioica (1 ml) gave no absorbance at 700nm.
3.3 Results of Chromatogram Development
Ethanolic extract TLC in solvent system of Chloroform: Methanol gave spots with Rf values
(0.05, 0.08, 0.3). Methanol extract TLC in Ethylacetate: Formic acid: Acetic acid: Water
solvent system brought about spots with Rf values (0.1724, 0.528, 0.66, 0.839, 0.94). While
during TLC of Chloroform extract in solvent system of Chloroform: Ethyl acetate spots with
Rf values (0.03, 0.144, 0.211, 0.65, 0.77, 0.88) were observed.In case of Ethyl acetate extract
TLC in solvent system of Toluene: Ethyl acetate shown spots with Rf values (0.117, 0.213,
0.3103, 0.48, 0.5655, 0.593, 0.724, 0.7586, 0.813, 0.9655).

Table 3.2 Interpretation of TLC plates of extracts Leaves ofTamarixDioica
Extract
Solvent
system
ά-Max and Absorption ,Visibility &Colours Naked Eye and under UV light of Spots
Visibility of Spots Colours of Spots
No of
spot
Rf
value
ά-Max Absorption
Naked
eye
At
366n
m
At
254nm
Iodine
Spray
Naked
eye
At
366nm
At 254
nm
Iodine
Spray
Ethyl
acetate
Toluene:
Ethyl
acetat 8:2
1 0.117 visible visibe visible visible Green Green Grey +
2 0.213 visible Green Grey
3 0.3103 visible +
4 0.48 visible visible Green Green Grey
5 0.5655 visibe visible Green Grey
6 0.593 visible Green
8 0.7586 visibe Green +
10 0.9655 visibe visible visible Green Grey +
Chlorofo
rm
Chloroform:
Ethyl acetate
4:6
1 0.03
221 0.427
visible visibe visible visible Green Orange Green +
307 0.160
2 0.144
221 0.400
visible visibe visible visible Green Orange Green +
307 0.140
3 0.211 206 0.597 visible visibe visible visible Green Orange Green +

266 0.174
4 0.65
221 0.780
visibe visible visible
Light
green
Green +
307 0.329
5 0.77
Straight line
visibe visible
Light
green
266 0.79
6 0.88 visibe visible Green Orange
Methano
l
Ethyl acetate:
Formic
acid:Aceticaci
d:Water
(100:11:11:27)
1 0.1724 350 0.121 visible visible visible
Dark
brown
Dark
brown
2 0.528 - - visible visibe visible visible
Light
brown
Light
brown
Light
brown
3 0.66 297 visibe
Light
brown
4 0.839 294 visible visibe visible visible
Light
brown
Light
brown
Light
brown
5 0.94
285 0.065
Visibe visibe visible visible
Dark
brown
Dark
brown
Dark
brown
564 0.036
Ethanol
Chloroform:
Methanol
9.9:0.1
1 0.05 visible visible
Light
yellow
Yellowi
sh
green
Light
brown
2 0.08 visibe Brown Brown
3 0.3 visible Brown Brown

TLC of ethanol extract in solvent system of Chloroform: Methanol gave spots with Rf values
(0.05, 0.08, 0.3). TLC of methanol extract in Ethylacetate: Formic acid: Acetic acid: Water
solvent system brought about spots with Rf values (0.1724, 0.528, 0.66, 0.839, 0.94). While
during TLC of Chloroform extract in solvent system of Chloroform: Ethyl acetate spots with
Rf values (0.03, 0.144, 0.211, 0.65, 0.77, 0.88) were observed. TLC of Ethyl acetate extract
in solvent system of Toluene: Ethyl acetate shown spots with Rf values (0.117, 0.213, 0.3103,
0.48, 0.5655, 0.593, 0.724, 0.7586, 0.813, 0.9655).
Visibilty in Iodine Spray is denoted by sign + ά-Max and Absorption in Chloroform:Ethyl
acetate 4:6 and Ethyl acetate: Formic acid:Aceticacid:Water (100:11:11:27)
Solvent systemwas checked by scratching each spot of concerned TLC plate and dissolving it
in methanol.
Fig 3.1: TLC P Late Of Chloroform Extract

Fig.3.2TLC Plate of Ethanol Extract Fig.3.3TLC Plate of Ethyl extract acetate
extract
Fig3.4TLC P Late Of Methanol Extract

Fig3.5 Absorption on UV Spectrum shown by scratched spots from TLC Plates
Fig 3.6 Anti oxidant activity of methanolic extract of leaves of TamarixDioica at 700nm

CONCLUSIONS
Presence of Saponin Glycosides, Tannins&Phenolic Compounds, Flavonoids, Sterols
&Triterpenoids, Proteins & Amino acids and Fats & Fixed Oils either in Methanolic or
aqueous extract or in both extract of leaves of TamarixDioica was confirmed by
Phytochemical analysis. Like wise methanolic extract of leaves of TamarixDioica showed
anti-oxidant activity. Presence of hepatoprotective constituents has proven the shrub leaves
extrats beneficial for liver injury.Active constituents such as flavonoids, triterpenoids and
anti-oxidant axiom have protective effect on liver due to its antioxidant properties.
ACKNOWLEDGEMENTS
First & foremost, praise to ALMIGHTY ALLAH, the merciful & benevolent who gave me
the power put my efforts together on this work.
All respect and gratitude for the prophet of Mercy, Hazrat Muhammad (PBUH) whose
teachings guide me throughout the life.
In fact, the process of completing this project has been an outcome for efforts and support
from a number of people whose direct and indirect contributions enabled me to accomplish
this task. I gratefully acknowledge the contribution of all my honorable teachers who taught
me and trained me the basic skills of knowledge.
I must place on record, my gratitude to respected vice chancellor. PROF. DR. SABIHA
MANSOOR, (LCWU) for her support and giving this opportunity.
I am highly obliged and my cordial gratitude to my honorable supervisior PROF. DR
MUHAMMAD HAFEEZ IKRAM, Head of pharmacy department, LCWU and who provided
the much needed confidence with encouragement in addition to his valuable suggestions on
the actual work. I am extremely grateful for his sincere and friendly cooperation throughout
research work.
My heartiest gratitude to my highly respectful and honourable co-supervisor PROF DR
MOBASHER AHMAD BUTT, Professor Of Pharmacology, University College of
Pharmacy, University of Punjab, Lahore without whom support it would not have been
possible for me to accomplish this task.
I am very gratefull to my highly respectful and honourable co-supervisor.

DR SHEHZAD HUSSAIN, Federal Government Analyst, National Institute of Health,
Islamabad without whom support it would not have been possible for me to accomplish this
task My special thanks to Kazi Muhammad Ashfaq , Assistant Scientific Officer chemistry
section , and miss Kokab from Chemistry section National Institute of Health, Islamabad and
their team Last but not the least, I pay my humble tribute to my parents & my caring and co-
operative husband who ever embraced me with their effective pray, motivation because of
which I was able to complete my laborious project.
REFERENCES
1. Lacuna-Richman, C. (2002). The socioeconomic significance of subsistence non-wood
forest Radha KD, Yogesh KC (2005). Herbal medicines for liver diseases. Digestive
Diseases and Sciences, 50: 1807–1812.
2. M. W. Beyerinck, Z (1889) TLC Contents. Phys. Chem. 3, Page: 110.
3. Myers N (1991). The world's forests and human population: the environmental
interconnections. Population and Development Review, 16: Page: 1-15.
4. U K Saad, M W Sultan, M. Subhan, Zahoor M, Kamal M and ShaheenTaj, 2009.
Department of Botany, University of Science and Technology, Bannu, Pakistan Pak. J.
Pl.Sci.15 (1): 81-85.
5. Said,H.M. (1969).Hamdard pharmacopeia of Eastern medicines,Hramdard National
Foundation of Pakistan.
6. Katewa S S&Galav P K (2006). Additions to the traditional folk herbal medicines from
Shekhawati Indian journal of traditional knowledge 5(4) : 499.
7. Lim, Y.Y. and Murtijaya, J. 2007. Antioxidant properties of Phyllanthusamarus extracts
as affected by different drying methods. LWT-Food Science and Technology 40:1664-
1669.

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