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79 | P a g e International Standard Serial Number (ISSN): 2319-8141

International Journal of Universal Pharmacy and Bio Sciences 6(2): March-April 2017

INTERNATIONAL JOURNAL OF UNIVERSAL


PHARMACY AND BIO SCIENCES
IMPACT FACTOR 2.96***
ICV 6.16***
Pharmaceutical Sciences RESEARCH ARTICLE !!!

PHYTOCHEMICAL SCREENING ON VARIOUS EXTRACTS (BENZENE,


ETHANOLIC AND AQUEOUS) OF STEM PARTS OF ZANTHOXYLUM RHETSA
(ROXB.) DC
K.Gopalasatheeskumar*, S.Parthiban, T.Manimaran, T.Boopathi,
1
KMCH college of Pharmacy, KovaiEstate ,Kalapatti Road, Coimbatore-641048, Tamil
Nadu, India.

KEYWORDS: ABSTRACT
The Zanthoxylum Rhetsa(Roxb.) DC.Is belongs with the family of
Zanthoxylumrhetsa,
Rutaceae. In this plant is the having the various chemical
Phytochemical Study,
constituents like terpenoid, xanthyletin and sesamin, alkaloids,
Flavonoids, Alkaloids.
flavonoids and sabinene etc. The chemical constituents are the
For Correspondence:
important in the medical activities of the plant. The various parts
K.Gopalasatheeskumar *
Address: (bark, fruits and spines) of the plants are undergoes the various
KMCH college of activities in the collected works. There is no studies on the stem
Pharmacy, KovaiEstate parts of this plant is not present, so that this study is aimed to that
,Kalapatti Road, the phytochemical studies of the various types of the stem extracts.
Coimbatore-641048, In this study aqueous extract, benzene extract and ethanolic extract
Tamil Nadu, India. of the stem part of Zanthoxylum Rhetsaare the used for the
phytochemical studies in this work. This work concluded that the
various extracts are having the various chemical constituents. In
this work is very useful to the further study of the stem part of
Zanthoxylumrhetsa.

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INTRODUCTION:
The Zanthoxylum Rhetsa(Roxb.) DC. Is belongs with the family of Rutaceae. The synonym of this
plant is Budrunga. A genus of herbs is well distributed in India, Bangladesh, bhutan, china etc. In
Tamil this plant is known as Veerasingi. [1,13] This plant contains the many of the chemical sustances
like terpenoid, xanthyletin and sesamin, alkaloids, flavonoids and sabinene. The different parts of
this species have been used in traditional medicine. The bark is having diuretic actions and used to
[2]
management of dental caries. In India old medicine, the bark has been used to treat cardiac,
respiratory diseases, tooth infection, stomach infection and rheumatism. The fruits are used as spice
and the essential oil extracted after the fruits is known as Mullilam oil used as anti-inflammatory,
antiseptic, anticholera, diarrhoea, hypo-cholesterolemic, mosquito repellent and soothing agent for
dental caries.[3,4] The Kanikkars family makes a paste of hard spines arranged by rubbing them
against rock with water and applies the extract to the breast of a nursing mother to aid pain and also
to increase milk supply. In the Phillippines, the powdered bark mixed with oil is a best formula to
treat stomach ache. In adding, the bark decoction is similarly taken to treat chest pain and chewed
bark applied as remedy for snake bites. The various parts of the plant is used for the various
medicinal values, there is many evidence is there for the study of bark, fruits and spines. [5]The
chemical constituents of the plants are the very important for the medicinal activities and there is no
evidence for the study of the stem of this plant, so that in this study aimed for the primary
phytochemical tests for the stem extracts are performed. In this study is very useful to the further
study of the various activities present in this stem part of this plant. [11]
II. MATERIALS AND METHODS
2.1 Plant material
The stem of Zanthoxylum rhesta (Roxb.) DC. Were collected from the area of Kollimalai hills in
Erode district, Tamilnadu, India. The stem of Zanthoxylum Rhetsa(Roxb.) DC. The plant material
was authenticated from Plant Anatomy Research Centre, Chennai, Tamilnadu, India and a voucher
specimen no: PARC/2014/2089.
2.1.1 Extraction of Plant Material:
The stem material was dried in the shade for two months. Then shade dried plant was subjected to
get coarse powder. The coarse powders were subjected to Soxhlet apparatus by using various
solvent according to their polarity. [6,12]
2.1.2 Materials Requirement
Shade dried stem powder, Benzene extract AR, Ethanol Extract AR, Aqueous Extract AR with
0.25% Chloroform water.

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2.2 Benzene Extract


The marc left after petroleum ether extraction was dried and extracted with 2-3 liters of benzene of
(79-81oC) by continuous hot percolation using Soxhlet apparatus. After completion of extraction it
was filtered and the solvent was removed by distillation under reduced pressure. The extract was
then stored in a desiccators. A brown color residue was obtained.
2.2.1 Ethanol Extract
The marc left acetone extraction was dried and extracted with 2-3 liters of ethanol 95% by
continuous hot percolation using Soxhlet apparatus. After completion of extraction, it was filtered
and the solvent was removed by distillation under reduced pressure. The extract was stored in
desiccators. A light brown color residue was obtained.
2.2.2 Aqueous Extract
The marc left after ethanol extraction was dried and macerated with 2-3 liters of chloroform water
(0.25%) in mouthed bottle for three days. After completion of extraction it was filtered and the
solvent was removed by distillation under reduced pressure. The extract was then store in
desiccators. A black color residue was obtained.
2.3 Phytochemical Test
The powder and various three types of extracts of the plant are focus to chemical tests for
identification of its active constituents. The following phytochemical tests are performed for the
extracts.[7,8,9]
2.3.1 Test for Alkaloids
All the three types of extracts are undergoes the alkaloid conformation tests are the following
a) A small portion of the solvent free Benzene, Ethanolic and Aqueous extracts were treated
separately with few drops ofDil. hydrochloric acid and filtered. The filtrateare tested
carefully with various alkaloidal reagents such as Mayers reagent, Dragondroffs reagent,
Hagers reagent and Wagners reagent.
b) Ammonia Reineckatetest: A saturated aqueous solution of Ammonia Reineckate slightly
acidified with hydrochloric acid.
2.3.2 Test for Amino Acids
a) Millons test:
All the three types of extracts are separately added with 2ml of millons reagent.
b) Ninhydrine test:
All the three types of extracts are separately added with ninhydrine solution and boiled.

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2.3.3 Test for Carbohydrates:


A small quantity of three types of extracts are dissolved separately in 5ml of distilled water and
filtered. The filtrates were subjected the following tests to detect the presence of carbohydrate.
a) Molischs test:
A solution added with Napthol, on addition of concentrated sulphuric acid along the sides of
the test tube.
b) Fehlings solution test:
The substance (0.5g) heated with diluted hydrochloric acid to hydrolyse a polysaccharide
the reaction mixture is neutralized by addition of sodium hydroxide solution and then
Fehlings solutions 1 and 2 are added.
c) Barfoeds test:
1ml of test solution is heated with 1ml of barfoeds reagent on water bath.
d) Seliwanoffs test: (test for ketones)
To the test solution add crystals of resorcinol and equal volume of Con. Hcl and heat on a
water bath.
e) Test for pentoses:
To the test solution is added with equal volume of Hydrochloric acid containing small
amount ofphloroglucinol and heat.
f) Osazone formation test:
Heat the test solution with solution of phenyl hydrazine hydrochloride, sodium acetate and
acetic acid. Examine the yellow crystals formed under microscope.
2.3.4 Test for Cellulose:
a) To the different extracts of stem of Zanthoxylum rhesta (Roxb.) DC is added with 1-2 drops
of iodinated zinc chloride solution, wait for a minute. Add 1 drop of iodine and then add 1
drop of sulphuric acid.
b) To the different extracts of stem of Zanthoxylum rhesta (Roxb.) added with 0.1M iodine
solution and sulphuric acid.
2.3.5 Test for Lignin:
a) To the three types of extracts added with conc. Hcl and phloroglucinol solution.
b) To the test different types of extracts are added with saffranine solution.
2.3.6 Test for Volatile Oil:
a) To the different types of extracts added with Sudan III solution red.
b) To the test solution added few drops of tincture alkana.

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2.3.7 Test for Fixed Oils and Fats


a) Small quantities of the various extracts were separately pressed between two filter Papers.
Appearance of oil stain on the paper indicates the presence of fixed oil. Few drops of 0.5
ethanolic potassium hydroxide was added a small quantity of various extracts along with a
drop of phenolphthalein. The mixture was heated on water bath for 1-2 hours.
b) Treat 5 drops of extracts with 1ml of 1%copper sulphate solution, then added 10% sodium
hydroxide solution.
2.3.8 Test for Flavonoids
a) Different types of extracts added with aqueous sodium hydroxide solution
b) Zinc hydrochloride test
To the extracts are added with a mixture of zinc dust and concentrated hydrochloric acid.
c) Shinodas test
The extracts are dissolved in ethanol to that piece of magnesium followed by concentrated
by hydrochloric acid drop wise are added and heated.
2.3.9 Test for Glycosides
a) Tollens test:
The extracted solutions are taken in mixture of pyridine and Ammonical silver nitrate.
b) Antimony tri-chloride test:
The extracted solutions are heated with antimony trichloride and trichloracetic acid.
c) Keller killani test:
The 1 gram of powdered stem of Zanthoxylum rhesta(Roxb.) DC extracted with 10 ml of
70% ethanol for few minutes and filtered. To 5 ml of filtrate 10 ml of hydrogen peroxide
and 0.5ml of strong solution of lead acetate is added. Precipitate thus obtained is filtered.
Filtrate is shaken with 5ml of chloroform layer is separated and to this 1ml of mixture of 1
volume of 5% ferric sulphate and 99 volumes of glacial acetic acid is added. To this mixture
1 or 2 drops of concentrated sulphuric acid is added.
2.3.10 Test for Anthraquinone Glycosides:
a) Borntrager'stest
Boil the different types of extracts with 1 ml of sulphuric acid in the test tube for few
minutes. Filter while hot. Cool the filtrate and shake with equal amount of chloroform.
Separate the lower layer of chloroform and shake it with half of its volume of dilute
ammonia.

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b) Modified Borntragers test


Boil 200 mg of extracts and added with 2ml of sulphuric acid, and treated with 2 ml of 5%
aqueous ferric chloride solution for 5 minutes, shake it with equal amount of chloroform
and continue the test above. As some plants contain anthraceneaglycone in reduce form, if
the ferric chloride is used during the extraction, oxidation to Anthraquinone takes place,
which shows response to Borntragers test. Added hydrolyzed ether extract with methanolic
magnesium.
2.3.11 Test for hydroxy- Anthraquinone:
The extract is added with potassium hydroxide solution.
2.3.12 Test for Cardiac Glycosides:
a) Kedde's test:
To the extracts added with one drop of 90% ethanol. Make alkaline with 20% sodium
hydroxide solution.
b) Keller killiani test (test for deoxy sugars):
To the extracts added with 0.4 ml of glacial acetic acid containing trace amount of ferric
chloride. Transfer to the small test tube; add carefully 0.5 ml of concentrated sulphuric acid
by the side of the test tube.
c) Legal's test:
Treat the extract solution is added with pyridine and added alkaline sodium nitroprusside
solution.
2.3.13 Test for Coumarin glycosides:
Place the small amount of extracts in the test tube and cover the test tube with the filter paper
moister with dilute sodium hydroxide solution. Place the covered test tube in the water bath for
several minutes. Remove the paper and expose to the ultraviolet light.
2.3.14 Saponin glycosides
Froth forming test: Place the 2 ml extract in water in a test tube and shake it well.
2.3.15 Test for Inulin
To the extract solution added with the solution of a naphthol and sulphuric acid.
2.3.16 Test for Mucilage
The extract solutions are added with ruthenium red solution.
2.3.17 Test for Pectin
To the extracts are added with 0.05 % toluidine blue solution.

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2.3.18 Test for Tannins


a) Goldbeater's skin test:
Add 2% hydrochloric acid to a small piece of goldbeater's skin, rinse it with distilled water and
place in the solution to be tested for five minutes. Then give wash of distilled water and transfer to
a 1% ferrous sulphate solution.
b) Ferric chloride test:
Treat the extract with ferric chloride solution.
2.3.19 Test for catechin
Dip a matchstick in the extract solution, dry it and lastly moisten with concentrated hydrochloric
acid. Then warm the stick near flame.
2.3.20 Test forchlorogenic acid
Treat the extract solution with aqueous ammonia and expose to air gradually.
2.3.21 Test for Proteins
a) Warming test:
Heat the extracted solution in the boiling water bath.
b) Test with trichloroacetic acid:
To the extract solutions added with trichloroacetic acid.
c) Biuret test:
To the extract solutions added with 2 ml of biuret reagent.
d) Hydrolysis test:
Hydrolyze the extract solutions with hydrochloric acid.
e) Xanthoproteic test:
To the 5 ml of extract solution, added with 1 ml of concentrated nitric acid and boil, yellow
precipitate is formed. After cool it, add 40 % sodium hydroxide solution.
2.3.22Steroids and triterpenoids
a) Libermann - Burchard test:
Treat the extracts with few drops of acetic anhydride, boil and cool. Then add concentrated
sulphuric acid with the side of the test tube.
b) Salkowski test:
Treat the extracts with few drops concentrated sulphuric acid.
2.3.23Naphthoquinones
Dam-Karrer test:
To the chloroformic plant extracts added 10% pot hydroxide solution.

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III.RESULTS
The results of the various extracts are phytochemical tests are given in the table 1, table 2 and table
3. The table 1 contains the all the positive and negative tests for the chemical constituents and table
2 and table 3 are contain only of the positive results of the chemical constituents.
Table 1: Phtyo-Chemical Tests for Benzene Extract of Zanthoxylum Rhetsa(Roxb.)Dc.
Compound Test Observation Inference
Mayers reagent Yellow precipitate
Alkaloids Dragondroffs Orange brown
reagent precipitate
Hagers reagent Yellow precipitate Presence of alkaloids
Wagners reagent Reddish brown
precipitate
Tannic acid test A precipitate
Ammonia Pink flocculent
Reineckatetest precipitate
Carbohydrates Molischs test No purple ring is
Fehlings solution test No Red precipitate
Barfoeds test No red cupric oxide
Seliwanoffs test No rose color is
produced Absence of
Test for pentoses No red color is carbohtdrates
formed.
Osazone formation No formation of
test osazone
Amino acids Millons test No white precipitate Absence of amino
Ninhydrine test No violet color acids
Iodinated chloride No blue color
Cellulose test Absence of cellulose
Iodine solution test No blue- violet
Lignin Phloroglucinol test Pink color Presence of lignin
Saffranine solution Pink color
Volatile Oil Sudan III solution Red color Presence of volatile

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Tincture alkana Red color oil


Fixed oils and fats Soap formation test Formation of soap Presence of fixed oil
Sodium hydroxide Yellow color
Flavonoids test Presence of
Zinc Hcl test Red color flavonoids
Shinodas test magenta color
Tollens test No Liberation of
silver mirror on the
Glycosides walls of tube Absence of
Antimony trichloride No blue color glycosides
test
Keller killani test No blue color
Inulin Naphthol, sulphuric No brownish red Absence of inulin
acid color
Mucilage Ruthenium red Pink color Presence of mucilage
Pectin Toluidine blue Reddish purple color Presence of pectin
Tannins Goldbeater's skin test No black color Absence of tannins
Ferric chloride test No blue color
Catechin Hcl Pink Presence of catechin
Chlorogenic acid Ammonia Green color Presence of
chlorogenic acid
Warming test Coagulated
Proteins Trichloroacetic acid Precipitate Presence of proteins
Biuret test Violet color
Xanthoproteic test Orange color

Starch Iodine No blue color Absence of iodine


Steroids and Libermann - Brown ring
triterpenoids Burchard test Presence of steroids
Salkowski test Yellow color
Naphthoquinones Dam-Karrer test No blue color Absence of
Naphthoquinones.

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Table 2: Phtyo-Chemical Tests for Ethanol Extract of ZanthoxylumRhetsa(Roxb.)Dc.


Compound Test Observation Inference

Mayers reagent Yellow precipitate

Dragondroffs Orange brown


reagent precipitate

Hagers reagent Yellow precipitate

Wagners reagent Reddish brown


Alkaloids Presence of alkaloids
precipitate

Tannic acid test A precipitate

Ammonia Pink flocculent


Reineckatetest precipitate

Amino acids Millons test White precipitate Presence of amino


acids
Ninhydrine test Violet color

Volatile Oil Sudan III solution Red color Presence of volatile


oil
Tincture alkana Red color

Fixed oils and fats Soap formation test Formation of soap Presence of fixed oil

Sodium hydroxide Yellow color


test
Flavonoids Presence of
Zinc Hcl test Red color flavonoids

Shinodas test Magenta color

Tollens test Liberation of silver


mirror on the walls
of tube
Glycosides Presence of
Antimony trichloride Blue color
glycosides
test

Keller killani test Blue color

Borntrager's test Rose pink to Red

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color

Anthraquinone Modified Pink color Presence of


Glycosides Borntragers test anthraquinone
glycosides
Hydroxyl Red color
Anthraquinone test

Mucilage Ruthenium red Pink color Presence of mucilage

Tannins Goldbeater's skin test Black color Presence of tannins

Ferric chloride test Blue color

Catechin Hcl Pink Presence of catechin

Chlorogenic acid Ammonia Green color Presence of


chlorogenic acid

Warming test Coagulated

Proteins Trichloroacetic acid Precipitate Presence of proteins

Biuret test Violet color

Xanthoproteic test Orange color

Steroids and Libermann - Brown ring Presence of steroids


triterpenoids Burchard test

Salkowski test Yellow colored

Table 3: Phtyo-Chemical Tests for Aqueous Extract of ZanthoxylumRhetsa(Roxb.)Dc.


Compound Test Observation Inference

Amino acids Millons test White precipitate Presence of amino


acids
Ninhydrine test Violet color

Volatile Oil Sudan III solution Red color Presence of volatile


oil
Tincturealkana Red color

Fixed oils and fats Soap formation test No Formation of soap absence of fixed oils

Flavonoids Sodium hydroxide test Yellow color Presence of


flavonoids
Zinc Hcl test Red color

Shinodas test Magenta color

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Mucilage Ruthenium red Pink color Presence of mucilage

Catechin Hcl Pink Presence of catechin

Chlorogenic acid Ammonia Green color Presence of


chlorogenic acid

Warming test Coagulated

Proteins Trichloroacetic acid Precipitate Presence of proteins

Biuret test Violet color

Xanthoproteic test Orange color

Starch Iodine Blue color Presence of starch

The tables are explains various extracts are having various chemical constituents. The benzene
extract contains the chemical constituents like alkaloids, lignin, volatile oils, fixed oils, flavonoids,
pectin and steroids. The ethanolic extract shows the positive results on alkaloids, amino acids,
volatile oils, fixed oils, flavonoids, glycosides, mucilage and steroids. And the aqueous extract is
shows the positive results of amino acids, volatile oils, fixed oils, flavonoids, mucilage, proteins
and starch. These are the results are the essential for the further studies on the stem part of
ZanthoxylumRhetsa(Roxb.)Dc.
III. CONCLUSION
In this present study the various chemical tests are performed in the three types of extracts of stem
part of ZanthoxylumRhetsa(Roxb.)Dc is performed from the results of this study, concluded that the
various extracts (benzene, ethanol, aqueous) are the having various chemical constituents. This
study is important in the study of the stem of ZanthoxylumRhetsa(Roxb.)Dc in the further medicinal
activity studies.
ACKNOWLEDGEMENT
I thankful to the almighty for blessings in successful completion of this work, my special thanks to
Mr.S.Parthiban and I would like to thank my friendsV.SanishDevan, M.Sanjay, A.Jeevanantham.
My deepest heartfelt gratitude and indebtedness belong to my Parents and Sisters who showed love,
affection, encouragement and finally made it possible to complete this work.
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