Abdul Latif et al / IJDFR volume 4 Issue 4 July-Aug. 2013
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ISSN 2229-5054
Research Article
INTERNATIONAL JOURNAL OF DRUG FORMULATION AND RESEARCH
Physiochemical Standardization of Market Sample of Gul-e-Zofa
(Nepeta bracteata Benth.)
Abdul Latif*, Zeenat Mahmood, Nazish Siddiqui, Abdur Rauf
Department of Ilmul Advia, Faculty of Unani Medicine, A.M.U., Aligarh
Received: 23 June 2013; Revised: 23 Jul. 2013; Accepted: 9 Aug. 2013; Available online: 5 Sep. 2013
ABSTRACT
Medicinal plants organize an effective source of folk and Modern Medicine. Correct identification, authentication and
quality control are essential to ensure safety, efficacy and reproducibility. Therefore, for the present study Gul- e- Zofa a
well known drug has been selected and its physicochemical standards were carried out for its correct identification,
authentication and quality control using standard methods. Previously Gul-e-Zofa was identified as flower of Hyssopus
officinalis Linn. by several authors, but sample of Gul- e- Zofa taken from local market was identified by National
Institute of Science Communication and Information Resources (NISCAIR), as Nepeta bracteata Benth.
(Reference: NICAIR/RHMD/Consult/-2011-12/1931/23) and physicochemical standardization is done on it .The
parameters studied are moisture content 8.4±0.748(1.673)% total ash 8.367±0.278(0.681)%, acid insoluble ash
1.59±0.126(0.309)%, water soluble ash 2.827±0.215(0.527)%, and sulphated ash 12.183±0.236(0.578)%, water soluble
extractive 4.86±0.0872(0.195)%, and alcohol soluble extractive 1±0.0707(0.158), Bulk Density 0.26±0.009(0.0203)
gm/ml, and loss on drying 11.02±0.146(0.327) %. On phytochemical analysis it revealed the presence of carbohydrates,
flavonoids, glycosides, phenols and proteins. Besides this determination of organoleptic character of powder drug,
extractive values in different organic solvent using soxhlet extractor, and thin layer chromatography and
fluorescence
analysis of successive extract of powder drug had also been done. This study will help in setting down Pharmacopoeial
standards in determining the quality and purity of
Nepeta bracteata Benth.
KEY WORDS: Nepeta bracteata Benth, physico-chemical Standard and TLC.
INTRODUCTION
The existence of several common names for the same plant species in different areas may
confuse end users
for selection and utilization of a genuine drug.The authenticated plant which is identified as Gul-e-Zofa is
Hyssopus officinalis Linn. The other plants substituted for Gul-e-Zofa are Nepeta bracteata, Nepeta ispahanica
and Nepeta micrantha[1]. We have taken Nepeta bracteata Benth for our study which is identified
by
National
Institute
of
Science
Communication
and
information
Resources
(NISCAIR)
(Reference no. NISCAIR/RHMD/Consult/2011-12/1931/23). It belongs to Lamiaceae family and is indigenous
to Iran[2]. It is brightly coloured shrub or sub shrub that ranges from 30-100cm in height. Found in western
temperate Himalayas from Garhwal to Kashmir at an altitudes of 1800-2400 metres. Leaves are ovate–obtuse.
During summer the plant produces bunches of pink blue and more rarely white fragrant flowers. Flowers are
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bisexual, zygomorphic, rarely sub actinomorphic and bracteolate
[4]
. Gul-e-Zofa is generally attributed to
Hyssopus officinalis, but this is not correct, as the flowers are in oblong spikes [5] and also it is confirmed by
NISCAIR as above. Its action are describe as stimulant, demulcent, expectorant, anti inflammatory,
carminative, resolvent emmenagogue , cleanser, laxative, antihelminthic and diaphoretic[ 6,7,8,9,10,11]. It is used
for chronic cough, chronic bronchitis, sore throat and asthma, in tooth ache, uterine or vesical affection,
induration of liver or spleen[3]. It is medicinally used in pneumonia, amenorrhoea, rheumatism influenza,
diphtheria, eye ailment ascitis, diarrhoea and sciatica [7, 8, 9, 10, 11, 12]. Medicinal properties of Nepeta species are
related to terpenoids and flavonoids. Compounds such as 1-8-cineale, are very common in
Nepeta and have
expectorant, antiseptic and anthelmintic activities.[13] The Nepeta bracteata Benth is medicinally important so
we have decided to standardize it in the light of W.H.O. guidelines.
Material and Method
Plant Material
The Gul-e-Zofa was procured from the local market of Baradari, Aligarh and Dawakhana Tibbiya College,
A.M.U., Aligarh and identified by National Institute of Science Communication and information Resources
(NISCAIR), as Nepeta bracteata Benth (Reference no. NISCAIR/RHMD/Consult/2011-12/1931/23). The
flowers were dried at
optimum temperature and further crushed and sieved to coarse powder mechanically
and stored in air tight container for physicochemical analysis. The Physicochemical parameter that were studies
included the organoleptic character of powder drug, determination of
extractive values of the test drug in
different organic solvent using soxhlet extractor, alcohol and water soluble contents, moisture content, ash
values, loss of weight on drying, pH values, phytochemical analysis, thin layer chromatography and
fluorescence analysis of successive extract of powder drug.
1. Extractive Values
The extractive values of the test drug in different organic solvents viz. petroleum ether,
diethyl ether,
chloroform, ethyl acetate, acetone, alcohol and distilled water were carried out by a soxhlet’s apparatus. The
heat was applied for six hours on a water bath for each solvent except water, which was heated directly on a
heating mantle. The extracts were filtered and after evaporation of the solvents; the extractive values were
determined with reference to the weight of drug. The procedures were repeated five times and the mean value
for each extract was calculated [14].
2. Water and Alcohol Soluble Contents
5 gm of powdered drug was taken into 100 ml of distilled water and alcohol, in a glass
Stoppard conical flask.
The mixture was carefully shaken frequently for 6 hours and then allowed standing for 18 hour. It was filtered
and 25ml was evaporated to dryness on a water bath. The residue was dried at 105ᵒC for 6 hours, cooled in
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desiccator for 30 minutes and weighed without delay. The percentage of water soluble matter was calculated
with reference to the amount of air dried drug . The percentage of alcohol soluble matter was determined as
above by using alcohol in place of water [14].
3. Moisture Content
The toluene distillation method was used for the determination of moisture content. 10 gm of drug was taken in
the flask of the apparatus and 75ml of distilled toluene was added to it. Distillation was carried out for 6 hours
and the process was repeated for five times. The
volume of water collected in receiver tube (graduated in ml)
was noted and the percentage of moisture calculated with reference to the weight of the air dried drug taken for
the process[15].
4. Ash Values
(A). Total Ash
2 gm of drug was incinerated in a silica crucible of a constant weight at a temperature not exceeding 450ᵒ C in a
muffle furnace until free from carbon, cooled and weighed and the percentage of ash was calculated by
subtracting the weight of crucible from the weight of crucible + ash. The percentage of total ash was calculated
with reference to the weight of drug taken [16].
(B). Water Soluble Ash
The obtained ash was boiled with 25ml of distilled water for 5 min. The insoluble matter was collected in an
ashless filter paper, (Whatman No. 42) washed with hot water and ignited in crucible, at a temp no more than
450 ᵒ C, the weight of insoluble ash was subtracted from the weight of total ash, giving the weight of water
soluble ash. The percentage of water soluble ash was calculated with reference to the air dried drug taken [16].
(C). Acid Insoluble Ash
The total ash was boiled with 25 ml of 10% hydrochloric acid for 5 min. The insoluble matter was collected on
ash less filter paper (Whatman No. 42), washed with hot water and ignited in crucible at a temperature not
exceeding 450ᵒC and weighed after cooling in desiccator. The percentage of acid-insoluble ash was calculated
with reference to the weight of drug taken [14].
A
(d) Sulphated Ash
sample of 2 gm of air dried powdered drug was incinerated in silica crucible at a temperature not exceeding dull
red (450ᵒC) in muffle furnance, until the drug substance is thoroughly charred. The residue was cooled and
moistened with 1-2ml of sulphuric acid and heated gently until white fumes are no longer evolved and ignited at
800ᵒC until all black disappear . The crucible was allowed to cool and few drops of sulphuric acid were added
and ignited as before. The percentage of sulphated Ash was calculated with reference to air dried drug [16].
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5. Loss of Weight on Drying
10 gm of drug was taken, spread uniformly and thin layered in a shallow petridish. It was heated at a regulated
temperature of 105ᵒC, cooled in a dessicator and weighed. The process was repeated many times till two
consecutive weights were found constant. The percentage of loss in weight was calculated with respect to initial
weight [15].
6. Bulk Density
A clean, dry and previously weighed bottle (25 ml) was taken. It was filled with known quantity of distilled
water and weighed, marked the water level and got the bottle emptied, rinsed with acetone and dried. The bottle
was filled with the drug, allowed it to settle overnight and again adjusted the level up to the mark and weighed.
The bulk density was determined from the weight of water and drug. The bulk density was calculated by the
following formula: [15].
Weight of the test drug
Bulk density of drug =
__________________________________
Weight of equivalent volume of Distilled water
7. pH Value
Determination of pH was carried out by a digital pH meter (model no. 335) equipped with a combined
electrode. The instrument was standardized by using buffer solution of 4.0, 7.0, and 9.20 to ascertain the
accuracy of the instrument prior to the experiment. The pH value of 1% solution and 10% of powder drug
solution was measured. [14].
8. Chromatographic Studies
Thin Layer Chromatography (TLC)
Thin layer chromatography was carried out on T.L.C. pre-coated aluminium plates with silica gel 60 of F254
(layer thickness 0.25mm) of diethyl ether extract. Taking petroleum ether: diethyl ether in 1:1 ratio as the
mobile phases. The Rf values of the spots were calculated by the following formula [17].
Rf value
Distance travelled by the spot
=
Distance travelled by the solvent
9. Phytochemical Evaluation [17, 18]
Nepeta bracteata Benth qualitatively analysed for the presence of various chemical
[17]:
1. Test for Alkaloids
constituent.
A drop of Dragendroff’s reagent in the extract was added. The brown precipitate shows
the presence of alkaloids.
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2. Test for Carbohydrate / Sugars[17]
i. Fehling’s solution Test: In the aqueous extract, a mixture of equal parts of Fehling’s
solution A and B
previously mixed was added and heated. A brick red precipitate of cuprous oxide indicates the presence of
reducing sugars.
ii. Molisch’s test: In an aqueous solution, α-napthol was added. Afterwards, concentrated sulphuric acid was
gently poured. A purple colour ring at the junction of the two solutions indicates the presence of the reducing
sugar.
3. Test for Flavonoids[18]: Magnesium ribbon was added to the ethanolic extract of the material followed by
drop wise addition of concentrated Hcl. Colour change from orange to red is a confirmatory test for flavonoid.
4. Test for Glycosides[17]: The test solution is to be filtered and sugar is removed by
fermentation with baker’s yeast. The acid is removed by precipitation with magnesium oxide or barium
hydroxide . The remaining ethanolic extract contains the glycosides which are
subsequently detected by the
following methods.
a. The hydrolysis of the solution is to be done with concentrated sulphuric acid and after the hydrolysis sugar is
determined with the help of Fehling’s solutions.
b. The Molisch’s test is done for sugar using α-napthol and concentrated sulphuric acid.
5. Test for Tannins: Ferric chloride solution was added in the aqueous extract of the drug. A bluish-black
colour, which disappeared on addition of dilute sulphuric acid followed by
a yellowish brown precipitate,
shows the presence of tannin.
6. Test for Proteins
Millon’s reaction: For the test solution, Millon’s reagent was mixed and white colour
precipitate showed
the presence of proteins.
7. Test for Starch[20]: 0.015 gm of Iodine and 0.015 gm of Potassium Iodide was added in 5 ml of distilled
water, 2 ml of this solution formed was added to 2 ml of aqueous test solution, the presence of blue colour
indicates the presence of starch.
8. Test for Phenol[17]: 5–8 drops of 1% aqueous solution of Lead acetate was added to aqueous or ethanolic
test solution. The presence of yellow colour precipitate indicates the presence of phenols.
9. Test for Sterol/Terpenes[17]
Salkowski reaction: In the test solution of chloroform 2 ml sulphuric acid (concentrated) was mixed from the
side of the test tube. The colour of the ring at the junction of the two layers was observed. A red colour ring
indicates
the
presence
[19]
10. Test for Amino Acids
80
of
the
: The ethanolic extract was mixed with ninhydrin solution
sterols/terpenes.
(0.1% in
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acetone). After heating gently on water bath for few minutes it gives a blue to red-violet colour that indicates
the presence of amino acids.
11.
Test
Resins[18]
for
The test solution was gently heated and acetic anhydride was added in it. After cooling, one drop of sulphuric
acid was mixed. A purplish red colour that rapidly changed to violet
indicates the presence of the resins.
10. Fluorescence Analysis of the successive extracts of drug sample
Successive extracts of all the drug sample viz. Petroleum ether, diethyl ether, chloroform, ethyl acetate, acetone,
ethanol and aqueous were observed.[19]
Result
and
Discussion
Nepeta bracteata Benth was subjected to systematic physiochemical and phytochemical screening by extracting
with various organic solvents. The data generated is helpful in
determining the quality and purity of a
powder drug. Correct identification, authentication and quality control are essential to ensure safety, efficacy
and reproducibility. The parameters
studied includes organoleptic characters of powder drug
summarised in table 1, determination of extractive values of the test drug in different organic solvents, alcohol
and water soluble contents, moisture content, ash values, loss of weight on drying, pH values and bulk density
as summerised in table 2, phytochemical analysis of the powder drug (in table 3), fluorescence analysis of
successive extract of the test drug given in table 4 and thin layer chromatography (TLC) given in table 5.
It was
Conclusion
concluded that now a days, many of the medicinal plants available in the market have ambiguous identification
along with adulteration and contamination. It is therefore necessary to work out physicochemical standards of
unani drugs. The physicochemical evaluation of the powder drug reveals the standard parameters for the quality
and purity of herbal drug and also gives information regarding the authenticity of crude drug.
Acknowledgement
Authors are thankful to DRS-I (UGC) Department of Ilmul Advia for providing assistance during the study.
REFERENCES
1. Joharchi MR, Avicenna Journal of Phytomedicine, March, 2012, Vol 2, pp.105-112
2.Mojab Faraz, B. Nickavar, H H. Tehrani, Iranian Journal of Pharmaceutical Sciences, 2009, 5(1), pp. 43-46.
3 .Kiritikar K.R. and Basu B.D., Indian Medicinal Plants, International Book Distributers Dehradun, 1987, Vol
4, p.1990
4.Bhatt Jalaluddin, Qudsia, Aslam M.,Interntional Journal Of Pharmacy and Life Sciences January -February
2012, Vol 2(1), pp 147-48
5.Dymock . “Pharmacographia Indica”A History of the principle of Drugs of vegetable origin met with British
India, The Institute of Health and Tibbi Research (Pakistan) 1891, Vol 3 p.359
6.Said H.M.,Medicinal Herbal, Hamdard Foundation Pakistan, 1996, Vol 1, pp.129-130
81
7.Nadkarni,A. K.,
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Abdul Latif et al / IJDFR volume 4 Issue 4 July-Aug. 2013
Indian Materia Medica, Popular book Depot, Bombay, India , 1954 , Vol 2 , p.673
8.Ibn Baitar, Al Jamiul Mufradatul Advia wal Aghzia ( Urdu ), 1999, Vol 3, p.185
9.Ibn e Sina, Al Qanoon Fit Tibb (Urdu translation by Ghulam Hussain Kantoori), Matba Nawal Kishore,
Lucknow,1887, Vol 2, p. 88
10.Hakeem, Bustanul Mufradat (Urdu translation), M.A Karkhana Jamitul Advia, Lucknow, India, 1893 pp.
83-188.
11. Lubhaya, R.H., Goswami Bayanul Advia, Goswami Kutub Khana Gali Qasim, Delhi, y.n.m , Vol 1, pp.303304.
12.Ghani, M.N., Khazainul Advia (Urdu translation), Sheikh Mohd Bashir and Sons, Urdu Bazar Lahore,
Pakistan, 1921, p. 768.
13. Naghibi Farzaneh, Mosaddegh M., Motamed S. M., Iranian Journal of Pharmaceutical Research 2005, Vol
2, pp. 63-79
14.Anonymous, British Pharmacopoeia, General Medicine Council, Pharmaceutical Press, Bloomsbury square
(London), 1968, pp.1276-77.
15.Jenkins G.L.,A.M. and Digangi F.E., Quantitative Pharmaceutical Chemistry, The Blackiston Division ,
McGraw Hill Book Company (U.S.A.), 1967, pp. 225, 235, 379,425 463
16.Anonymous, The Unani Pharmacopeia of India, 2009, Part 2, Vol 1, p.146
17.Afaq,S.H.,Tajuddin,Siddiqui, Standardization of Herbal Drugs, Publication Division, AMU (Aligarh), 1994
, pp. 66,100,143-146
18. Lalif Abdul, Thesis on Pharmacognostical and pharmacological studies of
Cardiospermum
halicacabum Linn seed, 1982, pp.22-26
19. Anonymous, Standardization of single drugs of Unani Medicine, New Delhi, 1992, part 2, pp.148-153.
20. Ali M., Text book of pharmacognosy, CBS Publishers & Distributor PVT. LTD.2010, p. 71
Table 1. Organoleptic Characters of powder of Nepeta bracteata Benth.
S. NO.
Parameter
Appearance
1.
Colour
Brownish green
2
Smell
Camphorous
3.
Taste
Slightly bitter
Table 2: Physicochemical Parameter of Nepeta bracteata Benth.
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S.NO. Physicochemical Parameter
Result: Mean±S.E.M. (S.D.)
1.
Loss of weight on drying at105 ˚C
11.02±0.146(0.327)
2.
Moisture content
8.4±0.748(1.673)
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3.
4.
Ash Value (in %)
Total Ash
8.367±0.278(0.681)
Acid Insoluble Ash
1.593±0.126(0.309)
Water Soluble Ash
2.827±0.215(0.527)
Sulphated Ashes
12.183±0.236(0.578)
PH Values (in %)
pH at 1%
6.434±0.008(0.017)
pH at 10%
5.44±0.009(0.020)
5.
Bulk Density (in gm/ml)
0.26±0.009(0.020)
6.
Solubility (in %)
7.
Alcohol Soluble extractive
1±0.0707(0.158)
Water Soluble extractive
4.86±0.0872(0.195)
Extractive values in different organic solvent (in%)
Pet Ether
3.586±0.215(0.480)
Diethyl Ether
14.548±0.858(1.918)
Chloroform
0.76 ±0.479(0.230)
Ethyl Acetate
1.46± 0.0509(0.230)
Acetone
2.87±0.0419(0.0938)
Alcohol
1.456±0.070(0.571)
Aqueous
18.84±0.727(0.325)
Table 3. Phytochemical analysis of Nepeta bracteata Benth.
S.No. Chemical Constituent
Test / Reagent
Inference
1.
Alkaloid
Dragendroff’s reagent
-ive
2.
Carbohydrate
Fehling solution Test
+ive
Benedict reagent Test
3.
Flavonoids
Mg ribbon Test
+ive
4.
Glycosides
Baker’s yeast Test
+ive
5.
Tannins
FeCl3 Test
+ive
6.
Protein
Millon reagent Test
+ive
7.
Starch
Iodine Test
-ive
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8.
Phenols
Lead Acetate Test
+ive
9.
Sterol
Salkowski reaction
+ive
10.
Amino acid
Ninhydrin Solution
-ive
11.
Resin
Acetic Anhydride Test
-ive
(+ive = Present;
- ive = absent)
Table 4. Fluorescence Analysis of the Successive extracts of Nepeta bracteata Benth.
Extract
Daylight
UV Short
UV long
Pet Ether
Green
Magenta
Green
Diethyl Ether
Straw Colour
Purple
Light Green
Chloroform
Transparent
Transparent
Transparent
Ethyl acetate
Transparent
Transparent
Transparent
Acetone
Light Green
Transparent
Transparent
Ethanolic
Transparent
Transparent
Transparent
Aqueous
Transparent
Transparent
Transparent
Table 5.
TLC of Gul e Zofa
S.NO. Extract
Solvent
Treatment
NO.
Sytem
Rf Values
of
spot
1.
Petroleum ether
PE:DE 1:1
Daylight
3
0.795 , 0.681 ,0.5
2.
Diethyl ether
PE:DE 1:1
UVlong
9
0.1137, 0.1819 ,0.25,
0.273, 0.340,0.398,
0.455 ,0.682, 0.807
UV Short
9
0.25, 0.204, 0.375,
0.466, 0.580, 0.557,
0.602,0.682, 0.807
(PE=Petroleum ether; DE= Diethyl ether)
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Fig.-1: TLC OF Nepeta bracteata Benth (a) Diethyl ether extracts (b) Pet. Ether extract in Day light
UV Long
Fig. 1(a)
UV Short
Diethyl ether extract in UV Long & UV Short
Fig. 1(b) Pet. Ether extract Day light
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Fig.2: Flowers of Nepeta bracteata Benth (Gul-e-Zofa)
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