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18 International Journal of Pharmacy and Pharmaceutical Science Research 2014; 4(1): 18-24
ISSN: 2249-0337
Original Article
DETECTION OF ADULTERATION AND AUTHENTICATION OF ALOE VERA
PRODUCTS BY ANALYTICAL AND PHARMACOGNOSTICAL TOOLS.
Dr.Vinay Sharma1, Dr. Renuka
2, Prof. P.K. Prajapati
3, Dr. V.J. Shukla
4, Dr. Harisha
5
M. Sc PhD (Medicinal plants) Department of RS & BK1, M. Pharm PhD Department of Pharmaceutical chemistry
2,M.D
PhD Department of RS & BK3,MSc. PhD Department of Pharmaceutical chemistry
4,MSc. PhD Department of
Pharmacognosy5.
E mail [email protected]
Received 15 February 2014; accepted 14 March 2014
Abstract
Analysis of botanicals is great challenge because they are complex mixture of compounds covering a broad range of
substance classes and exhibit natural variability. Amog all Aloe vera is found to be mostly adulterated one. The present
study was aimed to set the methods for detection of adulteration in Aloe formulation mainly aloe vera Gel and Kumari
Asava. For the desired purpose different samples were prepared and analysis was carried out taking Alion as component of
interest .To detect adulteration in both kind of formulation there are different sample prepared and analysis of main
chemical constituents has been done. HPTLC and UV spctrophotometric methods were used as detection tools. The results
shows that samples containing whole Aloe leaf shows presence of more Aloin content as compare to inner gel containing
samples. Also market samples were analyzed to detect and quantify the Aloin samples and results shows that out of 4
samples only one sample shows the presence of Aloin content and that is also in very low amount.
© 2013 Universal Research Publications. All rights reserved
INTRODUCTION Aloe vera (Kumari) is a well known Plant which is one of
the most discussed, studied, traded and paid plant. Aloe
vera is a natural product that is now a day frequently used
in the field of cosmetology, health drink and much more. It
is found that still modern science has to be worked on it
like Aloe has about 400 different species and different parts
of plant offer disparate health effects from laxative to
wound healing. The concept of whole leaf gel and inner gel
is still debating [1]
.
A. vera is the most commercialized aloe species and
processing of the leaf pulp has become a large worldwide
industry and Aloe Vera itself become a trade name for all
preparations. Currently Japan imports over fifty million
dollars per year of aloe to treat people with ulcers and
digestive problems [2]
. Over a hundred thousand plants have
been grown at one time in Florida, Texas, and Mexico for
market, and it has been estimated that annual sales in this
country amounts to millions of dollars. According to a
survey in $125 million dollars of Aloe raw material cost is
used in market [3]
.
The different cells of aloe leaf are responsible for different
by products e.g rind provides the active carbon which is
responsible for mannose and glucose formation [4]
. Alotic
cells secrete yellow colored, bitter liquid, as called Latex,
Sap or Aloe juice3. The dried juice of aloe in the form of
this sap later turns dark brown in color after drying[5]
. This
has anthraquinone glycosides which contain Aloin [6]
. This
colorless mucilaginous gel obtained from the parenchyma
cells called Aloe Gel [3]
. These cells have polysaccharides
and various essential components.
Adulteration is a practice of substituting original crude drug
partially or wholly with other similar looking substances
but the later is either free form or inferior in chemical and
therapeutics properties[7]
.
A drug shall be deemed to be spurious- (a) if it is imported
under a name which belongs to another drug; or (b) if it is
an imitation of , or a substitute for, another drug or
resembles another drug in a manner likely to deceive or
bears upon it or upon its label or container the name of
another drug unless it is plainly and conspicuously marked
so as to reveal its true character and its lack of identity with
such other drug ; or (c) if the label or the container bears
the name of an individual or company purporting to be the
manufacturer of the drug, which individual or company is
fictitious or does not exist; or (d) if it has been substituted
wholly or in part by another drug or substance; or (e) if it
purports to be the product of a manufacturer of whom it is
not truly a product[8]
9B.
According to Indian Penal Code 1860;Whoever adulterates
any drug or medical preparation in such a manner as to
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Universal Research Publications. All rights reserved
19 International Journal of Pharmacy and Pharmaceutical Science Research 2014; 4(1): 18-24
lessen the efficacy or change the operation of such drug or
medical preparation, or to make it noxious, intending that it
shall be sold or used for, or knowing it to be likely that it
will be sold or used for, any medicinal purpose, as if it had
not undergone such adulteration, shall be punished with
imprisonment of either description for a term which may
extend to six months, or with fine which may extend to one
thousand rupees, or with both[9]
.
It is one of the most marketed drugs. Aloe vera has been
presented in a variety of OTC drug products [10]
.A. vera
must be “biologically alive” and must be in bio-available
from if customers around the world tend to experience full
scope and range of benefits from this plant. However, over
95% of the Aloes on the market today still use only the
inner gel and Stabilize the Aloe in a high-heat process
degrades some of the enzymes, polysaccharides and
mucopolysaccharides. High heat (pasteurization and/or
autoclave methods) breaks down the constituents in Aloe,
which are the most valuable for healing. It also degrades
enzymes necessary for digestion [11-13]
. Even some of
chemical constituents degrade in direct sun light [14]
. Some
research works showed that there is very less or negligible
amount of Aloin present in market samples.
Adulteration represents a major concern for the aloe vera
market, mostly because of the high cost of the raw
materials. Historically, the most common substance used to
adulterate aloe gel powder is malt dextrin[15]
. Glucose,
glycerine and malic acid have also been reported [6-17]
.
Many methods have been developed to detect adulteration
and establish the authenticity of aloe gel powders. L-malic
acid and some phenolic compounds (aloesin, barbaloin, and
aloe-emodin,aloin) have been proposed as markers.
Present research work is amid to set the methods for
detection adulteration in most used and marketed
formulation aloe vera Gel and Kumari Asava. To detect
adulteration in both kind of formulation there are different
sample prepared and analysis of main chemical constituents
has been done. The detection was carried out by HPTLC
and UV spctrophotometric methods.
MATERIAL AND METHODS As per the proposed study an attempt has been made to
develop the quality standards for detection of adulteration
by estimation of main chemical constituent Aloin, in
different fermented formulations of Aloe vera. Asava and
Aristha are self-generated alcoholic preparations of
traditional Ayurvedic system. It comes in Sandhana
Kalpana which means liquid in which drugs kept for long
period, getting fermented. [18]
Kumaryasva is prepared as
per the reference of Ayurvedic formulary of India,
Kumaryasva “B”[19]
and Yoga Ratnakara Utrardha, Gulma
rogadhikar [20]
. During pharmaceutical study, Kumari inner
gel was fermented by adding Sandhana dravyas in other
words Kumari asava with inner gel was prepared without
adding Prakshep dravyas and Haritaki kwatha. Coded as
sample C. The samples were prepared in laboratory of
Department of RS&BK, IPGT&RA Gujarat Ayurved
University,Jamnagar. Two different techniques High
performance thin layer chromatographic and ultraviolet
visible spectroscopy were used for investigation of samples
along with the pharmacogonosy study.
DRUG SAMPLE Previously standardized samples (fermented and aqueous)
were taken and are mentioned below
G1 : Whole leaf gel (Kumari swarasa)
G2 : Inner gel (Kumari swarasa)
A : Whole leaf having Kumari asava
B : Inner gel having Kumari asava
C : Fermented Inner gel
MS1 : Market sample of Kumari asava
MS2 : Market sample of Kumari asava
MS3 : Market sample of Kumari swarasa
MS4 : Market sample of Kumari swarasa
Aloin standard was purchased from TOTAL HERB
SOLUTIONS, Mumbai, Maharashtra. All chemicals used
were of AR grade. METHODSFORADULTERATION DETERMINATION
Following methods were adopted for the detection of
adulteration in different samples of aloe vera formulation
HPTLC Analysis
Sample preparation The dried residues(5 mg weighed separately) of all the
samples were soaked in 2 ml of methanol and subjected for
extraction in ultrasonication bath for 30 minute. The
samples were filtered and volume was made up to 5 ml
with methanol in a standard flask to get final concentration
of 1mg/ml for each sample.
Preparation of standard Aloin solution Aloin standard (2mg) was accurately weighed and
transferred to 2ml of volumetric flask. Crystals were
dissolved in methanol and the volume was adjusted up to 2
ml with methanol (1mg/ml).The standard stock solution
was used to prepare solutions with concentration 2.5,5,
7.5μg/μl respectively.
Chromatography conditions and procedure 5.0 μl of each sample and standard aloin 2.5 μl,5 μl and 7.5
μl respectively were applied on E Merk Aluminium plate of
0.2 mm thickness pre-coated with 0.2mm layers of silica-
gel 60 F254. Sample was applied with Linomat IV
applicator. The mobile phase used for chromatography was
ethyl acetate-methanol-water (10: 1.35: 1.0 v/v/v). [21]
Development of the plate was performed to a distance of
8.0 cm in chamber previously saturated for 5 min. Plate
was scanned at 254 nm and 359 nm using Deuterium
tungsten lamp. The Rf value and percentage area of each
spot were calculated.
Calibration curve for Aloin Calibration graph was found to be linear over the
concentration range 2.5-7.5μg/ μl. Linearity was evaluated
by determining three standard working solutions . The peak
area and concentration was subjected to least square linear
regression analysis to determine the correlation coefficient.
UV Profiling
Instrumental condition Instrument: Double beam UV spectrophotometer Shimadzu
Working wavelengths: 357 nm
Sample cell: Quartz cuvette
Monochromator: Grating
Detector: Phototube
Sample preparation Samples of 1mg/ml concentration were prepared by similar
20 International Journal of Pharmacy and Pharmaceutical Science Research 2014; 4(1): 18-24
method as that for HPTLC sample preparation.
Standard Preparation Standard Aloin solution with conc. 1 mg/ml was prepared.
Determination of Absorbance value for standard and
sample 3 ml sample and standard solutions were taken separately
in UV Quartz cell and subjected to UV spectrophotometer.
The sample was read out at wavelengths 357 nm[22]
and
absorbance was measured.
Determination of linearity in Aloin standard Standard Aloin solutions in concentration range of 20-
100μg/ml were prepared and absorbance was measured at
357 nm taking methanol as blank. The resultant absorbance
values were used to generate calibration curve taking
absorbance and concentration on respective axis.
Correlation coefficient was calculated by using the linear
equation. The graph was found to be linear in range of 20-
100μg/ml. The coefficient of determination was 0.990.
(Plate: 9; Fig: 3)
Microscopy of Kumari Swarasa and Kumari Asava The samples Kumari swarasa and Kumari asava were
subjected for microscopy study.The samples were analyzed
under trinocular microscope for presence of typical
identifying characters. And it was found that Acicular
crystals were present in both Kumari swarasa as well as
Kumari asava sample.
RESULT AND DISCUSSION The chromatography method described utilizes silica gel
GF 254 HPTLC plates as stationary phase and ethyl
acetate-methanol-water (10: 1.35: 1.0 v/v/v) as mobile
phase which gives good separation of Aloin (Rf.=0.55)
standard. Table 5.4 shows the no. of spots and Rf at short
UV (254nm) and long UV (366). In Sample A (A-A4) 4
spots were detected at short UV and 5 spots were detected
at long UV. Sample B (B-B4) shows 3 spots at short UV
and 4 spots at long UV. 2 spots were detected in sample C
at short UV while 3 spots at long UV. Sample G1 shows 7
spots at short UV while 8 spots were detected at long UV.
Reference standard Aloin shows 3 spots under short UV
while 5 spots were detected at long UV (Table 1 and Fig.
1).
Sample MS1 shows 5 spots at both short and long UV.
MS2 shows 2 spots at short UV and 3 spots at long UV. In
MS3 at short long UV1 spot and at long UV 4 spots were
detected. MS4 was aloin free sample it was taken as blank
track. Reference standard Aloin shows 3 spots at short UV
and 5 spots were detected at long UV (Table 5.5).The
HPTLC fingerprint results reveals the presence of Aloin in
all samples(Table 2 fig. 2) .
The HPTLC fingerprint study was also carried out to
determine the amount of Aloin in all the samples including
market samples of Kumari gel and Kumari asava. The
results shows that when comparison was done between
sample G1 & G2 sample G1 shows more Aloin content as
compare to sample G2 the reason in sample G1 whole leaf
was used that contains more Alotic cells as compare to
inner gel which results in presence of more Aloin content in
G1(Table 3).
For sample A & B, Aloin was found to be more in sample
A then sample B the reason is whole leaf was used in this
preparation.
When comparison was carried between A, B & C, sample
C Shows more Aloin content the reason is there was no
prakshep dravya used in this preparation while in A & B
prakshep dravya were used which creates matrix
environment. As the matrix increases the Amount of
interfering components also increase this interferes with the
target components. Matrix is more in sample A & B so it’s
difficult to measure the Aloin content (Table 3).
Four marketed samples of Kumari gel and Kumari asava
coded as MS1, MS2, MS3 and MS4 were taken for the
study to find out the amount of Aloin and the results shows
that out of all 4 samples, Aloin was detected only in sample
MS2 and that is also in very low amount as compare to
laboratory prepared samples. The sample contains aloin in
the range as that of asav prepared from inner gel .So
Amount of Aloin gives an idea that by which part asava is
prepared. This tool can be used for detection adulteration
and authentication of product (Table 3).
The findings for UV study (Table 4) shows presence of one
component with ʎmax value the 357nm which shows the
presence of Aloin in all samples. The UV study was also
carried out to determine the amount of Aloin in all the
samples and to detect the presence of matrix and its affects.
When comparison was carried out between sample G1 and
G2 results shows that sample G1 contains more Aloin
content as compare to sample G2 the reason is same
presence of more Alotic cells in sample G1 then sample
G2.comparitive results for sample A and B shows more
Aloin content in sample A then sample B, reason is sample
A was prepared from whole leaf containing more Alotic
cells. When samples A,B and C were compared for UV
analysis results shows more Aloin content in sample C as
compare to sample A and B. it is due to reason that
prakshep dravya were used in sample A and B which
creates matrix environment while no prakshep dravya had
been used in sample C.
The samples were also studied out for the affect of heat on
the Aloin content. Table. 5. shows effect of heat at below
boiling point (800C). Before heating, Aloin was found more
(0.22) as compared after heating (0.18).The results directly
indicates that Aloin is heat sensitive content and it degrades
as the temperature increases which may result in reducing
the quality of product.
The samples were also studied to find out the matrix affect
in asav samples in context of recovery parameter (Table
no.6). The recovery study was carried out by standard
addition method. Known amount of standard 80%, 100%
and 120% was added. Samples of10 μg/ml was taken and to
this standard was added. The results show that Sample G1
shows 88% recovery. Sample G2 88% recovery and sample
A, B and C showed 75%, 80%, 98% recovery respectively.
A method is said to accurate if recovery is more than 90%.
Sample C passed the recovery parameter while recovery
result for sample A and B were not within acceptable limit.
The reason is presence of more matrixes that interferes the
response factor of target analyte Aloin. More the matrix it
will be difficult to measure out the active principle. The
results show practically interference of matrix component
over the response factor of target analyte that is Aloin.
21 International Journal of Pharmacy and Pharmaceutical Science Research 2014; 4(1): 18-24
Table 1: Results for HPTLC of Samples of Kumari swarasa and Kumari asava and Aloin
Mobile phase: Ethyl acetate :Methanol :Water(10:1.35:1.0v/v/v)
Track
No. Samples
254 nm 366 nm
No. of
spots Rf value No. of spots Rf value
1 Sample A1 3 0.03,0.30,0.35 5 0.03,0.06,0.13,0.27,0.33
2 Sample A2 2 0.30,0.34 3 0.12, 0.27, 0.43
3 Sample A3 3 0.30,0.34,0.93 3 0.12,0.30,0.78
4 Sample A4 4 0.28,0.30,0.33,0.93 2 0.13, 0.28
5 Sample B1 2 0.31,0.34 2 0.15,0.31
6 Sample B2 2 0.31,0.35 3 0.14,0.29,0.34
7 Sample B3 2 0.31,0.34 2 0.12,0.31
8 Sample B4 3 0.31,0.34,0.56 2 0.29,0.55
9 Sample A 4 0.8,0.31,0.34,0.49 3 0.6,0.29,0.48
10 Sample B 2 0.31,0.34 4 0.12,0.16,0.28, 0.60
11 Sample C 2 0.30,0.33 2 0.30,0.66
12 Sample G1 7 0.30,0.33,0.10,0.23,0.27,0.
36,0.54 4 0.19, 0.33, 0.49,0.60
13 Sample G2 6 0.01,0.10,0.28,0.32,
0.66,0.74 8
0.07,0.17,0.24,0.30,
0.58,0.70, 0.73,0.78,
14 Aloin 4 0.32,0.66,0.72 6 0.01,0.26,0.31,0.34,0.40,0.62
Table 2: Results for HPTLC of Marketed sample of Kumari swarasa and Kumari asava
Mobile phase-Ethyl acetate: Methanol: Water (10:1.35:1.0v/v/v)
Track no. Samples
254 nm 366 nm
No. of
spots Rf value No of spots Rf value
1 MS 1 5 0.09,0.60,0.66,0.71,0.81 5 0.09,0.15,0.66,0.89,0.89
2 MS2 2 0.09,0.31 3 0.39,0.55,0.90
3 MS 3 1 0.43 4 0.05,0.13,0.41,0.94
4 MS 4 -- -- -- --
5 Aloin 3 0.02,0.16,0.31 5 0.02,0.07,0.15,0.47,0.63
Table 3: Quantitative estimation of Aloin by HPTLC
Sample Amount of Aloin in μg/5 μg of methanol extract
G1 2.5
G2 2.0
A 1.21
B 0.22
C 0.46
MS 1 ND
MS 2 0.26
MS3 ND
MS4 ND
Table 4: Quantitative estimation of Aloin by UV spectrophotometer
Samples Average Absorbance at 357 nm Quantity of Aloin in μg/5μg
G1 0.177 1.9
G2 0.040 0.44
A 0.351 3.9
B 0.248 2.7
C 0.455 5.0
22 International Journal of Pharmacy and Pharmaceutical Science Research 2014; 4(1): 18-24
Table 5: Effect of heat on Aloin in Kumari swarasa
Condition Amount of Aloin in μg/5 μg of methanol extract
Before heating 0.22
After heating 0.18
Table 6: Results for Recovery study
Accuracy and recovery for Sample G1
Amount taken(µg/ml) Amount added (µg) Absorbance % recovery
10 0 0.087 -
10 80 0.470 70%
10 100 0.589 79%
10 120 0.715 83%
Accuracy and recovery for Sample G2
Amount taken(µg/ml) Amount added (µg) Absorbance % recovery
10 0 0.115 -
10 80 0.387 81%
10 100 0.572 85%
10 120 0.681 88%
Accuracy and recovery for Sample A
Amount taken(µg/ml) Amount added (µg) Absorbance % recovery
10 0 0.183 -
10 80 0.598 69%
10 100 0.822 75%
10 120 0.451 59%
Accuracy and recovery for Sample B
Amount taken(µg/ml) Amount added (µg) Absorbance % recovery
10 0 0.119 -
10 80 0.451 73%
10 100 0.621 80%
10 120 0.623 80%
Accuracy and recovery for Sample C
Amount taken(µg/ml) Amount added (µg) Absorbance % recovery
10 0 0.217 -
10 80 0.492 97%
10 100 0.639 97%
10 120 0.704 98%
Fig. 1: HPTLC fingerprint Kumari swarasa and asav
23 International Journal of Pharmacy and Pharmaceutical Science Research 2014; 4(1): 18-24
Fig. 2: HPTLC of Market Samples of Kumari Swarasa and
Kumari Asava
Fig 3. A) Graph Area of Aloin Standard, B) Graph Height of
Aloin Standard
Fig.4: Linearity graph for Aloin (UV)
Fig.5: Acicular Crystals in Kumari Gel
Fig.6: Acicular Crystals in Kumari Asava
When microscopy of Kumari gel and Kumari asava were
analyzed acicular crystal were present as it can be seen in
parenchymatous region in T.S of Kumari leaf. In asava
sample the Kumari gel was used which is the main
ingredient of Kumari asava. Because of this acicular
crystals seen under microscopy. It can be a key character
for authentication and detection of adulteration in Kumari
asava if acicular crystals found in drug it is assured that
asava is prepared by Aloe gel (Fig.5 & 6).
CONCLUSION UV Spectroscopy can be used for detection of particular
chemical constituent in compound formulations. Recovery
falls within the acceptable limit as per ICH guidelines
method so method is said to be accurate. Presence of
acicular crystals and amount of Aloin can be a
characteristic element to detect adulteration and
authentication. Based upon the all results and findings it
can be concluded that the formulation in which whole aloe
is used will contain more Aloin content as compare to inner
gel formulations.
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Source of support: Nil; Conflict of interest: None declared