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Quantification of allicin by high performance liquid chromatography-ultraviolet analysis with effect of post-ultrasonic sound and microwave radiation on fresh garlic cloves

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  • Sanaka Educational Trust's Group of Institutions
  • Gupta College of Technological Sciences, Asansol
Article

Quantification of allicin by high performance liquid chromatography-ultraviolet analysis with effect of post-ultrasonic sound and microwave radiation on fresh garlic cloves

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Background: Garlic (Allium sativum L.) has been accepted universally to be applied in food, spice and traditional medicine. The medicinal and other beneficial properties of garlic are attributed to organosulfur compounds. Objective: As of today no simultaneous analysis has been performed; hence the transformation of allicin to its degraded products during cultivation and storage is open into doubt. Materials and methods: In our present work, we have tried to develop a sensitive and reproducible analytical method to measure allicin by high performance liquid chromatography-ultraviolet analysis with effect of post-acoustic waves and microwave radiation on fresh garlic cloves. Results: The process revealed the effect of different radiation techniques on fresh garlic retains the principle component, allicin in its pure form and generated higher yield than the conventional way of extraction. Conclusion: Therefore, materializing these techniques in the pharmaceutical industry will definitely be proved beneficial in term of time as well as money. Most interestingly, the methods ruled out possibilities of degradation of organosulfur compounds as well.
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S288 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
Quantification of allicin by high performance liquid
chromatography‑ultraviolet analysis with effect of
post‑ultrasonic sound and microwave radiation on fresh
garlic cloves
Sankhadip Bose, Bibek Laha
1
, Subhasis Banerjee
2
Departments of Pharmacognosy,
1
Pharmaceutics, and
2
Pharmaceutical Chemistry, Gupta College of Technological Sciences,
College of Pharmacy, Asansol, West Bengal, India
Submitted: 30‑07‑2013 Revised: 29‑08‑2013 Published: 28‑05‑2014
Address for correspondence:
Dr. Sankhadip Bose, Department of Pharmacognosy,
Gupta College of Technological Sciences, College of Pharmacy,
Ashram More, G.T. Road, Asansol - 713 301, West Bengal, India.
E‑mail: sankha.bose@gmail.com
Background: Garlic (Allium sativum L.) has been accepted universally to be applied in food, spice
and traditional medicine. The medicinal and other benecial properties of garlic are attributed to
organosulfur compounds. Objective: As of today no simultaneous analysis has been performed;
hence the transformation of allicin to its degraded products during cultivation and storage
is open into doubt. Materials and Methods: In our present work, we have tried to develop a
sensitive and reproducible analytical method to measure allicin by high performance liquid
chromatography-ultraviolet analysis with effect of post-acoustic waves and microwave radiation
on fresh garlic cloves. Results: The process revealed the effect of different radiation techniques
on fresh garlic retains the principle component, allicin in its pure form and generated higher yield
than the conventional way of extraction. Conclusion: Therefore, materializing these techniques
in the pharmaceutical industry will denitely be proved benecial in term of time as well as
money. Most interestingly, the methods ruled out possibilities of degradation of organosulfur
compounds as well.
Key words: Acoustic waves, allicin, garlic, high performance liquid chromatography-ultraviolet
analysis, microwave radiation
Access this article online
Website:
www.phcog.com
DOI:
10.4103/0973‑1296.133279
Quick Response Code:
INTRODUCTION
Garlic (Allium sativum Linné) is a widely distributed plant
and is used in all parts of the world not only as a spice
and food, but also as a popular remedy.
[1]
Numerous
studies have previously demonstrated that garlic may be
useful for the prevention of carcinogenesis, cardiovascular
and age‑related diseases.
[2]
Especially, it has been strongly
suggested that its medicinal and benecial properties
are attributed to specic organosulfur compounds.
[3‑6]
It
is known that garlic contains three ç‑glutamyl peptides,
that is, ç‑L‑glutamyl‑S‑(2‑propenyl)‑L‑cysteine,
ç‑L‑glutamyl‑S‑(trans‑1‑propenyl)‑L‑cysteine and
ç‑L‑glutamyl‑S‑methyl‑L‑cysteine; their corresponding
sulfoxide derivatives, viz., (+)‑S‑(2‑propenyl)‑L‑cysteine
sulfoxide (alliin), (+)‑S‑(trans‑1‑propenyl)‑L‑cysteine sulfoxide
(isoalliin), (+)‑S‑methyl‑L‑cysteine sulfoxide (methiin) and
(1S,3R,5S)‑5‑methyl‑1,4‑thiazane‑3‑carboxylic acid 1‑oxide
(cycloalliin) [Figure 1].
Sulfoxides have been reported to have some medicinal
properties; for example, alliin shows anticancer effects
[4,5]
and alliin and cycloalliin present lipid‑lowering effects.
[3,5]
In
contrast, ç‑glutamyl peptides have been reported to lower
blood pressure
[4‑6]
and to have cholesterol‑lowering effect.
[4,6]
However, once garlic is crushed, these compounds are
transformed into other compounds such as allicin, ajoene,
dithiins and diallylpolysuldes.
[6,7]
Therefore, it is important
to control sample preparation to minimize articial errors.
The biosynthetic pathway of organosulfur compounds
in garlic has been proposed to involve transformation of
ç‑glutamyl peptides into their corresponding sulfoxides
by ç‑glutamyl transpeptidase and oxidase.
[8‑10]
Moreover,
it has been reported that, the amount of organosulfur
compounds do not remain same during cultivation and
storage
[10‑13]
[Figure 2].
ORIGINAL ARTICLEPHCOG MAG.
ABSTRACT
Bose, et al.: Quantication of allicin with effect of radiations
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S289
Therefore, an analytical method for the simultaneous
determination of all sulfoxides and their precursors,
ç‑glutamyl peptides, in a garlic sample is required to
evaluate the quality of garlic even after different cooking
or extraction methods where microwave radiation and
acoustic waves are used. Our present study aims at
determining the allicin content by high performance
liquid chromatography‑ultraviolet (HPLC‑UV) analysis.
Chromatograms obtained after normal extraction,
microwave extraction, acoustic waves effect by the probe
and bath sonication and quantitative result of the amount
of allicin in a commercial garlic sample were reported.
Microwave radiation and acoustic waves are of importance
because many food materials and medical formulations
are prepared by using them. Therefore, it is important
to determine the stability and quantity of allicin after
conducting various extraction processes.
MATERIALS AND METHODS
Sources of reagents
Sodium chloride was purchased from Qualigens, Mumbai,
Dichloromethane from Merck Specialties Pvt. Ltd.,
Mumbai, Methanol and water of HPLC grade were
procured from Merck Specialties Pvt. Ltd., Mumbai.
Fresh garlic cloves (A. sativum L.) were obtained from the
cultivated elds of Asansol area in West Bengal, India
which was authenticated (Authentication no. CNH/1‑1/
(201)/2013/Tech. II/35) by head, Shibpur Botanical
Garden, Shibpur, Howrah, West Bengal, India.
Standard allicin
The synthesis of allicin was performed following the
method proposed by Bocchini et al.
[10]
According to this,
an equimolar amount of perbenzoic acid in 20 ml of
dichloromethane, was slowly added to a solution of allyl
disulde (1.46 g in100 ml of dichloromethane), under
rapid magnetic agitation while cooling at −10°C. The
reaction mixture was allowed to stand at room temperature
for 1 h. The excess of acid was removed by washing
the mixture with a sodium bicarbonate solution. The
dichloromethane solution was rinsed with distilled water,
dried over anhydrous sodium sulfate and the solvent was
removed by rotary‑evaporation. The dried product was
weighed and standard solution was made by dissolving the
pure allicin in methanol. Data obtained out of HPLC‑UV
analysis showed that allicin accounted for >99% of the
standard.
Garlic samples
A sample of 30 g of garlic cloves in 300 ml of distilled
water were crushed for 1 min in a blender mixer.
Microwave extraction
[14]
The above mixture was transformed to a specially designed
round bottom ask with which a condenser was tted.
The entire assembly was nally kept inside the microwave
extractor (Catalyst System, Pune, Model No. CATA 2R).
It was reuxed for 10 min with the percentage power of
20 at 140 watt (41°C) and subsequently processed to get
the pure allicin. The generalized purication process has
been given just next to all extraction process.
Probe sonication
This was done in a frontline probe sonicator (Frontline,
Ahmedabad, Model No. Sonicator FS 600) taking the
same amount of mixture that of earlier in an Erlenmayer
ask. The process was repeated twice for 20 min with the
transducer frequency of 20 kHz at an interval of 10 min
between each sonication.
Bath sonication
Bath ultrasonic assisted extraction was carried out in an
ultrasonic bath (Enertech Electronics Pvt. Ltd., Mumbai,
Model No. 2K 205035). Equal amount of the same
mixture was transferred to an Erlenmayer ask and kept
in an ultrasonic bath twice for 20 min with the transducer
frequency of 20 kHz at an interval of 10 min between each
sonication. The temperature was kept constant (25 ± 1°C)
by periodical adding of ice in the bath.
Figure1: Chemical structure of organosulfur compounds in garlic
Figure2: Simplified mechanism of allicin formation
Bose, et al.: Quantication of allicin with effect of radiations
S290 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
Cold maceration
The earlier amount of the mixture was kept for cold
maceration in a well closed container separately for 3 h
with frequent mechanical stirring.
Purication of each extract
Each aqueous extract was ltered and diluted to 500 ml
with water. The diluted extract (10 ml) was saturated with
sodium chloride and subsequently extracted with 10 ml of
dichloromethane (3 times). All the fractions were pooled,
dried over anhydrous sodium sulfate and dessicated by
means of rotary‑evaporation.
UV spectroscopic analysis of allicin
A preliminary analysis of standard allicin using a
UV‑visible detector (Thermo Scientic, UK, Model:
Evaluation 201, Sl. No. SA30240002) with spectral
range 200‑600 nm was made under the chromatographic
conditions. The UV spectrum was recorded for each
puried extract in order to ascertain the presence of
pure allicin.
HPLC analysis of allicin
The HPLC system consisted of the following
components: A pump (Lachrom Hitachi, Mumbai,
Model no. L‑7400, pump no. L‑7110), a rheodyne
injector (Cotati, CA, USA, Model 7725i), a kromasil
C18 column (250 × 4.6 mm i.d., 5 µm particle size).
The column was operated in isocratic mode (50:50
MeOH: H
2
O) at a ow rate of 0.5 ml/min. Unless
otherwise specied, the UV detector was set at 254 nm
for all operations.
RESULTS AND DISCUSSION
The λ
max
obtained of all the extracts including the standard
allicin was marginal, i.e. 220 nm. Therefore, the inference
can be drawn from the above fact that irrespective of the
extraction method, one of the principle component allicin
remains unchanged despite of microwave or acoustic waves
exposure [Figure 3].
The chromatogram obtained by analyzing a solution
of standard allicin (10 µg/ml) was shown [Figure 4].
The response was linear in the range 1‑20 µg/ml. The
linear regression equation was: y = 98173x, (y = area
in arbitrary units, x = concentration in µg/ml), with
R
2
= 1.
The UV detector response (254 nm), with acoustic
waves and microwave radiation effect of allicin, resulted
higher than that obtained when standard allicin was
used [Figure 5]. The higher response was ascribed to
changes in the absorption spectrum due to the presence
of more amount of allicin in original state. Figures 6 and
7 clearly demonstrated the newer techniques not only
generated a high amount of allicin but also unaltered
allicin chemically. Three replicate injections of a standard
solution of allicin (10 µg/ml) yielded a relative standard
deviation of 1.3%.
The HPLC data given in Table 1 clearly suggested that the
allicin was not at all degraded by the effect of ultra‑sonic
and microwave radiation [Figures 5‑7]. Even, from
the different area and peak height, it was easily visible
that the amount of allicin was more if the probe, bath
ultra‑sonication and microwave radiation were applied for
a specic time as described above.
According to the manufacturer, it may require
sonication at the time of preparation of different
garlic formulations. However, it was not confirmed
earlier that whether that ultra‑sonic radiation will create
degradation of different organo‑sulphur compounds
especially allicin or not. Our present study clearly
indicates, despite of different extraction methods the
Figure3: Ultraviolet spectra of allicin from (a) normal maceration extract, (b) probe sonication extract, (c) bath sonication extract and
(d) microwave extract. Wave length in X axis and absorbance in Y axis
b
a
c d
Bose, et al.: Quantication of allicin with effect of radiations
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S291
allicin content was unaltered, the yield was much higher
in compare with the conventional way of extraction
as well. The above fact in turn supports microwave
cooking system too.
Quantitative data on the amount of allicin in garlic were
obtained using the standard calibration curve and are
summarized in Table 2 as micrograms of allicin per milliliter
puried extracts obtained from different sonication and
microwave extraction ± standard deviation.
Microscopic studies of normal garlic clove and after
treatment of acoustic wave and microwave radiation for
20 min were also performed to observe the fracture of cell
wall, which may responsible for getting more amounts of
allicin and other organosulfur compounds. The transverse
sections was done by using microtome instrument (Model
no. SP‑1120 from Sipcon Optical Industries, Haryana,
India) and was observed under ×40 eyepiece of a projection
compound microscope (Model no. 521328 from Magnus,
New Delhi, India) and photographs were taken through a
microscope attached camera (Model no. SIN S009906M
from Moticam of 1.3 megapixel live resolution). It was
clearly visible that the cell wall of fresh garlic cloves were
dissolved properly in case of ultrasonic and microwave
radiations [Figures 8 and 9] than the normal transverse
section of garlic [Figure 10].
With different types of radiations so far employed on
the extraction of allicin garlic cloves clearly indicated the
presence of pure allicin with a higher yield, therefore,
materializing these techniques in the pharmaceutical
industry denitely prove benecial in terms of time as
well as money. Most interestingly, the methods ruled out
possibilities of degradation of organosulfur compounds
as well.
ACKNOWLEDGMENT
It’s a great pleasure to the authors to express their gratitude
to Dr. Kalyan Kumar Sen, Principal, Gupta College of
Technological Sciences, Asansol, West Bengal, India for
his valuable suggestions and constant support throughout
the course of study. The authors are also indebted to
Prof. Debesh Chandra Majumdar, Chairman, Trinity
Trust, Asansol, West Bengal, India for providing necessary
facilities to carry out this work. The grant from All India
Council of Technical Education as MODROBS (Ref.
No‑8024/RIFD/MOD‑32 (Pvt.) dated 20.07.2013) for
the “Microwave Extractor” is immensely acknowledged.
A sense of gratitude goes to the West Bengal University
of Technology, Salt Lake, Kolkata for affiliating our
Institution.
Figure5: Chromatogram of standard solution of allicin (10 μg/ml) as obtained by high performance liquid chromatography and ultraviolet analyzer
Figure4: Calibration curve of standard allicin as obtained by high
performance liquid chromatography-ultraviolet analysis
Table1: HPLC output of allicin(standard, purified
extracts obtained from different sonication
and microwave extraction±standard deviation,
triplicate analysis)
Component R
t
value
(min)
Peak
area
RSD
(%)
Peak
height
Standard allicin 5.07 979821 0.42089 956
Normal maceration extract 5.07 87068 0.40887 252
Probe sonication extract 5.09 816799 0.50704 1953
Bath sonication extract 5.09 935588 0.69736 1872
Micro-wave extract 5.06 837369 0.75486 436
HPLC: High performance liquid chromatography; RSD: Relative standard deviation
Table2: Quantitative data of allicin(µg/ml
purified extracts obtained from different
sonication and microwave extraction±standard
deviation, triplicate analysis)
Allicin extracts Quantitative data of allicin (µg/ml)
Normal maceration extract 3.33±0.1
Bath sonication extract 8.32±0.01
Probe sonication extract 9.53±0.02
Micro-wave extract 8.5±0.1
Bose, et al.: Quantication of allicin with effect of radiations
S292 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
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Figure 10: Transverse section of garlic clove in normal condition
Figure6: Chromatogram of purified extracts of garlic (10 µg/ml) as
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applied
b
a
Figure7: Chromatogram of purified extracts of garlic (10 µg/ml) as
obtained by high performance liquid chromatography and ultraviolet
detection where (a) microwave radiation was applied and (b) normal
maceration was performed
b
a
Figure9: Transverse section of garlic clove after effect of microwave
radiation
Figure8: Transverse section of garlic clove after effect of ultrasonic
radiation
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Cite this article as: Bose S, Laha B, Banerjee S. Quantication of allicin by
high performance liquid chromatography-ultraviolet analysis with effect of
post-ultrasonic sound and microwave radiation on fresh garlic cloves. Phcog
Mag 2014;10:288‑93.
Source of Support: Nil, Conict of Interest: None declared.
... The analysis of alliin was developed by Bose, et al. [14], where alliin was detecte using an HPLC with ultraviolet-visible detection (Perkin Elmer Series 200, United State of America). The column temperature controller was set to 30 °C, and the wavelengt detection was set to 254 nm. ...
... The analysis of alliin was developed by Bose, et al. [14], where alliin was detected using an HPLC with ultraviolet-visible detection (Perkin Elmer Series 200, Waltham, MA, USA). The column temperature controller was set to 30 • C, and the wavelength detection was set to 254 nm. ...
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... In the last few decades, single subgroups of botanical agents such as vegetables, fruits, herbs, and nuts, have been taken into consideration to use as nutrient supplements and recognized for their numerous health benefits, including the prevention of diseases [3][4][5]. Among these, garlic (Allium sativum) has been one of the most important dietary sources not only for health benefits but also as a traditional medicinal all over the world [6][7][8][9][10]. Despite processing plans (Table 1). ...
... In the last few decades, single subgroups of botanical agents such as vegetables, fruits, herbs, and nuts, have been taken into consideration to use as nutrient supplements and recognized for their numerous health benefits, including the prevention of diseases [3][4][5]. Among these, garlic (Allium sativum) has been one of the most important dietary sources not only for health benefits but also as a traditional medicinal all over the world [6][7][8][9][10]. Despite processing plans (Table 1). ...
... In addition, N-α-(1-deoxy-d-fructose-1-yl)-l-arginine (Fru-Arg) is another promising antioxidant found in aged garlic extract and not present in raw or heated treatment preparations (Bose et al. 2014). Furthermore, the steamdistilled oil of garlic is made of allyl methyl, diallyl, and dimethyl mono-to hexasulfides. ...
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... In the last few decades, single subgroups of botanical agents such as vegetables, fruits, herbs, and nuts, have been taken into consideration to use as nutrient supplements and recognized for their numerous health benefits, including the prevention of diseases [3][4][5]. Among these, garlic (Allium sativum) has been one of the most important dietary sources not only for health benefits but also as a traditional medicinal all over the world [6][7][8][9][10]. Despite processing plans (Table 1). ...
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