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© September 2015 | IJIRT | Volume 2 Issue 4 | ISSN: 2349-6002
IJIRT 142573 INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH IN TECHNOLOGY 3
EXTRACTION, CRYSTALLIZATION,
PRESERVTION, PELLETIZING AND
QUANTIFICATION OF HYDROXY CITRIC ACID
FROM GARCINIA CAMBOGIA
E. A. H. E. Edirisinghe, S. B. Nawarathna, R. A. U. J. Marapana, J. Jaysinghe
Department of Food Science and Technology, Faculty of Applied Sciences,
University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka.
Abstract— Hydroxycitric acid (HCA), both free acid and
lactone forms is present in the fruit rind, of Garcinia
species and it is known to help to prevent obesity. HCA
is isolated from dehydrated Garcinia cambogia fruit rind
at 600C, by the defined most efficient extraction method
of refluxing for 5 hours in soxhelt apparatus using 99%
ethanol at 600C. acid base titration with 0.5M KOH(eth)
determined The total acid content of the extracted
solutions was 0.50 – 0.51g g per 1 g. most stabilized
precipitate of the salt of HCA was formed with 0.05M
ethanolic KOH and it was able to crystallized into a dry
form by drying in a rotary vacuum evaporator, below
400C. The formed dry precipitate was further preserved
by incorporating anti caking agent at the ratio of 1:3
(vacuum dried potassium salt of HCA: corn starch). The
amount of HCA in the isolated precipitate and the
extract was analyzed by HPLC and UV-Visible
Spectroscopy and obtained graph was compared with
standard potassium salt of HCA. The λ max was obtained
at 208 nm when the solution of isolated HCA was scanned
between 200 – 400 nm. Content of HCA present in the
plant extract has been found to be in the range of 42 -
44% by using HPLC with 0.01M hydrochloric acid as
mobile phase with a flow rate of 0.5ml/min using UV
detection at 208 nm. Retention time of HCA was
observed at 4.535 min in selected mobile phase. Pellets
were obtained by mixing aqua edible gum solution with
the preserved powder of HCA salt in 1: 4 ratio and
drying at 600C for 12 hours after molding in to pellet
shape.
Index Terms— Garcinia cambogia, High Performance
Liquid Chromatography (HPLC), Hydroxy citric acid
(HCA) , Potassium hydroxide (KOH), Ultra Violet (UV)
I. INTRODUCTION
Acid hydroxycitric (HCA) is a major component in the
fruit rind, of Garcinia cambogia, which grown quite
popular in Southeast Asia, to use as spice for cooking.
The plant contains various chemical constituents such
as Xanthones, Benzophenones and plant acids like
Hydroxy Citric Acid, Maleic acid, Citric acid. [8]
Hydroxycitric acid is used for weight reduction,
particularly for overweight people. The acid is known
to be soluble in water and alcohol and the free acid is
unstable and is converted to its more stable lactone
form. For consumer products, the free acid is often
stabilized by forming salts of hydroxycitric acid [6].
Traditionally its dried fruits are used as anti obesity
agent to reduce the cholesterol level, prevent the fat
accumulation and improve the blood lipid level by
boosting the excessive glucose metabolism in the body
into lipid. The novel researches have discovered that
the Hydroxycitric acid in the fruit is the active
compound act on increases glycogenesis,
gluconeogenesis, fat oxidation, [2] suppresses the fatty
acid synthesis, food intake, and induced weight loss.
(1)
Effectiveness of the HCA in weight lost has been
studied as invitro and invivo on human. The dosages
of G. cambogia extract in clinical trials ranged from
1,500 to 4,667 mg/day (25 to 78 mg/kg/day). The
equivalent hydroxycitric acid (HCA) dose in the trials
ranged from 900 to 2,800 mg/day (15 to 47
mg/kg/day). Significant effectiveness was observed
among these studies when 2800mg dosage was
administrated. Decrease in Body weight and BMI (5-
6%), Food intake, total cholesterol, low-density
© September 2015 | IJIRT | Volume 2 Issue 4 | ISSN: 2349-6002
IJIRT 142573 INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH IN TECHNOLOGY 4
lipoproteins, triglycerides , serum leptin levels and
increase in high-density lipoprotein levels and
excretion of urinary fat metabolites were observed
within 8 weeks [7].
Therefore objective of the present work is to identify
the productive extraction method for HCA from
Garcinia cambogia, crystallization, preservation and
quantification of the HCA amount in the extract and a
pelletizing method.
II. MATERIALS AND METHODS
Preparation of Garcinia cambogia for HCA
extraction
Non salted, non-smoked ripen Garcinia fruits were
taken and the fleshy part was removed. Then the rind
was taken and cut into small pieces and dried in
dehydrator at 600C for 24hours.
Identification of a productive extraction method
for HCA from Garcinia cambogia and
quantification of total acid content
Extraction was carried out in different methods to
identify the most productive extraction method.
Subsequent extraction method: Hot water (at 600C)
and cold water (room temperature) was used as the
medium for the extraction process. In the process, 8g
of Garcinia pieces were added to 50ml of distilled
water and kept for 1hour. Then the extract was filtered
out and the filter cake was undertaken to the same
process 3 times. Extraction process was repeated
separately for the two temperature conditions and they
were maintained in the same temperature throughout
the extraction time periods. The extracts were
collected separately after filtering through filter paper.
Soxhlet extraction: Exactly 12.5g of dried Garcinia
pieces were taken and was transferred to extraction
thimble and plugged the end of the thimble with cotton
wool. It was placed in soxhlet extraction apparatus
which was fixed to round bottom flask, filled with
100ml of distilled water and allowed to reflux for 5
hours at 600C.
Distillation in pressure cooker: Exactly 10g of dried
Garcinia rind pieces were taken and was cooked with
about three volumes of water in an autoclave (10
lb/in2) for 15 minutes. The resulting extract was
filtered through a paper filter and the extract was
collected.
After extracting the acid by the above mentioned
procedures, the total acid content of the extracted
solutions were determined by acid base titration with
0.5M KOH(aq) solution. Same procedure was repeated
to the extracts, which were pectin precipitated by
alcohol precipitation. By the results obtained the most
productive method was identified.
Ethanol extraction: According to the results of the
above extraction methods, the most productive method
was identified and acid extraction was carried out
under same condition using the solvent as 99%
absolute ethanol. The total acid content was
determined by acid base titration with 0.05M ethanolic
KOH solution and the pH was measured to determine
the end point of the titration.
Crystallization of HCA
The soxhelt extracted solutions of Garcinia c. in
ethanol medium and water medium were treated with
NaOH, KOH and CaCl2 solutions in aqua medium and
ethanolic medium at 0.5M and 0.05M concentrations
to obtain the precipitate. Then the characteristics and
the yield of the precipitate were observed.
Preservation of the crystallized salt of HCA
The obtained most stabilized precipitate was treated in
different ways to identify the best preservation
method.
Under normal atmosphere: The precipitate was kept
under normal atmosphere.
In desiccator: The precipitate was kept in desiccator
with silica gel.
Oven dried at 60C0: The precipitate was dried in a
dehydrator at 60C0 for 12 hours
Dried under vacuum: The precipitate was dried in a
rotary vacuum evaporator, below 400C
Quantification of HCA content
By UV-Visible Spectroscopy
The solution of 800ppm concentration of standard
potassium hydroxy citrate (Potassium salt of HCA)
and solution of 1014ppm concentration extracted
potassium hydroxy citrate (Potassium salt of HCA)
from Garcinia cambogia fruit extract (vacuum dried
for precipitate) in 0.1M hydrochloric acid was scanned
between 190 – 400 nm against blank disk separately.
The absorption and vibration bands were observed at
various frequencies and reported graph (Fig.IV) was
compared with standard graph (Fig V) [5]
Content analysis by High Performance Liquid
Chromatography
The solution of 800ppm concentration of standard
potassium hydroxy citrate (Potassium salt of HCA)
and solution of 1014ppm concentration extracted
© September 2015 | IJIRT | Volume 2 Issue 4 | ISSN: 2349-6002
IJIRT 142573 INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH IN TECHNOLOGY 5
potassium hydroxy citrate (Potassium salt of HCA)
from Garcinia cambogia fruit extract (vacuum dried
for precipitate) in 0.01N hydrochloric acid were
prepeared. Exactly 10 μl solutions of standard and test
sample were injected in HPLC instrument using C18
column as stationary phase and 0.1M hydrochloric
acid as mobile phase at a flow rate of 0.5 ml/min with
UV detection at 208 nm.[3][4] The resultant graph was
shown at Fig VI. The percentage content of HCA in
fruit extract was calculated.
Pelletizing of salt of HCA
The preserved precipitates were further analyzed by
incorporating anti caking agent (corn starch) in order
to form a dry powder form salt, for pelletizing
purposes and more stabilized salt, to store under
normal atmospheric conditions. For the purpose corn
starch was used with different ratios to determine the
best combination for the preservation.
Pelletizing of the precipitate contains HCA salt with
corn starch, was carried out using gelatin as an edible
gum. The gum was mixed with water (in 1:1 ratio) and
the prepared gum solution was mixed with the
precipitate contains HCA salt with corn starch. (In 1:
4 ratio). Here the amount of precipitate added was
calculated according to the content of HCA and the
amount of HCA needed to be contained within the
tablet. Then the mixture was molding into the shape or
formed into a thin sheet and cut into different shapes
using a mold. Then the formed pellets were dried in a
dehydrator at 600C for 12 hours.
III. RESULTS AND DISCUSSION
Identification of a productive extraction method
for HCA from Garcinia cambogia
According to the functional groups in the HCA, it
contains 3 acid groups and therefore it can combine
with 3 hydroxyl groups.
In the water extraction method, color and the acid level
in the extracted solutions were decreased among the
subsequence 3 extracted solutions. According to the
results of the extraction methods used, highest acid
amount per 1g of dried Garcinia rind was obtained
through soxhelt extraction method.
Table 1: Total acid content in Garcinia c. extract,
obtained by different extraction methods in water
medium
Extract
ion
method
Water
extracti
on (hot)
Water
extracti
on
(cold)
Soxhelt
extracti
on
(water)
Pressu
re
cookin
g
With
pectin (g
of acid
extracte
d per 1g
of
Garcini
a)
0.0355
0.0323
0.2884
0.2006
Without
pectin (g
of acid
extracte
d per 1g
of
Garcini
a)
0.0320
0.0289
0.2408
0.1698
According to the result of the above experiment, most
productive extraction method was soxhelt extraction
method and therefore it was used for the ethanol
extraction process. During the titration process of the
ethanol extract of the Garcinia c., with 0.05M
ethanolic KOH, precipitate was observed throughout
the titration process and the pH changes determined
that the amount of acid extracted is 0.513g g per 1 g of
dried Garcinia c. rind.
+ 3 KOH
Potassium salt
of
hydroxylcitrat
e
Hydroxycitric acid
Hydroxycitric
© September 2015 | IJIRT | Volume 2 Issue 4 | ISSN: 2349-6002
IJIRT 142573 INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH IN TECHNOLOGY 6
Figure 1: pH change of the ethanolic Garcinia c. Solution during the titration with 0.05M, ethanolic KOH
Crystallization of HCA
Water extract of Garcinia c. : At 0.05M concentration
of aqua KOH, NaOH and CaCl2 precipitate were not
observed. At higher concentrations of aqua KOH and
NaOH precipitate was obtained. But due to the
hygroscopic nature of the formed salt, it dissolved
rapidly by absorbing moisture from the medium. No
precipitate was obtained in the addition of aqua CaCl2,
at the higher concentrations. In the addition of
ethanolic KOH, NaOH and CaCl2 same results were
observed but the precipitate dissolved rapidly in the
presence of water in the medium.
Ethanol extract of Garcinia c.: At low concentrations
of ethanolic KOH and NaOH, precipitate was
observed with small sized crystals with light pink
color. At higher concentrations, precipitate was form
as cluster. Crystals were red-brown crystals due to the
present of color pigments in Garcinia C. extract. (Fig:
3) When the precipitate washed with ethanol the color
removes gradually. It was also hygroscopic and
dissolves in the presence of moisture (Dissolve by
absorbing moisture in air). The precipitate formed
with ethanolic KOH was more stable than the
precipitate formed with ethanolic NaOH under the
normal atmospheric condition. But the stability of the
precipitate formed in ethanol medium was higher than
the precipitate formed in water medium. No
precipitate was obtained in the
addition of ethanolic CaCl2, neither at the higher or
lower concentrations.
Figure II: Formation of crystals at different
concentrations of KOH (a)- at higher
concentrations / (b)- at lower concentrations
Preservation of the crystallized salt of HCA
Due to the highly hygroscopic nature of the formed
crystals, it rapidly dissolved by absorbing moisture
from the atmosphere. The same action was observed
with the precipitate kept in desiccator at lower rate.
The precipitate dried in a dehydrator at 60C0 was a
light brown color solid with a hard sticky texture.
Light pink / white colour powder form solid
precipitate was obtained by drying the precipitate
under vacuum. The best form of precipitate was
formed by rotary vacuumed evaporation and it was
stabilized under vacuum conditions. All the formed
perecipitates were dissolved within few seconds under
normal atmospheric condition by absorbing moisture.
(a)
(b)
© September 2015 | IJIRT | Volume 2 Issue 4 | ISSN: 2349-6002
IJIRT 142573 INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH IN TECHNOLOGY 7
Figure III : Preservation of precipitate under
different conditions a- Under normal atmosphere
/ b- dried in a desiccator / c- dried in a dehydrator
at 60C0/ d- dried using rotary vacuumed
evaporation
Quantification of HCA content
Qualitative Analysis By UV-Visible Spectroscopy
The absorbance maximum was observed at 208nm; for
the UV-Visible Spectrum for standard potassium salt
of Hydroxy citric acid and it was 0.333A. For the test
sample the absorbance was 2.523A at the same
wavelength.
Figure IV: UV-Visible Spectrum for standard
potassium salt of Hydroxy citric acid
Figure V; UV-Visible Spectrum for extracted
potassium salt of Hydroxy citric acid from Garcinia
cambogia fruit extract
Content analysis by High Performance Liquid
Chromatography
The absorbance maximum was observed at 208nm;
hence 208 nm was used for HPLC detection. Retention
time of HCA was observed at 4.535 min in selected
mobile phase. Some other minor peaks were also
observed which may be due to other acids present in
plant. HCA was resolved as single peak and confirmed
by spiking with standard HCA. The content of
potassium salt of HCA in the precipitate was around
1300 – 1400 mg/g and the amount of HCA in the
precipitate was Content of HCA in fruit extract was
found to be 48 - 49%.
(a)
(b)
(c)
(d)
© September 2015 | IJIRT | Volume 2 Issue 4 | ISSN: 2349-6002
IJIRT 142573 INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH IN TECHNOLOGY 8
Figure VI:. HPLC graph for extracted potassium salt of Hydroxy citric acid from Garcinia cambogia fruit
extract
Pelletizing of salt of HCA
After treating the dried precipitate with the anti-caking
agent (corn starch) the stability was increased and the
precipitate was able to preserve without dissolving by
moisture.
The oven dried precipitate was not mixed well with
corn starch and the formed a cluster natured precipitate
among the corn starch powder.
Figure VII: Precipitate formed by incorporating
of oven dried precipitate and anti-caking agent
(corn starch)
The precipitate kept under normal atmosphere and
desiccators formed a sticky nature precipitate at lower
ratios of corn starch and powder form precipitate at
higher ratios of corn starch. But the ratio of corn starch
needed to be incorporated to form the powder form
was higher than 1: 6 (oven dried precipitate: corn
starch).
Figure VIII: Preservation of desiccators dried
precipitate using different ratios of anti-caking
agent (ratio between corn starch: desiccators
dried precipitate)
The best preserved precipitate was observed in
Vacuum dried precipitate with corn starch. At lower
ratios of corn starch, precipitate was sticky in nature
and tends to absorb moisture with time. At higher
ratios of corn starch, the precipitates formed in dry
powder nature and it was stable under normal
atmospheric conditions. The most stabilized dry
powder precipitate with lowest corn starch amount
gave at the ratio of 1:3 (vacuum dried precipitate: corn
starch).
(a)
1:2 ratio
(b)
1:3 ratio
(c)
1:4 ratio
min
(a) - 1:3 ratio
(b) – 1:6 ratio
© September 2015 | IJIRT | Volume 2 Issue 4 | ISSN: 2349-6002
IJIRT 142573 INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH IN TECHNOLOGY 9
Figure IX: Preservation of vacuum dried
precipitate using different ratios of anti-caking
agent (ratio between corn starch: vacuum dried
precipitate)
After mixing the edible gum solution with the
precipitate preserved by vacuum drying and
incorporated with corn starch it showed a non-sticky
and non hygroscopic in nature. After molding and
drying the pellets were in exact shape with rigid
nature.
Figure X: Pellets formed by preserved precipitate
and edible gum.
IV CONCLUSION
In summary, hydroxycitric acid was extracted from the
dehydrated Garcinia c. rinds by using soxhelt
extraction method. Extraction using 99% ethanol as
solvent was defined as the most productive method by
analyzing the total acid content and the amount of acid
extracted is 0.513g g per 1 g of dried Garcinia c. rind.
Most stabilized precipitate or crystals of potassium salt
of HCA was obtained by treating the extract with
0.05M KOH(eth) . The obtained crystals were highly
hygroscopic in nature and therefore it was preserved
by drying under vacuum, below 400C and
incorporation of anti caking agent (corn starch) in 1:3
ratio. Pellets were obtained by mixing aqua edible gum
solution with the preserved powder of HCA salt in 1:
4 ratio and drying at 600C for 12 hours after molding
in to pellet shape. The amount of HCA in the isolated
precipitate and the extract was analyzed by HPLC and
UV-Visible Spectroscopy and obtained graph was
compared with standard potassium salt of HCA. The λ
max was obtained at 208 nm when the solution of
isolated HCA was scanned between 200 – 400 nm.
Absorbance values were 0.333A and 2.523A for
potassium salt of Hydroxy citric acid and test sample
respectively. Content of HCA present in the plant
extract has been found to be in the range of 42 - 44%
by using HPLC with 0.01M hydrochloric acid as
mobile phase with a flow rate of 0.5ml/min using UV
detection at 208 nm. Retention time of HCA was
observed at 4.535 min in selected mobile phase.
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