Weight loss abstract
The present invention provides methods of suppressing appetite
and causing weight loss by administering to a patient hydroxy citric
acid in the form of a potassium salt extracted from Garcinia fruit.
Methods of inhibiting cytoplasmic citric lyase and increasing fat
metabolism in a patient are also described.
Weight loss claims
We claim:
1. A method for suppressing appetite in a patient in need of such
effect comprising administering to said patient an appetite suppressing
effective amount of potassium hydroxycitric acid composition comprising
less than 2% by weight of potassium hydroxycitric lactone, based
on the combined amount of potassium hydroxycitric acid and potassium
hydroxycitric lactone.
2. A method for inhibiting cytoplasmic citric lyase in a patient
in need of such inhibition comprising administering to said patient
a citrate lyase inhibiting effective amount of potassium hydroxycitric
acid composition comprising less than 2% by weight of potassium
hydroxycitric lactone, based on the combined amount of potassium
hydroxycitric acid and potassium hydroxycitric lactone.
3. A method of increasing fat metabolism in a patient in need of
such effect comprising administering to said patient a fat metabolism
increasing effective amount of potassium hydroxycitric acid composition
comprising less than 2% by weight of potassium hydroxycitric lactone,
based on the combined amount of potassium hydroxycitric acid and
potassium hydroxycitric lactone.
4. A method for causing weight loss in a patient in need of such
an effect comprising administering to said patient a weight loss
effective amount of potassium hydroxycitric acid composition comprising
less than 2% by weight of potassium hydroxycitric lactone, based
on the combined amount of potassium hydroxycitric acid and potassium
hydroxycitric lactone.
5. A method as recited in claim 4 wherein the potassium hydroxycitric
acid is non-hygroscopic.
6. A method for suppressing appetite in a subject in need of such
effect comprising administering an appetite suppressing effective
amount of potassium hydroxycitric acid composition made by a process
comprising:
a) extracting Garcinia fruit with an alkyl alcohol at least three
times to obtain a first extract, a second extract and a third extract;
b) combining said first extract, said second extract and said third
extract to obtain a combined extract;
c) treating said combined extract with potassium hydroxide and
reflux to form potassium hydroxy citrate precipitate;
d) filtering said precipitate to obtain filtered precipitate;
e) washing said filtered precipitate with an alkyl alcohol to obtain
washed precipitate;
f) drying said washed precipitate under vacuum to obtain dried
precipitate; and
g) milling, sifting, blending and packing said dried precipitate
under nitrogen to obtain said potassium hydroxycitric acid composition.
7. A method for inhibiting cytoplasmic citric lyase in a patient
in need of such inhibition comprising administering to said patient
a citrate lyase inhibiting effective amount of potassium hydroxycitric
acid composition made by a process comprising:
a) extracting Garcinia fruit with an alkyl alcohol at least three
times to obtain a first extract, a second extract and a third extract;
b) combining said first extract, said second extract and said third
extract to obtain a combined extract;
c) treating said combined extract with potassium hydroxide and
reflux to form potassium hydroxycitrate precipitate;
d) filtering said precipitate to obtain filtered precipitate;
e) washing said filtered precipitate with an alkyl alcohol to obtain
washed precipitate;
f) drying said washed precipitate under vacuum to obtain dried
precipitate; and
g) milling, sifting, blending and packing said dried precipitate
under nitrogen to obtain said potassium hydroxycitric acid composition.
8. A method of increasing fat metabolism in a patient in need of
such effect comprising administering to said patient a fat metabolism
increasing effective amount of potassium hydroxycitric acid composition
made by a process comprising:
a) extracting Garcinia fruit with an alkyl alcohol at least three
times to obtain a first extract, a second extract and a third extract;
b) combining said first extract, said second extract and said third
extract to obtain a combined extract;
c) treating said combined extract with potassium hydroxide and
reflux to form potassium hydroxycitrate precipitate;
d) filtering said precipitate to obtain filtered precipitate;
e) washing said filtered precipitate with an alkyl alcohol to obtain
washed precipitate;
f) drying said washed precipitate under vacuum to obtain dried
precipitate; and
g) milling, sifting, blending and packing said dried precipitate
under nitrogen to obtain said potassium hydroxycitric acid composition.
9. A method for causing weight loss in a patient in need of such
an effect comprising administering to said patient a weight loss
effective amount of potassium hydroxycitric acid composition made
by a process comprising:
a) extracting Garcinia fruit with an alkyl alcohol at least three
times to obtain a first extract, a second extract and a third extract;
b) combining said first extract, said second extract and said third
extract to obtain a combined extract;
c) treating said combined extract with potassium hydroxide and
reflux to form potassium hydroxycitrate precipitate;
d) filtering said precipitate to obtain filtered precipitate;
e) washing said filtered precipitate with an alkyl alcohol to obtain
washed precipitate;
f) drying said washed precipitate under vacuum to obtain dried
precipitate, and
g) milling, sifting, blending and packing said dried precipitate
under nitrogen to obtain said potassium hydroxycitric acid composition.
Weight loss description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is directed to a new process for making hydroxy citric
acid in a form that is stable and biologically active. Compositions
containing the potassium hydroxy citric acid are useful as natural
appetite suppressants.
2. Description of Related Art
During the 1970s, scientists at Brandeis University and at Hoffman
LaRoche demonstrated that synthetic hydroxycitric acid, when blended
with the diet, had a marked suppressive effect on weight gain in
rats. The researchers noted that the HCA-treated rats tended to
eat less; food consumption was suppressed by 10% or is more on optimal
HCA intakes, that is, when HCA constituted 1% or more of the diet.
The mechanism by which the HCA affected weight gain was not known.
Since the brain uptake of HCA appeared to be negligible, scientists
speculated that the appetite-suppressive effect of HCA was exerted
not through central nervous system (CNS) action, but rather by directly
affecting the metabolic processes of the organism.
One of the most metabolically active organs in the body is the
liver. One important function of the liver is to insure that the
blood maintains adequate concentrations of glucose to fuel the body's
energy requirements. The liver can store dietary glucose in the
form of the polysaccharide glycogen, and release glucose when blood
glucose levels are low. The liver can also synthesize glucose in
a complex process known as gluconeogenesis, from either amino acids
or lactic acid as starting material. This newly synthesized glucose
can either be released into the blood stream to supply energy requirements
of body tissues, or can be stored as glycogen for future use.
The direct parasympathetic connection between the liver and the
CNS monitors the level of glucose and glycogen in the liver. A high
level of glycogen, as a result of high glucose supply, is translated
by the CNS as the state of satiety, which results in decreased craving
for food.
Since increased glycogen in the liver aids satiety, the effect
of HCA on gluconeogenesis in rat liver has been studied. It has
been found that the rate of gluconeogenesis, from lactate or the
amino acid alanine, was approximately doubled in HCA-treated rats.
This result provides support for the idea that HCA causes the observed
appetite suppression via altering the rate of gluconeogenesis.
However, it appears unlikely that reduction of food intake can
entirely account for the substantial reductions in weight gain seen
in HCA-treated rats. For example, in one study, the net reduction
in food consumption during the 80 day study period amounted to only
4%--and yet the rats had gained 78% less weight than the controls
over this period. Other studies, providing less dramatic results,
suggested that the reduction of weight gain was disproportionately
large compared to the reduction in food consumption.
Because of this discrepancy between considerable weight loss versus
a meager appetite suppression, it has been postulated that HCA exerts
a mechanism which increases fat burning, which in turn could decrease
body weight, in addition to affecting gluconeogenesis.
Fat burning, or oxidation, plays a prominent role in liver metabolism.
Liver metabolic activity accounts for over a quarter of the total
body oxygen consumption in a subject at rest. The substantial energy
needs of the liver are met largely by oxidation of fat. The dietary
fat is absorbed by liver cells, which oxidize or burn it for energy
in the mitochondria. The fats are transported from the cell cytoplasm
into the liver mitochondria, by linking them to the special transporting
molecule L-carnitine. This reaction is facilitated by the enzyme
carnitine acyltransferase.
Carnitine acyltransferase is inhibited by malonyl CoA, which can
be obtained from conversion of acetyl CoA. Malonyl CoA, can not
only inhibit the fat burning process, but also increase the body
fat synthesis, since it is the direct precursor for the synthesis
of fat and cholesterol. The acetyl CoA is synthesized in mitochondria,
but it has to be transported to the cell cytoplasm to exert its
biochemical action. However, it cannot be transported to the cytoplasm
from mitochondria before it is converted to citric acid. Thus, citric
acid is a transportable form of acetyl CoA. Citric acid, once in
the cytoplasm, is converted to acetyl CoA with the help of the enzyme--citrate
lyase. HCA was found to be an extremely potent competitive inhibitor
of citrate lyase (K.sub.i =0.15 .mu.M). The affinity of the enzyme
for HCA was over a hundred times greater than the affinity of the
enzyme for citric acid. This action was afforded only by a HCA in
a pure acid form, but not in the lactone form.
The significance of citrate lyase inhibition by HCA is that without
active citrate lyase, little acetyl CoA could reach the cytoplasm.
This in turn would limit the availability of malonyl CoA and slow
the synthesis of fats and cholesterol, while disinhibiting the metabolic
breakdown of fat, or oxidation of fat.
In light of the considerations noted above, it is likely that the
ability of HCA to promote fat loss in humans results primarily from
the stimulation of fat oxidation.
Activation of fat oxidation in the liver also tends to stimulate
gluconeogenesis, primarily due to increased activity of the key
enzyme pyruvate carboxylase. This in turn may replenish the stores
of liver glycogen, and send a message of satiety to the brain center.
The drawbacks of HCA use as a weight loss compound stem from the
following problems:
1. The poor technology of HA extraction from the fruit of Garcinia
cambogia often provides HCA in lactone form, which is inactive,
or less active, in inhibiting the citrate lyase;
2. The HCA, if not stabilized chemically, has natural propensity
to be converted to the lactone form in aqueous solutions and in
the gastrointestinal tract, i.e., without absorption of HCA in pure
acid form, the HCA can not inhibit the citrate lyase; and
3. High concentrations of HCA, that is, 1% or more (by weight)
of the daily dietary intake, are required to exert the metabolic
activity, because of poor cellular uptake. Without absorption of
HCA, and the presence of HCA in the cytoplasm in pure acid form,
HCA can not inhibit citrate lyase and exert its inhibitory activity
on acetyl CoA formation.
In the past, it has been difficult to isolate hydroxy citrate in
a form which is both stable and biologically active. Hydroxy citric
acid exists in two forms, the free acid form and the lactone form.
The free acid form is biologically active and the lactone form is
inactive. However, the free acid form is not stable and gets converted
to its lactone form, which is stable but inactive.
One prior art isolation procedure, that of Y. S. Lewis et al.,
in Phytochem 1965, Vol. 4, pp. 610-625, results in the isolation
of hydroxy citric acid lactone.
I. WATER EXTRACT OF (-) HYDROXYCITRIC ACID FROM FRUIT OF GARCINIA
CAMBOGIA (Lewis, Y. S. and Neelakantan, S., phytochemistry (1965)
Vol. 4; pp. 619-625)
The prior art procedure to obtain (-)HCA from Garcinia cambogia
on a large scale included the following procedure:
1. The dried rind was cooked with about three volumes of water
in an autoclave (10 lb/in.sup.2) for 15 minutes;
2. The resulting extract was filtered through a cloth and then
through a paper filter;
3. The obtained filtrate was concentrated to a small volume, and
the alcohol precipitation method removed pectin contamination;
4. The clear filtrate was then treated with potassium hydroxide
(alkali) to form viscous, dark, heavy liquid; this treatment resulted
in formation of a hygroscopic material consisting of potassium salt
of hydroxycitric acid;
5. The clear supernatant was decanted off and the oily liquid washed
with 60% alcohol several times;
6. By repeated treatment with absolute alcohol, the material could
be dried to a pale yellow hygroscopic powder, which formed pure
alkali salt;
7. Aqueous solutions of the alkali salt were passed through a cation-exchange
resin (Zeocarb 215) for recovery of the acid;
8. The obtained (-)HCA was chemically unstable, and upon evaporation
formed lactone.
Another process for isolation of lactone was reported by Y. S.
Lewis.
II. ACETONE EXTRACT OF (-) HYDROXYCITRIC ACID FROM THE FRUIT OF
GARCINIA CAMBOGIA (Lewis, Y. S. (1981) Methods in Enzymology, Vol.
77; Published by Academic Press; pp. 613-619).
1. One kg of fruit of Garcinia cambogia is kept in 1500 ml of acetone
in an overnight;
2. The fruit is re-extracted in a similar manner;
3. Acetone is removed from the combined extracts by distillation;
4. The viscous residue is stirred with 1 liter of water at 45.degree.-50.degree.
C.;
5. The mixture is filtered through cheesecloth;
6. The precipitated insoluble material is removed by filtration;
7. The reddish brown filtrate is treated with activated charcoal
at 80.degree.-90.degree. C. and concentrated to a thick syrup;
8. The syrup is "seeded" with a few crystals of the lactone
and left overnight;
9. The yield is vigorously extracted with 3 liters of ether;
10. The combined extracts are dried over anhydrous sodium sulfate;
11. Ether is then removed, and the remaining material is white
solid, consisting mainly of lactone. The yield is approximately
150 gm.
SUMMARY OF THE INVENTION
The principle of the present invention is to provide technology
for extraction of HCA in pure acid form, and technology for chemical
modification of HCA to afford chemically stable product, which will
not convert into lactone form, which will not be hygroscopic, and
which is soluble in aqueous solutions and easily absorbable by the
gastrointestinal tract.
The invention provides HCA by combining it with potassium into
potassium hydroxycitrate--a water soluble salt. Potassium is an
ion primarily found in the cell cytoplasm, and it can easily cross
from outside the cell to inside the cell. The cell membrane permeability
for potassium is 100 times higher than for sodium and 25 times higher
than for chloride. Potassium salt of HCA acts as a transporter of
HCA inside the cell, where the biochemical action of HCA is exerted.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an infrared spectrum of potassium hydroxy citrate.
FIG. 2 is a thermogram of potassium hydroxy citrate.
FIG. 3 is a NMR spectrum of potassium hydroxy citrate.
FIG. 4 is an HPLC chromatogram of Potassium Hydroxy Citrate
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Process Outline
The process of the present invention is used to isolate hydroxy
citric acid as potassium hydroxy citrate from a natural source of
Garcinia species. Preferred sources include Garcinia cambogia and
Garcinia indica.
Briefly, fruit of the Garcinia species is extracted with an alkyl
alcohol. Preferred alcohols include methyl alcohol, ethyl alcohol,
propyl alcohol, and isopropyl alcohol. Especially preferred is methanol.
The extract is treated with a suitable alkali to precipitate the
potassium hydroxy citrate. Preferred alkalis include potassium hydroxide,
potassium carbonate, etc. Most preferred alkali is potassium hydroxide.
The general process includes the following steps. Garcinia fruit
is extracted with an alkyl alcohol at above ambient temperature.
This is done at or above atmospheric pressure. The extract is collected.
The extraction step is repeated at least three times. The extracts
are combined and treated with an alcoholic solution containing alkali.
The resultant mass is heated to above ambient temperature and pH
is adjusted to make the solution alkaline. The pH of the solution
is normally between 8 to 11.5. The product is filtered and washed
with alcohol. The product is dried at or above 25.degree. C. under
vacuum or at atmospheric pressure or under inert atmosphere, like
nitrogen. The dried product is milled, sifted, blended and packed
under nitrogen blanket to obtain product. The yield from 500 kgs
of garcinia fruit ranges from 60 to 150 kgs of potassium hydroxy
citrate based on the hydroxy citric acid content present in the
fruit.
Unique Aspects of our Process
Hydroxy citric acid exists in two forms, i.e., free acid form and
lactone form. The free acid form is biologically active and the
lactone form is inactive. However, the free acid form is not stable
and this gets converted to its lactone form, which is stable but
inactive. In our process, the free acid form is isolated and stabilized
as potassium salt to retain the activity. This is one of the unique
aspects of our process.
Another unique aspect of our process is that our potassium hydroxy
citrate is water soluble and therefore, it is readily available
in the biological system for its bioefficacy.
EXAMPLE
The detailed procedure used to obtain the product trademarked at
CITRIN.RTM.-K is as follows:
1. The 500 kg of Garcinia fruit is extracted with 1500 1 of methanol
at about reflux temperature for 3 hours;
2. This is filtered through the cloth filter to collect the first
extract;
3. Additional 1500 l of methanol is added to the Garcinia fruit
and refluxed for about 3 hours;
4. This is filtered to collect the second extract;
5. The 1500 l of methanol is added again to the Garcinia fruit
and refluxed for 3 hours;
6. This is filtered, and the third extract is collected;
7. All the three extracts are combined;
8. The combined extracts are treated with methanolic potassium
hydroxide at pH 10;
9. This is again refluxed for about 3 hours to attain constant
pH 10 to precipitate potassium hydroxycitrate;
10. The precipitate is filtered and washed with 500 l of methanol;
11. The precipitate is dried under vacuum at about 70.degree. C.;
12. The dried product is milled, sifted, blended and packed under
nitrogen blanket to obtain product trademarked at CITRIN.RTM.-K;
13. The methanolic mother solution is distilled to recover methanol;
14. The yield from 500 kg of Garcinia fruit is about 150 kg of
potassium hydroxycitrate.
The specifications of the product CITRIN.RTM.-K is given below:
Specifications
Molecular Structure
Molecular Formula C.sub.6 H.sub.5 K.sub.3 O.sub.8 .multidot.H.sub.2
O
Molecular weight 340.41
Description Beige to pale brown colored powder
Solubility Soluble in water, acids and aqueous alcohols. Insoluble
in solvents like methanol, alcohol, chloroform, benzene, etc.
Loss on Drying Not less than 3% and not more than 6.0% pH of 5%
solution 7.0 to 9.0 in water
Specific Rotation -18.degree. to -25.degree. on anhydrous basis
Potassium content not less than 30% by weight on anhydrous basis
Hydroxy citric Not less than 50% on anhydrous basis acid content
Lactone content (HPLC) less than 2% by weight
Identification
a) By IR Spectrum
The infrared absorption spectrum of a potassium bromide dispersion
of potassium hydroxy citrate, previously dried, exhibits maxima
only at the same wavelength as that of similar preparation of Working
Standard. IR Spectrum of Potassium Hydroxy Citrate Working Standard
is shown in FIG. 1.
b) For Potassium
Produces yellow or orange-yellow precipitate with sodium cobaltinitrite
solution.
Dissolve 50 mg of 1 ml of water, add 1 ml of dilute acetic acid
and 1 ml of freshly prepared 10% w/v solution of sodium cobaltinitrite.
A yellow or orange-yellow precipitate forms immediately.
c) For Citrate
Dissolve 0.5 g in a mixture of 10 mL of water and 2.5 mL of 2N
nitric acid. Add 1 mL of mercuric sulphate solution heat to boiling,
and add 1 mL of potassium permanganate solution: a white precipitate
is formed.
d) By Paper Chromatography
Mobile Phase
Butanol (4): Acetic Acid (1): Water (5)
Prepare 100 mL of mobile phase in separator and mix well. Allow
it to separate and use the upper layer as mobile phase.
Stationary Phase:
Whatman filter Paper No. 1
Sample Preparation
Dissolve 100 mg of the sample in 1 mL of water and dilute to 10
mL with methanol in a volumetric flask.
Standard Preparation
Dissolve 100 mg of the Working Standard in 1 mL of water and dilute
to 10 mL with methanol in a volumetric flask.
Procedure
Apply separately equal volume (10 .mu.l) of sample and standard
preparation and develop the chromatogram in the chamber previously
saturated with mobile phase. After developing the chromatogram to
3/4, the paper is removed and dried in a current of air.
Detection
The paper is sprayed with sodium metavanadate solution (5% w/v)
and observed for the orange spot. The Rf value of the spot obtained
from the sample solution is same as that of the Standard solution.
LOSS ON DRYING
Limit: Not less than 3.0% and not more than 6.0%
The material shows weight loss of about 5% when dried at 150.degree.
C. under vacuum for four hours. This weight loss is due to the release
of water of hydration from the molecule.
THERMAL ANALYSIS
Potassium hydroxy citrate is analyzed by Thermogravimetry. This
technique is used to estimate the presence of water of hydration
in the product. The details of the methods are given below:
In this method, the sample is heated under nitrogen/argon atmosphere
and the weight loss is recorded continuously.
Limit: The weight loss is not more than 6.0%
Analysis is carried out using about 3 mg of the sample accurately
weighed. The temperature setting is from 30.degree. C. to 400.degree.
C. with the rate of heating as 10.degree. C. per minute. The heating
of the sample is done under nitrogen/argon atmosphere flowing at
a flow rate of 40 mL/min.
From the TGA thermogram, it is observed that there is weight loss
between 180.degree. C. and 250.degree. C. to a level of about 5%
which indicates the presence of water of hydration.
The percentage loss corresponds to one molecule of water.
A typical thermogram is given in FIG. 2.
pH OF SOLUTION
pH of 5.0% w/v solution is between 7.0 and 9.0.
Dissolve 2.5 g in 50 ml of water and determine the pH using suitable
calibrated pH meter.
SPECIFIC ROTATION
Between -18.0.degree. and -25.0.degree. calculated on anhydrous
basis.
Weigh accurately about 1 g of the sample and transfer into a 100
ml volumetric flask, dissolve in water, dilute to volume and mix.
Measure the rotation using suitable polarimeter at about 25.degree.
C.
ASSAY
Assay of the product is estimated by estimating the content of
HYDROXY CITRIC ACID and POTASSIUM.
For determination of HYDROXY CITRIC ACID, the following methods
are employed:
i) TITRATION METHOD
ii) HPLC METHOD
The details of the methods are given below: Limit: Content of HCA
is not less than 50.0% calculated on anhydrous basis
TITRATION METHOD
Weigh accurately about 200 mg of the sample and transfer into a
beaker. Add 100 ml of water and dissolve. Pass the solution through
cation ion exchange resin column and collect the affluent into a
1 L flask. Rinse the beaker with water and pass the washings through
the column. Wash the column with distilled water until the elute
shows a pH of 4.0 to 4.5. Adjust the volume to about 500 ml and
titrate with 0.1N sodium hydroxide solution using phenolphthalein
solution as indicator.
Perform a blank titration after eluting 500 ml of water through
the column.
Calculation:
Note 1 Column Preparation and Regeneration:
About 75 g of cation exchange is packed in a column of 2 cm diameter.
Soak the column for 30 minutes in 2N Hcl. Wash thoroughly with distilled
water to get a pH of 4.0 to 4.5
After the analysis, the cation exchange resin is soaked with 2N
Hcl for 3 hours. It is then washed well with distilled water until
the pH of the washings shows 4.0 to 4.5
Note 2 The above method is based on the published research paper
titled "Chemical Constituents of Kokum Fruit Rind" by
CFTRI, Mysore.
Note 3 Specification of the cation exchange resin is given in FIG.
3
Note 4 The factor of 0.006933 is arrived at by the following calculation
[2 moles of HCA].
416 g of HCA.ident.6000 ml of 1N NaOH or
6000 ml of 1N NaOH.ident.416 g of HCA ##EQU1## HPLC METHOD
In this method, normally, (-) Threo Hydroxy Citric Acid Ethylene
Diamine Salt (Fluka Standard) is used as a Standard to estimate
Hydroxy Citric Acid content in Potassium Hydroxy Citrate. This Standard
is not readily available, and therefore an alternate standard, Potassium
Hydroxy Citrate is preferred. A pure sample of Potassium Hydroxy
Citrate has been synthesized and validated against the Fluka Standard
(FIG. 4). In the method given below, Potassium Hydroxy Citrate is
used as a Working Standard (WS).
Mobile Phase
Prepare 0.01N sulfuric acid, filter and degas.
Sample Preparation
50 mg of the sample is accurately weighed, dissolved in water and
diluted to 25 ml with water.
Standard Preparation
50 mg of Potassium Salt of Hydroxy Citric Acid (WS) is dissolved
in 10 ml of water, and diluted to 25 ml with water.
Chromatographic system
The liquid chromatograph is equipped with 210 nm detector and a
4.6.times.250 mm organic acid column (Vydac make). The flow rate
is about 1 ml per minute. Chromatograph the standard preparation
and calculate the Relative Standard Deviation (RSD) for replicate
injections. The RSD is not more than 2.0%.
Procedure
Separately inject equal volume (20 .mu.l) of sample and standard
preparation and record the responses obtained for the major peaks.
Calculation: ##EQU2## ESTIMATION OF POTASSIUM
This estimation is done by two methods:
i) FLAME PHOTOMETRY
ii) ATOMIC ABSORPTION
The details of the methods are given below:
Limit of Potassium: Not less than 30.0% calculated on anhydrous
basis
FLAME PHOTOMETRY
Standard Stock Solution
Weigh accurately about 1.84 g of Potassium Chloride, previously
dried at 105.degree. C. for 2 hours and transfer into a 250 ml volumetric
flask, add water to volume and mix.
Lithium Diluent Solution
Transfer 1.04 g of Lithium Nitrate to a 1000 ml volumetric flask,
add a suitable nonionic surfactant, add water to volume and mix
Standard Preparation
Pipette 5 ml of stock solution into a 50 ml of volumetric flask,
dilute to volume with water and mix. Transfer 5 ml of this solution
to a 100 ml volumetric flask and dilute with lithium diluent solution
to volume and mix.
Assay Preparation
Weigh accurately about 3 g of the sample and transfer into a 250
ml volumetric flask, add water to dissolve and dilute to volume
and mix. Pipette 5 ml of this solution into a 50 ml volumetric flask,
add water to volume and mix. Transfer 5 ml of this to a 100 ml volumetric
flask, dilute with lithium diluent solution to volume and mix.
Procedure
Using a suitable flame photometer adjust to read zero with lithium
diluent solution concomitantly determine the emission readings for
Standard and Sample preparations at about 766 nm
Calculate the content of Potassium as follows: ##EQU3## ATOMIC
ABSORPTION Potassium Stock Solution
Dissolve 190.7 mg of potassium chloride, previously dried at 105.degree.
C. for 2 hours, in water. Transfer to a 500 ml volumetric flask,
dilute with water to volume and mix. transfer 5 ml of this solution
to a 100 ml volumetric flask, dilute to volume with water and mix.
Standard Preparation
To separate 100 ml volumetric flask, transfer 10, 15 and 20 ml
respectively of Potassium stock solution. To each flask, add 2 ml
of sodium chloride solution (1 in 5) and 1 ml of hydrochloric acid,
dilute with water to volume and mix.
Assay Preparation
Weigh accurately about 1 g of the sample and transfer into a 500
ml volumetric flask dissolve in water, dilute to volume and mix.
Transfer 5 ml of this to a 100 ml volumetric flask, dilute to volume
with water and mix. Transfer again 5 ml of this solution to a 100
ml volumetric flask, add 2 ml of sodium chloride solution (1 in
4) and 1 ml of hydrochloric acid, Dilute with water to volume and
mix.
Procedure
Concomitantly determine the absorbencies of the Standard preparations
and assay preparation at the potassium emission line of 766.5 nm,
with a suitable atomic absorption spectrophotometer equipped with
a potassium hollow cathode lamp and an air acetylene flame, using
water as the blank. Plot the absorbance of standard preparation
versus concentration in .mu.g per ml of potassium and draw the straight
line best fitting the three plotted points. From the graph so obtained,
determine the concentration, in .mu.g per ml of potassium in the
assay preparation.
Calculate the content of potassium in mg as follows: 200.times.C
where `C` is concentration in .mu.g per ml
Calculate the percentage of potassium as follows: ##EQU4## MICROBIAL
ASSAY
Total Plate Count, E. coli, Salmonella, yeasts and molds are estimated
as per procedures described in "OFFICIAL METHODS OF ANALYSIS--ASSOCIATION
OF OFFICIAL ANALYTICAL CHEMISTS" (14th Edition, 1990)
The limits are given below:
______________________________________ Total Plate Count 10000
cfu/g E. coli Absent Salmonella Absent Yeasts/Molds 1000 cfu/g ______________________________________
Aflatoxins are estimated by the following procedure, which is based
on the methods described in "OFFICIAL METHODS OF ANALYSIS--ASSOCIATION
OF OFFICIAL ANALYTICAL CHEMISTS" (15th Edition 1990)
Limit: Aflatoxins--Not more than 20 parts per billion
Procedure for estimated Aflatoxins
Apparatus
High speed stirrer (1400-1600 rpm with stainless steel shaft and
propeller blade)
Ultra Violet Light
Long wave UV with intensity of 430 .mu. watt/cm.sup.2 at 15 cm
at 365 nm
Minicolumn
Borosilicate std wall tubing, Ca 6(id).times.200 nm tapered at
one end to ca 2 mm
Densitometer
With fluorometry attachment
Reagents
Distilled water or deionized water may be used.
a) Solvents
CHCl.sub.3 and acetone AR grade must be used
b) Potassium hydroxide wash solution
0.02N KOH with 1% Kcl. Dissolve 1.12 g KOH pellets and 10 g Kcl
in 1 L H.sub.2 O
c) Sodium hydroxide solution
0.02N 8.00 g of NaOH/LH.sub.2 O
d) Sulphuric acid solution-0.03%
Dilute 0.3 ml H.sub.2 SO.sub.4 to 1 L
e) Precipitating reagents
i) Copper carbonate, basic
ii) Ferric chloride slurry: Mix 20 g anhydrous
FeCl.sub.3 with 300 ml H.sub.2 SO.sub.4
f) Diatomaceous earth
Hyflo Super-Cel or equivalent
g) Column Packing
Silica Gel G 60-100 mesh; Florisil 100-200 mesh; Alumina neutral
80-200 mesh; CaSO.sub.4 anhydrous 20-40 mesh
h) Aflatoxin Standard solutions
Standards from Sigma Chemicals, USA, are used.
Preparation of Mini Column
Tamp small plug of glass wool into tapered end of column. To column,
add to height indicated in following order: 30 mm silica gel; 10
mm neutral alumina and 10 mm CaSO.sub.4. Tamp small plug of glass
wool on top of column. Tamp column after each addition to settles
packing and maintain interfaces as level as possible. After packing,
apply pressure to top glass wool plug with 5 mm diam. rod. Activate
packed columns at 110.degree. C. for 1-2 hours and store in vapor
tight container.
Extraction of Aflatoxins
Weigh 50 g sample into stirrer, add 250 ml CHCl.sub.3 --H.sub.2
O(85+15) and stir it for 30 minutes. Filter through Whatman No.
4 filter paper. Collect 150 ml filtrate and transfer to 500 ml beaker.
Purification
To 50 ml beaker, add 170 ml 0.2 N NaOH and 30 ml FeCl.sub.3 slurry
and mix well. Add 3 g basic CuCO.sub.3 to sample extract in 500
ml beaker and mix well, add both 1 and 2 mixtures and mix well.
Filter the mixture through Whatman No. 4 filter paper in a Buchner
funnel using Hyflo supercel bed.
Transfer 150 ml filtrate to 500 ml separator, add 150 ml 0.03%
H.sub.s SO.sub.4 and 10 ml CHCl.sub.3. Shake vigorously for 5 minutes
and allow to stand for 30 minutes. Transfer lower CHCl.sub.3 layer
(13-14 ml) to 125 ml separator. Add 100 ml KOH wash solution, swirl
gently for 30 seconds and allow to stand. (If emulsion occurs, drain
emulsion into 10 ml test tube add 1 g anhydrous Na.sub.2 SO.sub.4,
stopper, shake 30 seconds and allow to stand (CHCl.sub.3 phase need
not be completely clear). If emulsion is not broken, transfer emulsion
to 125 separator and wash with 50 ml 0.03% H.sub.2 SO.sub.4. Collect
3 ml CHCl.sub.3 layer in 10 ml test tube.
Column Chromatography
Transfer 2 ml ChCl.sub.3 solution (extract) to minicolumn, using
5 ml syringe. Hold the column vertically and apply slight air pressure
(with the help of a rubber bulb) to force solvent through column
at rate .ltoreq.10 cm/min until solvent appears at tip. Remove rubber
bulb and add about 5 ml of elution solvent containing CHCl.sub.3
--acetone (9:1). Collect the fractions.
Examine column under UV lamp for blue fluorescent band at top of
Florisil layer (Ca 2.5 cm from bottom of column) indicative of aflatoxin.
Collect the fractions corresponding the blue band separately and
concentrate to a residue.
TLC
Dissolve the residue in minimum quantity of CHCl.sub.3 and carry
out the TLC testing along with authentic sample of Aflatoxins. Solvent
system--Benzene: Methanol: Acetic Acid (95:5:5). Quantify the aflatoxin
by using TLC densitometer.
STABILITY
The stability of the product was evaluated in solid state and in
aqueous solution in temperature and humidity conditions as specified
below. The following parameters of the product were considered:
physical appearance, specific rotation, HCA content by HPLC, lactone
content by HPLC.
1. In solid state:
a) Room temperature,
b) 37.degree. .+-.2.degree. C. and 75%.+-.2 relative humidity
c) 45.degree. .+-.20.degree. C. and 75%.+-.2 relative humidity
2. In solution form (5% in water)
a) Room temperature
b) 37.degree. .+-.20.degree. C.
c) 45.degree. .+-.2.degree. C.
Conclusion
The product is found to be stable under stress conditions (higher
temperature and higher humidity) for a minimum of 90 days. These
results indicate that the product will be stable for about 5 years
under normal storage conditions. |