Note: Descriptions are shown in the official language in which they were submitted.
CA 02430277 2003-05-28
ONC-129 Patent application
The alginate having low molecular weight, methods of preparation
and its use
Field of Invention
This invention is about the low molecular weight alginate salts obtained from
alginic acid
by depolymerization methods. The average molecular weight of the alginates is
between 5000
and 20,000 Da, the intrinsic viscosity measured by Ubbelohde viscometer is
between l and 20,
most of the alginates have molecular weights of between 1,000 and 30,OOODa.
This invention
also includes preparation methods of such low molecular weight alginates and
their applications
to prevent and treat hypertension, primary hyperaldosteronism and to reduce
blood sugar level.
Background
Natural alginic acid is a very common material that can be extracted from
marine plants
such as brown algae like kelp. It has been documented for a long time that
using natural alginic
acid and its derivatives as drugs to decrease blood pressure and to prevent
hypertension.
Kelp is a popular species and widely cultivated in many places along the
coastline. In
some coast regions of China, kelp root has been used to relieve hypertension
symptom; an intake
of 12 g root daily was showed to exhibit certain level of therapeutic
effectiveness for phase I or
phase II primary hypertension. Alginic acid or its salts have been extracted
from marine plants
such as kelp and used for many applications, for example, an intake of 20 g
sodium alginate
daily for adults could temporarily decrease the blood pressure. It was also
reported that
potassium alginate had an effect on animal in a rat study for SHR primary
hypertension,
however, it was shown to be not effective for hypertension patients who took
high molecular
weight potassium alginate. On the other hand, an intake of alginate salts with
much lower
molecular weight would not give a positive effect on decreasing blood pressure
for a long time.
For instance, JP 6-237783 reported a preparation method for oligomers of
alginate acid
polysaccharide that had an effect on prevention of hypertension and other
health benefits.
Alginate lyase that could depolymerize polysaccharides was used to decompose
natural alginic
acid to potassium alginate that was an oligosaccharide with a degree of
polymerization at 2 - 5.
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However, the product was only used for prevention (inhibition) hypertension
and no treatment
effects on hypertension were mentioned.
Patent CN1097307 reported an anti-obesity alginate agent, the preparation
method was as
follows: the commercially available alginic acid (pharmaceutical grade) was
swelled and soaked
in water, degraded to a polysaccharides with lower degree of polymerization by
adding 6 - 7 N
hydrochloride, then converted to potassium alginate by adding a saturated
solution of potassium
hydroxide in ethanol to pH 8, dried, and grounded to yield a powder form of
anti-obesity
alginate. However, the patent did not give details about the molecular weight
of the product and
the effect on decreasing blood pressure, and also did not mention the
treatment effects on
hypertension and aldosteronism.
SU1821470 reported a method for extraction of polysaccharides from seaweeds:
polysaccharides were extracted under alkaline condition from pretreated
seaweed at elevated
temperatures. HCl or HZS04 was added to the extract, then the material was
hydrolyzed at 80-
100 °C and pH 0-1; the hydrolyzate was cooled, neutralized caustic soda
solution. The
depolymerized alginate was precipitated and dried to yield a product with
viscosity of 1.5-4.0
cps. The patent did not mention the application of this product as an anti-
hypertension agent.
W09320826 A1 reported a pharmaceutical formulation based on alginate for
prevention
and treatment of uncomfortableness of gastrointestinal system.
US5460957 reported novel alginate oligosaccharide salts. The alginates were
calcium or
potassium but not sodium salt; they were used as a food additive for lowering
blood pressure.
The alginates were prepared by treatment of potassium or sodium alginates with
polysccharide
lyase and then exchanged sodium or potassium ions with calcium ion. The
products had the
degree of polymerization at 2 -5 and a lower molecular weight.
KR9200242 reported a method to prepare low molecular weight alginate. They
used
ultrasonic to break the polymer chain, however, the molecular weight of the
product was greater
than 35,000 Da. Repetitively ultrasonic treatment only yielded products with
molecular weight
of 20,000 Da or above. The patent did not mentioned effects on prevention and
treatment of
hypertension.
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KR9105768 reported a method for extraction of sodium alginate with a molecular
weight
of 60,000 - 300,000 Da from brown algae.
US5283076 reported sodium alginate-containing health foods and beverages that
could
be used for prevention of obesity and diabetic. The molecular weight of the
alginate was 10,000
-15,000 Da.
JP3273002 reported depolymerized products of alginic acid with a molecular
weight of
1,000 -1,500 Da. The products could be used as stabilizers for foods or drugs.
US4104460 reported a method to extract alginic acid from seaweed. It included
adding
hydrogen peroxide to alginic acid, then treatment with a base such as sodium
carbonate,
ammonium carbonate and sodium hydroxide. However the patent did not mention
the usages of
the products as drugs.
So far, the alginates obtained through the existing approaches mainly
restrained the
further increase of elevated blood pressure in a short time, but they normally
did not have a
treatment effect.
Detailed Description of the Invention
The purpose of this invention is to provide alginic acid and its derivatives
(alginate salts),
that have remarkable and long-lasting effects on prevention and treatment of
hypertension and
hyperaldosteronism, and that can also decrease blood sugar level.
Based on several years of research work, the inventors found that natural
alginic acid
(alginate salt) could be depolymerized to a certain degree, i.e., the average
molecular weight was
in a specific range, by using oxidation or enzyme digestion methods. The
products had a notable
effect on the treatment of hypertension and hyperaldosteronism, and also could
reduce blood
sugar level.
There were three stages of this work. In the beginning, the alginate salts
prepared had an
average molecular weight of 6.8 x 104 Da, and most of molecules had molecular
weight of 2,000
-100,000 Da. The alginate was fiu~ther refined by removing molecules with
molecular weight
over 50,000 Da, and the rest was used in the clinical study at Xiyuan
Hospital, Chinese Academy
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of Traditional Chinese Medicine. A daily dose of 5-8 g salts showed remarkable
effect on
primary hypertension patients, an overall effective rate was around 83.3 %.
The further clinical
investigation of the molecular weight distribution on hypertension patients
revealed that alginate
salts with molecular weight of above 30,000 Da, mainly in the range of 30,000 -
100,000 Da,
had no remarkable anti-hypertension effect. During the second stage, low
molecular weight
alginate salts were made. They had an average molecular weight of 3.5 x 104 Da
and most of
molecules were in the range of 2,000 - 58,000 Da. A daily intake of 4-7 g
showed a total
effective rate at 85 % for primary hypertension patients. In the third stage,
lower molecular
weight alginate were prepared. The average molecular weight of alginate was
5,000 - 20,000
Da, and preferably to be in the range of 7,000 -12,000 Da, especially the
average molecular
weight was 6,000 -10,000 Da. The most preferred products were molecules with
average
molecular weight in the range of 7,000-9,000 Da. The molecular size
distribution for most of the
molecules were in the range of 1,000 - 30,000 Da, better to be at 1,600 -
20,000 Da, preferably
at 1,600 -12,000 Da, and better at 1,600 -10,000 Da. The most preferred
products are the
alginates with molecular weight at 1,800 -10,000 Da. The intrinsic viscosity
values measured
by Ubbelohde viscometer for the alginates prepared were 1 - 20, and preferably
3-14. When
alginates that had an average molecular weight of 6,000 - 9,000 Da and most of
molecules had
an molecular weight of 1,600 -10,000 Da, were given 0.5 - 3 g per day to
hypertension patients,
the overall effective rate reached to 88 %. Moreover, when the alginate
treatment was removed
after a week of administration, the blood pressures of hypertension patients
could still be
maintained at a normal level for 2 -3 days. So during this stage our invention
was completed.
This invention provides low molecular weight alginates. Specifically, the
alginates were obtained
from the staring material alginic acid by the combination of depolymerization
methods and
membrane separation techniques. The average molecular weight of the alginates
was at 5,000 -
20,000 Da, preferably at 7,000 -12,000 Da and most preferred at 7,500 - 8,500
Da. The
molecular size distribution of the most molecules was at 1,000 - 30,000 Da,
preferably at 1,600 -
20,000 Da and most preferred at 1,600 -10,000 Da. Their intrinsic viscosity
values measured by
Ubbelohde viscometer were 1 - 20, the cations of alginate salt were chromium
(III) ion or other
pharmaceutically acceptable cations.
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This invention also provided preparation methods and uses of the low molecular
weight
alginate for prevention and treatment of hypertension and primary
hyperaldosteronism, as well as
for reducing blood sugar level.
The depolymerization approaches in above mentioned methods included physical
methods (ultrasonic and high speed cutting), photochemical methods (light
radiation and
catalysis), chemical methods (strong acid/base and inorganic/organic oxidant)
and immobilized
enzyme method.
Depolymerization by oxidation required the use of organic and inorganic
oxidants, such
as hydrogen peroxide, peracetic acid, potassium peroxide, potassium
peroxycarbonate, potassium
(sodium) perborate, potassium permangonate, ammonium persulfate, potassium
hydrogen
persulfate, sodium chlorite, and potassium chlorite, etc.
After degradation, ultrafiltration and nanofiltration membrane techniques were
used in
order to obtain products with required molecular weight. DHFM ZBS 1 and ZBS 3
hollow fiber
ultrafiltration membranes (manufactured by Dalian Institute of Chemical
Physics, Chinese
Academy of Sciences) were used to cut off molecules with molecular weight
10,000 Da and
30,000 Da, so that the alginate molecules with molecular weight greater than
10,000 Da and
30,000 Da were removed. After that the Bo Te NF4040 nanofiltration membrane
(produced by
the Membrane Engineering Centre of the Dalian Institute of Chemical Physics)
was used to
remove alginate salts with molecular weight smaller than 1,000 Da or 1,200 Da
(light pressure
applied).
It must be explained here for the meaning of word "most" we used in the phase
"most of
the alginic acid (alginate salt) molecules had molecular weights of ": because
the pore sizes of
ultrafiltration and nanofiltration membranes were not absolutely uniform,
there were small
amount of holes with greater or smaller pore size, also the sizes could be
changed under
pressure; so we used the word "most" in this situation. For example, when we
used
nanofiltration membrane to do a filtration, the membrane was designed to
retain molecules with
molecular weight above 1,000 Da, however, molecules with molecular weight
slightly greater
than 1,000 Da (such as molecular weight of 1,200 Da) could also pass through
the membrane.
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This invention provided a preparation method for low molecular weight
alginates. It
included: (1) Depolymerization: the starting material alginic acid with high
molecular weight
was depolymerized to a product with an average molecular weight of 20,000 -
80,000 Da. The
product was dissolved in an alkaline solution; (2) Ultrafiltration: the step
used to remove
molecules with molecular weight greater than 30,000 Da and retain alginate
molecules with
molecular weight smaller than 30,000 Da; (3) Nanofiltration: the membrane was
used to remove
alginate molecules with molecular weight smaller than 1,000 Da. The average
molecular weight
of the alginate product was 5,000 - 20,000 Da, and most of the alginate
molecules had molecular
weight of 1,000 - 30,000 Da. The product was dried at the end of the process.
This invention provided a preparation method for low molecular weight
alginates. It
included: (1) Depolymerization: the starting material alginic acid with high
molecular weight
was depolymerized to a product with an average molecular weight of 20,000 -
80,000 Da; (2)
Alkaline treatment of the product to convert it into a soluble salt; (3) Water
or water-containing
media was added while stirring, then oxidant was used to further depolymerize
the alginate; (4)
Ultrafiltration method was used to remove molecules with molecular weight
greater than 30,000
Da and retain alginates with molecular weight smaller than 30,000 Da; (5)
Nanofiltration
membrane was used to remove alginate molecules with molecular weight smaller
than 1,000 Da.
The average molecular weight of product was 5,000 - 20,000 Da, and most of the
alginate
molecules had molecular weight of 1,000 - 30,000 Da. The product was dried at
the end of the
process.
This invention provided a preparation method for low molecular weight
alginates. It
included: (1) Depolymerization: the starting material alginic acid with high
molecular weight
was depolymerized to a product with an average molecular weight of 10,000 -
70,000 Da, and
then product was dissolved in an alkaline solution; (2) Ultrafiltration method
was used to remove
molecules with molecular weight greater than 10,000 Da and retain alginates
with molecular
weight smaller than 10,000 Da; (3) Nanofiltration membrane was used to remove
alginates
molecules with molecular weight smaller than 1,200 Da. The average molecular
weight of the
product was 5,000 - 8,000 Da, and most of the alginate salt molecules had
molecular weight of
1,200 -10,000 Da. The product was dried at the end of the process.
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This invention provided a preparation method for low molecular weight
alginates. It
included: ( 1 ) Depolymerization: the starting material alginic acid with high
molecular weight
was depolymerized to a product with an average molecular weight of 10,000 -
70,000 Da; (2)
Alkaline treatment of the product to convert it into a water soluble salt; (3)
Water or water-
containing media was added while stirring, then oxidant was used to further
depolymerize the
alginates; (4) Ultrafiltration method was used to remove molecules with
molecular weight
greater than 10,000 Da and retain alginates with molecular weight smaller than
10,000 Da; (5)
Nanofiltration membrane was used to remove alginate molecules with molecular
weight smaller
than 1,200 Da. The average molecular weight of the product was 5,000 - 7,000
Da, and most of
the alginate molecules had molecular weight of 1,200 -10,000 Da. The product
was dried at end
of the process.
The depolymerization step in above mentioned included physical methods
(ultrasonic
and high speed cutting), photochemical methods (light radiation and
catalysis), chemical
methods (strong acid/base and inorganic/organic oxidant) and immobilized
enzyme method.
Only the small molecule alginates could get into blood vessels by intestinal
mucosa
absorption so that they were the major effective components against
hypertension and primary
hyperaldosteronism. Small amount of alginates with molecular weight above
12,000 Da could
still pass through and be absorbed by intestinal mucosa; however, the higher
molecular weight
of alginates were, the less the amount could be absorbed. Only the potassium
salt of alginate that
had high molecular weight exhibited certain effectiveness, in this case, the
only explanation
could be the alginates binding with sodium ion in intestines and then
regulated blood pressure
through a sodium-potassium ion exchange effect. In other word, the molecular
weight played an
important role and alginates had to be absorbed in intestines so that to be
more effective. It was
worth to mention that alginates with much smaller molecular weight (such as
lower than 900 Da)
could be easily absorbed by intestinal mucosa to generate an anti-hypertension
effect, however,
the effect could only last for a short period of time.
The alginate containing chromium could reduce blood sugar level. The content
of
chromium in alginate was normally 0.01% - 0.05% (w/w), a daily intake of 6 - 8
g chromium
containing alginate would notably reduce blood sugar level.
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The alginic acid used in this invention was extracted from brown algae,
especially from
kelp which was available abundantly and economically. In order to obtain the
alginates that had
the specific average molecular weight in this invention, two staring materials
were used: the
commercial alginic acid or alginate with a high molecular weight, or alginic
acid or alginate that
was extracted by normal procedures and had the average molecular weight of
30,000 - 300,000
Da. The differences of extraction procedures for seaweeds did not affect the
alginic acid or
alginate yielded if the average molecular weight and the molecular weight
distribution were in
the above range.
The alginate salts mentioned in this invention refer to any salts that can be
used as drugs,
i.e., pharmaceutically acceptable salts. Cations included alkali metal,
alkaline-earth metal, Fe,
Zn, Cr(III), Mn and ammonium ion. Water-soluble salts were preferable and
potassium salt was
the most preferred for the purpose.
The "molecular weight" mentioned here was measured by column chromatography:
HL-2
constant flow pump and BSZ-100 fraction collector (Shanghai Huxi Instrument
Factory), model
222 spectrophotometer detector (Shanghai Analytical Instrument Factory).
Because alginic acid
and its salts are polysaccharides, so L-Dextran with different molecular
weights was used as the
reference. The average molecular weights of Dextran T4-6, T8-12, T60-90, T100
and T200-300
were 5,000, 10,200, 68,300, 100,000 and 266,000 Da, respectively (manufactured
by Phamacia
Sweden and provided by Farco Chemical Supplier HongKong). The packing material
for the
column was Sephacryls-300. Chromatography condition: mobile phase was 0.1 M
sodium
chloride aqueous solution (50 mL), flow rate =1 mL/min; collection volume was
1 mL/tube.
Detection: 1,3,5-trihydoxylbenzene in hydrochloric acid was added to a
solution of the sample
( 10 mg) in water ( 1 mL), the mixture was heated to boiling and the color
turned to reddish
purple. The sample was a complex of the potassium salts of mannuronic acid and
guluronic acid
polymers, so it could react with 1,3,5-trihydoxylbenzene in hydrochloric acid
to form a reddish
purple products. We then could detect the colourful compound by a
spectrophotometer, or
measure the absorbance of alginates at 230 nm with Shimadzu spectrophotometer.
Dextran T4-6, T8-12, T60-90, T100 and T200-300 (5 mg) were weighed accurately,
dissolved in the mobile phase (0.5 mL) and then applied to the column
separately under the same
condition. Low molecular weight alginates (5 mg) were also weighed accurately,
dissolved in the
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mobile phase (0.5 mL) and then applied to the column. Dextran reference
eluting curve and the
invented sample eluting curve were obtained, and the average molecular weight
of low molecular
weight alginate was measured by comparison of the two curves. It had to be
noted that the
insoluble alginates were first converted to soluble form of salt (such as
sodium or potassium
salts) by ion exchange, then the molecular weight was measured. For instance,
calcium alginate
was converted to alginic acid by precipitation through adding hydrochloric
acid, and then
dissolved in sodium hydroxide solution to form sodium alginate.
The intrinsic viscosity was measured with an Ubbelohde viscometer according to
China
Pharmacopoeia 1995 volume II, appendix VI G: viscosity measurement method, the
concentration of alginate was 0.2 wt %.
List of Figures
Figure 1: IR spectrum of potassium alginate with low molecular weight in
Example 1
Figure 2: IR spectrum of starting material alginic acid with high molecular
weight
Figure 3: 1H-NMR spectrum of potassium alginate with low molecular weight in
Example 1
Figure 4: 1H-NMR spectrum of starting material alginic acid with high
molecular weight
The following is a set of examples best representing this invention:
Example 1: Ultrasonic depolymerization
100 g alginic acid (average molecular weight 200,000, and most of molecules
had
molecular weight of 35,000 - 300,000 Da. It was manufactured by China Dalian
Alginate
Industry Co., and was approved as pharmaceutical grade) was added to water
(10% to be alginic
acid) and the water suspension was stirred and depolymerized by ultrasoonic
for 2 h (Sonic &
Materials 1500, 1500W, operation frequency 20 KHz). Potassium hydroxide
aqueous solution
was added to keep the pH at 5 - 9 during the ultrasonic treatment period, and
the alginates
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obtained were with an average molecular weight of 35,000 Da. DHFM-ZBS 3 hollow
fiber
ultrafiltration membrane (manufactured by Dalian Institute of Chemical
Physics, Chinese
Academy of Sciences; operating pressure 0.15 Mpa) was then employed to remove
alginates
with molecular weight greater than 30,000 Da. The alginate solution with
molecular weight
smaller than 30,000 Da was dehydrated and alginate molecules smaller than
1,200 Da were
removed by using Bo Te NF4040 nanofiltration membrane (0.15 Mpa operating
pressure,
manufactured by Dalian Institute of Chemical Physics, Chinese Academy of
Sciences). So a
product that had a molecular weight distribution at 1,200 - 30,000 Da was
obtained after two
steps of filtrations. A low molecular weight alginate with average molecular
weight of16,000 Da
(measured by column chromatography) was thus prepared through precipitation by
adding 95%
ethanol (2 folds in volume) and dried.
The removed portion of potassium alginates with molecular weight greater than
30,000
Da could be re-used as part of the starting material along with the original
and commercial
alginic acid and repeat above operations to produce low molecular weight
alginates.
From the comparison of the IR (Figure l and 2) and'H-NMR (Figure 3 and 4)
spectra of
the low molecular weight alginate product obtained from Example 1 to that of
the starting
material alginic acid, it would be clear that basic molecular structure was
not changed by the
depolymerization.
Example 2: H~gen peroxide depolymerization
The first stage: S00 g of alginic acid (pharmaceutical grade, manufactured by
China
Dalian Alginate Industry Co.) was suspended in distilled water (alginic acid
content 9%),
homogenized the mixture by stirring at 40 °C. Hydrogen peroxide
solution (30 %) was added
dropwise while stirring until the final concentration of hydrogen peroxide in
the system reached
1 % (w/w). The reaction mixture was stirred for 2 h, and then the temperature
was increased
above 80 °C in order to decompose and remove the unreacted hydrogen
peroxide. Potassium
hydroxide aqueous solution (30 %) was added dropwise to the reaction mixture,
suitable amount
of distilled water was added to dissolve product thoroughly. The system pH was
kept at 6-7 to
finally obtain a product with an average molecular weight of 65,000 Da. The
intrinsic viscosity
values of the products were listed in table 1.
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The second stage: DHFM-ZBS 1 hollow fiber ultrafiltration membrane (0.15 Mpa
operating pressure) was used to remove potassium with molecular weight above
10,000 Da, and
obtained a solution of potassium alginate with molecular weight smaller than
10,000 Da.
Followed by the application of Bore NF4040 nanofiltration membrane (0.11 Mpa
operating
pressure) to dehydrate the alginate solution and remove alginate molecules
with molecular
weight smaller than 1,000 Da. Thus a product that had a molecular weight
distribution at 1,000 -
10,000 Da was obtained after two steps of filtration and dried. The average
molecular weight
was determined to be 7,500 Da by column chromatography.
The potassium alginates with molecular weight greater than 10,000 Da removed
in the
step mentioned above could be re-used as a part of the starting material along
with the original
alginic acid commercially acquired and repeat the operations as described.
Examples 3-10:
Operations described in Example 2 were repeated under different reaction
temperatures
and with different final concentrations of hydrogen peroxide as shown in table
1.
Table 1: Experiment results
Example Reaction Temp. Final H202 Intrinsic viscosity
(C) concentration of
__,_ (%) otassium alginates
2 40 1 15-19
3 40 2 13-17
4 40 3 12-16
60 1 12-16
6 60 2 11-15
7 60 3 10-14
8 80 1 9-13
9 80 2 8-12
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80 3 7-11
Example 11: Combination of hydrogen peroxide and ultrasonic depolymerization
First, the same operations as described in the first stage in Example 2 were
repeated.
The product obtained was depolymerized further with ultrasonic treatment at 40
°C for 1
h (Sonic & Materials 1500, 1,SOOW, operating frequency 20 KHz), and potassium
hydroxide
aqueous solution (30%) was added dropwise during the ultrasonic operation
period until pH was
about 7.3.
Then DHFM-ZBS 1 hollow fiber ultrafiltration membrane was used to remove
alginate
molecules with a molecular weight above 10,000 Da to obtain a potassium
alginate solution with
molecular weight less than 10,000 Da. Following that, Bo Te NF4040
nanofiltration membrane
was employed for dehydration and further removal of alginate molecules with
molecular weight
smaller than 1,000 Da. A product that had a molecular weight distribution at
1,000 -10,000 Da
was thus obtained after two steps of filtration, precipitation from 95%
ethanol (2 folds in
volume), and finally dried. The average molecular weight of the product was 7,
000 Da measured
with column chromatography and the intrinsic viscosity was 3.
Potassium alginate molecules with molecular weight greater than 10,000 Da that
had
removed in the ultrafiltration step described above could be re-used as a part
of the starting
material along with the original alginic acid commercially acquired, and
repeated the operations
as described.
Example 12: Potassium peroxide K~O~I oxidation
Nine experiments were carried out parallelly. 100 g of alginic acid was
suspended and
soaked in 400 mL distilled water, and potassium peroxide aqueous solution
(20%) was added
with stirring under three different reaction temperatures (40°C,
60°C and 80°C). Under each
reaction temperature, three experiments with different final K202
concentration (2%, 4% and
6%) were tried. The reaction mixture was stirred for 2.5 h after completing
the addition of
potassium peroxide. The obtained colloidal product was potassium alginate with
a lower level of
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polymerization. The product was treated with distilled water and adjusted the
pH to 7 by using
potassium hydroxide aqueous solution (20%) and adjusted the concentration of
potassium
alginate to be 0.8%. DHFM ZBS 1 hollow fiber ultrafiltration membrane was used
to remove
alginate molecules with molecular weight larger than 10,000 Da, and then
NF4040 nanofiltration
membrane was used to remove alginates with molecular weight smaller than 1,000
Da. The
solution was dehydrated and concentrated to give a 5% syrup, and 95% ethanol
(two folds in
volume) was added to precipitate potassium alginate. The yielded product was
dried, and its
average molecular weight was 8,000 Da.
The potassium alginates removed from ultrafiltration with molecular weight
greater than
10,000 Da could be re-used as a part of the starting material along with the
original commercial
alginic acid.
Table 2: Experiment results using K202
Reaction K202 concentration Intrinsic viscosity
tem erature (%) of
(C) otassium al inate
40 2 12-16
4 10-14
6 8-12
60 2 8-10
4 7-9
6 6-8
80 2 5-8
4 4-7
6 3-6
The product made at 80°C with 6% K202 was determined to have an average
molecular
weight of 6,000 Da by a column chromatography method.
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Example 13: Potassium.peroxycarbonate oxidation
The procedures and conditions used were similar to those in Example 12 except
for using
potassium peroxycarbonate aqueous solution (30%) instead of using potassium
peroxide (20%).
The intrinsic viscosity of the obtained products were very close to that shown
for
Example 12.
Example 14: Hydrogen fluoride
100 g of alginic acid was ground and dried under vacuum, then transferred into
a reaction
vessel. The pressure in the reaction kettle was reduced to 5 mmHg and then
returned to the
atmosphere by filling in HF gas till the adsorbed HF was about 32 - 40 % (wlw)
of the alginic
acid. The reaction mixture was stirred at 10 - 20°C for 1 h. The inert
gas tetrachloroethane was
used to desorb HF in the kettle at 80 -100°C, and alginic acid with a
low level of polymerization
was then obtained. The desorbed HF gas could be recycled and used repeatedly.
The alginic acid obtained above was neutralized with potassium hydroxide
aqueous
solution (10%) to about pH 7.3, water was added to form a potassium alginate
solution. ZBS 1
and NF4040 membranes were employed for the ultrafiltration and nanofiltration
as described
previously and fluoride as an impurity was removed by the filtration process
as well. The product
was precipitated from 95 % ethanol (2 folds in volume), dried and yielded
potassium alginate
with an average molecular weight of 7,000 Da.
Example 15: Depolymerization with immobilized enzyme
Immobilized enzyme technique could increase the efficiency of enzyme in
depolymerization. The method by A.N. Emery CChem. Eng., 71, 1972) was improved
by using
polyporous sodium glass beads (with a pore size 600 - 800 ~, and diameter 1
mm). The beads
was soaked in TiCl4 aqueous solution (5%) at 45°C for 24 h, washed with
distilled water to
remove unabsorbed TiCl4. The glass beads were then transferred into a 2 %
solution of alginic
acid lyase (See JP 6-237783), soaked at 2°C for 18 h, then applied onto
a column. Potassium
alginate aqueous solution (8%) was loaded on the column while the column
temperature was
kept at 40 - 55 °C and pH at 7.0 - 7.3. It took 6 hours to obtain
alginate solution eluting out from
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the column after loading the sample, and low molecular weight potassium
alginate was yielded
with a molecular weight distribution at 800-35,000 Da.
Example 16: Photochemical denolymerization
Titanium oxide powder was coated on the surface of argil ball and burned at
1250°C to
form titanium oxide beads (bead diameter 2 mm). The beads were mixed with
alginic acid
containing 25 % water, and then put into a sealed container. Oxygen gas was
introduced, and the
mixture was exposed to Vis/UV for 20h. to yield alginic acid with a low level
of polymerization.
The reaction mixture was neutralized to pH = 7.3 with 10% potassium hydroxide
aqueous
solution and then water was added to obtain a potassium alginate solution. ZBS
1 and NF4040
membranes were employed for the ultrafiltration and nanofiltration as
described previously.
Potassium alginate was precipitated from the solution by adding 95% ethanol (2
folds in
volume), and then dried. The average molecular weight of this product is 7,000
Da. Titanium
oxide beads could be recycled and used repeatedly.
Example 17: Preparation of low molecular weight calcium al irate
A similar method as described above was used. The starting material alginic
acid was
depolymerized by hydrogen peroxide. The amount of hydrogen peroxide had to be
1 % to 3%
(w/w) of the sum of alginic acid and water. Oxidation was carried out at 40 -
80°C for 2 h. The
oxidized alginic acid was converted to low molecular weight sodium alginate by
adding sodium
hydroxide aqueous solution (30%). ZBS 1 and NF 4040 membranes were used for
the
ultrafiltration and nanofiltration as described above and sodium alginate with
an average
molecular weight of 8,000 Da was obtained. Finally, insoluble calcium alginate
was precipitated
out from the sodium alginate solution by adding calcium chloride aqueous
solution (S%). The
precipitate was washed with distilled water until the filtrate became neutral
to remove
hydrochloride byproduct, then dried.
Example 18: Preparation of chromium-containing ~ginates
Repeated the operations in the Example 17 by using chromium (III) ion aqueous
solution
(such as CrCl3) instead of calcium chloride.
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Example 19: Preparation of low molecular weimt alginic acid
100 g low molecular weight alginate obtained in Example 2 was treated with 10%
HCl
and the precipitate was washed with distilled water and dried to yield low
molecular weight
alginic acid.
Pharmaceutical formulations:
- Capsule:
Alginate (0.5 g) with a molecular weight of 8,000 Da was encapsulated into a
glutin
capsule. A daily intake of about four capsules was recommended.
Alginic acid (0.5 g) with low molecular weight that was made in Example 19 was
put
into a glutin capsule. A daily intake of about four capsules was recommended.
- Granule:
Alginate (1 g) with a molecular weight of 8,000 Da was packed in a small
plastic bag,
and one or two bags daily intake was recommended.
Alginate (2 g) with a molecular weight of 18,000 Da was packed in a small
plastic bag,
and one or two bags daily intake was recommended.
- Tablet:
Alginate with an average molecular weight of 8,000 Da was mixed with
commercially
available starch (pharmaceutical grade) at a ratio of 3:1, and the mixture was
pressed to tablets
(0.7 g). A daily intake of four tablets was recommended.
It was recommended that the capsule, granule and tablet described above were
taken after
meals.
The results of hypertension treatment with alginates:
Clinical study was conducted at Xiyuan Hospital, Chinese Academy of
Traditional
Chinese Medicine, Beijing. 30 subjects with primary hypertension were observed
by taking low
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molecular weight potassium alginate {molecular weight 5,000-30,000 Da) with a
dosage of 4 -7
g per day. The total effective rate was revealed to be around 85%.
Specialists on cardiovascular diseases at Dalian Medical University used
potassium
alginate (daily dose 1-4 g) with even lower molecular weight (1,000 -10,000
Da) to treat 30
hypertension patients, and a total effective rate was above 88%.
The result on treatment of hyperaldosteronism with al~inates~
Low molecular weight potassium alginate could be used to treat hypertension
patients
with hyperaldosteronism.
By using radioimmunoassay kits, ten volunteers were measured aldosterone (ALD)
levels
in their blood plasma before and after taking low molecular weight potassium
alginate. The
subjects were advised to keep normal diet, and phlebotomized in a standing
position. With a
daily dose of 6-8 g (divided into two administrations) for 15 days, the
aldosterone levels in blood
plasma of the subjects before and after taking alginates were measured by
using a
radioimmunoassay (RIA) method.
Table 3: Aldosterone levels in standing position
Volunteer A B C D E F G H I J
Before treatment 265 250 225 195 185 205 300 290 250 185
After treatment 200 125 180 195 130 145 180 200 180 185
Statistical analysis of the data showed that the mean ALD level before
treatment was 235
ng/L, whereas that level reduced to 172 ng/L after the treatment, there was an
average decrease
of 63 ng/L in ALD level.
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The result of lowering blood sugar level with al 'gmates:
30 hypertension subjects accompanying by higher blood sugar level (average
plasma
sugar level 7.0 mmol/L) were treated with chromium-containing alginate
(chromium content was
0.01 % of total weight of the alginate) at a daily dose of 6-8 g for 15 days.
The blood sugar level
was reduced to 4.7 mmol/L in average.
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