CA2430277A1 - The alginate having low molecular weight, methods of manufacturing it and its use - Google Patents
The alginate having low molecular weight, methods of manufacturing it and its use Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
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Abstract
The alginic acid or the salts thereof having average molecular weight of between about 5,000 -20,000 daltons and exhibiting intrinsic viscosity of between 1 - 20 measured by the Ubbelohde viscometer are provided. Most of the alginates have molecular weight of between 1,000 - 30,000 daltons. They are prepared form a high molecular weight alginic acid by means of decomposition, ultrafiltration and nanofiltration membran. they are of uses for prevention and treatment of hypertension, primary hyperaldosteronism and for reducing blood sugar.
Description
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.
Page 2 of 22 ONC-129 Patent application 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.
Page 3 of 22 ONC-129 Patent application 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 Page 4 of 22 ONC-129 Patent application 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.
Page 5 of 22 ONC-129 Patent application 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.
Page 6 of 22 ONC-129 Patent application 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.
Page 7 of 22 ONC-129 Patent application 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.
Page 8 of 22 ONC-129 Patent application 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:
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 Page 9 of 22 ONC-129 Patent application 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 Page 10 of 22 ONC-129 Patent application 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.
Page 11 of 22 ONC-129 Patent application 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 Page 12 of 22 ONC-129 Patent application 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 Page 13 of 22 ONC-129 Patent application 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 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.
Page 14 of 22 ONC-129 Patent application 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 Page 15 of 22 ONC-129 Patent application 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.
Page 16 of 22 ONC-129 Patent application 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 Page 17 of 22 ONC-129 Patent application 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.
Page 18 of 22 ONC-129 Patent application 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.
Page 19 of 22
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.
Page 2 of 22 ONC-129 Patent application 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.
Page 3 of 22 ONC-129 Patent application 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 Page 4 of 22 ONC-129 Patent application 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.
Page 5 of 22 ONC-129 Patent application 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.
Page 6 of 22 ONC-129 Patent application 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.
Page 7 of 22 ONC-129 Patent application 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.
Page 8 of 22 ONC-129 Patent application 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:
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 Page 9 of 22 ONC-129 Patent application 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 Page 10 of 22 ONC-129 Patent application 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.
Page 11 of 22 ONC-129 Patent application 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 Page 12 of 22 ONC-129 Patent application 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 Page 13 of 22 ONC-129 Patent application 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 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.
Page 14 of 22 ONC-129 Patent application 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 Page 15 of 22 ONC-129 Patent application 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.
Page 16 of 22 ONC-129 Patent application 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 Page 17 of 22 ONC-129 Patent application 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.
Page 18 of 22 ONC-129 Patent application 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.
Page 19 of 22
Claims (11)
1. A low molecular weight alginic acid or its salts (therapeutically accepted ions or chromium ion (III)) prepared from the starting material alginic acid through the process involves depolymerization methods and membrane separation techniques, with an average molecular weight between 5,000 and 20,000 Da, and most of the alginate molecules have molecular weight of 1,000 - 30,000 Da, and an intrinsic viscosity at 1- 20 measured by the Ubbelohde viscometer.
2. A low molecular weight alginic acid or its salts described in claim 1, the average molecular weight was between 7,000 and 12,000 Da, and most of the alginate molecules have molecular weight of 1,000 - 20,000 Da, and an intrinsic viscosity of 3 - 14 measured by the Ubbelohde viscometer.
3. A low molecular weight alginic acid or its salts described in claim 2, the average molecular weight was between 7,000 and 9,000 Da, and most of the alginate molecules have molecular weight of 1,200 -10,000 Da.
4. A preparation method for low molecular weight alginate, which includes: (1) The depolymerization of high molecular weight starting material alginic acid to a product with an average molecular weight of 20,000 - 80,000 Da, and the use of alkaline solution to dissolve the product; (2) The removal of molecules with molecular weight larger than 30,000 Da and retaining of alginates with molecular weight less than 30,000 Da by ultrafiltration method; (3) The removal of molecules with molecular weight less than 1,000 Da and obtaining after dried alginates with average molecular weight of 5,000- 20,000 Da, and most of the alginate molecules are with molecular weight of 1,000-30,000 Da by using nanofiltration membrane.
5. A preparation method for low molecular weight alginate, which includes: (1) The depolymerization of high molecular weight starting material alginic acid a product with a molecular weight of 20,000 - 80,000 Da; (2) The treatment of product from the previous step into a soluble alginate with alkaline solution; (3) The further depolymerization of this product to a alginate with even lower molecular weight, by adding water or water-containing media, and then an oxidant under a stirring to the above mentioned solution; (4) The removal of molecules with molecular weight larger than 30,000 Da and retaining of alginates with molecular weight less than 30,000 Da by ultrafiltration method; (5) The removal of molecules with molecular weight less than 1,000 Da and obtaining after dried alginates with average molecular weight of 5,000- 20,000 Da, and most of the alginate molecules are with molecular weight of 1,000-30,000 Da by using nanofiltration membrane.
6. A preparation method for low molecular weight alginate, which includes: (1) The depolymerization of high molecular weight starting material alginic acid to a product with an average molecular weight of 10,000 - 70,000 Da, and the use of alkaline solution to dissolve the product; (2) The removal of molecules with molecular weight larger than 10,000 Da and retaining of alginates with molecular weight less than 10,000 Da by ultrafiltration method; (5) The removal of molecules with molecular weight less than 1,200 Da and obtaining after dried alginates with average molecular weight of 5,000- 8,000 Da, and most of the alginate molecules are with molecular weight of 1,200-10,000 Da by using nanofiltration membrane.
7. A preparation method for low molecular weight alginate, which includes: (1) The depolymerization of high molecular weight starting material alginic acid a product with a molecular weight of 10,000 - 70,000 Da; (2) The treatment of product from the previous step into a soluble alginate with alkaline solution; (3) The further degradation of the product into alginates with a even lower molecular weight by adding water or water-containing medium, and then oxidants under stirring; (4) The removal of molecules with molecular weight larger than 10,000 Da and retaining of alginates with molecular weight less than 10,000 Da by ultrafiltration method;
(5) The removal of molecules with molecular weight less than 1,200 Da and obtaining after dried alginates with average molecular weight of 5,000- 7,000 Da, and most of the alginate molecules are with molecular weight of 1,200-10,000 Da by using nanofiltration membrane.
(5) The removal of molecules with molecular weight less than 1,200 Da and obtaining after dried alginates with average molecular weight of 5,000- 7,000 Da, and most of the alginate molecules are with molecular weight of 1,200-10,000 Da by using nanofiltration membrane.
8. A depolymerization method including physical, photochemical, and immobilized enzyme, or chemical method according to any of claims 4-7.
9. According to claim 8, the physical method includes ultrasonic and high speed cutting;
photochemical method employs light radiation and catalysis; chemical method involves strong acid/base and inorganic/organic oxidant.
photochemical method employs light radiation and catalysis; chemical method involves strong acid/base and inorganic/organic oxidant.
10. A method to prepare alginic acid by treating the low molecular weight alginate products obtained according to any of claims 4-9 with aqueous acid, and the low molecular weight alginic acid precipitate washed and dried.
11. Uses of the low molecular weight alginic acid and its salts according to any of claims 1-3 in prevention and treatment of hypertention, primary hyperaldosteronism and also for reducing blood sugar.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN1999/000202 WO2001040315A1 (en) | 1999-11-30 | 1999-11-30 | The alginate having low molecular weight, methods of manufacturing it and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2430277A1 true CA2430277A1 (en) | 2001-07-06 |
Family
ID=4575155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002430277A Abandoned CA2430277A1 (en) | 1999-11-30 | 1999-11-30 | The alginate having low molecular weight, methods of manufacturing it and its use |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU1502700A (en) |
| CA (1) | CA2430277A1 (en) |
| WO (1) | WO2001040315A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010187659A (en) * | 2009-01-20 | 2010-09-02 | Kao Corp | Purified alginic acid or salt thereof |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310954C (en) * | 2003-04-15 | 2007-04-18 | 哈尔滨工程大学 | Preparation method of chromium alginate |
| CN101104649B (en) * | 2006-07-12 | 2011-05-18 | 谭攸恒 | Potassium alginate and composition thereof |
| WO2009086685A1 (en) * | 2007-12-29 | 2009-07-16 | Dalian Yaweite Bioengineering Co., Ltd. | An alginic acid with low molecular weight, its salts, uses, preparative methods, pharmaceutical compositions and foods |
| CN104311696B (en) * | 2014-09-26 | 2017-04-26 | 大连雅威特生物技术股份有限公司 | Application of oligomeric sodium alginate in preparation of medicines, health products and salt products |
| CN112037931B (en) * | 2020-07-15 | 2023-10-20 | 新疆维吾尔自治区人民医院 | Aldosteronism prediction system for use in suspected primary aldosteronism |
Family Cites Families (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1010654B (en) * | 1984-08-10 | 1990-12-05 | 麦克公司 | The preparation method of the solid-state herbicidal composition of bipyridilium quaternary salt |
| JPS63101302A (en) * | 1986-10-17 | 1988-05-06 | Meiji Seika Kaisha Ltd | Culture of plant |
| IT1213384B (en) * | 1986-11-24 | 1989-12-20 | Lab Derivati Organici Mediolan | PROCESS FOR THE CONTROLLED PREPARATION OF LOW MOLECULAR WEIGHT GILCOSAMINOGLICANS. |
| JPS63214192A (en) * | 1987-03-03 | 1988-09-06 | Meiji Seika Kaisha Ltd | Production of alginic acid oligosaccharide |
| US4792452A (en) * | 1987-07-28 | 1988-12-20 | E. R. Squibb & Sons, Inc. | Controlled release formulation |
| JPH02225422A (en) * | 1989-02-28 | 1990-09-07 | Kimitsu Kagaku Kogyo Kk | Medicine composition |
| JPH03273002A (en) * | 1990-03-22 | 1991-12-04 | Otsuka Chem Co Ltd | Alginic acid decomposition product |
| JP3024994B2 (en) * | 1990-10-31 | 2000-03-27 | マルハ株式会社 | Production method and purification method of alginate oligosaccharide |
| JP2643669B2 (en) * | 1990-12-28 | 1997-08-20 | 共成製薬株式会社 | Algin-containing foods |
| JP2663408B2 (en) * | 1991-05-09 | 1997-10-15 | 明治製菓株式会社 | Method for promoting plant morphogenesis or differentiation |
| JPH0517315A (en) * | 1991-07-03 | 1993-01-26 | Sankin Kogyo Kk | Alginic acid salt impression material composition for dental use |
| JPH05304974A (en) * | 1992-03-06 | 1993-11-19 | Maruha Corp | Calcium aliginate oligosaccharide and its production |
| GB2266532B (en) * | 1992-04-28 | 1996-09-04 | Taiyo Fishery Co Ltd | Alginate oligosaccharides methods for their production and their use in foodstuffs |
| CN1089619A (en) * | 1993-01-06 | 1994-07-20 | 山东烟台西苑制药厂 | A kind of production method of propylene glycol alginate sodium sulfate |
| JP2986324B2 (en) * | 1993-11-26 | 1999-12-06 | 明治製菓株式会社 | Diet / Diabetic Food |
| JP3825069B2 (en) * | 1994-07-14 | 2006-09-20 | マルハ株式会社 | Human epidermal keratinocyte activator |
| CN1081194C (en) * | 1996-12-18 | 2002-03-20 | 谭攸恒 | Process for preparing potassium alginate and its use |
| JPH1180204A (en) * | 1997-09-05 | 1999-03-26 | Nikka Chem Co Ltd | Method for hydrolyzing alginic acid and hydrolyzed material |
| JP3617912B2 (en) * | 1997-10-08 | 2005-02-09 | 日本原子力研究所 | Plant growth promoter |
-
1999
- 1999-11-30 WO PCT/CN1999/000202 patent/WO2001040315A1/en active Application Filing
- 1999-11-30 AU AU15027/00A patent/AU1502700A/en not_active Abandoned
- 1999-11-30 CA CA002430277A patent/CA2430277A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010187659A (en) * | 2009-01-20 | 2010-09-02 | Kao Corp | Purified alginic acid or salt thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001040315A9 (en) | 2001-12-06 |
| AU1502700A (en) | 2001-06-12 |
| WO2001040315A8 (en) | 2002-03-21 |
| WO2001040315A1 (en) | 2001-06-07 |
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