Method for isolating fraction having anti-inflammatory or osteoarthritis-inhibiting effects by using oyster shell chips
09937210 ยท 2018-04-10
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K35/618
HUMAN NECESSITIES
International classification
A61K35/12
HUMAN NECESSITIES
Abstract
The present invention relates to a method for isolating a fraction having anti-inflammatory or osteoarthritis-inhibiting effects by removing impurities and washing oyster shells, which are produced during a process of producing oysters, drying same, forming a powder from the oyster shells, and adding anhydrous citric acid to same for a reaction, obtaining a reaction product, and isolating a supernatant, which is obtained by centrifugally separating the reaction product, by means of ultrafiltration. By adding the anhydrous citric acid to the oyster shell chips for the reaction at a relatively low temperature, the present invention provides superior advantages, such as significantly reducing energy costs and isolating the fraction having the anti-inflammatory or osteoarthritis inhibiting effects through a simple method.
Claims
1. A method for isolating a fraction having an anti-inflammatory effect using oyster shell, characterized in that the said method comprises: a) a preliminary step of removing foreign materials attached to the oyster shell, washing the oyster shell with water and then drying and grinding the washed oyster shell to make a powder; b) a reaction step of reacting the oyster shell powder obtained from the said preliminary step with an anhydrous citric acid to obtain a reaction product; c) a supernatant-recovering step of centrifuging the reaction product obtained from the said reaction step to recover a supernatant; and d) an isolation step of isolating the supernatant obtained from the said supernatant-recovering step using ultra-filtration membrane and then drying the same to isolate a fraction having an anti-inflammatory effect.
2. The method for isolating a fraction having an anti-inflammatory effect using oyster shell according to claim 1, characterized by adding an anhydrous citric acid to the oyster shell powder during the reaction step in a ratio of 1: 0.19211.9212 parts by weight and conducting the reaction at 4100 C. for 124 hours.
3. The method for isolating a fraction having an anti-inflammatory effect using oyster shell according to claim 2, characterized in that, in the said supernatant-recovering step, the ultra-filtration membrane has a size of 10 kDa.
4. The method for isolating a fraction having an anti-inflammatory effect using oyster shell according to claim 2, characterized in that, in the said supernatant-recovering step, the ultra-filtration membrane has a size of 3 kDa.
5. A method for isolating a fraction having an osteoarthritis-inhibiting effect using an oyster shell, characterized in that the said method comprises: a) a preliminary step of removing foreign materials attached to the oyster shell, washing the oyster shell with water and then drying and grinding the washed oyster shell to make a powder; b) a reaction step of adding anhydrous citric acid to the oyster shell powder obtained from the said preliminary step in a ratio of 1: 0.19211.9212 parts and conducting the reaction at 4100 C. for 124 hours; c) a supernatant-recovering step of centrifuging the reaction product obtained from the said reaction step to recover a supernatant; and d) an isolation step of isolating the supernatant obtained from the said supernatant-recovering step using ultra-filtration membrane having a size of 3 KDa and then drying the same to isolate a fraction having an osteoarthritis-inhibiting effect.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
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DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
(9) Hereinafter, specific embodiments of the present invention are described in detail, including preferred examples and comparative example. However, they are intended to describe the invention in detail such that the invention can be easily practiced by a person with ordinary skill in the technical field to which the invention pertains, and are not intended to mean that the technical idea and range of the present invention are limited thereto.
(10) In accordance with the present invention, the method for isolating a fraction having an anti-inflammatory effect using oyster shell comprises:
(11) 1) a preliminary step of removing foreign materials attached to the oyster shell, washing the oyster shell with water and then drying and grinding the washed oyster shell to make a powder;
(12) 2) a reaction step of reacting the oyster shell powder obtained from the said preliminary step with an anhydrous citric acid to obtain a reaction product;
(13) 3) a supernatant-recovering step of centrifuging the reaction product obtained from the said reaction step to recover a supernatant; and
(14) 4) an isolation step of isolating the supernatant obtained from the said supernatant-recovering step using an ultra-filtration membrane and drying the same to isolate a fraction having anti-inflammatory effect.
(15) First, the preliminary step of removing foreign materials attached to the oyster shell, washing the oyster shell with water and then drying the washed oyster shell to make a powder, is conducted. When an oyster shell generated in the oyster farm is collected, various foreign materials are attached to the surface thereof. Therefore, these foreign materials are removed using a knife, etc. Then, the oyster shell is completely washed with water, naturally dried and dry-ground in 50300 mesh of a suitable size using an air mill in order to facilitate the reaction of the oyster shell with the anhydrous citric acid.
(16) The anhydrous citric acid is added to the oyster shell obtained in the said preliminary step and their reaction is conducted. The reaction step to obtain a reaction product is one of the important steps in the methods for isolating fraction having anti-inflammatory or osteoarthritis-inhibiting effects using oyster shell according to the present invention.
(17) As the reactants to obtain the reaction product of the present invention, anhydrous citric acid has the molecular formulae, C.sub.6H.sub.8O.sub.7 or HOC(CO.sub.2H)(CH.sub.2CO.sub.2H).sub.2. When the anhydrous citric acid meets with calcium (Ca), it reacts with the calcium, losing a hydrogen ion (H.sup.+), to form a compound called calcium citrate. During this process, water (H.sub.2O) and carbon dioxide (CO.sub.2) gas are generated. One molecule of citric acid can react with up to three calcium atoms. The chemical formulae associated with this are shown in the following Chemical Formulae (1) to (3):
HOC(CO.sub.2H)(CH.sub.2CO.sub.2H).sub.2+CaCO.sub.3.fwdarw.HOC(CO.sub.2Ca)(CH.sub.2CO.sub.2H).sub.2+CO.sub.2+H.sub.2O(1)
HOC(CO.sub.2Ca)(CH.sub.2CO.sub.2H)(CH.sub.2CO.sub.2H)+CaCO.sub.3.fwdarw.HOC(CO.sub.2Ca)(CO.sub.2Ca)(CH.sub.2CO.sub.2H)+CO.sub.2+H.sub.2O(2)
HOC(CO.sub.2Ca)(CH.sub.2CO.sub.2Ca)(CH.sub.2CO.sub.2H)+CaCO.sub.3.fwdarw.HOC(CO.sub.2Ca)(CO.sub.2Ca).sub.2+CO.sub.2+H.sub.2O(3)
(18) When all the three calcium are bonded to citric acid, the chemical formula can be expressed as in the following Chemical Formula (4).
HOC(CO.sub.2H)(CH.sub.2CO.sub.2H).sub.2+3CaCO.sub.3.fwdarw.HOC(CO.sub.2Ca)(CH.sub.2CO.sub.2Ca).sub.2+3CO.sub.2+3H.sub.2O(4)
(19) The bonding of calcium and citric acid is a principle wherein citric acid groups surround calcium atoms bonding to calcium to form macromolecules, thereby causing precipitation to occur. Therefore, two citric acid molecules and three calcium carbonates are reacted. The molecular weight of citric acid is 192.12 and the molecular weight of calcium carbonate is 100.09.
(20) To the oyster shell powder obtained in the preliminary step, anhydrous citric acid is added in a ratio of 1:0.19211.9212 parts by weight and their reaction is preferably conducted at a relatively low temperature of 4100 C. for 124 hours. Anhydrous citric acid is dissolved in water and used as a citric acid solution. 10 l of 0.11.0 M citric acid aqueous solution (0.19211.9212 kg of citric acid) is added to 1 kg of oyster shell powder, and they are sufficiently stirred and then reacted.
(21) When the amount of anhydrous citric acid is less than 0.1921 parts by weight, a sufficient reaction with oyster shell does not occur. When the amount of anhydrous citric acid exceeds 1.9212 parts by weight, there occurs a problem that a residual amount of citric acid is generated, thereby lowering the purity of calcium citrate. Furthermore, when the reaction temperature is 4 C. and the reaction time is less than 1 hour, the reaction rate slows, thereby not conducting a sufficient reaction. When the reaction temperature is 100 C. and the reaction time exceeds 24 hours, there occurs a problem that the reaction rate is no longer increased and the energy cost is higher.
(22) In the supernatant-recovering step of centrifuging the reaction product obtained from the said reaction step to recover a supernatant, the oyster shell powder is reacted with citric acid. In the reaction product thus produced, calcium citrate, water, carbon dioxide and the like are mixed. The carbon dioxide is a gas and thus is discharged into the air. The remaining mixed solution is centrifuged to isolate a pellet form of calcium citrate which is the supernatant and the precipitate, thereby recovering the supernatant. At this time, centrifugation is conducted at 8,000 rpm at room temperature for 20 minutes, and then the supernatant and the pellet precipitate are isolated from each other.
(23) The supernatant recovered in the supernatant-recovering step is isolated using an ultrafiltration machine. The ultrafiltration membrane uses two types having cutoff molecular weights of 10 kDa and 3 kDa. Hence, the fraction isolated at more than 10 kDa (below, called fraction 1), the fraction isolated between 3 kDa and 10 kDa (below, called fraction 2) and the fraction isolated at not more than 3 KDa (below, called fraction 3) are obtained. Subsequently, the fractions 1, 2 and 3 thus obtained can be dried in a vacuum dryer to isolate the fractions.
(24) Further, the method for isolating a fraction having an osteoarthritis-inhibiting effect using oyster shell in accordance with the present invention is compared with the method for isolating a fraction having an anti-inflammatory effect using the oyster shell. The only difference is that the present invention uses only the ultra-filtration membrane with a size of 3 kDa instead of the ultra-filtration membrane with sizes of 10 kDa and 3 kDa to obtain the fraction 3. Therefore, detailed description thereof will be omitted.
(25) Below, the present invention shall be more specifically described through the following examples and comparative examples.
EXAMPLE 1
(26) An oyster shell generated in the oyster farm was collected and various foreign materials attached on the surface thereof were removed. The oyster shell was then completely washed with water, naturally dried and then dry-ground by 50300 mesh using an air-mill. 0.9606 kg of anhydrous citric acid was dissolved in water to make 10 l of 0.5M citric acid aqueous solution to which 1 kg of oyster shells were added and completely mixed. The mixed solution was then reacted at 30 C. for 12 hours. The resulting reaction product was centrifuged with 8,000 rpm at 4 C. for 20 minutes. The supernatant and the pellet precipitate were isolated from each other. The isolated supernatant was isolated using an ultra-filtration machine. Herein, the ultra-filtration membrane used two types having the fraction molecular weights of 10 kDa and 3 kDa.
(27) The fractions recovered through this process were dried in a vacuum dryer. As a result, the three fractions, i.e., 0.3 g of the fraction 1 (recovery rate 0.014%) isolated at more than 10 kDa, 0.6 g of the fraction 2 (recovery rate 0.029%) isolated between 3 kDa and 10 kDa and 24.5 g of the fraction 3 (recovery rate 2.45%) isolated at not more than 3 kDa were combined to obtain a total of 25.4 g.
EXAMPLE 2
(28) Among the fractions obtained in Example 1, a fraction with a molecular weight of not more than 3 kDa was isolated with ultra-filtration membrane to obtain 24.6 g of the fraction 3.
COMPARATIVE EXAMPLE 1
(29) Indomethacin which is one of the drugs that strongly inhibit cyclo-oxygenase (COX) has an anti-inflammatory activity and so is used mainly for the treatment of osteoarthritis. In the present invention, 35.779 g of 10 mM indomethacin was used as a comparative drug.
(30) Hereinafter, various experiments were conducted for the fractions 1, 2, 3 and indomethacin obtained in the Examples 1 and 2 and Comparative Example 1.
EXPERIMENT 1
Inhibitory Activity of Cyclo-Oxygenase (COX)-2
(31) In order to simply and rapidly measure the anti-inflammatory activity of the fractions 1, 2 and 3 recovered in Example 1 and indomethacin obtained in Comparative Example 1, the inhibitory activity was measured using COX-2 kit manufactured and sold by US Cayman company. In the experimental method, 10 l of heme, 10 l of assay buffer (100 mM Tris-HCl, pH 8.0), and 10 l of solvent (solvent which dissolved an inhibitor) were added to 2 well of a 96 well plate. For the background well, 10 l of heme, 10 l of assay buffer, and 10 l of solvent were added to 2 wells. For the measurement of 100% initial activity, 10 l of heme, 10 l of enzyme (COX-2) and 10 l of solvent were added to 2 wells. For the measurement of COX inhibitory activity of sample (inhibitor), 10 l of heme, 10 l of enzyme (COX-2) and 10 l of sample were added to 2 wells. As the standard inhibitor, indomethacin was treated by dissolving in dimethyl sulfoxide. The sample was dissolved in 100 mM Tris-HCl (pH 8.0) and then treated according to concentration. 200 l of assay buffer was added to every well and reacted at room temperature. 10 l of chemiluminescent substrate was then added to every well with one dispenser using two dispensers to which the emission analyzer was attached. Immediately, 50 l of arachidonic acid was added using another dispenser and then the relative luminescence unit (RLU) was measured with a luminescence analyzer.
(32) As a result, the COX-2 inhibitory activity was identified as 79% for 10 mM indomethacin, 25% for the fraction 1, 7% for the fraction 2 and 42% for the fractions 3 as shown in Table 1. It has been identified that the fractions 1 and 3 of Example 1 exhibit a high COX-2 inhibiting activity even at a low concentration as compared with indomethacin of Comparative Example 1. In particular, the fraction 3 of the fractions exhibited the most excellent activity.
(33) TABLE-US-00001 TABLE 1 Inhibitory Activity of Cyclo-oxygenase (COX)-2 COX-2 inhibitory Concentration (g) activity (%) Comparative Example 1 35.779 79 12 Example 1, Fraction 1 10.0 25 9 Example 1, Fraction 2 10.0 71 12 Example 1, Fraction 3 10.0 42 18 *All experimental date were mean SD of triple determinations
EXPERIMENTAL EXAMPLE 2
(34) Anti-inflammatory effect of the fraction 3 from Raw 264.7 macrophages induced by LPS
(35) (1) Measurement of Nitric Oxide (NO) Yield
(36) The cells were injected by 210.sup.4 cells/ml per well in 96-well plate and incubated for 2 hours. The fraction 3 of Example 2 was treated according to concentration and incubated for 2 hours. Lipo poly saccharide (LPS) was added in an amount of 1 g/ml to induce a stress. After incubation for 20 hours, the supernatant was taken and measured. The NO content of the supernatant was measured by Griess reaction. To 50 l of the supernatant, the same amount of the Griess reagent, i.e., 0.1% N-(1-naphthyl)-ethylenediamine, 1% sulfanilamide in 5% phosphoric acid] was introduced, agitated, and incubated at room temperature for 10 minutes. The absorbance at 550 nm was measured and the results are shown in Table 2 below. When only one cell was incubated and the NO yield was considered to be 100%, the cells treated with LPS were stimulated, thus increasing the NO yield to 196.7%. The NO yields increased through LPS stimulation were measured as 125%, 120%, 110%, 101.7%, respectively, at the treatment concentration 10100 g/ml of fraction 3. Therefore, it can be seen that as the concentration increased, the NO yield significantly decreased (p<0.05).
(37) (2) Measurement of Reactive Oxygen Standard (ROS Level) in Cells
(38) Dichlorofluorescein diacetate (DCF-DA) was measured by a principle wherein acetyl group was removed in cells and quantitatively reacted with an intracellular radical (mainly, hydrogen peroxide) to change to DCF fluorescence material. DCF-DA measures the generation of reactive oxygen in the oxidative stress. If the cells were in the confluence state, the cells were seeded with 2104 cells/ml per well in a 96-well plate and incubated for 2 hours. The samples were then treated according to concentration and incubated for 2 hours to which LPS (1 g/ml) was added and incubated for 20 hours, thus inducing stress. The culture media was then removed and the cells were washed twice in phosphate buffered saline (PBS). The cells were introduced with 100 M of DCF-DA, incubated for 15 minutes and again incubated at room temperature for 60 minutes. The rate of increase of DCF-DA fluorescence was measured by flow cytometer. The results are shown in
(39) The reactive oxygen yield induced by LPS in RAW 264.7 macrophage was 202.3%. When the fraction 3 was pre-treated in a concentration of 10100 g/ml, the reactive oxygen yield was decreased in a concentration-dependent manner. In particular, at the concentration of 100 g/ml, the reactive oxygen yield was 107.8% which does not represent a significant difference (p<0.05) as compared with the control group, thus leading to a high reactive oxygen yield.
(40) (3) Measurement of Lipid Peroxide
(41) The degree of lipid peroxidation was measured according to Fraga method by thiobarbituric acid reactive substance yield (TBARS). In 96-well plate, the cells were introduced by 2104 cells/ml per well and incubated for 2 hours. The samples were treated according to concentration and incubated for 2 hours. LPS (1 g/ml) was then added to induce a stress. After the incubation for 20 hours, 200 l of media supernatant per well was put in a corning tube and incubated for 1 hour to which 400 l of TBARS solution was added, agitated and then boiled at 95 C. for 20 minutes. After cooling and mixing again, the absorbance at 532 nm was measured with the supernatant which was centrifuged at 4,000 rpm for 10 minutes. Lipid peroxides were calculated by converting into the amount of malonic dialdehyde (MDA).
(42) If the fraction 3 was treated by inducing stress with LPS in RAW 264.7 cells, the influence on the degree of lipid peroxidation was investigated. The results were shown in
EXPERIMENTAL EXAMPLE 3
(43) Inhibitory Effects of Arthritis in Animal Models of Osteoarthritis
(44) (1) Experiment Animals and Breeding
(45) 40 eight week-old animal models of osteoarthritis C57BL/6J mouse were purchased from Central Laboratory Animal Inc. (Seoul, Korea), adapted with a general diet for 1 week and then used in the experiments. The temperature and humidity of the breeding room were maintained at 202 C., 5010%, the lightening conditions were such that the light was turned on and off every 12 hours and the light was kept on from 07:0019:00. During the experiment, the feedstuff (Orient Bio, Seoul, Korea) and water were to be freely taken.
(46) (2) Preparation of Model of Osteoarthritis
(47) The osteoarthritis was induced by the method given in the test guidelines of the effect of the herbal preparation presented by the Korean Food and Drug Administration. Papain (type IV, double crystallized, 15 units/mg, Sigma, USA) was adjusted to a concentration of 1.0% (w/v) in which 0.03 ML-cystein HCl (Sigma, USA) was supplemented for the activation of papain and then passed through the patellar ligament. Physiological saline 6 was administered in the right knee joint to induce osteoarthritis.
(48) (3) Classification of Experiment Group
(49) The experiment animals were adapted in a lab environment for 1 week and then the body weight was measured. Eight animals were assigned to each group to have even body weight. In the normal group, the physiological saline 6 was injected in the right knee joint and then the physiological saline 0.2 ml was orally administered once a day for 20 days. In the treated groups, i.e., a control group, a drug-administered group and a fraction 3-administered group, papain 6 was injected in the right knee joint to induce osteoarthritis. The physiological saline in the control group, Diclofenac sodium (DS, 2 mg/kg/bw) in the drug-administered group, and 100 mg/kg/bw {below, called fraction 3 (100)} and 200 mg/kg/bw {below, called fraction 3 (200)} in the fraction 3-administered group of Example 2 were orally administered once daily at a predetermined time for 20 days.
(50) (4) Sample Treatment
(51) All subjects were lightly anesthetized with ether at the experiment end day to take a heart blood sample. The blood was centrifuged to isolate a serum. Immediately after taking a heart blood sample, the right hip joint and ankle joint were isolated and removed so as not to damage the knee joint. The tibial of the right knee joint was fixed in 10% neutral formalin solution for 24 hours and then used for the visual observation of the joints.
(52) (5) Visual Observation
(53) The cartilage surrounding the fixed right knee joint was cleaned to take a picture. The damage level of the joint cartilage surface was visually confirmed. The difference between the groups for the knee joint surfaces is shown in
(54) The DS-treated group exhibited a visual appearance similar to that of the papain-administered group, but the scope of the discoloration was less and the elimination of visual cartilage surface was not observed. The fraction 3-treated group exhibited a reduced scope of discoloration and an increased luster of the surface as compared with a papain-administered group. Such a visual diagnosis was similar to that for the DS-treated group.
(55) (6) Evaluation of Osteoarthritis Index
(56) The osteoarthritis index for the observed histopathological lesions in the articular cartilage and synovial membrane was digitalized in accordance with a method of Rudolphi, at al. and is shown in Table 2 below.
(57) TABLE-US-00002 TABLE 2 Matrix Structure Cellularity 0 Normal 0 Normal 1 Surface irregularities 2 Reduced 3 Superficial fibrillation 5 Strongly reduced 6 Clefts in deep zone 8 Total loss of cartilage and cells 8 Massive loss of cartilage Subchondral Bone Synovial Membrane 0 Normal 0 Normal 3 Remodeling processes 3 Mild hyperplasia 8 Thickening strong sclerosis 5 Severe hyperplasia 8 Hyperplasia with infiltration of inflammatory cells
(58) The osteoarthritis index according to the histopathological opinions per subject observed in the papain-administered group and treated group is shown in Table 3. The DS-treated group and the fraction 3-treated group exhibited a significantly lower osteoarthritis index (p<0.05) than the papain-administered control group. The fraction 3 (200)-treated group had a lower osteoarthritis index than the fraction 3 (100)-treated group, but there was no significant difference.
(59) TABLE-US-00003 TABLE 3 The osteoarthritis index according to histopathological opinions per subject Group and Identifica- Sum of tion Matrix Cellu- Subchondral Synovial osteoarthric No. structure larity bone membrane score Papain 1 3 5 8 8 24 2 3 5 3 5 16 3 8 2 0 0 10 4 6 5 3 8 22 5 8 5 0 5 18 6 3 8 3 8 22 7 3 8 8 8 27 8 6 2 8 3 19 Mean 5.0 5.0 4.1 5.6 19.8 Papain + 1 1 2 3 0 6 DS 2 1 2 0 1 4 3 3 2 3 3 11 4 3 2 0 0 5 5 3 2 0 3 8 6 6 2 0 3 11 7 3 2 3 0 8 8 1 2 0 3 6 Mean 2.6 2.0 1.1 1.5 7.4 Papain + 1 3 2 0 3 8 Fraction 2 6 5 3 5 19 3 (100) 3 3 2 3 3 11 4 3 2 3 3 11 5 6 5 5 3 19 6 8 5 3 3 19 7 3 2 3 3 11 8 5 5 3 3 16 Mean 4.6 3.5 2.9 3.3 14.3 Papain + 1 1 2 0 3 6 Fraction 3 2 3 2 3 3 11 (200) 3 1 0 0 3 4 4 6 2 3 4 14 5 8 5 3 3 19 6 6 2 3 0 11 7 3 0 3 3 9 8 6 5 3 5 19 Mean 4.3 2.3 2.3 3.1 11.6
(60) (7) Measurement of Cytokine Content in Blood
(61) The contents of tumor necrosis factor- (TNF-), interleukin-1 (IL-1) and interleukin-6 (IL-6) were measured using enzyme-linked immunosorbent assay (ELISA) kit, respectively.
(62) {circle around (1)} TNF- Content in Blood
(63) The effect of fraction 3 of the inflammatory cytokine TNF- produced in large quantities in animals where arthritis was induced by injection of papain, was measured by ELISA method. The measured results are shown in
(64) {circle around (2)} Interleukin-1 Content in Blood
(65) The effect of fraction 3 of the inflammatory cytokine IL-1 produced in large quantities in animals where arthritis was induced by injection of papain was measured by ELISA method. The measured results are shown in
(66) {circle around (3)}Interleukin-6 Content in Blood
(67) The difference of IL-6 content in mice where arthritis was induced by papain injection is shown in Table 8 below. IL-6 content in blood in the papain-administered group is significantly higher than that in the normal group. DS and fraction 3-treated groups are significantly lower than the papain-administrated control group. In particular, the fraction 3 (200)-treated group exhibited a significantly lower IL-6 content (p<0.05) than the fraction 3 (100)-treated group.