A compound algal extract, its preparation method and application

Abstract

This invention belongs to the field of active ingredient extraction technology, and relates to a compound algae extract, its preparation method, and its application. The preparation method includes: S1. Taking algae raw materials and soaking them in a citrate-citrate buffer solution with a pH of 3-5 at 60-80°C; S2. Cooling the obtained treated material to 50-55°C, and then performing enzymatic hydrolysis and enzyme inactivation to obtain an enzymatic hydrolysate; S3. Filtering the enzymatic hydrolysate and collecting the filtrate; S4. Performing ultrafiltration on the filtrate to obtain a concentrated solution; S5. Sterilizing and drying the concentrated solution to obtain the compound algae extract. This invention utilizes a citrate-citrate buffer solution with a specific pH range combined with enzymatic hydrolysis and ultrafiltration to extract active ingredients from algae while removing residual heavy metals, achieving a high yield of algae extract and a high heavy metal removal rate. This compound algae extract can play a dual role in anti-inflammatory and tablet adhesion in bone and joint health care compositions.

Description

Technical Field

[0001] This invention belongs to the field of active ingredient extraction technology, especially the extraction of active ingredients from algae, and specifically relates to a compound algae extract, its preparation method and application. Background Technology

[0002] Algae are a type of photosynthetic organism, widely studied for their rich nutritional value and bioactivity. Algae extracts are substances extracted from algae using various methods. They contain a variety of bioactive substances and possess multiple health benefits, including antioxidant, anti-inflammatory, antibacterial, and immune-regulating properties.

[0003] Currently, there are many methods for preparing algal extracts. For example, CN119700600A discloses a method for preparing algal extracts and its applications. This method first defatts the algae, which on the one hand separates the high-value-added polyunsaturated fatty acids in the algae, improving the utilization rate of raw materials, and on the other hand reduces the inconvenience caused by oil in subsequent processing. Secondly, water extraction is performed on the defatted algal tissue, which dissolves various water-soluble substances in the defatted algal tissue into water, so as to obtain algal extracts rich in nucleic acids, sugars, amino acids and inorganic salts. Finally, the algal aqueous solution is concentrated to help obtain product dosage forms that are easy to store and use. Another example is CN105237614A, which discloses a method for extracting bioactive substances from algae. This method uses inorganic salt solutions of different concentrations for salting out. First, the algae are subjected to cell wall disruption, and then mixed with inorganic salts of different concentrations for multiple salting out processes. This allows for the simultaneous extraction of phycobiliproteins and polysaccharides from the algae.

[0004] However, algae, due to their large surface area, viscosity, and highly selective cell membranes, have a much higher capacity for adsorbing heavy metals than typical marine plants; their adsorption and concentration capabilities are tens of times greater than those of terrestrial plants. Heavy metals are difficult to biodegrade but can accumulate thousands of times within organisms through the food chain, which negatively impacts the application of algal extracts. None of the extraction methods mentioned above address the removal of heavy metals from algae.

[0005] Existing methods for removing heavy metals from algal extracts, such as the process disclosed in CN119638864A for the stepwise extraction of seaweed protein, alginate, and fucoidan from brown algae, mention the use of polyethersulfone (PES) composite membrane filtration to remove free heavy metals and small molecule impurities. However, this method is not very effective at removing heavy metals, and the residues remain significant.

[0006] For example, CN114681495A discloses a method for preparing seaweed extract that can efficiently remove heavy metals. This method combines soaking the seaweed in acetic acid during the raw material processing stage with resin adsorption during the extraction stage. This combination not only efficiently, conveniently, and quickly removes heavy metals from seaweed extracts and their preparations, but also offers several drawbacks. Using non-specific adsorption resins may result in the adsorption of active ingredients, reducing the yield. While using specific adsorption resins increases the yield, the regeneration of these resins is difficult and costly. Furthermore, the extract's therapeutic effect in treating joint inflammation is not ideal. Summary of the Invention

[0007] The purpose of this invention is to provide a compound algae extract, its preparation method, and its application, so as to solve the problems existing in the above-mentioned algae extract extraction methods.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing a complex algae extract, comprising the following steps:

[0010] S1. Take algae raw materials and soak them in 100-150 times the volume of citrate-citrate buffer solution with pH 3-5, and process them at 60-80℃.

[0011] S2. The material obtained in step S1 is cooled to 50-55℃ and then enzymatically hydrolyzed. After the enzymatic hydrolysis is completed, the enzyme is inactivated to obtain the enzymatic hydrolysate.

[0012] S3. After filtering the enzymatic hydrolysate, collect the filtrate;

[0013] S4. The filtrate is subjected to ultrafiltration to obtain a concentrated solution;

[0014] S5. The concentrate is sterilized and spray-dried to obtain a compound algae extract.

[0015] In the above-described scheme of the present invention, a combination of citric acid-citrate buffer solution with a specific pH range and enzymatic hydrolysis and ultrafiltration is used to extract active ingredients from algae while removing residual heavy metals from algae, and to achieve a high yield of algae extract and a high heavy metal removal rate.

[0016] Specifically, at pH 3-5, the carboxyl group (-COOH) on the citric acid molecule partially ionizes into a carboxylate group (-COO). - These negatively charged groups are effective against heavy metal cations (such as Pb). 2+ Cd 2+ Hg2+ As 3+ (etc.) possesses extremely strong coordination ability, forming stable, water-soluble citric acid-heavy metal chelates. This pH range is the optimal window for chelation efficiency: if the pH is too low (<3), H... + Excessive concentration leads to competition for binding sites with heavy metal ions, reducing chelation capacity. High pH (>5) may cause some heavy metals to form hydroxide precipitates, making them more difficult to remove through chelation. Furthermore, chelates are unstable under high pH conditions, resulting in poor removal. Additionally, a weakly acidic environment combined with moderately high temperature (60-80℃) effectively softens, swells, and even partially destroys the tough cell walls and cell membrane structures of algae (especially components like cellulose and hemicellulose). This reduces the barrier effect of the cell wall on internal substances, creating favorable conditions for subsequent enzymatic hydrolysis. Simultaneously, high temperature increases molecular motion speed, promoting the chelation reaction.

[0017] Building upon S1, specific enzymes are used for enzymatic hydrolysis, breaking down cell walls and intracellular structural macromolecules (such as cellulose networks and protein linkages), thus fully releasing the target active ingredients from the algal cells into the solution. Simultaneously, the enzymatic hydrolysis process disrupts organelle structures and biomolecules, potentially exposing or releasing "bound" heavy metals that were previously encapsulated within the cell or tightly bound to proteins and polysaccharides. These newly released heavy metals may exist in ionic form or bound to organic fragments, facilitating removal.

[0018] The solid-liquid separation in S3 removes the algal residue (mainly insoluble substances such as recalcitrant cell wall fragments) that remains after treatments S1 and S2 and has not been enzymatically hydrolyzed. At this point, the filtrate contains the target active ingredient, the citric acid-heavy metal chelate formed in S1, the active ingredient released in S2, and possibly the accompanying heavy metal ions / complexes.

[0019] S4's ultrafiltration achieves efficient separation of active macromolecules from small molecule impurities (including heavy metals), and after sterilization and drying, a stable extract is obtained.

[0020] The preparation method of the above-mentioned citrate-citrate buffer combined with enzymatic hydrolysis and ultrafiltration has at least three key aspects. First, the pH value of the citrate-citrate buffer is crucial; only within this specific range can citrate-citrate remove heavy metals adsorbed on the algal surface through chelation. Second, the order of the citrate-citrate buffer impregnation and enzymatic hydrolysis is also critical. Experimental results show that only by performing citrate-citrate buffer impregnation first, followed by enzymatic hydrolysis, can both the algal extract yield and heavy metal removal rate be significantly improved. Third, the citrate-citrate buffer impregnation temperature (60-80℃) is essential. Controlling the temperature at 60-80℃ significantly accelerates molecular motion, increases the collision frequency and reaction rate between citrate ions and heavy metal ions, and makes the chelation reaction faster and more complete. Simultaneously, this temperature can more effectively soften, swell, and even partially hydrolyze the tough cell wall components of algae, disrupting cell membrane fluidity and significantly reducing its barrier effect on internal substances, laying the foundation for subsequent enzymatic hydrolysis. Too low a temperature (<60℃) will result in insufficient pretreatment effect; too high a temperature (>80℃) may lead to the degradation of some heat-sensitive active ingredients or excessive charring / cross-linking of cell walls, which is not conducive to subsequent enzymatic hydrolysis.

[0021] In summary, the above preparation method ensures high extraction yield and effectively removes heavy metals. Furthermore, it has been found that the algae extract obtained by this method has superior effects on chondrocyte repair and anti-inflammatory properties, making it more suitable for bone and joint health care.

[0022] Preferably, the pH of the citrate-citrate buffer solution is 4.

[0023] Preferably, the concentration of the citrate-citrate buffer solution is 0.1-0.3 mol / L.

[0024] Preferably, the processing time in step S1 is 1-2 hours.

[0025] Preferably, in step S4, the ultrafiltration membrane used for ultrafiltration has a size of 3000-5000 Da.

[0026] Preferably, the enzymatic hydrolysis is a three-step process performed sequentially. The first step uses one or a mixture of two of pectinase, alginate, and agarase; the second step uses cellulase; and the third step uses one or a mixture of two of bromelain, papain, and β-glucanase. In this scheme, through stepwise enzymatic hydrolysis, the outer layer of algal gelatinous substances is first removed, then cellulase is used to break down the cell wall to release internal substances, and the third step uses protease to remove impurities. This allows for a more complete release of the algal active ingredients and facilitates the subsequent separation and collection of the algal active ingredients.

[0027] Preferably, the algae raw material is one or more of red algae, brown algae, and green algae.

[0028] Preferably, the filtrate is diluted with 1-4 times its volume of water before ultrafiltration. The main purpose of diluting the filtrate with 1-4 times its volume of water is to reduce the viscosity and salt ion concentration / osmotic pressure of the filtrate. High viscosity (rich in polysaccharides and proteins) and high salt concentration affect the flux (filtration rate) of the ultrafiltration membrane and increase concentration polarization (solute accumulation on the membrane surface), reducing filtration efficiency. Dilution helps maintain high membrane flux and separation efficiency.

[0029] Preferably, the enzyme inactivation conditions are at a temperature of 70-80℃.

[0030] Preferably, the sterilization conditions are 130-140℃ and 3-4s.

[0031] Preferably, the inlet air temperature of the spray dryer is 170-185℃ and the outlet air temperature is 70-85℃.

[0032] Secondly, the present invention provides a compound algae extract, which is obtained by the above-described preparation method.

[0033] Thirdly, the present invention provides the application of the above-mentioned compound algae extract in the preparation of a composition for bone and joint health care.

[0034] Fourthly, the present invention provides a composition for bone and joint health care, comprising the above-mentioned algae extract.

[0035] Fifthly, the present invention provides an application of the above-mentioned composite algae extract in the preparation of pharmaceutical adhesives.

[0036] By implementing the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention utilizes a citrate-citrate buffer solution with a specific pH range, combined with enzymatic hydrolysis and ultrafiltration, to effectively remove residual heavy metals from algae while extracting active ingredients. It achieves high algae extract yield and heavy metal removal rate, and the resulting algae extract exhibits superior effects in chondrocyte repair and anti-inflammatory properties, making it more suitable for bone and joint health maintenance. Furthermore, when used in bone and joint health maintenance compositions, this algae extract also acts as a binder, eliminating the need for adhesives when the composition is formulated into tablets. Detailed Implementation

[0038] The present invention will be further described in detail below through specific embodiments.

[0039] It should be noted that the following embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0040] Example 1

[0041] This embodiment provides a method for preparing a low-heavy-metal complex algae extract, comprising the following steps:

[0042] S0. Fresh algae raw materials (red algae, partridge, brown algae, green algae = 1:1:2) are put into a cooking pot, and 25 times the weight of the raw materials are added to water and stirred and washed.

[0043] S1. The washed algae raw material was impregnated with 150 times its volume of 0.1 mol / L citrate-citric acid buffer solution and treated at 65°C for 1.5 h.

[0044] S2. The processed material obtained in step S1 is cooled to 50°C, and then subjected to stepwise enzymatic hydrolysis (pectinase is added for 1 hour of enzymatic hydrolysis, cellulase is added for 1 hour of enzymatic hydrolysis, bromelain is added for 1 hour of enzymatic hydrolysis). After enzymatic hydrolysis is completed, the temperature is raised to 70°C, stirring is stopped, and the enzyme is inactivated for more than 1 hour to obtain the enzymatic hydrolysate.

[0045] S3. After filtering the enzymatic hydrolysate, collect the filtrate;

[0046] S4. Add water with a volume of 3 times the filtrate to the filtrate and perform ultrafiltration (3000kD ultrafiltration membrane) to obtain a concentrated solution;

[0047] S5. The concentrate is instantaneously sterilized under the following conditions: 130°C for 4 seconds. After circulating for half an hour, it is spray-dried. The inlet air temperature of the spray dryer is set to 175°C and the outlet air temperature to 75°C to obtain a low-heavy-metal complex algae extract.

[0048] To investigate the effect of pH on the results, the same batch of algal raw materials was used. Based on Example 1, the pH of the citrate-citrate buffer was adjusted, and the removal rate of heavy metals and the yield of algal extract were statistically analyzed. The results are shown in Table 1.

[0049] Methods for detecting heavy metal content: Lead GB5009.12, Cadmium GB5009.15, Mercury GB5009.17, Arsenic GB5009.11.

[0050] Yield of algal extract:

[0051] Table 1. Effect of different pH values ​​of citrate-citrate buffer on the results.

[0052]

[0053] Finished product yield = (Spray-dried finished product / Fresh raw material) * 100%

[0054] As shown in Table 1, the removal rates of heavy metals vary significantly under different pH citrate-citrate buffer conditions. Between pH 3 and 6, the removal rates of lead, arsenic, mercury, and cadmium are all above 85%, approximately 90%, approximately 99%, and approximately 80%, with a final product yield of over 2.5%. Among these, the pH 4 condition yields the best results in both heavy metal removal and algal extract yield.

[0055] The following experiments were conducted using a citrate-citrate buffer solution with a pH of 4.

[0056] To investigate the effect of immersion temperature in step S1 on the results, the same batch of algae raw materials was used, based on Example 1, with the immersion temperature varied. The results are shown in Table 2.

[0057] Table 2 Effect of different impregnation temperatures on the results

[0058]

[0059] As can be seen from the results shown in Table 2, under the condition of pH 4, the heavy metal removal rate is relatively high when the impregnation temperature is between 60-80℃. Considering both the heavy metal removal rate and the product yield, the impregnation temperature is selected to be between 60-80℃.

[0060] Comparative Example 1

[0061] This comparative example uses a pure water system (pH adjusted with hydrochloric acid) instead of a citrate-citrate buffer system. That is, the citrate-citrate buffer system in step S1 of Example 1 is replaced with a hydrochloric acid solution with a pH of 4 (other conditions are the same as in the example).

[0062] Comparative Example 2

[0063] This comparative example uses a pure water system (pH adjusted with acetic acid) instead of a citric acid-citrate buffer system. That is, the citric acid-citrate buffer system in step S1 of Example 1 is replaced with an acetic acid solution with a pH of 4 (other conditions are the same as in the example). The heavy metal residue in the finished product after treatment is compared, and the results are shown in Table 3.

[0064] Comparative Example 3

[0065] The difference from Example 1 is that the citrate-citrate buffer system is added together with the enzymatic hydrolysis, and the specific steps are as follows:

[0066] S0. The same raw materials as in Example 1 are put into a cooking tank, and 25 times the weight of the raw materials are added to water for stirring and washing.

[0067] S1. The washed algae raw material was soaked in 150 times the volume of citrate-citrate buffer, and the system temperature was controlled at 50℃. Then, stepwise enzymatic hydrolysis was carried out (pectinase was added for 1 hour of enzymatic hydrolysis, cellulase was added for 1 hour of enzymatic hydrolysis, bromelain was added for 1 hour of enzymatic hydrolysis). After the enzymatic hydrolysis was completed, the temperature was raised to 90℃, the pH was adjusted to 9, stirring was stopped, and the enzyme was inactivated for more than 1 hour to obtain the enzymatic hydrolysate.

[0068] S2. After filtering the enzymatic hydrolysate, collect the filtrate;

[0069] S3. Add water with a volume of 3 times the filtrate to the filtrate and perform ultrafiltration to obtain a concentrated solution;

[0070] S4. The concentrate is instantaneously sterilized under the following conditions: 130°C for 4 seconds. Then, it is spray-dried with the inlet air temperature of the spray dryer set to 175°C and the outlet air temperature set to 75°C to obtain a low-heavy-metal complex algae extract.

[0071] The heavy metal residues in the finished products after each comparative treatment were compared with those in Example 1 (with a pH of 4 in the citric acid-citrate buffer solution). The results are shown in Table 3.

[0072] Table 3

[0073]

[0074] As shown in Table 3, it can be seen that in Comparative Examples 1 and 2, although the pH of the system was also adjusted to 4, the addition of the citric acid-citrate buffer system resulted in a significantly reduced heavy metal removal effect compared to Example 1. This is because citric acid is a tricarboxylic acid containing three carboxyl groups (-COOH) and one hydroxyl group (-OH), all of which are strong coordinating groups (providing lone pairs of electrons to bind with metal ions). Multiple coordination sites can form cyclic chelates (such as five-membered or six-membered rings) with heavy metal ions, and the stability of this structure is far greater than that of simple ionic bonds or monodentate coordination compounds. In contrast, hydrochloric acid has no coordinating groups and can only dissolve some heavy metal compounds under acidic conditions, but it cannot form complexes with heavy metal ions, and has almost no removal ability for bound heavy metals (the part bound to biomolecules); while acetic acid contains only one carboxyl group, and the acetate ion (CH3COO)... -Algae have weak coordination ability and form complexes with heavy metals with low stability (mostly monodentate coordination), making it difficult for them to compete with heavy metals bound to biomolecules in algae. In Comparative Example 3, the citrate-citrate buffer system was added at different times, simultaneously with the enzymatic hydrolysis step. This showed that while the heavy metal removal effect was somewhat improved compared to Comparative Examples 1 and 2, the removal rates of lead, cadmium, mercury, and arsenic all showed a significant downward trend compared to Example 1. This is because in Comparative Example 3, the algae were not pre-soaked in the buffer, and the free heavy metals attached to the surface were not removed. During the enzymatic hydrolysis stage, these metals may rapidly bind with the simultaneously released active ingredients (such as hydroxyl groups of polysaccharides and sulfhydryl groups of proteins), forming more stable complexes. This makes subsequent chelation competition with the buffer more difficult, resulting in higher residual heavy metal levels. Simultaneously, some active ingredients suffered structural damage due to repeated dissociation and binding, leading to decreased product activity and purity.

[0075] In this invention, the effective components in the compound algae extract were also detected, and the detection methods and results are as follows.

[0076] Detection method:

[0077] Total sugar and organic sulfate group detection: SC / T 3404.

[0078] Glucuronic acid detection: Carbazole-sulfuric acid method.

[0079] Table 4. Detection results of active ingredients in different compound algae extracts

[0080] Total sugar (calculated as fucose), % Organic sulfate group, % Glucuronic acid, % Comparative Example 1 58.8 10.4 18.8 Comparative Example 2 65.1 14.1 23.6 Comparative Example 3 67.4 18.6 25.1 Example 1 70.6 22.4 28.4

[0081] The main active components of algae are polysaccharides (such as alginic acid, fucoidan, carrageenan, agar, and Ulva prolifera polysaccharides), accounting for 20%-70% of their dry weight. Algal polysaccharides possess rich biological activities, such as anti-inflammatory, antioxidant, immunomodulatory, and moisturizing effects. Total sugar is the core indicator for measuring the total amount of these polysaccharides, and its content reflects the potential for efficacy: the higher the total sugar content, the richer the total amount of active polysaccharide components in the extract, and the more solid the foundation for its above-mentioned efficacy.

[0082] Sulfate groups are active "synergistic groups" that can significantly enhance the biological activity of polysaccharides. Studies have found that artificial sulfation modification can make polysaccharides that originally did not contain sulfate or had low sulfate content exhibit strong antiviral activity.

[0083] Glucuronic acids (such as D-mannuronic acid and L-guluronic acid in alginic acid, and glucuronic acid in hyaluronic acid analogues) are characteristic building blocks of acidic polysaccharides from algae, and their content directly affects the physicochemical properties and biological activity of polysaccharides.

[0084] Application Examples

[0085] In this application example, a combination of compound algae extract (obtained in Example 1, using a citrate-citrate buffer system at pH 4) with glucosamine and calcium carbonate is used to create a joint health-promoting composition. Glucosamine replenishes the raw materials needed for cartilage matrix repair, the algae extract can alleviate inflammatory responses, and calcium carbonate enhances bone density, thus providing joint care from different angles. The joint health-promoting effect is further enhanced after adding the compound algae extract.

[0086] Safety experiment of compound algae extract on chondrocytes

[0087] Design compositions with and without algae extracts, and with different amounts of algae extracts added, and test the survival rate of chondrocytes under these treatments.

[0088] The method for treating chondrocytes with the composition is as follows:

[0089] 1) Take normally cultured C28 / I2 human chondrocytes in the logarithmic growth phase, digest them with 0.25% Typsin + 0.02% EDTA, centrifuge at 1000 rpm for 5 min, count them using a counting chamber, seed them into 96-well plates, with 6 replicates per group, and add 5 × 10⁶ cells to each well. 3 Each cell.

[0090] 2) After the plated cells adhered overnight, add 1 mg / mL of different drugs according to the groups and incubate in an incubator for 72 h.

[0091] 3) After drug treatment, add 10% CCK8 detection solution to each well, react at 37℃ in the dark for 1 h, and read the OD value of each well at 450 nm.

[0092] 4) The experimental results are calculated using the following formula:

[0093] Cell viability (%) = experimental group (OD450) / blank control group (OD450) × 100%.

[0094] The results are shown in Table 5.

[0095] Table 5

[0096] 60% glucosamine + 40% calcium citrate 60% Glucosamine + 40% Calcium Citrate + 10% Complex Algae Extract 60% Glucosamine + 40% Calcium Citrate + 20% Complex Algae Extract Survival rate <![CDATA[96.71±3.31 c ]]> <![CDATA[105.45±4.72 b ]]> <![CDATA[118.18±4.97 a ]]>

[0097] Note: The weight percentage of the compound algae extract is based on the total weight of glucosamine and calcium citrate. Different letters indicate significant differences (P < 0.05).

[0098] Conclusion: Table 5 shows that the compound algae extract has no toxic side effects on chondrocytes, has good affinity, and the cell survival rate is increased to a certain extent compared with that without the compound algae extract, indicating that the compound algae extract of the present invention has a certain protective effect on cells.

[0099] Experiment on the repair of chondrocytes under inflammatory conditions by the composition

[0100] 1) Take normally cultured C28 / I2 human chondrocytes in the logarithmic growth phase, digest them with 0.25% Typsin + 0.02% EDTA, centrifuge at 1000 rpm for 5 min, count them using a counting chamber, seed them into 96-well plates, with 6 replicates per group, and add 5 × 10⁶ cells to each well. 3 Each cell.

[0101] 2) Three groups were set up: a blank control group, a model control group, and a drug intervention group. The blank control group consisted of normally cultured human chondrocytes without any treatment. The model control group and the drug intervention group were induced with 10 ng / mL IL-1β for 24 h. Then, the model control group was cultured in ordinary culture medium (without the compound) for 72 h, and the drug intervention group was treated with the set dose of drug for 72 h. Cell viability of each group was measured by CCK8 at the observation endpoint.

[0102] Cell viability (%) = experimental group (OD450) / blank control group (OD450) × 100%.

[0103] The results are shown in Table 6.

[0104] Table 6

[0105] Blank control Model comparison 60% glucosamine + 40% calcium citrate 60% Glucosamine + 40% Calcium Citrate + 10% Complex Algae Extract 60% Glucosamine + 40% Calcium Citrate + 20% Complex Algae Extract Survival rate <![CDATA[100.01±3.97 b ]]> <![CDATA[76.50±3.33 d ]]> <![CDATA[93.71±3.55 c ]]> <![CDATA[101.28±2.38 b ]]> <![CDATA[108.84±2.56 a ]]>

[0106] Note: Different letters indicate significant differences (P < 0.05).

[0107] Conclusion: Table 6 shows that, compared with the model group, the cell survival rate of the group with the addition of the compound algae extract of the present invention was significantly improved, especially in the 20% addition group, indicating that the compound algae extract of the present invention has a significant effect on enhancing the proliferation activity of chondrocytes under inflammatory conditions.

[0108] Experiment on the effect of the composition on inflammatory factors in an inflammatory state

[0109] Experimental methods:

[0110] 1) Take normally cultured C28 / I2 human chondrocytes in the logarithmic growth phase, digest them with 0.25% Typsin + 0.02% EDTA, centrifuge at 1000 rpm for 5 min, count them using a counting chamber, seed them into 96-well plates, with 6 replicates per group, and add 5 × 10⁶ cells to each well. 3 Each cell.

[0111] 2) Three control groups were set up: a blank control group, a model control group, and a drug intervention group. The blank control group consisted of normally cultured human chondrocytes without any treatment. The model control group and the drug intervention group were induced with 10 ng / mL IL-1β for 24 h. Then, the model control group was cultured in ordinary culture medium (without the compound) for 72 h, and the drug intervention group was treated with the set dose of drug for 72 h. Cell supernatants were collected, and the levels of TNF-α and IL-6 were detected using an ELISA kit.

[0112] The results are shown in Table 7.

[0113] Table 7

[0114] Blank control Model comparison 60% glucosamine + 40% calcium citrate 60% Glucosamine + 40% Calcium Citrate + 10% Complex Algae Extract 60% Glucosamine + 40% Calcium Citrate + 20% Complex Algae Extract TNF-α <![CDATA[6.55±0.49 c ]]> <![CDATA[10.80±1.12 a ]]> <![CDATA[8.68±0.52 b ]]> <![CDATA[6.96±0.31 c ]]> <![CDATA[6.35±0.44 c ]]> IL-6 <![CDATA[13.17±1.18 bc ]]> <![CDATA[16.27±0.40 a ]]> <![CDATA[14.35±0.87 b ]]> <![CDATA[13.13±0.09 bc ]]> <![CDATA[12.65±0.52 c ]]>

[0115] Note: Different letters indicate significant differences (P < 0.05).

[0116] Conclusion: Table 7 shows that, compared with the model group and the group without the compound algae extract, the inflammatory factors TNF-α and IL-6 were decreased in the group with the compound algae extract of the present invention, indicating that the compound algae extract of the present invention has a significant anti-inflammatory effect on chondrocytes.

[0117] Comparative experiment on the effects of compound algae extracts obtained under different processing conditions

[0118] This experiment uses the composition: glucosamine + calcium citrate + 10% compound algae extract as an example. The compound algae extract is the extract obtained in Example 1 and Comparative Examples 1-4.

[0119] The experimental methods were as described above. The experimental results are shown in Table 8 (chondrocyte repair results under inflammatory conditions) and Table 9 (the influence of inflammatory factors under inflammatory conditions).

[0120] Table 8

[0121] Composition Survival rate Example 1 <![CDATA[101.28±2.38 a ]]> Comparative Example 1 <![CDATA[85.48±3.65 c ]]> Comparative Example 2 <![CDATA[91.82±1.77 b ]]> Comparative Example 3 <![CDATA[97.54±1.67 a ]]> Blank control <![CDATA[100.01±3.97 a <!-- 8 -->]]> Model comparison <![CDATA[76.50±3.33 d ]]>

[0122] Note: Different letters indicate significant differences (P < 0.05).

[0123] As shown in Table 8, the composite algae extract obtained in Example 1 of this invention exhibits the best repair effect on chondrocytes under inflammatory conditions. In Comparative Examples 1 and 2, purified water was used instead of the citrate-citrate buffer system. Although the pH of the system was controlled at 4, the results showed that the number of chondrocytes under inflammatory conditions was much lower than in Example 1. This indicates that the citrate-citrate buffer treatment not only affects the heavy metal content but also the repair effect of the composite algae extract on chondrocytes. In Comparative Example 3, the citrate-citrate buffer system was added together with enzymatic hydrolysis. Although the cell repair effect was improved compared to Comparative Examples 1 and 2, it was still lower than that in Example 1. This suggests that the timing of the citrate-citrate buffer treatment also affects the repair effect of the composite algae extract on chondrocytes.

[0124] Table 9

[0125] Composition TNF-α IL-6 Example 1 <![CDATA[6.96 d ±0.31]]> <![CDATA[13.13±0.09 d ]]> Comparative Example 1 <![CDATA[8.77±0.54 b ]]> <![CDATA[14.82±0.45 b ]]> Comparative Example 2 <![CDATA[7.79±0.23 c ]]> <![CDATA[14.58±0.31 b ]]> Comparative Example 3 <![CDATA[7.68±0.23 c ]]> <![CDATA[13.84±0.13 c ]]> Blank control <![CDATA[6.55±0.49 d ]]> <![CDATA[13.17±1.18 d ]]> Model comparison <![CDATA[10.80±1.12 a ]]> <![CDATA[16.27±0.40 a ]]>

[0126] Note: Different letters indicate significant differences (P < 0.05).

[0127] As shown in Table 9, the composite algae extract obtained in Example 1 of this invention, under inflammatory conditions, exhibited lower levels of inflammatory factors compared to the model control that did not use the composition, indicating a repairing effect on inflammation. In Comparative Examples 1 and 2, purified water was used instead of the citrate-citrate buffer system. Although the pH of the system was controlled at 4, the results showed that the content of inflammatory factors under inflammatory conditions was higher than in Example 1. This indicates that treatment with the citrate-citrate buffer not only affects the heavy metal content but also influences the effect of the composite algae extract on eliminating inflammation in chondrocytes. In Comparative Example 3, the citrate-citrate buffer system was added together with enzymatic hydrolysis. Although the content of inflammatory factors was somewhat lower than in Comparative Examples 1 and 2, it was still higher than in Example 1. This suggests that the timing of the citrate-citrate buffer treatment also affects the effect of the composite algae extract on eliminating inflammation in chondrocytes.

[0128] Experiment on the adhesive effect of compound algae extract in the composition

[0129] 1) Accurately weigh the corresponding materials according to the formula, and sieve them through a 20-mesh standard sieve to remove large particulate impurities.

[0130] 2) Place the sieved material into the mixer, set the mixing speed to 100-150 r / min, and the mixing time to 10-15 min to ensure that the material is mixed evenly.

[0131] 3) Slowly add a certain amount of wetting agent to the well-mixed material while stirring until the material forms a moist soft mass (it should be able to be formed into a ball when squeezed in the hand and crumble easily when touched).

[0132] 4) Place the soft material into the pellet mill and granulate it through a 20-mesh sieve. Collect the resulting wet pellets.

[0133] 5) Spread the wet granules evenly on a tray (with a thickness not exceeding 1cm), and place it in a 60℃ oven to dry for 30 minutes. During this period, turn the granules over every 10 minutes to ensure even drying.

[0134] 6) Take out the dried granules and cool them to room temperature (about 30 minutes). Then, granulate them through a 20-mesh sieve. Collect the granulated granules and weigh them, recording the total granule mass.

[0135] 7) Pass the granulated particles through a 100-mesh sieve again, collecting the coarse powder on the sieve and the fine powder that passes through. Accurately weigh and record the mass of the coarse powder and the mass of the fine powder using an electronic balance. Calculate the coarse powder ratio (mass of coarse powder / total particle mass × 100%). The higher the coarse powder ratio, the better the bonding effect.

[0136] 8) Take the granules from each group after granulation and determine their density and compactness according to the Chinese Pharmacopoeia method.

[0137] 9) Take the granules from each group after granulation and put them into a tablet press for tableting. Set the tableting pressure to 5-10 MPa (adjust according to the tablet hardness requirements) and press tablets with a diameter of 5-10 mm. Press 50 tablets per group.

[0138] 10) Hardness test: Randomly select 10 tablets and use a hardness tester to measure the hardness of each tablet, and calculate the average value. The hardness of tablets is generally required to be 3-6 kgf. The higher the hardness, the better the adhesion effect.

[0139] 11) Disintegration time test: Following the method specified in the Chinese Pharmacopoeia, take 6 tablets, place them in a disintegration time tester, record the disintegration time of each tablet, and calculate the average value. The disintegration time of ordinary tablets should be within 15 minutes; excessive adhesion may prolong the disintegration time.

[0140] 12) Friability test: Take 20 tablets, remove surface powder, weigh them, and place them in a friability tester. Rotate the tablets 100 times, remove the powder, weigh them again, and calculate the friability (loss of mass / initial mass × 100%). The friability should not exceed 1%. The lower the value, the stronger the tablet's abrasion resistance and the better its adhesion.

[0141] Table 10

[0142] 60% glucosamine + 40% calcium citrate 60% glucosamine + 40% calcium citrate + 2% sodium carboxymethyl cellulose 60% Glucosamine + 40% Calcium Citrate + 2% Complex Algae Extract wetting agent water water water Wetting agent dosage, w / w% 15 13 12 <![CDATA[Apparent density, g / cm 3 > 0.41 0.62 0.61 <![CDATA[Density, g / cm 3 > 0.65 0.85 0.85 Coarse powder ratio, % 80 72 87 Liquidity, ° 38.42 33.14 32.25 Tablet weight, g / tablet 0.79 0.8 0.8

[0143] Note: The amount of sodium carboxymethyl cellulose / complex algae extract added is calculated based on the total amount of glucosamine and calcium citrate.

[0144] Table 11

[0145] 60% glucosamine + 40% calcium citrate 60% glucosamine + 40% calcium citrate + 2% sodium carboxymethyl cellulose 60% Glucosamine + 40% Calcium Citrate + 2% Complex Algae Extract Compressibility Smooth Adhesive punch Smooth Hardness, N 35 105 126 Friability, % fragment 0.38 0.12 Disintegration time limit, min 5 15 12

[0146] Note: The amount of sodium carboxymethyl cellulose / complex algae extract added is calculated based on the total amount of glucosamine and calcium citrate.

[0147] Conclusion: As can be seen from the results shown in Tables 10 and 11, the compound algae extract of the present invention has the effects of reducing the amount of wetting agent, improving particle flowability, and improving the mechanical properties of tablets.

Claims

1. A method for preparing a complex algae extract, characterized in that, Includes the following steps: S1. Take algae raw materials and soak them in 100-150 times the volume of citrate-citrate buffer solution with pH 3-5, and process them at 60-80℃. S2. The processed material obtained in step S1 is cooled to 50-55℃, and then enzymatically hydrolyzed. After enzymatic hydrolysis, enzyme inactivation treatment is performed to obtain the enzymatic hydrolysate. The enzymatic hydrolysis is a three-step enzymatic hydrolysis performed sequentially. The first step enzymatic hydrolysis uses one or a mixture of two of pectinase, alginate, and agarase. The second step enzymatic hydrolysis uses cellulase. The third step enzymatic hydrolysis uses one or a mixture of two of bromelain, papain, and β-glucanase. S3. After filtering the enzymatic hydrolysate, collect the filtrate; S4. The filtrate is subjected to ultrafiltration to obtain a concentrated solution; S5. The concentrate is sterilized and spray-dried to obtain a compound algae extract.

2. The method for preparing a compound algae extract according to claim 1, characterized in that, The pH of the citric acid-citrate buffer solution is 4.

3. The method for preparing a compound algae extract according to claim 1, characterized in that, The algae raw material is one or more of red algae, brown algae, and green algae.

4. The method for preparing a compound algae extract according to claim 1, characterized in that, Before ultrafiltration, dilute the filtrate with 1-4 times its volume of water.

5. The method for preparing a compound algae extract according to claim 1, characterized in that, The inlet air temperature for spray drying is 170-185℃, and the outlet air temperature is 70-85℃.

6. A compound algae extract, characterized in that, It is obtained by the preparation method described in any one of claims 1-5.

7. The use of the compound algae extract as described in claim 6 in the preparation of a composition for bone and joint health care.

8. The use of the compound algae extract as described in claim 6 in the preparation of pharmaceutical adhesives.

9. A composition for bone and joint health care, characterized in that, It contains the complex algae extract as described in claim 6.

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