Composite algae extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of active ingredient extraction, and relates to a composite algae extract as well as a preparation method and application thereof, the preparation method comprises the following steps: S1, taking an algae raw material, and carrying out immersion treatment by adopting a citric acid-citrate buffer solution with the pH value of 3-5 at 60-80 DEG C; s2, cooling the treated material obtained in the step S2 to 50-55 DEG C, and then performing enzymolysis and enzyme deactivation to obtain enzymatic hydrolysate; s3, after the enzymatic hydrolysate is filtered, filtrate is collected; s4, carrying out ultrafiltration treatment on the filtrate to obtain a concentrated solution; and S5, sterilizing and drying the concentrated solution to obtain the composite algae extract. According to the method, the citric acid-citrate buffer solution with a specific pH value range is combined with enzymolysis and ultrafiltration, so that residual heavy metals can be removed while active components in the algae are extracted, and the method has relatively high algae extract yield and heavy metal removal rate and is suitable for industrial production. The composite algae extract can play a dual role in anti-inflammation and tablet adhesion in the bone joint health-care preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of active ingredient extraction, in particular to the extraction of active ingredients from algae, and specifically relates to a composite algae extract and a preparation method and application thereof. Background Art

[0002] Algae, a type of photosynthetic organism, has been extensively studied for its rich nutritional value and biological activity. Algae extracts are substances extracted from algae through various methods. They contain a variety of bioactive substances with various health benefits, including antioxidant, anti-inflammatory, antibacterial, and immune-modulating properties.

[0003] Currently, there are many methods for preparing algae extracts. For example, CN119700600A discloses a method for preparing an algae extract and its application. This method first defattes the algae, which can separate the high-value-added polyunsaturated fatty acids in the algae, improve the utilization rate of the raw materials, and reduce the inconvenience caused by oil in the subsequent processing process. Secondly, the defatted algae tissue is subjected to water extraction, which can dissolve various water-soluble substances in the defatted algae tissue into water, so as to obtain an algae extract rich in nucleic acids, sugars, amino acids and inorganic salts. Finally, the algae aqueous solution is concentrated to help obtain a product dosage form that is easy to store and use. Another example is a method for extracting bioactive substances from algae disclosed in CN105237614A. This method uses inorganic salt solutions of different concentrations for salting out. First, the algae is broken into pieces, and then mixed with inorganic salts of different concentrations for multiple salting out. In this way, phycobiliproteins and polysaccharides can be extracted from the algae at the same time.

[0004] However, due to their large surface area, sticky structure, and highly selective cell membranes, algae have a much higher capacity for heavy metal adsorption than typical marine plants, with adsorption and concentration capabilities dozens of times greater than those of terrestrial plants. Heavy metals are difficult to biodegrade, but can accumulate hundreds of times within organisms through the food chain, negatively impacting the application of algae extracts. None of the aforementioned extraction methods address the removal of heavy metals from algae.

[0005] Existing methods for removing heavy metals from algae extracts, such as CN119638864A, disclose a process for the stepwise extraction of algal protein, alginate, and fucoidan from brown algae, which involves filtration through a polyethersulfone (PES) composite membrane to remove free heavy metals and small molecule impurities. However, this method is not very effective in removing heavy metals, and significant residual amounts remain.

[0006] Another example is CN114681495A, which discloses a method for preparing a seaweed extract capable of efficiently removing heavy metals. This method involves soaking the raw materials in acetic acid during the preparation phase and adsorbing the extract with a resin during the extraction phase. This combination of methods not only efficiently, conveniently, and quickly removes heavy metals from seaweed extracts and their preparations, but also offers a good heavy metal removal rate. However, using a nonspecific adsorption resin for adsorption can result in the effective ingredients also being absorbed and removed, reducing yield. Using a specific adsorption resin improves yield, but regeneration is difficult and costly. Furthermore, the extract is not ideal for treating joint inflammation. Summary of the Invention

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

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

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

[0010] S1. Take the algae raw material, soak it in 100-150 times the volume of citric acid-citrate buffer at pH 3-5, and treat it at 60-80°C;

[0011] S2. The treated material obtained in step S1 is cooled to 50-55°C and then subjected to enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated to obtain an enzymatic solution;

[0012] S3. After filtering the enzymatic hydrolyzate, collecting the filtrate;

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

[0014] S5. sterilizing and spray-drying the concentrated solution to obtain a composite algae extract.

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

[0016] Specifically, at pH 3-5, the carboxyl group (-COOH) on the citric acid molecule is partially ionized to carboxylate (-COO - ), these negatively charged groups are resistant to heavy metal cations (such as Pb 2+ , Cd 2+ , Hg2+ , As 3+ etc.) have strong coordination ability to form stable, water-soluble citric acid-heavy metal chelates. This pH range is the best window for chelation efficiency: pH is too low (<3), H + If the concentration is too high, it will compete with heavy metal ions for binding sites, reducing chelation ability. If the pH is too high (>5), some heavy metals may begin to form hydroxide precipitates, making them difficult to chelate and remove. Furthermore, under high pH conditions, chelates are unstable, resulting in poor removal efficiency. Furthermore, a weakly acidic environment combined with moderately high temperatures (60-80°C) can effectively soften, swell, and even partially destroy the tough algae cell walls and cell membrane structures (especially components such as cellulose and hemicellulose). This reduces the cell wall's barrier effect on internal substances, creating favorable conditions for subsequent enzymatic hydrolysis steps. Furthermore, high temperatures increase the speed of molecular movement, promoting chelation reactions.

[0017] Building on the S1 method, enzymatic hydrolysis uses specific enzymes to hydrolyze the cell wall and intracellular structural macromolecules (such as the cellulose network and protein connections), fully releasing the target active ingredients within the algae cells into the solution. Simultaneously, the enzymatic hydrolysis process disrupts organelle structures and biomacromolecules, potentially exposing or releasing "bound" heavy metals previously encapsulated within the cell or tightly bound to proteins, polysaccharides, and other substances. These newly released heavy metals may exist as ions or bound to organic fragments, facilitating their removal.

[0018] Solid-liquid separation in S3 removes any remaining, unenzymatically digested algal residue (primarily insoluble matter such as refractory cell wall fragments) after treatment in S1 and S2. The filtrate now contains the target active ingredient, the citric acid-heavy metal chelate formed in S1, the active ingredient released in S2, and any accompanying heavy metal ions / complexes.

[0019] S4's ultrafiltration achieves efficient separation of active macromolecules from small molecule impurities (including heavy metals), and then sterilizes and dries to obtain an extract with stable properties.

[0020] The above-mentioned preparation method combining citric acid-citrate buffer with enzymatic hydrolysis and ultrafiltration has at least three key factors. The first is the pH value of the citric acid-citrate buffer. Only citric acid-citrate within this specific range can remove heavy metals adsorbed on the algae surface through chelation. The second is the order of citric acid-citrate buffer immersion treatment and enzymatic hydrolysis treatment, which is also a key factor affecting the results. Experimental results show that only by first performing citric acid-citrate buffer immersion treatment and then enzymatic hydrolysis can both the algae extract yield and heavy metal removal rate be improved. Furthermore, the citric acid-citrate buffer immersion temperature (60-80°C) is important. Controlling the temperature at 60-80°C 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. At the same time, this temperature can more effectively soften, swell, and even partially hydrolyze the tough cell wall components of the algae, disrupting the fluidity of the cell membrane and significantly reducing its barrier effect on internal substances, laying the foundation for subsequent enzymatic hydrolysis. If the temperature is too low (<60°C), the pretreatment effect will be insufficient; if the temperature is too high (>80°C), it may cause degradation of some heat-sensitive active ingredients or excessive charring / cross-linking of the cell wall, which is not conducive to subsequent enzymatic hydrolysis.

[0021] In summary, the above preparation method, on the one hand, ensures the extraction yield, and on the other hand, efficiently removes heavy metals. It is also found that the algae extract obtained by the above method has better effects on cartilage cell repair and anti-inflammatory effects, and is more suitable for bone and joint health care.

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

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

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

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

[0026] Preferably, the enzymatic hydrolysis is a three-step enzymatic hydrolysis performed sequentially, wherein the first step uses one or a mixture of pectinase, alginate, and agarase; the second step uses cellulase; and the third step uses one or a mixture of bromelain, papain, and β-glucanase. In this solution, the algae's outer gelatin is first removed through step-by-step enzymatic hydrolysis, followed by cellulase breaking down the cell wall to release the internal substances. In the third step, protease removes impurities, allowing for more complete release of the algae's active ingredients and facilitating subsequent separation and collection of the algae's active ingredients.

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

[0028] Preferably, dilute the filtrate with 1-4 times its volume of water before ultrafiltration. Adding 1-4 times its volume of water to dilute the filtrate primarily reduces its viscosity and salt concentration / osmotic pressure. High viscosity (rich in polysaccharides and proteins) and high salt concentration can affect ultrafiltration membrane flux (filtration rate) 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 condition is a temperature of 70-80°C.

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

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

[0032] In a second aspect, the present invention provides a composite algae extract obtained by the above-mentioned preparation method.

[0033] In a third aspect, the present invention provides an application of the above-mentioned composite algae extract for use in a bone and joint health care composition.

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

[0035] In a fifth aspect, the present invention provides a use of the above-mentioned composite algae extract as a pharmaceutical adhesive.

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

[0037] The present invention utilizes a citric acid-citrate buffer solution in a specific pH range in combination with enzymatic hydrolysis and ultrafiltration, and can effectively remove heavy metals remaining in the algae while extracting active ingredients from the algae. The method also has a high algae extract yield and heavy metal removal rate. The obtained algae extract has better effects on cartilage cell repair and anti-inflammatory effects, and is more suitable for use in bone and joint health care. At the same time, the algae extract can also act as an adhesive when used in a bone and joint health care composition, eliminating the need for a binder when the composition is made into tablets. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below through specific examples.

[0039] It should be noted that the following implementation cases are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned implementation cases, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned implementation cases, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the implementation cases of the present invention.

[0040] Example 1

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

[0042] S0. Place fresh algae raw materials (red algae partridge, brown algae kelp, and green algae enteromorpha in a ratio of 1:1:2) into a cooking pot and add 25 times the weight of the raw materials in water, stirring and washing.

[0043] S1. The washed algae material was immersed in 150 volumes of 0.1 mol / L citric acid-citrate buffer at 65°C for 1.5 h;

[0044] S2. The treated material obtained in step S1 was cooled to 50°C and then subjected to step-by-step enzymatic hydrolysis (pectinase was added for 1 hour, cellulase was added for 1 hour, bromelain was added and hydrolyzed for 1 hour). After the enzymatic hydrolysis was completed, the temperature was raised to 70°C, stirring was stopped, and the enzymes were inactivated for more than 1 hour to obtain an enzymatic hydrolyzate;

[0045] S3. After filtering the enzymatic hydrolyzate, collecting the filtrate;

[0046] S4. The filtrate was added with 3 times the volume of water and ultrafiltration (3000kD ultrafiltration membrane) was performed to obtain a concentrate;

[0047] S5. The concentrated solution is instantaneously sterilized at 130°C for 4 seconds. After circulating for half an hour, spray drying is performed. The spray dryer inlet temperature is set at 175°C and the outlet temperature is set at 75°C to obtain a low-heavy metal composite algae extract.

[0048] In order to explore the effect of the pH of the citric acid-citrate buffer on the results, the same batch of algae raw materials was used. On the basis of Example 1, the pH of the citric acid-citrate buffer was adjusted, and the removal rate of heavy metals and the finished product yield of the algae extract were statistically analyzed. The results are shown in Table 1.

[0049] Detection method for heavy metal content: lead GB5009.12, cadmium GB5009.15, mercury GB5009.17, arsenic GB5009.11.

[0050] Yield of algae extract:

[0051] Table 1 Effect of different pH citric acid-citrate buffer on the results

[0052]

[0053] Finished product yield = spray-dried finished product / fresh raw material * 100%

[0054] From the results shown in Table 1, it can be seen that under different pH citric acid-citrate buffer conditions, the removal rates of heavy metals are significantly different. Between pH 3-6, the lead removal rate is above 85%, the arsenic removal rate is above about 90%, the mercury removal rate is above about 99%, and the cadmium removal rate is above about 80%. The finished product yield can reach more than 2.5%. Among them, under the condition of pH 4, both the heavy metal removal rate and the yield of algae extract are optimal.

[0055] The pH of the citric acid-citrate buffer solution was 4 for subsequent experiments.

[0056] In order to explore the effect of the immersion temperature in step S1 on the results, the same batch of algae raw materials was used, based on Example 1, and the immersion temperature was changed. The results are shown in Table 2.

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

[0058]

[0059] From the results shown in Table 2, it can be seen that under the condition of pH 4, the heavy metal removal rate is relatively high when the immersion temperature is 60-80°C. Considering the heavy metal removal rate and the finished product yield, the immersion temperature is selected to be 60-80°C.

[0060] Comparative Example 1

[0061] In this comparative example, a pure water system (hydrochloric acid was used to adjust the pH) was used instead of a citric acid-citrate buffer system. That is, the citric acid-citrate buffer system in step S1 of Example 1 was replaced with a hydrochloric acid solution having a pH of 4 (other conditions were the same as those in the example).

[0062] Comparative Example 2

[0063] In this comparative example, a pure water system (acetic acid was used to adjust the pH) was used instead of a citric acid-citrate buffer system. That is, the citric acid-citrate buffer system in step S1 of Example 1 was replaced with an acetic acid solution having a pH of 4 (all other conditions were the same as those in the example). The heavy metal residues in the finished products after treatment were compared. The results are shown in Table 3.

[0064] Comparative Example 3

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

[0066] S0. The same raw materials as in Example 1 were put into a cooking pot, and 25 times the weight of the raw materials was added with water and stirred for washing.

[0067] S1. The washed algae material was impregnated with 150 volumes of citric acid-citrate buffer at 50°C. The system was then subjected to stepwise enzymatic hydrolysis (pectinase was added for 1 hour, followed by cellulase for 1 hour, and finally bromelain for 1 hour). After the hydrolysis was complete, the temperature was raised to 90°C, the pH was adjusted to 9, stirring was stopped, and the enzymes were inactivated for at least 1 hour to obtain an enzymatic hydrolyzate.

[0068] S2. After filtering the enzymatic hydrolyzate, collecting the filtrate;

[0069] S3. The filtrate was added 3 times the volume of water and ultrafiltration was performed to obtain a concentrate;

[0070] S4. The concentrated solution was instantaneously sterilized at 130°C for 4 seconds, and spray-dried by setting the spray dryer inlet temperature to 175°C and outlet temperature to 75°C to obtain a low-heavy metal composite algae extract.

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

[0072] Table 3

[0073]

[0074] From the results 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 citric acid-citrate buffer system was not added, showing that the heavy metal removal effect was significantly reduced compared with Example 1. Since citric acid is a tricarboxylic acid, the molecule contains 3 carboxyl groups (-COOH) and 1 hydroxyl group (-OH), and these groups are all strong coordination groups (can provide lone pairs of electrons to bind to metal ions). Multiple coordination sites can form cyclic chelates (such as five-membered rings or six-membered rings) with heavy metal ions. The stability of this structure is much higher than that of simple ionic bonds or monodentate coordination compounds. In contrast, hydrochloric acid has no coordination groups and can only dissolve some heavy metal compounds under acidic conditions, but cannot form complexes with heavy metal ions, and has almost no removal ability for bound heavy metals (the part bound to biological macromolecules); while acetic acid contains only 1 carboxyl group, and the acetate group (CH3COO -) has weak coordination capacity, resulting in low stability of the complexes formed with heavy metals (mostly monodentate coordination), making it difficult to compete for heavy metals bound to biomolecules in algae. In Comparative Example 3, the citric acid-citrate buffer system was added at different times and performed simultaneously with the enzymatic hydrolysis step. While the heavy metal removal efficiency improved somewhat compared to Comparative Examples 1 and 2, the removal rates of lead, cadmium, mercury, and arsenic all showed a significant decrease compared to Example 1. This is because in Comparative Example 3, the samples were not pre-soaked in buffer, so free heavy metals attached to the surface were not removed. During the enzymatic hydrolysis stage, they could quickly bind to the simultaneously released active ingredients (such as polysaccharide hydroxyl groups and protein sulfhydryl groups), forming more stable complexes. This makes subsequent chelation competition with the buffer more difficult, resulting in higher residual heavy metal levels. Furthermore, the repeated dissociation and binding of some active ingredients damaged their structures, reducing the activity and purity of the product.

[0075] In the present invention, the active ingredients in the composite algae extract were also tested, and the testing method and results are as follows.

[0076] Detection method:

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

[0078] Uronic acid detection: carbazole-sulfuric acid method.

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

[0080] Total sugar (as fucose), % Organic sulfate groups, % Uronic 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 primary active ingredients in algae are polysaccharides (such as alginic acid, fucoidan, carrageenan, agar, and Enteromorpha polysaccharides), which account for 20%-70% of their dry weight. Algal polysaccharides possess a wealth of biological activities, including anti-inflammatory, antioxidant, immunomodulatory, and moisturizing properties. Total sugars are a key indicator of these polysaccharide components, and their content reflects their potential therapeutic benefits: a higher total sugar content indicates a greater abundance of polysaccharide active ingredients in the extract, and a more robust foundation for these benefits.

[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 do not contain sulfate groups or have low sulfate content exhibit stronger antiviral activity.

[0083] Uronic acid (such as D-mannuronic acid and L-guluronic acid in alginic acid, and glucuronic acid in hyaluronic acid analogs) is a characteristic component unit of algal acid polysaccharides, and its content directly affects the physicochemical properties and biological activity of polysaccharides.

[0084] Application Examples

[0085] In this application example, a composite algae extract (obtained in Example 1, using a citric acid-citrate buffer system at pH 4) is combined with glucosamine and calcium carbonate to form a bone and joint health care composition. Glucosamine replenishes the raw materials needed for cartilage matrix repair, the algae extract alleviates inflammation, and the calcium carbonate enhances bone density, providing joint care from multiple perspectives. The addition of the composite algae extract enhances joint health.

[0086] Safety experiment of compound algae extract on chondrocytes

[0087] Compositions without adding algae extract or adding different amounts of algae extract were designed, and the survival rate of chondrocytes under the treatments was tested.

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

[0089] 1) Normally cultured C28 / I2 human chondrocytes in the logarithmic phase were digested with 0.25% Typsin + 0.02% EDTA, centrifuged at 1000 rpm for 5 min, counted under a counting plate, and plated on a 96-well plate. Each group had 6 replicate wells, and 5×10 3 cells.

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

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

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

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

[0094] See Table 5 for the results.

[0095] Table 5

[0096]

[0097] Note: The weight percentage of the composite 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 composite algae extract has no toxic side effects on chondrocytes and has good affinity. In addition, compared with the extract without composite algae extract, the cell survival rate is increased to a certain extent, indicating that the composite algae extract of the present invention has a certain protective effect on cells.

[0099] Experimental study on the repair of chondrocytes under inflammatory conditions using the composition

[0100] 1) Normally cultured C28 / I2 human chondrocytes in the logarithmic phase were digested with 0.25% Typsin + 0.02% EDTA, centrifuged at 1000 rpm for 5 min, counted under a counting plate, and plated on a 96-well plate. Each group had 6 replicate wells, and 5×10 3 cells.

[0101] 2) A blank control group, a model control group, and a drug intervention group were established. The blank control group cultured human chondrocytes normally without any treatment. The model control and drug intervention groups were first induced with 10 ng / mL IL-1β for 24 hours. The model control group was then cultured in standard culture medium (without the drug combination) for 72 hours. The drug intervention group was treated with the prescribed dose of drug for 72 hours. Cell viability was measured using CCK8 at the endpoint of each observation.

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

[0103] See Table 6 for the results.

[0104] Table 6

[0105]

[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 supplemented with the composite algae extract of the present invention was significantly improved, especially in the 20% addition group, indicating that the composite algae extract of the present invention has a significant effect on improving the proliferation activity of chondrocytes under inflammatory conditions.

[0108] Experiment on the effect of the composition on inflammatory factors under inflammatory conditions

[0109] Experimental methods:

[0110] 1) Normally cultured C28 / I2 human chondrocytes in the logarithmic phase were digested with 0.25% Typsin + 0.02% EDTA, centrifuged at 1000 rpm for 5 min, counted on a microplate, and plated on a 96-well plate. Each well had 6 replicates, and 5 × 10 3 cells.

[0111] 2) A blank control group, a model control group, and a drug intervention group were set up. The blank control group cultured human chondrocytes normally without any treatment. The model control and drug intervention groups were first induced with 10 ng / mL IL-1β for 24 hours. The model control group was then cultured in standard culture medium (without the drug combination) for 72 hours. The drug intervention group was treated with the prescribed drug dose for 72 hours. Cell supernatants were collected and TNF-α and IL-6 levels were measured using ELISA kits.

[0112] See Table 7 for the results.

[0113] Table 7

[0114]

[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 addition of the composite algae extract, the inflammatory factors TNF-α and IL-6 in the group with the addition of the composite algae extract of the present invention were both reduced, indicating that the composite 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 process conditions

[0118] This experiment takes the composition of glucosamine+calcium citrate+10% compound algae extract as an example, wherein the compound algae extract is the extract obtained in Example 1 and Comparative Examples 1-4 respectively.

[0119] The experimental method was as described above, and the experimental results are shown in Table 8 (results of chondrocyte repair under inflammatory conditions) and Table 9 (effects 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 ]]> Model comparison <![CDATA[76.50±3.33 d ]]>

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

[0123] The results shown in Table 8 show that the composite algae extract obtained in Example 1 of the present invention has the best effect on chondrocyte repair under inflammatory conditions. In Comparative Examples 1 and 2, pure water was used instead of the citric acid-citrate buffer system. Although the pH of the system was controlled at 4, the results showed that the chondrocytes under inflammatory conditions were much lower than those in Example 1, indicating that treatment with the citric acid-citrate buffer not only affects the heavy metal content but also the repair effect of the composite algae extract on chondrocytes. In Comparative Example 3, the citric acid-citrate buffer system was added together with the 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 shows that the timing of treatment with the citric acid-citrate buffer system also affects the repair effect of the composite algae extract on chondrocytes.

[0124] Table 9

[0125]

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

[0127] The results shown in Table 9 show that the composite algae extract obtained in Example 1 of the present invention reduced the content of inflammatory factors under inflammatory conditions compared to the model control using the composition, indicating a repairing effect on inflammation. In Comparative Examples 1 and 2, pure water was used instead of the citric acid-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 that in Example 1, indicating that the citric acid-citrate buffer treatment not only affected the heavy metal content but also the anti-inflammatory effect of the composite algae extract on chondrocytes. In Comparative Example 3, the citric acid-citrate buffer system was added together with the enzymatic hydrolysis. Although the content of inflammatory factors was somewhat reduced compared to Comparative Examples 1 and 2, it was still higher than that in Example 1. This indicates that the timing of the citric acid-citrate buffer treatment also affects the anti-inflammatory effect of the composite algae extract on chondrocytes.

[0128] Experimental study on the adhesive effect of composite algae extract in composite materials

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

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

[0131] 3) Slowly add a certain amount of wetting agent to the evenly mixed material, stirring while adding, until the material forms a moist soft material (it is best if it can be held in the hand and falls apart when touched lightly).

[0132] 4) Place the soft material into a granulator, granulate it through a 20-mesh screen, and collect the resulting wet granules.

[0133] 5) Spread the wet granules evenly on a tray (no more than 1 cm thick) and dry them in a 60°C oven for 30 minutes, turning the granules every 10 minutes to ensure even drying.

[0134] 6) Take out the dried granules, place them at room temperature to cool to room temperature (about 30 minutes), and then pass them through a 20-mesh sieve to granulate them. Collect the granules after granulation and weigh them to record them as the total granule mass.

[0135] 7) Pass the granules through a 100-mesh sieve. Collect the coarse powder on the 100-mesh sieve and the fine powder below. Accurately weigh the coarse powder and fine powder using an electronic balance and record the mass. Calculate the coarse powder ratio (coarse powder mass / total granule mass × 100%). A higher coarse powder ratio indicates better bonding performance.

[0136] 8) Take each group of granules after whole granulation and measure the bulk density and compactness according to the Chinese Pharmacopoeia method.

[0137] 9) Take each group of granules after granulation and place 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 measure the hardness of each tablet using a hardness tester. Calculate the average hardness. Tablet hardness is generally required to be between 3-6 kgf. The higher the hardness, the better the bonding effect.

[0139] 11) Disintegration Time Test: According to the method specified in the Chinese Pharmacopoeia, take six tablets and place them in a disintegration time tester. Record the disintegration time for each tablet and calculate the average. The disintegration time of ordinary tablets should be within 15 minutes. Excessive binding may cause the disintegration time to be prolonged.

[0140] 12) Friability Test: Take 20 tablets, remove surface powder, weigh them, place them in a friability tester, rotate them 100 times, remove them, remove the powder, weigh them again, and calculate the friability (loss mass / initial mass × 100%). The friability should not exceed 1%. The lower the value, the greater the tablet's wear resistance and the better the bonding effect.

[0141] Table 10

[0142]

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

[0144] Table 11

[0145]

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

[0147] Conclusion: From the results shown in Tables 10 and 11, it can be seen that the composite algae extract of the present invention has the effect of reducing the amount of wetting agent, improving the flowability of particles, and improving the mechanical properties of tablets.

Claims

1. A method for preparing a composite algae extract, characterized in that: The steps include: S1. Take the algae raw material, soak it in 100-150 times the volume of citric acid-citrate buffer at pH 3-5, and treat it at 60-80°C; S2. The treated material obtained in step S1 is cooled to 50-55°C and then subjected to enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated to obtain an enzymatic solution; S3. After filtering the enzymatic hydrolyzate, collecting the filtrate; S4. The filtrate is subjected to ultrafiltration to obtain a concentrate; S5. sterilizing and spray-drying the concentrated solution to obtain a composite algae extract.

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

3. The method for preparing a composite algae extract according to claim 1, characterized in that: The enzymatic hydrolysis is a three-step enzymatic hydrolysis performed sequentially, wherein the first step enzymatic hydrolysis uses one or a mixture of pectinase, alginate, and agarase; the second step enzymatic hydrolysis uses cellulase; and the third step enzymatic hydrolysis uses one or a mixture of bromelain, papain, and β-glucanase.

4. The method for preparing a composite 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.

5. The method for preparing a composite algae extract according to claim 1, characterized in that: Before ultrafiltration, dilute the filtrate with 1-4 volumes of water.

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

7. A composite algae extract, characterized in that: The method is as described in any one of claims 1 to 6.

8. The use of the composite algae extract according to claim 7, characterized in that: Composition for bone and joint health care.

9. The use of the composite algae extract according to claim 7, characterized in that: Used as a drug adhesive.

10. A bone and joint health care composition, characterized in that: Comprising the composite algae extract according to claim 7.

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