Plant extract composite oral liquid containing nannochloropsis powder and preparation method thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of plant extract composite oral liquid containing parachlorella powder and a preparation method thereof.The oral liquid contains parachlorella powder and turmeric extract;Optionally, it can further include perilla stem extract, wherein the weight ratio of parachlorella powder to turmeric extract is (3-5):1.In vitro cell experiments show that under the above ratio, the compound shows synergistic effect on the inhibition of TNF-alpha, IL-6, and the improvement of SOD activity and total antioxidant capacity.In preparation, hydroxypropyl-beta-cyclodextrin is used to include turmeric extract, and xanthan gum and sodium carboxymethyl cellulose are used as suspending agents.The physical stability of the obtained oral liquid is improved, the bitterness is reduced, and the taste acceptance is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a compound oral liquid containing microalgae powder and its preparation method. Background Technology

[0002] With the fast pace of modern life and increasing environmental pollution, the risk of oxidative stress and inflammation in the human body has significantly increased. Long-term chronic inflammation and oxidative damage are widely recognized as important contributing factors to various chronic diseases (such as cardiovascular disease, diabetes, and neurodegenerative diseases) and aging. Therefore, maintaining health through supplementation with natural plant extracts with antioxidant and anti-inflammatory effects has become a research hotspot in the health food industry.

[0003] *Nannochloropsis* is a marine microalga whose algal powder is rich in omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA), as well as abundant bioactive substances such as fucoxanthin and chlorophyll, exhibiting significant anti-inflammatory and lipid-regulating effects. The main active components of turmeric extract are curcuminoids, including curcumin, demethoxycurcumin, and didemethoxycurcumin, which possess broad-spectrum anti-inflammatory, antioxidant, and antitumor pharmacological activities. Its anti-inflammatory mechanism involves inhibiting multiple signaling pathways such as NF-κB and MAPK. Perilla stem is the dried stem of the Lamiaceae plant *Perilla frutescens*. Traditional Chinese medicine believes it has the effects of regulating qi, relieving chest congestion, and calming the fetus. Modern pharmacological studies have shown that perilla stem extract is rich in flavonoids and phenolic acids such as rosmarinic acid and apigenin, possessing antioxidant and anti-inflammatory bioactivities.

[0004] In the prior art, patent CN118716617A (Shenzhen Aosa Pharmaceuticals) discloses a composition consisting of Spirulina, Micrococcus pluvialis, chia seeds, and vitamin K for bone and joint protection. This patent, used for bone and joint protection, verified through the Jinzhengjun Q-value method that microalgae, plant seeds, and vitamin K have a synergistic effect in inhibiting pro-inflammatory cytokines and osteoclasts. This composition mainly works through microalgae nutritional supplementation and the vitamin K-dependent osteocalcin pathway, and its bone and joint protective effect has been verified in animal experiments. Patent CN119564775B (By-Health) discloses the use of Micrococcus pluvialis oil combined with water-soluble tomato concentrate in assisting in lowering blood lipids. The Micrococcus pluvialis used in this patent is in the form of an oil extract, and its efficacy is positioned in the field of cardiovascular health. In addition to containing eicosapentaenoic acid (EPA), Micrococcus pluvialis powder is also rich in natural polysaccharides and proteins, but there are no research reports in the prior art on the use of this whole algae powder combined with specific plant extracts for immune regulation and antioxidant effects. Most existing antioxidant and anti-inflammatory health foods are in tablet or capsule form, which have problems such as difficulty in swallowing and low bioavailability, and poor compliance, especially for the elderly and children. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a plant extract compound oral liquid containing *Micrococcus pseudochlorella* powder and its preparation method. The plant extract compound oral liquid containing *Micrococcus pseudochlorella* powder uses specific active ingredients and proportions, and through specific excipient ratios and preparation processes, the resulting oral liquid has synergistic antioxidant and anti-inflammatory effects, while improving the stability of fat-soluble components in aqueous systems and the taste of the oral liquid.

[0006] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution: In a first aspect, the present invention provides a binary active ingredient composition comprising *Micrococcus pseudocarpa* powder and turmeric extract, wherein the weight ratio of *Micrococcus pseudocarpa* powder to turmeric extract is (3-5):1. As one optional technical solution, the weight ratio is (3.6-4.4):1. As another optional technical solution, the weight ratio is 4:1.

[0007] In a second aspect, the present invention provides a ternary active ingredient composition comprising the binary active ingredient composition described in any one of the first aspects and perilla stem extract.

[0008] As one of the optional technical solutions, the weight ratio of the *Microcystis aeruginosa* powder, turmeric extract, and perilla stem extract is (3-5):1:1.

[0009] As one of the optional technical solutions, the perilla stem extract is an extract obtained by extracting perilla stems with an ethanol-water solution.

[0010] Thirdly, the present invention provides a plant extract compound oral liquid containing microchlorella powder, comprising microchlorella powder, turmeric extract and perilla stem extract, wherein the weight ratio of microchlorella powder to turmeric extract is (3-5):1, and the turmeric extract is added to the oral liquid in the form of hydroxypropyl-β-cyclodextrin inclusion complex.

[0011] As one of the optional technical solutions, the compound oral liquid also includes any one or more of suspending agents, wetting agents, preservatives, flavoring agents, and water.

[0012] As one of the optional technical solutions, the suspending agent is a combination of xanthan gum and sodium carboxymethyl cellulose, and the wetting agent is glycerin.

[0013] As one of the optional technical solutions, based on 1000mL of oral solution, the component dosage is as follows: 20-30g of *Micrococcus pseudocarpa* powder, 4-6g of turmeric extract, 4-6g of perilla stem extract, 1-2g of xanthan gum, 2-4g of sodium carboxymethyl cellulose, 30-50g of glycerin, 0.8-1.5g of potassium sorbate, 1-2g of vitamin C, 0.5-1g of vitamin E, 0.2-0.6g of sucralose, 1-2mL of strawberry flavoring, and purified water to make up to 1000mL, and the pH is adjusted to 4.8-5.2 with citric acid.

[0014] Fourthly, the present invention provides a method for preparing the compound oral liquid according to any one of the third aspects, comprising the following steps: Curcuma extract was encapsulated with hydroxypropyl-β-cyclodextrin to obtain a curcuma extract inclusion complex. Perilla stems were extracted by reflux with an ethanol-water solution. The extract was then concentrated and dried to obtain perilla stem extract. Micrococcus pseudochlorella powder, the turmeric extract inclusion complex from step (1) and the perilla stem extract from step (2) were added to glycerol and stirred to obtain suspension A; The suspending agent was dissolved in heated purified water and dispersed by shearing to obtain colloidal solution B; Add suspension A to colloidal solution B and mix. Then add preservatives, flavoring agents and other excipients, mix well, dilute to volume with purified water, adjust pH, filter, fill and sterilize to obtain the final product.

[0015] As one of the optional technical solutions, the volume fraction of the ethanol aqueous solution in step (2) is 60% to 80%, the material-liquid ratio is 2:10 to 1:20, and the reflux extraction is performed 1 to 3 times, each time for 1 to 2 hours.

[0016] As one of the optional technical solutions, the stirring speed in step (3) is 400-800 rpm. As one of the optional technical solutions, the purified water in step (4) is heated to 35-50°C, and the shear dispersion speed is 5000-10000 rpm.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The plant extract compound oral liquid containing *Micrococcus pseudochlorella* powder described in this invention, based on the screening of active ingredient combinations, determined that *Micrococcus pseudochlorella* powder and turmeric extract at a weight ratio of (3-5):1, especially 4:1, can produce synergistic anti-inflammatory and antioxidant effects. In vitro cell experiments showed that at this ratio, the increase in TNF-α and IL-6 inhibition rates, as well as SOD activity and total antioxidant capacity, exceeded the expected summation when each component was used alone, suggesting that the complementary regulation of the two components in the NF-κB and Nrf2 pathways may enhance the overall cell-protective effect. Furthermore, a ternary composition consisting of *Perilla frutescens* stem extract was introduced, further improving the above four activity indicators and demonstrating even better comprehensive antioxidant and anti-inflammatory potential.

[0018] This invention employs hydroxypropyl-β-cyclodextrin to encapsulate turmeric extract, combined with a xanthan gum and sodium carboxymethyl cellulose composite suspension system, significantly improving the physical stability of fat-soluble curcumin in aqueous oral solutions. Accelerated testing results show that the product remained in a homogeneous suspension after 30 days of storage at 60°C, with a curcumin content retention rate of 98.4%, while the unencapsulated control sample showed significant stratification and a decrease in content during the same period. This indicates that the formulation process helps delay the sedimentation and oxidation of the active ingredient, supporting the product's quality stability over its shelf life.

[0019] Furthermore, through inclusion masking and the combined use of sucralose, strawberry flavoring, and citric acid, the oral liquid of this invention effectively reduces the bitterness of turmeric extract, resulting in a higher sensory evaluation and a better taste. The oral liquid dosage form itself facilitates swallowing and absorption; combined with the inclusion technology improving curcumin dissolution, it is expected to enhance patient compliance and the bioavailability of the active ingredient. Detailed Implementation

[0020] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and are therefore only examples and should not be used to limit the scope of protection of the present invention.

[0021] Special note: The materials used in the following experiments were as follows: *Nannochloropsis sp.* powder was whole algae powder obtained by spray drying, with a moisture content ≤7%; *Curcuma longa L.* and *Perilla frutescens (L.) Britt.* were purchased from a traditional Chinese medicine market and used after identification. Rosemary extract (from dried leaves of *Rosmarinus officinalis L.* extracted with ethanol), grape seed extract (from dried seeds of *Vitis vinifera L.* extracted with ethanol), *Ganoderma lucidum* (from fruiting bodies of *Ganoderma lucidum* (Leyss. ex Fr.) Karst. extracted with water), and perilla seed extract (from dried mature seeds of *Perilla frutescens (L.) Britt.* extracted with ethanol) were all commercially available raw materials and prepared in the laboratory. Specific preparation methods are briefly described in Experiment Example 2. Hydroxypropyl-β-cyclodextrin (HP-β-CD, degree of substitution DS≈0.6) was purchased from a pharmaceutical grade excipient supplier; xanthan gum, sodium carboxymethyl cellulose, glycerin, potassium sorbate, vitamin C, vitamin E, sucralose, citric acid, strawberry flavoring, and other excipients were all food-grade or pharmaceutical-grade, and purified water was prepared in the laboratory. Unless otherwise specified, the amounts of each component in the following examples are based on dry weight or raw material. Example

[0022] The purpose of this embodiment is to provide a method for preparing turmeric extract inclusion complexes, the specific steps of which are as follows: (1) Ingredients: Accurately weigh 120 g of turmeric extract powder and 150 g of hydroxypropyl-β-cyclodextrin. The turmeric extract powder is prepared according to the following method: The dried turmeric rhizomes were crushed into coarse powder, and extracted twice with 70% ethanol (10 times the mass of the coarse powder) for 2 hours each time. The extracts were filtered, the filtrates were combined, and the extracts were concentrated under reduced pressure to a thick paste with a relative density of about 1.20 (measured at 60℃). The paste was then vacuum dried and crushed through an 80-mesh sieve to obtain turmeric extract powder.

[0023] (2) Dissolution: Dissolve 150 g of hydroxypropyl-β-cyclodextrin in 2000 mL of purified water, heat to 60°C, and stir at 300 rpm until completely dissolved to prepare a saturated aqueous solution.

[0024] (3) Inclusion: Add the turmeric extract powder in three equal portions to the above saturated aqueous solution at 60°C, with a 5-minute interval between each addition. Shear the mixture at 5000 rpm for 30 minutes using a shear emulsifier, then cool it down to 40°C and continue stirring at 300 rpm for 2 hours to include the mixture.

[0025] (4) Drying: After inclusion, the mixture was vacuum dried. The vacuum drying parameters were: drying temperature 60℃, vacuum degree -0.09MPa, and drying time 24 hours. The dried solid was pulverized and passed through an 80-mesh sieve to obtain the turmeric extract inclusion complex with a product mass of 257 g. This product was used for subsequent experiments.

[0026] The purpose of this experiment is to explore the synergistic anti-inflammatory and antioxidant effects of *Micrococcus pseudocarpa* powder and turmeric extract at different ratios, and to evaluate the synergistic effect using the Jinzhengjun Q-value method.

[0027] Experimental methods: (1) Experimental grouping: The experiment included a blank control group, a model control group, a group using only *Chlorella vulgaris* powder, a group using only turmeric extract, and groups using different ratios of combined administration. The total amount of *Chlorella vulgaris* powder and turmeric extract (based on turmeric extract raw material) was fixed at 36 μg / mL (based on the final concentration of cell culture medium). In all the above groups, turmeric extract was added in the form of an inclusion complex of turmeric extract, and the weight ratio of the two was set to 1:1, 2:1, 3:1, 4:1, and 5:1, for a total of five ratio groups. (The mass fractions or mass ratios are based on turmeric extract raw material).

[0028] (2) Cell culture: RAW264.7 mouse macrophage cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. It was cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37℃ in a 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments.

[0029] (3) Anti-inflammatory activity test: RAW264.7 cells were subjected to an inoculum concentration of 5 × 10⁻⁶ cells. 5 Cells were seeded per well in 24-well plates and cultured for 24 hours. Except for the blank control group, all other groups received 1 μg / mL LPS to induce an inflammatory response, along with the corresponding drugs, and were cultured for another 24 hours. The cell culture supernatant was collected, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured according to the ELISA kit instructions. The inhibition rate was calculated using the following formula: Inhibition rate (%) = [(Content in model control group - Content in drug treatment group) / (Content in model control group - Content in blank control group)] × 100%.

[0030] Each group has 6 replicate wells, and the experiment is repeated 3 times.

[0031] (4) Antioxidant activity test: RAW264.7 cells were subjected to an oxidation state of 5 × 10⁻⁶ cells / mL. 5Cells were seeded per well in 24-well plates and cultured for 24 hours. Except for the blank control group, all other groups were treated with 200 μmol / L H2O2 to induce oxidative stress, along with the corresponding drugs. Cells were then cultured for another 12 hours. Cells were collected, sonicated, and superoxide dismutase (SOD) activity was measured according to the instructions of the WST-1 method SOD assay kit. Simultaneously, total antioxidant capacity (T-AOC) was measured according to the instructions of the total antioxidant capacity (T-AOC) assay kit.

[0032] SOD activity enhancement rate calculation: Enhancement rate (%) = [(SOD activity in the treatment group - SOD activity in the model control group) / SOD activity in the model control group] × 100%.

[0033] The improvement rate of T-AOC value is calculated as follows: Improvement rate (%) = [(T-AOC value of the treatment group - T-AOC value of the model control group) / T-AOC value of the model control group] × 100%.

[0034] Each group of experiments had 6 replicate wells, and the experiment was repeated 3 times.

[0035] (5) Evaluation of synergistic effect: The Jin Zhengjun Q-value method was used to evaluate the synergistic effect. The calculation formula is as follows: Q = E(A+B) / (EA + EB-EA×EB / 100); Where E(A+B) is the effect value (inhibition rate or enhancement rate) of the combined drug administration group, EA is the effect value of the single-use group of Micrococcus pseudochlorella powder at the corresponding dose, and EB is the effect value of the single-use group of Curcuma longa extract at the corresponding dose.

[0036] Experimental results were defined as follows: Q > 1.15 indicates a synergistic effect, 0.85 ≤ Q ≤ 1.15 indicates an additive effect, and Q < 0.85 indicates an antagonistic effect.

[0037] The test results are shown in Table 1.

[0038] Table 1. Synergistic effects of different ratios of *Microcystis aeruginosa* powder and turmeric extract (x̄±s, n=3)

[0039] Table 1 shows that the Q values ​​of *Micrococcus pseudocarpa* powder and turmeric extract differ significantly at different weight ratios.

[0040] At a 1:1 ratio, the Q values ​​of the various indicators were not entirely consistent. TNF-α (Q=0.89), IL-6 (Q=0.88), and T-AOC (Q=0.88) were within the additive range of 0.85–1.15, while SOD's Q value (0.84) was slightly lower than 0.85, showing a slight antagonistic trend. At a 2:1 ratio, the Q values ​​of the four indicators were TNF-α 1.13, IL-6 1.12, SOD 1.11, and T-AOC 1.13, all within the additive range of 0.85–1.15. Overall, in the lower ratio range of 1:1 to 2:1, the two active ingredients did not generally exhibit synergistic complementarity; their effects were mainly a simple additive of independent activities, with SOD even showing slight antagonism at extremely high curcumin ratios. This may be related to the excessively high relative proportion of curcumin extract within this range. The high content of curcumin compounds is sufficient to exert a near-saturated anti-inflammatory and antioxidant effect in the selected cell model. At this point, the additional contribution of microalgae powder is masked by the dominant effect of curcumin extract, making it difficult to demonstrate synergistic characteristics. In fact, it may even exhibit weak antagonism at the SOD activity level due to the possible pro-oxidative effect of excessive curcumin.

[0041] When the ratio was increased to 3:1, the Q values ​​of all four indicators significantly exceeded the synergistic threshold of 1.15 (TNF-α 1.39, IL-6 1.32, SOD 1.58, T-AOC 1.33), entering the synergistic effect range. The synergistic peak of SOD activity enhancement occurred at 3:1 (Q=1.58). When the ratio was further increased to 4:1, the synergistic strength of the anti-inflammatory indicators (TNF-α, IL-6) and the total antioxidant capacity indicator (T-AOC) reached its peak (Q values ​​were 1.56, 1.51, and 1.63, respectively); although the Q value of SOD slightly decreased to 1.52 compared to 3:1, it still maintained a high synergistic level. This synergistic effect is presumably due to the different targets or pathways of polyunsaturated fatty acids such as EPA and active ingredients such as fucoxanthin in the *Chlorella vulgaris* powder compared to curcumin-like compounds in their anti-inflammatory and antioxidant mechanisms. The two complement each other at multiple stages of the inflammatory response and at different levels of the oxidative defense system, thus producing a superimposed effect that exceeds the individual effects when they work together.

[0042] It is worth noting that when the ratio was further increased to 5:1, the Q values ​​of all indicators decreased to varying degrees compared to 4:1, but the four indicators still maintained a synergistic effect with Q values ​​greater than 1.15. This trend suggests that as the proportion of *Chlorella pseudochlorella* powder continues to increase, the relative content of curcumin extract in the system is too low, which may not be sufficient to fully support the optimal regulatory effect of its antioxidant active ingredients, leading to a decrease in synergistic strength; however, each pathway can still maintain a synergistic state, which is presumably related to the direct contribution of anti-inflammatory and antioxidant components such as EPA in *Chlorella pseudochlorella* powder, and a certain tolerance to the decrease in curcumin concentration.

[0043] In summary, the synergistic effect of *Micrococcus pluvialis* powder and turmeric extract is not a simple dose-dependent relationship, but rather exhibits an optimal ratio window. At a 1:1 ratio, all indicators except SOD showed weak antagonism were additive; at a 2:1 ratio, all four indicators showed additive effects; the synergistic window opened between 3:1 and 5:1, with 4:1 being the optimal ratio, where the synergistic strength of most indicators reached its highest level and overall consistency was best; beyond 4:1, the synergistic effect weakened due to insufficient relative concentration of curcumin, but all indicators remained within the synergistic range. These results indicate that the complementarity of the two in their anti-inflammatory and antioxidant mechanisms is the basis for synergistic effects, and 4:1 is precisely the optimal balance point to achieve this complementary effect.

[0044] Based on the experimental results of Example 1 above, this embodiment provides a binary compound composition in which the weight ratio of *Micrococcus pseudocarpa* powder to turmeric extract is (3-5):1, preferably (3.6-4.4):1, and most preferably 4:1. This ratio of composition provides the most complete complementary effect on anti-inflammatory and antioxidant pathways, and its synergistic effect is the strongest.

[0045] Based on the optimal active ingredients screened in the aforementioned Experimental Example 1 and Example 4, this example further prepares a plant extract compound oral liquid containing Micrococcus pseudochlorella powder.

[0046] The formula for 1000mL oral solution is as follows: 24g of *Micrococcus pseudochlorella* powder, 12.85g of turmeric extract inclusion complex prepared according to the method of Example 1, 6g of perilla stem extract prepared in Example 4, 1.5g of xanthan gum, 3.0g of sodium carboxymethyl cellulose, 40g of glycerin, 1.2g of potassium sorbate, 1.5g of vitamin C, 0.8g of vitamin E, 0.4g of sucralose, 1.5mL of strawberry flavoring, appropriate amount of citric acid, and purified water added to 1000mL.

[0047] The preparation method is as follows: (1) Wetting and dispersing: 24g of Micrococcus pseudochlorella powder, 12.85g of turmeric extract inclusion complex and 6g of perilla stem extract were added to 40g of glycerol and stirred at 600rpm for 15 minutes until homogeneous to obtain suspension A.

[0048] (2) Colloid preparation: Heat about 800 mL of purified water to 40 °C, mix 1.5 g of xanthan gum with 3.0 g of sodium carboxymethyl cellulose, and slowly add the mixture to the purified water in batches. Shear and disperse the mixture at 8000 rpm for 15 minutes to ensure full swelling and uniformity, thus obtaining colloidal solution B.

[0049] (3) Mixing: Slowly add suspension A to colloidal solution B and stir at 400 rpm for 20 minutes until homogeneous; then add 1.2 g potassium sorbate, 1.5 g vitamin C, 0.8 g vitamin E, 0.4 g sucralose and 1.5 mL strawberry flavoring, and continue stirring to dissolve for 15 minutes.

[0050] (4) Volume adjustment and pH adjustment: Add purified water to a volume of 1000 mL, adjust the pH value to 5.0±0.1 with 10% citric acid solution, filter through a 200-mesh sieve, fill into 10 mL brown oral liquid bottles, seal the caps, and autoclave at 121℃ for 15 minutes to obtain the product.

[0051] The purpose of this embodiment is to provide a method for preparing perilla stem extract, the specific steps of which are as follows: (1) Raw material processing: Select dried perilla stems (produced in Jiangsu Province, harvested in autumn), crush them with a universal pulverizer, and pass them through a 40-mesh sieve to obtain coarse perilla stem powder.

[0052] (2) Extraction: Weigh 100g of coarse perilla stem powder, add 1500mL of 70% ethanol solution, place in a reflux extraction device, heat and reflux twice for 1.5 hours each time, and combine the two extracts. Maintain a gentle boil during the extraction process and reflux the cooled water.

[0053] (3) Concentration: The combined extracts are filtered through a 200-mesh sieve, and the filtrate is concentrated under reduced pressure at 60°C and a vacuum of -0.08MPa to a paste with a relative density of 1.10 to 1.15 (measured at 60°C).

[0054] (4) Drying and pulverizing: The extract is placed in a vacuum drying oven and dried to constant weight (moisture content ≤ 5%) at 60℃ and vacuum degree -0.09MPa. It is then pulverized by a universal pulverizer and passed through an 80-mesh sieve to obtain perilla stem extract powder.

[0055] The purpose of this experiment is to screen for a third active ingredient that can produce the best synergistic effect with Micrococcus pseudochlorella powder and turmeric extract.

[0056] Experimental methods: (1) Experimental groups: The experiment included a blank control group, a model control group, a baseline group consisting of two components, and five groups with three different additives. The model control group received an equal volume of culture medium and the corresponding stimulant (1 μg / mL LPS or 200 μmol / L H2O2), without any test drug, to verify the modeling effect. The baseline group was administered *Micrococcus pseudocarpa* powder and turmeric extract at a 4:1 ratio, with a total dosage of 36 μg / mL (i.e., 28.8 μg / mL of *Micrococcus pseudocarpa* powder and 7.2 μg / mL of turmeric extract; the actual amount of inclusion complex added was based on the turmeric extract raw material). Each of the three-component groups maintained the 4:1 ratio of *Microcystis aeruginosa* powder to turmeric extract, and additionally introduced perilla stem extract, perilla seed extract, rosemary extract, grape seed extract, or *Ganoderma lucidum* extract as a third component. The ratio of the three components in each group was 4:1:1, and the total dosage was 36 μg / mL (i.e., 24 μg / mL of *Microcystis aeruginosa* powder, 6 μg / mL of turmeric extract, and 6 μg / mL of the third component; the actual amount of inclusion complex added to the turmeric extract was based on the raw materials of each extract). The total dosage volume and administration method remained consistent across all groups.

[0057] The preparation method of the perilla seed extract is briefly described as follows: Perilla seeds are crushed and passed through a 40-mesh sieve. The extract is then extracted twice by reflux with a 70% ethanol solution at a material-to-liquid ratio of 1:12, for 1.5 hours each time. The extracts are combined, concentrated under reduced pressure at 60°C, vacuum dried, and then crushed through an 80-mesh sieve to obtain the final product.

[0058] The preparation method of this rosemary extract is briefly described as follows: Rosemary leaves are crushed and passed through a 40-mesh sieve. The extract is then extracted twice by reflux with a 75% ethanol solution at a material-to-liquid ratio of 1:12, for 1 hour each time. The extracts are combined, concentrated under reduced pressure at 60°C, dried under vacuum, and crushed through an 80-mesh sieve to obtain the final product.

[0059] The preparation method of this grape seed extract is briefly described as follows: Grape seeds are crushed and passed through a 40-mesh sieve. The extract is then extracted twice by reflux with a 60% ethanol solution at a material-to-liquid ratio of 1:12, for 1 hour each time. The extracts are combined, concentrated under reduced pressure at 60°C, vacuum dried, and crushed through an 80-mesh sieve to obtain the final product.

[0060] The preparation method of this Ganoderma lucidum extract is briefly described as follows: Ganoderma lucidum fruiting bodies are crushed and passed through a 40-mesh sieve. The extract is then boiled twice with purified water at a material-to-liquid ratio of 1:12, each time for 2 hours. The decoctions are combined, concentrated under reduced pressure at 60°C, vacuum dried, and crushed through an 80-mesh sieve to obtain the extract.

[0061] (2) Activity test: Using the same RAW264.7 cell model and detection method as in Example 1, TNF-α, IL-6, SOD, and T-AOC levels were measured. Based on the baseline group of the binary composition, the rate of change of each of the three-component addition groups relative to the baseline group was calculated. The formula for calculating the rate of change is: Change rate (%) = [(Addition group measurement value - Baseline group measurement value) / Baseline group measurement value] × 100%.

[0062] The model control group data is used to confirm the success of the model and is not included in the direct calculation of the rate of change.

[0063] Each group had 6 replicate wells, and the experiment was repeated 3 times. The experimental results are shown in Table 2.

[0064] Table 2 Results of the third component screening experiment (x̄±s, n=3)

[0065] Table 2 shows that, under the premise of a fixed total dosage, the antioxidant and anti-inflammatory activities of the complex showed significant differences after the introduction of different third components.

[0066] The synergistic effect of rosemary extract was generally weak, showing only a slight positive regulation of SOD activity and TNF-α, with negligible contributions to IL-6 and total antioxidant capacity (T-AOC). Grape seed extract, on the other hand, exhibited a clear antioxidant bias, moderately increasing SOD activity and T-AOC, but its anti-inflammatory effect was very limited. Ganoderma lucidum extract showed a dual effect; while increasing T-AOC, it led to a decrease in IL-6 levels, demonstrating its complex regulation of immune pathways rather than simple inhibition.

[0067] The addition of perilla seed extract showed a significant synergistic effect, particularly in inhibiting IL-6 and enhancing T-AOC, but its ability to activate the activity of endogenous SOD enzymes was relatively insufficient.

[0068] In comparison, perilla stem extract exhibited the optimal and most balanced synergistic effect. Among the four indicators, it achieved the highest values ​​in T-AOC, TNF-α inhibition, and SOD activity enhancement. Although its increase in IL-6 was slightly less than that of perilla seeds, its increase in SOD activity was significantly higher (approximately 30%), effectively compensating for the latter's weakness in activating endogenous antioxidant enzymes. Given that perilla stem extract is rich in rosmarinic acid, apigenin, and other components, its combination with *Micrococcus pluvialis* powder and turmeric extract demonstrates a more comprehensive and efficient synergistic biological activity than that of perilla seeds.

[0069] In summary, the perilla stem extract exhibits the best broad-spectrum and synergistic effect in the antioxidant and anti-inflammatory pathways, and has been identified as the optimal third active component of this invention.

[0070] This comparative example provides a compound oral liquid of plant extracts, the formulation and preparation method of which are basically the same as those of Example 3, except that: instead of using the turmeric extract inclusion complex prepared in Example 1, an equal amount of unincluded turmeric extract powder is directly added (i.e., 6g of turmeric extract powder is directly added to replace the 12.85g of turmeric extract powder in the inclusion complex prepared in Example 1), and the other excipients and process parameters are the same as those of Example 3.

[0071] This experiment investigated the stability and taste of the oral liquids prepared in Example 3 and Comparative Example 1 to observe the effect of the encapsulation process on the product stability and taste.

[0072] 3.1 Stability testing method: After the samples were filled and sealed, they were placed in a 60℃ constant temperature incubator for accelerated testing, with samples taken at 0 days, 10 days, and 30 days. The evaluation indicators included: Appearance characteristics: Visually inspect the sample for color, uniformity, and precipitation. pH value: Measured using a pH meter; Curcumin content retention rate: Curcumin content was determined by HPLC, and the retention rate was calculated with the content at day 0 as 100%. Three batches of samples were taken at each time point, and each batch was measured three times.

[0073] 3.2 Taste evaluation method: Twenty sensory evaluation volunteers (10 men and 10 women, aged 22-45) were recruited. A 5-point scoring method was used (5 points - good taste, no off-flavor; 4 points - good taste, slightly medicinal taste; 3 points - average taste; 2 points - poor taste, bitter taste; 1 point - poor taste, strong bitter taste). At room temperature, 10 mL of the sample was held in the mouth for 10 seconds and then spat out. The mouth was rinsed with water. The next sample was evaluated after a 5-minute interval.

[0074] The average score was recorded for all experimental results, and the test results are shown in Table 3: Table 3. Results of stability and mouthfeel tests

[0075] Note: Taste ratings are only evaluated on day 0 (n=20); other indicators are evaluated on day n=3.

[0076] Comparing the two sets of data, Example 3 outperformed Comparative Example 1 in terms of physical stability, curcumin chemical stability, and taste score. After 30 days of accelerated testing, the system in Example 3 was homogeneous, with no precipitation or stratification, a curcumin retention rate of 98.4%, and a stable pH of around 5.0, indicating good product stability. In contrast, Comparative Example 1, due to the lack of inclusion of curcumin extract, showed uneven suspension and a grainy texture in its oral liquid system as early as day 0. After 30 days, it exhibited obvious stratification, with hardened sediment at the bottom, a darkened color, and a curcumin retention rate of only 85.3%, indicating poor stability and a significant decrease in pH. Regarding taste, Example 3 received an average score of 4.6, indicating a good taste; Comparative Example 1 received an average score of 2.8, mainly characterized by a pronounced bitterness and a prominent curcumin flavor.

[0077] The results indicate that the process of encapsulating turmeric extract with hydroxypropyl-β-cyclodextrin and combining it with xanthan gum and sodium carboxymethyl cellulose as a suspending agent helps improve the stability and taste of the oral liquid.

[0078] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A binary active ingredient composition, characterized in that, It is composed of microalgae powder and turmeric extract, wherein the weight ratio of microalgae powder to turmeric extract is (3-5):

1.

2. The binary active ingredient composition according to claim 1, characterized in that, The weight ratio of the *Micrococcus pseudocarpa* powder to the turmeric extract is (3.6–4.4):

1.

3. The binary active ingredient composition according to claim 2, characterized in that, The weight ratio of the *Micrococcus pseudocarpa* powder to the turmeric extract is 4:

1.

4. A ternary active ingredient composition, characterized in that, The composition comprises the binary active ingredient composition according to any one of claims 1-3 and perilla stem extract.

5. The ternary active ingredient composition according to claim 4, characterized in that, The perilla stem extract is an extract obtained by extracting perilla stems with an ethanol-water solution.

6. A compound oral liquid containing plant extracts of *Micrococcus pseudocarpa* powder, characterized in that, The oral liquid contains microalgae powder, turmeric extract and perilla stem extract, wherein the weight ratio of microalgae powder to turmeric extract is (3-5):1, and the turmeric extract is added in the form of hydroxypropyl-β-cyclodextrin inclusion complex.

7. The compound oral liquid according to claim 6, characterized in that, It also includes any one or more of suspending agents, wetting agents, preservatives, flavoring agents, and water.

8. The compound oral liquid according to claim 7, characterized in that, The suspending agent is a combination of xanthan gum and sodium carboxymethyl cellulose, and the wetting agent is glycerin.

9. The compound oral liquid according to claim 7, characterized in that, Based on 1000mL of oral solution, the components and dosages are as follows: 20-30g of *Micrococcus pseudocarpa* powder, 4-6g of turmeric extract, 4-6g of perilla stem extract; 1-2g of xanthan gum, 2-4g of sodium carboxymethyl cellulose, 30-50g of glycerin, 0.8-1.5g of potassium sorbate, 1-2g of vitamin C, 0.5-1g of vitamin E, 0.2-0.6g of sucralose, 1-2mL of strawberry flavoring, and purified water to make up to 1000mL. The pH is adjusted to 4.8-5.2 with citric acid.

10. A method for preparing a plant extract compound oral liquid containing *Micrococcus pseudocarpa* powder as described in any one of claims 6-9, characterized in that, Includes the following steps: (1) Turmeric extract was included with hydroxypropyl-β-cyclodextrin to obtain turmeric extract inclusion complex; (2) Take perilla stems and extract them by reflux with ethanol aqueous solution. The extract is concentrated and dried to obtain perilla stem extract; (3) Add the microalgae powder, the turmeric extract inclusion complex from step (1) and the perilla stem extract from step (2) to glycerol and stir to obtain suspension A; (4) Dissolve the suspending agent in heated purified water and disperse it by shearing to obtain colloidal solution B; (5) Add suspension A to colloidal solution B and mix, then add preservatives, flavoring agents and other excipients, mix evenly, make up to volume with purified water, adjust pH, filter, fill and sterilize to obtain the final product.

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