Preparation process and application of uric acid-reducing anoectochilus formosanus extract

Abstract

The invention discloses an extraction process of a uric acid-reducing anoectochilus roxburghii extract and application of the uric acid-reducing anoectochilus roxburghii extract in preparation of uric acid-reducing medicines or health-care products. According to the method, the anoectochilus formosanus is subjected to water extraction and alcohol extraction synergic extraction, so that multi-component functional substances such as polysaccharide, amino acid, kinsenoside and flavone can be enriched at the same time, and the yield of the functional components and the uric acid reducing effect of the functional components can be remarkably improved. The process is simple, efficient and suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine extracts, and particularly relates to a preparation process and application of a uric acid-lowering roxburghii extract. Background Art

[0002] Hyperuricemia not only causes gout, but also induces a variety of diseases such as hypertension, diabetes, coronary heart disease and renal failure, and has received widespread attention in the medical community.

[0003] Hyperuricemia is related to uric acid levels in the body. Under normal circumstances, the body's daily production and excretion of uric acid maintain a dynamic balance. Any factors that affect uric acid production or excretion can lead to elevated blood uric acid levels. The kidneys are a key organ for uric acid excretion. A decrease in renal creatinine clearance by 5% to 20% can lead to hyperuricemia.

[0004] At present, the main ways to reduce uric acid levels in the human body include: 1) reducing the production of uric acid from the source and inhibiting the activity of key enzymes such as xanthine oxidase (XOD); 2) increasing the excretion of uric acid and regulating the induced expression of urate transporters.

[0005] Currently, while commonly used medications for the prevention and treatment of HUA can significantly lower blood uric acid levels, long-term use is associated with widespread toxic side effects such as liver and kidney damage and allergic reactions. Patient compliance is poor, and relapses are common after discontinuation. Screening for highly effective, safe, and nontoxic uric acid-lowering active ingredients from natural products has become a new research direction, possessing significant scientific significance and practical value for the prevention and improvement of HUA.

[0006] Anoectochilus roxburghii is a perennial herb of the genus Anoectochilus in the Orchidaceae family. It can effectively improve the kidney function of hyperuricemia mice, accelerate the excretion of uric acid, and has a certain preventive and therapeutic effect on gout and hyperuricemia. Summary of the Invention

[0007] The present invention aims to provide a new process for preparing a uric acid-lowering extract of Anoectochilus roxburghii. The present invention also provides the use of the extract obtained by the process in preparing a drug or health product for treating hyperuricemia.

[0008] According to the purpose of the present invention, a new preparation process of the above-mentioned uric acid-lowering Anoectochilus roxburghii extract is proposed, comprising the following steps:

[0009] A. Raw material pretreatment: Take the whole plant of Anoectochilus roxburghii, dry it, grind it into ultrafine powder, sieve it and set aside;

[0010] B. The raw material obtained in step A is extracted with an alcohol solution at a material-liquid mass-to-volume ratio of 1:15-45, and subjected to heating and reflux extraction for 1-3 times, each time for 0.5-3 hours. The extract is filtered and the filtrate is combined;

[0011] C. Add water to the residue obtained after filtering the extract in step B, heat and extract 1 to 3 times, each time for 1 to 3 hours, and combine the filtrates;

[0012] D. The filtrate obtained in steps B and C is subjected to membrane separation, the permeate is collected, and spray-dried.

[0013] In some embodiments of the present invention, in the preparation method, in step A of sieving, the mesh number of the sieve is 16 to 25 meshes, preferably, the mesh number of the sieve is 20 meshes;

[0014] In some embodiments of the present invention, in step B, the concentration of the extraction medium alcohol solution is 70-90%, preferably, the concentration of the alcohol solution is 80%; the mass-to-volume ratio of the material to the liquid is 1:20-40, preferably, the mass-to-volume ratio of the material to the liquid is 1:30; heating reflux extraction is performed twice, each time for 1 hour;

[0015] In some embodiments of the present invention, in the preparation method, in step C, the mass volume ratio of the material to the liquid is 1:20 to 40, preferably, the mass volume ratio of the material to the liquid is 1:30; the heating temperature is 100° C., and the extraction time is 100 minutes;

[0016] In some embodiments of the present invention, in step D of the preparation method, the membrane is a 100 kDa ultrafiltration membrane, which retains target components with a molecular weight of 1-10 kDa.

[0017] In some embodiments of the present invention, the preparation method may encapsulate the components obtained in step D by nanoliposomes.

[0018] The roxburghii extract of the present invention obtained by the above-mentioned preparation process is added with various conventional excipients required for preparing different dosage forms, such as disintegrants, lubricants, adhesives, etc., and prepared into any commonly used oral preparations, such as tablets, capsules, etc., using conventional Chinese medicine preparation methods, and can be used to treat hyperuricemia.

[0019] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: compared with the prior art, the present invention simultaneously enriches multiple active ingredients such as polysaccharides, amino acids, anoectochilus glycosides and flavonoids through the synergistic extraction of water extraction and alcohol extraction, which not only greatly improves the extraction rate of the obtained extract, but also significantly improves its uric acid-lowering effect and liver and kidney protection effect. This shows that the extraction process of the present invention has lower cost and less energy consumption, and is more conducive to large-scale industrial production. At the same time, the product obtained by the extraction process of the present invention is more effective in treating hyperuricemia. DETAILED DESCRIPTION

[0020] Example 1

[0021] The whole plant of Anoectochilus roxburghii is dried (to a water content of ≤8%), ultrafinely ground to obtain ultrafine powder, and passed through a 20-mesh sieve; 100 g of the Anoectochilus roxburghii ultrafine powder is added to 3000 mL of 80% ethanol, heated for extraction at 70°C±2°C, extracted twice for 1 hour each time, filtered, and the two filtrates are combined to obtain an alcohol extract for standby use; purified water is then added to the alcohol-extracted medicinal residue at a material-liquid ratio of 1:30, heated for extraction at 100°C±5°C, extracted twice for 100 minutes each time, filtered, and the two filtrates are combined to obtain a water extract for standby use; the alcohol extract and the water extract are combined, and membrane separation and purification is performed using an ultrafiltration system, the permeate (target component with a molecular weight of 1-10 kDa) is collected, and spray-dried to obtain a light yellow powder, i.e., the extract of the present invention, wherein the polysaccharide retention rate is ≥95% and the yield is 36.85%.

[0022] Example 2

[0023] The whole plant of Anoectochilus roxburghii is dried (to a water content of ≤8%), ultrafinely ground to obtain ultrafine powder, and passed through a 25-mesh sieve; 100 g of the Anoectochilus roxburghii ultrafine powder is added to 4000 mL of 75% ethanol, heated for extraction at 60°C±2°C, extracted three times for 0.5 hour each time, filtered, and the filtrates from the three extractions are combined to obtain an alcohol extract for standby use; purified water is then added to the alcohol-extracted medicinal residues at a material-liquid ratio of 1:20, heated for extraction at 100°C±5°C, extracted for 3 hours, filtered, and the filtrate is collected to obtain an aqueous extract for standby use; the alcohol extract and the aqueous extract are combined, membrane separation and purification is performed using an ultrafiltration system, the permeate (target component with a molecular weight of 1-10 kDa) is collected, spray-dried to obtain a light yellow powder, i.e., the extract of the present invention, wherein the polysaccharide retention rate is ≥95% and the yield is 35.12%.

[0024] Example 3

[0025] The whole plant of Anoectochilus roxburghii is dried (to a water content of ≤8%), ultrafinely ground to obtain ultrafine powder, and passed through a 16-mesh sieve; 100 g of the Anoectochilus roxburghii ultrafine powder is added to 2000 mL of 90% ethanol for heating extraction at 70°C±2°C for 0.5 hour, filtered, and the filtrate is collected to obtain an alcohol extract for later use; purified water is then added to the alcohol-extracted medicinal residue at a material-liquid ratio of 1:40, and the extraction is performed three times by heating at 100°C±5°C for 1 hour, filtered, and the three filtrates are combined to obtain a water extract for later use; the alcohol extract and the water extract are combined, and membrane separation and purification is performed using an ultrafiltration system, and the permeate (target component with a molecular weight of 1-10 kDa) is collected, spray-dried to obtain a light yellow powder, and encapsulated by nanoliposomes to obtain the extract of the present invention, wherein the polysaccharide retention rate is ≥95% and the yield is 38.39%.

[0026] Example 4

[0027] Comparative test on the extraction yield of extracts from Anoectochilus roxburghii obtained by different methods

[0028] Water extraction process: take the whole plant of Anoectochilus roxburghii and dry it (to a moisture content of ≤8%), then ultrafinely grind it to obtain ultrafine powder, and pass it through a 16-mesh sieve; take 100g of Anoectochilus roxburghii ultrafine powder, add 4000ml of water, and heat extract at an extraction temperature of 100℃±5℃, extract twice, each time for 100 minutes and boil for 1.5 hours, and pour out the extract; combine the two filtrates to obtain a water extract for use; use an ultrafiltration system for membrane separation and purification, collect the permeate (target component with a molecular weight of 1-10kDa), spray dry to obtain a light yellow powder, i.e., Sample 1, with a yield of 20.96%.

[0029] Alcohol extraction process: take the whole plant of Anoectochilus roxburghii and dry it (to a moisture content of ≤8%), then ultrafinely grind it to obtain ultrafine powder, and pass it through a 16-mesh sieve; take 100 g of Anoectochilus roxburghii ultrafine powder, add 2000 mL of 80% ethanol, and heat and extract at 70°C ± 2°C, extract twice, each for 1 hour, filter, and combine the two filtrates to obtain an alcohol extract, use an ultrafiltration system for membrane separation and purification, collect the permeate, and spray dry to obtain a light yellow powder, i.e., Sample 2, with a yield of 15.14%.

[0030] Water-alcohol composite extraction process: Take the sample of Example 3 to obtain Sample 3.

[0031] After calculation, the extraction yields of each extraction method of Anoectochilus roxburghii are shown in Table 1.

[0032] Table 1 Comparison of extraction yields of various extraction methods of Anoectochilus roxburghii

[0033] Sample 1 (water extraction) Sample 2 (alcohol extraction) Sample 3 (water-alcohol combined extraction) Yield 20.96％ 15.14％ 38.39％

[0034] The results in Table 1 above show that different extraction methods of gold thread lead to very obvious differences in extraction yields. The extraction process of the present invention has a higher extraction yield than that of water extraction or alcohol extraction alone.

[0035] Example 5

[0036] The extracts obtained by different extraction methods of Anoectochilus roxburghii in Example 4 were used to conduct a comparative test on the uric acid reduction rate of each extract on hyperuricemic zebrafish

[0037] 1. Experimental Animals

[0038] AB wild-type zebrafish were maintained under standard conditions. Adult zebrafish were maintained in a centralized breeding and rearing system at 28°C with a daily 14:10 light-dark cycle. Zebrafish were maintained in a flowing water environment with a conductivity of 550-650 μs / cm and a pH of 7.2-7.6. Collected zebrafish embryos were incubated in a 28°C constant-temperature incubator with programmable lighting. Adult zebrafish were fed at least twice daily with hatched, molted brine shrimp. Embryonic stages are expressed in hours post-fertilization (hpf) or days post-fertilization (dpf).

[0039] 2. Drug configuration

[0040] Weigh 156.136 mg of potassium oxonate and 696.36 μg of xanthine sodium salt, add them to 80 mL of fish culture water, and heat and stir until dissolved.

[0041] 3. Experimental methods

[0042] 5dpf wild-type AB strain zebrafish were randomly selected in a 12-well plate, with four replicates per well (15 tails / well) and a capacity of 4mL per well. Different concentrations of drugs were treated simultaneously with 10mmol / L potassium oxonate and 50μmol / L xanthine sodium salt for 20h. The model group was treated only with potassium oxonate and xanthine sodium salt. Except for the normal control group, the other experimental groups were given potassium oxonate and xanthine sodium salt in water to establish a zebrafish hyperuricemia model. The normal group, model group, and drug group were administered by water-soluble immersion (drug concentration: low concentration of 62.5μg / mL, medium concentration of 125μg / mL, high concentration of 250μg / mL) and treated in a 28°C incubator for 20h. The following tests were performed:

[0043] A. Uric acid test kit

[0044] A uric acid content detection kit was used to collect data using a multifunctional microplate reader software to analyze the uric acid content in zebrafish, and the uric acid-lowering efficacy was evaluated based on the statistical analysis results of this indicator.

[0045] The absorbance measurement method is as follows: Place the reaction solution in a 1 mL glass cuvette and measure the absorbance at 505 nm. Record these values ​​as A, A control, A standard, and A blank. Calculate ΔA as = A - A control, and ΔA standard = A standard - A blank. Note: A control tube is required for each sample; the blank tube only needs to be measured once or twice.

[0046] Establishment of standard curve: Using 0.25, 0.2, 0.1, 0.05, 0.025, and 0.0125 μmol / mL as the abscissa (x) and the corresponding AA standard as the ordinate (y), obtain the standard equation y = kx + b. Substitute the AA determination into the equation to obtain x (μmol / mL). Calculate uric acid content according to tissue sample mass:

[0047]

[0048] (Note: V: total volume of the sample to be tested, 1 mL; W: sample weight, g; MUA: molecular weight of uric acid, 168)

[0049] The uric acid content was determined and calculated according to the above method. The test results are shown in Table 2.

[0050] Table 2 Uric acid reduction rate of extracts obtained from different extraction methods of Anoectochilus roxburghii in hyperuricemic zebrafish

[0051]

[0052] From the experimental results in Table 2, it can be seen that the extract obtained by the extraction process of the present invention has a significantly higher uric acid reduction rate in hyperuricemia zebrafish than the extract obtained by only water extraction or alcohol extraction, p<0.05, which is a significant difference. That is, the product obtained by the extraction process of the present invention is significantly superior to other products in the treatment of hyperuricemia.

[0053] B. RT-PCR detection of related gene expression

[0054] RNA was extracted from zebrafish brain tissue using Trizol reagent. Total RNA was reverse transcribed into cDNA using a reverse transcription kit. The reaction was performed on a real-time PCR instrument using a SYBR premix Taq kit. -△△Ct The relative mRNA expression levels of related genes in zebrafish were calculated using the PCR method. Primers were designed using Primer5 software, and β-actin was used as an internal reference. The experimental results are shown in Table 3.

[0055] Table 3 RT-PCR detection results of GLUT9 gene of each sample

[0056]

[0057] As shown in Table 3, compared with the model group, the transporter GLUT9 values ​​of all sample concentration groups were significantly reduced. The transporter GLUT9 (glucose transporter 9) is involved in the reabsorption of urate by the renal tubules, allowing urate to be reabsorbed from the renal tubular cell membrane back into the blood, thereby causing an increase in blood uric acid concentration; therefore, abnormal expression of GLUT9 is closely related to the occurrence of hyperuricemia.

[0058] In summary, Anoectochilus roxburghii can significantly reduce uric acid in zebrafish with hyperuricemia model, and its mechanism may be related to reducing GLUT9 expression and promoting uric acid excretion.

Claims

1. A preparation process of a uricosuric acid-lowering roxburghii extract, comprising the following steps: A. Raw material pretreatment: Take the whole plant of Anoectochilus roxburghii, dry it, grind it into ultrafine powder, sieve it and set aside; B. The raw material obtained in step A is extracted with an alcohol solution at a material-liquid mass-to-volume ratio of 1:15-45, and subjected to heating and reflux extraction for 1-3 times, each time for 0.5-3 hours. The extract is filtered and the filtrate is combined; C. Add water to the residue obtained after filtering the extract in step B, heat and extract 1 to 3 times, each time for 12 to 3 hours, and combine the filtrates; D. The filtrate obtained in steps B and C is subjected to membrane separation, the permeate is collected, and spray-dried to obtain a light yellow powder.

2. The preparation process according to claim 1, wherein In step A, the mesh number of the sieve is 16 to 25 meshes, and preferably, the mesh number of the sieve is 20 meshes.

3. The preparation process according to claim 1, wherein In step B, the concentration of the extraction medium alcohol solution is 70-90%, preferably, the concentration of the alcohol solution is 80%; the mass volume ratio of the material to the liquid is 1:20-40, preferably, the mass volume ratio of the material to the liquid is 1:30; heating reflux extraction is performed twice, each time for 1 hour.

4. The preparation process according to claim 1, wherein In step C, the mass-to-volume ratio of the material to the liquid is 1:20-40, preferably, the mass-to-volume ratio of the material to the liquid is 1:30; the heating temperature is 100° C., and the extraction time is 100 minutes.

5. The preparation process according to claim 1, wherein In step D, the membrane is a 100 kDa ultrafiltration membrane, which retains target components with a molecular weight of 1-10 kDa.

6. The preparation process according to claim 1, wherein The components obtained in step D are encapsulated by nanoliposomes.

7. The uric acid-lowering Roxburghii extract obtained by the preparation process according to claim 1.

8. Use of the Roxb. roxb. extract according to claim 7 in the preparation of drugs or health products for treating hyperuricemia.

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