Gastrodia elata and medlar fruit extract, and preparation method and application thereof

Abstract

The application discloses a Gastrodia elata-Wolfberry extract as well as a preparation method and application thereof, and belongs to the field of traditional Chinese medicine. The preparation method of the Gastrodia elata-Wolfberry extract comprises the following steps: (1) mixing Gastrodia elata and Wolfberry, crushing into powder, sieving, and obtaining a mixture powder; (2) adding water and a compound enzyme I, performing enzymolysis extraction under high-intensity ultrasonic waves with a power of 900-1800 W, obtaining a first enzymolysis liquid, centrifuging, and obtaining a first supernatant; (3) adding a compound enzyme II, performing enzymolysis extraction under high-intensity ultrasonic waves with a power of 900-1800 W, obtaining a second enzymolysis liquid, centrifuging, and obtaining a Gastrodia elata-Wolfberry extract liquid; and (4) freeze-drying to obtain the Gastrodia elata-Wolfberry extract. The compound enzyme I and the compound enzyme II are used to perform double enzymolysis on the Gastrodia elata and the Wolfberry, and high-intensity ultrasonic waves with a power of 900-1800 W are used to assist in the enzymolysis process, so that the nutritional components and effective active components in the Gastrodia elata and the Wolfberry can be effectively extracted, the enzyme consumption is reduced, and the extraction time is shortened.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, and in particular to a gastrodia-lycium extract, its preparation method, and its application. Background Technology

[0002] Hypertension (HTN) is a chronic disease characterized by persistently elevated systemic arterial blood pressure and is a major risk factor for cardiovascular and cerebrovascular diseases (CCVDs), leading in global mortality and morbidity. HTN patients often experience vascular damage, which frequently serves as an early sign of structural changes in target organs such as the heart, brain, kidneys, and large blood vessels, causing further harm. Therefore, treating vascular damage caused by hypertension and protecting blood vessels is crucial for reducing the risk of complications such as myocardial infarction, stroke, and cardiovascular death caused by HTN. In Western medicine, ACE inhibitors are the main antihypertensive drugs with vascular-protective effects. Besides lowering blood pressure, these drugs also improve ventricular remodeling, reduce endothelin and NO levels, thereby protecting blood vessels. However, these drugs have adverse reactions such as dry cough and laryngeal edema, and are contraindicated in cases of renal insufficiency and renal artery stenosis. In contrast, traditional Chinese medicine is highly praised for its natural, environmentally friendly, low-toxicity, fewer side effects, and good drug resistance.

[0003] Gastrodia elata and Lycium barbarum are two medicinal herbs widely used to treat cardiovascular and cerebrovascular diseases, and they are often included in compound preparations for lowering blood pressure. In the extraction and preparation of traditional Chinese medicine, methods such as decoction or soaking are often used, which are not only time-consuming and labor-intensive, but also have low extraction efficiency. Some effective active ingredients cannot be extracted, and in some cases, prolonged decoction or soaking can cause the medicinal effects to dissipate, resulting in a significant reduction in the effectiveness of treating hypertension and protecting blood vessels. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a Gastrodia elata-Lycium barbarum extract, its preparation method and application, to overcome the problems of time-consuming and laborious preparation of traditional Chinese medicine extracts and low extraction efficiency of effective active ingredients in the prior art.

[0005] In a first aspect, the present invention provides a method for preparing a Gastrodia elata-Lycium barbarum extract, comprising the following steps:

[0006] (1) Mix Gastrodia elata and Lycium barbarum, crush them into powder, and sieve them to obtain the mixture powder;

[0007] (2) After adding water and compound enzyme I to the mixture powder, enzymatic hydrolysis extraction is performed under high intensity ultrasound of 900~1800W to obtain the first enzymatic hydrolysate, and centrifugation is performed to obtain the first supernatant.

[0008] (3) After adding compound enzyme II to the first supernatant, enzymatic hydrolysis extraction is performed under high intensity ultrasound of 900-1800W to obtain the second enzymatic hydrolysate. After centrifugation, the second supernatant obtained is the Gastrodia elata-Lycium barbarum extract.

[0009] The complex enzyme II includes at least two of α-L-arabinofuranase, α-glucuronidase, and papain.

[0010] (4) Freeze-dry the Gastrodia elata-Lycium barbarum extract to obtain Gastrodia elata-Lycium barbarum extract.

[0011] Compared with existing technologies, this invention first pulverizes two traditional Chinese medicinal materials, Gastrodia elata and Lycium barbarum, then adds compound enzyme I for cell wall breaking extraction. After enzymatic hydrolysis, a specific type of compound enzyme II is added to perform a second enzymatic hydrolysis of the extract. The synergistic effect of α-L-arabinofuranylase, α-glucuronidase, and papain enables deep enzymatic hydrolysis of Gastrodia elata and Lycium barbarum cells. This double enzymatic hydrolysis not only fully releases the effective active ingredients from the cells of Gastrodia elata and Lycium barbarum, but compound enzyme II also further breaks down large molecular nutrients that are difficult to absorb, facilitating nutrient absorption. Furthermore, this invention incorporates high-intensity ultrasonic treatment (900-1800W) during both enzymatic hydrolysis processes, further improving hydrolysis efficiency, reducing enzyme dosage, significantly shortening extraction time, and lowering costs.

[0012] Furthermore, the mass ratio of Gastrodia elata to Lycium barbarum is (1~3):1.

[0013] The above technical solution further limits the mass ratio of Gastrodia elata and Lycium barbarum. Within the above mass ratio range, especially when Gastrodia elata and Lycium barbarum are combined at a mass ratio of 2:1, the two have the best synergistic effect, and the resulting Gastrodia elata-Lycium barbarum extract has a significantly enhanced protective effect on blood vessels.

[0014] Furthermore, compound enzyme I includes cellulase and pectin methyl esterase; the amount of cellulase added is 0.02%~0.05% of the dry weight of the medicinal material, and the amount of pectin methyl esterase added is 0.01%~0.02% of the dry weight of the medicinal material.

[0015] The above technical solution specifies the specific types and amounts of compound enzyme I. Cellulase and pectin methyl esterase can separate medicinal plant cells from the tissue, decompose cell wall cellulose into proteins containing oligosaccharides or monosaccharides, improve the extraction rate of effective active ingredients, and at the same time reduce the content of suspended undecomposed cell wall fragments and other high molecular weight substances in the extract, reduce viscosity, reduce aggregation and precipitation, thereby enhancing the physical stability of the extract.

[0016] Furthermore, in step (2), the pH of the enzymatic hydrolysis is 3-5, the temperature is 4-10℃, and the time is 60-180s;

[0017] The high-intensity ultrasound processing mode is as follows: under the conditions of 20~40kHz and 40%~60% amplitude, work for 3~5 seconds and then pause for 2~3 seconds.

[0018] The above technical solution further specifies the enzymatic hydrolysis reaction conditions of compound enzyme I and the high-intensity ultrasound treatment mode. Under the above conditions, the enzymatic hydrolysis reaction can ensure the enzyme activity and catalytic efficiency of cellulase and pectin methyl esterase. High-intensity ultrasound improves the enzymatic hydrolysis efficiency of compound enzyme I through cavitation and microfluidic effects. At the same time, the intermittent treatment time is limited. Under the above intermittent treatment mode, on the one hand, the damage of high-intensity ultrasound to the effective active ingredients in the medicinal materials can be effectively avoided, and on the other hand, the best enzymatic hydrolysis effect can be ensured, so that the active components of the enzymatic catalytic product are extracted more completely.

[0019] Furthermore, the amount of α-L-arabinofuranosidase added is 0.02%~0.05% of the dry weight of the medicinal material;

[0020] The amount of α-glucuronidase added is 0.02%~0.05% of the dry weight of the medicinal material;

[0021] The amount of papain added is 0.02%~0.05% of the dry weight of the medicinal material.

[0022] The above technical solution limits the amount of compound enzyme II to be added. Within the above-mentioned addition range, α-L-arabinofuranylase, α-glucuronidase and papain can better release the effective active ingredients from the cells, further improve the extraction efficiency of the effective active ingredients, decompose protein macromolecules and non-starch polysaccharides that cannot be absorbed by the human body, and facilitate the absorption of nutrients by the human body.

[0023] Furthermore, in step (3), the pH of the enzymatic hydrolysis is 6.5~7.5, the temperature is 4~10℃, and the time is 60~180s;

[0024] The high-intensity ultrasound processing mode is as follows: under the conditions of 20~40kHz and 40%~60% amplitude, work for 3~5 seconds and then pause for 2~3 seconds.

[0025] The above technical solution further defines the enzymatic hydrolysis reaction conditions of compound enzyme II, providing a suitable reaction environment for the enzymatic hydrolysis of compound enzyme II. The cavitation and microjet effects generated by high-intensity ultrasound further improve the enzymatic hydrolysis efficiency of compound enzyme II.

[0026] Furthermore, freeze drying includes the following steps:

[0027] (1) Pre-freeze the Gastrodia elata-Lycium barbarum extract at -4~4℃ in a low-temperature freezer for one day;

[0028] (2) After pre-freezing, the Gastrodia elata-Lycium barbarum extract in the low-temperature freezer is transferred to a vacuum freeze dryer. The vacuum degree in the vacuum freeze dryer is 0~1.0 Pa, the temperature of the partition is 45~60℃, and the heating rate of the partition is 5~7.5℃ / h.

[0029] (3) Dry in a vacuum freeze dryer for 2 to 4 days to obtain Gastrodia elata-Lycium barbarum extract.

[0030] Compared with existing technologies, the above technical solution specifies the concrete steps for freeze-drying the Gastrodia elata-Lycium barbarum extract to obtain the Gastrodia elata-Lycium barbarum extract. The freeze-drying process involves low temperature-sublimation-drying, without high-temperature operations, thus avoiding the loss of amino acids and other effective active ingredients due to excessively high temperatures. This effectively improves the quality of the Gastrodia elata-Lycium barbarum extract while preserving its nutritional components and effective active ingredients. Furthermore, the Gastrodia elata-Lycium barbarum extract powder obtained through the above freeze-drying process is fine, residue-free, and uniform in particle size, exhibiting good flowability and solubility.

[0031] Secondly, the present invention provides a Gastrodia elata-Lycium barbarum extract, prepared by any of the above methods, wherein the active ingredients of the Gastrodia elata-Lycium barbarum extract include one or more of gastrodin, p-hydroxybenzyl alcohol, barisonin A, Lycium barbarum polysaccharide, luteolin or gallic acid.

[0032] Compared with existing technologies, the active ingredients such as gastrodin and p-hydroxybenzyl alcohol in the Gastrodia elata-Lycium barbarum extract of this invention may exert their effects through core targets such as PPARG, NFkB, TNF, JAK2, and BAX, thereby achieving a hypotensive effect. Among them, NFkB has good binding activity with all six core components and may be the core target for the hypotensive effect. Detailed Implementation

[0033] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0034] In a first aspect, embodiments of the present invention provide a method for preparing a Gastrodia elata-Lycium barbarum extract, comprising the following steps:

[0035] S1. Mix Gastrodia elata and Lycium barbarum, crush them into powder, and sieve to obtain the mixture powder.

[0036] In the above steps, the mass ratio of Gastrodia elata and Lycium barbarum is (1~3):1. For example, it can be 1:1, 2:1 or 3:1, preferably 2:1. When the mass ratio of Gastrodia elata and Lycium barbarum is (1~3):1, especially 2:1, the two show a synergistic effect in the treatment of hypertension and vascular damage, and the therapeutic effect is significantly higher than that of using Gastrodia elata alone, using Lycium barbarum alone, or other combinations of Gastrodia elata and Lycium barbarum.

[0037] In the above steps, an ultra-fine pulverizer can be used to pulverize Gastrodia elata and Lycium barbarum. When sieving, a 200-400 mesh sieve can be used to facilitate the subsequent enzymatic hydrolysis reaction.

[0038] S2. After adding water and compound enzyme I to the above-mentioned mixed powder, enzymatic extraction is performed under high-intensity ultrasound at 900~1800W to obtain a first enzymatic hydrolysate. Centrifugation is then performed to obtain a first supernatant. For example, the power of the above-mentioned high-intensity ultrasound can be 900W, 1100W, 1300W, 1500W or 1800W, preferably 1100~1500W.

[0039] The amount of water added in the above steps is 10 to 30 times the dry weight of the medicinal materials.

[0040] Furthermore, the complex enzyme I in the above steps includes cellulase and pectin methylesterase.

[0041] The amount of cellulase added is 0.02% to 0.05% of the dry weight of the medicinal material. For example, it can be 0.02%, 0.03%, 0.04%, or 0.05% of the dry weight of the medicinal material, preferably 0.03% to 0.04% of the dry weight of the medicinal material.

[0042] The amount of pectin methyl esterase added is 0.01%~0.02% of the dry weight of the medicinal material. For example, it can be 0.01% or 0.02% of the dry weight of the medicinal material.

[0043] In the above steps, the cellulase and pectin methyl esterase in compound enzyme I work together to break down cell walls, making the intercellular matrix more loose, separating the medicinal plant cells from the tissue, thereby improving the extraction rate of effective active ingredients and the stability of the extract.

[0044] Furthermore, in the above steps, the enzymatic hydrolysis is performed at a pH of 3-5, a temperature of 4-10℃, and a time of 60-180 seconds; the high-intensity ultrasound treatment mode is as follows: operating at 20-40 kHz and 40%-60% amplitude, with a 2-3 second interval every 3-5 seconds. It should be understood that the enzymatic hydrolysis and high-intensity ultrasound are performed simultaneously.

[0045] For example, the specific operation steps of high-intensity ultrasound in the above steps can be as follows: immerse the amplitude transformer of the ultrasonic generator into the liquid surface by 10-20 mm, and perform ultrasonic treatment for 60-180 seconds in a mode of working for 3-5 seconds and intermittent for 2-3 seconds under the conditions of 20-40 kHz, 900-1800 W, and 40%-60% amplitude.

[0046] A pH value of 3-5 and a temperature of 4-10℃ provide a suitable enzymatic hydrolysis environment for compound enzyme I. Combined with high-intensity ultrasound in intermittent working mode, the efficiency of enzymatic extraction can be further improved, thereby reducing the amount of enzyme used, shortening the enzymatic extraction time, and reducing costs.

[0047] In the above steps, after obtaining the first enzymatic hydrolysate, centrifuge at 6000~10000 r / min for 8~12 min to obtain the first supernatant.

[0048] S3. After adding compound enzyme II to the first supernatant, enzymatic extraction is performed under high-intensity ultrasound at 900-1800W to obtain a second enzymatic hydrolysate. The second supernatant obtained after centrifugation is the Gastrodia elata-Lycium barbarum extract. For example, the power of the high-intensity ultrasound can be 900W, 1100W, 1300W, 1500W, or 1800W, preferably 1100-1500W.

[0049] In the above steps, complex enzyme II includes at least two of α-L-arabinofuranylase, α-glucuronidase, and papain.

[0050] The amount of α-L-arabinofuranosidase added is 0.02% to 0.05% of the dry weight of the medicinal material. For example, it can be 0.02%, 0.03%, 0.04%, or 0.05% of the dry weight of the medicinal material, preferably 0.03% to 0.04% of the dry weight of the medicinal material.

[0051] The amount of α-glucuronidase added is 0.02% to 0.05% of the dry weight of the medicinal material. For example, it can be 0.02%, 0.03%, 0.04%, or 0.05% of the dry weight of the medicinal material, preferably 0.03% to 0.04% of the dry weight of the medicinal material.

[0052] The amount of papain added is 0.02% to 0.05% of the dry weight of the medicinal material. For example, it can be 0.02%, 0.03%, 0.04%, or 0.05% of the dry weight of the medicinal material, preferably 0.03% to 0.04% of the dry weight of the medicinal material.

[0053] The α-L-arabinofuranase, α-glucuronidase, and papain in the above technical solution have a synergistic effect. They can further extract the effective active ingredients in the medicinal materials by deep enzymatic hydrolysis of the first supernatant. At the same time, they can further decompose large protein molecules and non-starch polysaccharides that cannot be absorbed by the human body, and the resulting small molecule nutrients such as amino acids are more easily absorbed by the human body.

[0054] Furthermore, in the above steps, the pH of the enzymatic hydrolysis is 6.5~7.5, the temperature is 4~10℃, and the time is 60~180s;

[0055] The high-intensity ultrasound treatment mode is as follows: operating at 20–40 kHz and 40%–60% amplitude, with a 2–3 second interval every 3–5 seconds. It should be understood that the enzymatic digestion and high-intensity ultrasound are performed simultaneously.

[0056] For example, the specific operation steps of high-intensity ultrasound in the above steps can be as follows: immerse the amplitude transformer of the ultrasonic generator into the liquid surface by 10-20 mm, and perform ultrasonic treatment for 60-180 seconds in a mode of working for 3-5 seconds and intermittent for 2-3 seconds under the conditions of 20-40 kHz, 900-1800 W, and 40%-60% amplitude.

[0057] The above technical solution provides a suitable enzymatic hydrolysis environment for the enzymatic hydrolysis of compound enzyme II, ensuring the enzyme activity and hydrolysis efficiency of compound enzyme II. At the same time, the cavitation and microjet effect of high-intensity ultrasound further accelerates the enzymatic hydrolysis reaction, enabling the extraction of effective active ingredients from medicinal materials in a shorter time.

[0058] After obtaining the second enzymatic hydrolysate through the above steps, centrifuge at 6000~10000 r / min for 8~12 min. The resulting second supernatant is the Gastrodia elata-Lycium barbarum extract.

[0059] S4. Freeze-dry the above-mentioned Gastrodia elata-Lycium barbarum extract to obtain Gastrodia elata-Lycium barbarum extract. The specific steps are as follows:

[0060] S41. Pre-freeze the Gastrodia elata-Lycium barbarum extract in a low-temperature freezer at -4~4℃ for one day.

[0061] The above steps allow the water in the Gastrodia elata-Lycium barbarum extract to form ice crystals, thereby maintaining the integrity of the structure and components of the extract.

[0062] S42. After pre-freezing, transfer the Gastrodia elata-Lycium barbarum extract from the low-temperature freezer to a vacuum freeze dryer. The vacuum degree in the vacuum freeze dryer is 0~1.0 Pa, the partition temperature is 45~60℃, and the heating rate of the partition is 5~7.5℃ / h.

[0063] The above steps set the vacuum level in the vacuum freeze dryer to 0~1.0 Pa, which is an extremely low vacuum environment that can effectively lower the boiling point of water, allowing ice to directly transform from a solid state to a gaseous state (i.e., sublimation) at a relatively low temperature without having to pass through a liquid state.

[0064] The temperature of the partition is set at 45~60℃. This temperature range can ensure rapid sublimation of water while reducing damage to heat-sensitive components in the Gastrodia elata-Lycium barbarum extract.

[0065] The heating rate of the partition is controlled at 5~7.5℃ / h, which can provide heat energy gradually and evenly, avoiding structural damage or component denaturation of the extract due to excessive heating.

[0066] S43. Dry in a vacuum freeze dryer for 2-4 days to obtain the Gastrodia elata-Lycium barbarum extract.

[0067] Under the conditions defined in step S42 above, after the Gastrodia elata-Lycium barbarum extract is dried in a vacuum freeze dryer for 2 to 4 days, most of the water can be removed through the sublimation process, leaving the dried extract while maintaining its original effective active ingredients and nutritional value.

[0068] The freeze-drying process described above yields a gastrodia elata-goji berry extract with high stability, which is easy to store for a long time and suitable for preparing products that require the preservation of active ingredients.

[0069] Secondly, embodiments of the present invention provide a Gastrodia elata-Lycium barbarum extract, prepared using the above method, wherein the active ingredients in the Gastrodia elata-Lycium barbarum extract include one or more of gastrodin, p-hydroxybenzyl alcohol, barisonin A, Lycium barbarum polysaccharide, luteolin, or gallic acid.

[0070] The six active ingredients in the aforementioned technical solution—gastrodin, p-hydroxybenzyl alcohol, barisonin A, Lycium barbarum polysaccharide, luteolin, and gallic acid—may exert their effects through core targets such as PPARG, NF-κB, TNF, JAK2, and BAX, thereby achieving a hypotensive effect by influencing ligand-receptor interactions. Among these, NF-κB exhibits good binding activity with all six active ingredients and is likely a core target for the hypotensive effect. Furthermore, gastrodin and gallic acid have a synergistic effect, exerting their hypotensive effect by modulating the sphingolipid signaling pathway.

[0071] To better illustrate the technical solution of the present invention, the following specific embodiments are also provided. It should be understood that, unless otherwise specified, all raw materials used in the following embodiments are commercially available.

[0072] Example 1

[0073] This embodiment provides a method for preparing Gastrodia elata-Lycium barbarum extract, including the following steps:

[0074] (1) Weigh 10g of Gastrodia elata and 5g of Lycium barbarum, mix them, crush them into powder using an ultra-micro pulverizer, and sieve them through a 300-mesh sieve to obtain the mixture powder.

[0075] (2) Add 20 times the dry weight of the medicinal materials of water, 0.03% of the dry weight of the medicinal materials of cellulase and 0.01% of the dry weight of the medicinal materials of pectin methyl esterase to the above-mentioned mixture powder, adjust the pH of the solution to 4 and the temperature to 7°C. Then, immerse the amplitude transformer of the ultrasonic generator 15 mm below the surface of the above liquid, and perform ultrasonic enzymatic hydrolysis for 120 s in a mode of working for 4 s and intermittent for 2 s under the conditions of 30 kHz, 1300 W and 50% amplitude. Then, centrifuge the enzymatic hydrolysate at 8000 r / min for 10 min to obtain the first supernatant.

[0076] (3) Adjust the pH of the first supernatant to 7, and add 0.03% of the dry weight of the medicinal material α-L-arabinofuranase, 0.03% of the dry weight of the medicinal material α-glucuronidase and 0.03% of the dry weight of the medicinal material papain. Then, immerse the amplitude transformer of the ultrasonic generator 15 mm below the surface of the liquid, and perform ultrasonic enzymatic hydrolysis for 120 s at 30 kHz, 1300 W and 50% amplitude with a working mode of 4 s and an intermittent mode of 2 s. Then, centrifuge the hydrolysate at 8000 r / min for 10 min to obtain the Gastrodia elata-Lycium barbarum extract.

[0077] (4) The above-mentioned Gastrodia elata-Lycium barbarum extract was pre-frozen at 0°C for one day in a low-temperature freezer. The vacuum degree of the vacuum freeze dryer was set to 0.5 Pa, the partition temperature was set to 52°C, and the heating rate was controlled at 6°C / h. The pre-frozen Gastrodia elata-Lycium barbarum extract was transferred to the vacuum freeze dryer and dried for 3 days to obtain Gastrodia elata-Lycium barbarum extract.

[0078] Example 2

[0079] This embodiment provides a method for preparing Gastrodia elata-Lycium barbarum extract, including the following steps:

[0080] (1) Weigh 15g of Gastrodia elata and 5g of Lycium barbarum, mix them, crush them into powder using an ultra-micro pulverizer, and sieve them through a 300-mesh sieve to obtain the mixture powder.

[0081] (2) Add 20 times the dry weight of the medicinal materials of water, 0.02% of the dry weight of the medicinal materials of cellulase and 0.01% of the dry weight of the medicinal materials of pectin methyl esterase to the above-mentioned mixture powder, adjust the pH of the solution to 3 and the temperature to 4℃. Then, immerse the amplitude transformer of the ultrasonic generator 15 mm below the surface of the above liquid, and perform ultrasonic enzymatic hydrolysis for 180 s in a mode of working for 5 s and intermittent for 2 s under the conditions of 20 kHz, 900 W and 40% amplitude. Then, centrifuge the enzymatic hydrolysate at 8000 r / min for 10 min to obtain the first supernatant.

[0082] (3) Adjust the pH of the first supernatant to 6.5, and add 0.02% of the dry weight of the medicinal material α-L-arabinofuranase, 0.05% of the dry weight of the medicinal material α-glucuronidase and 0.02% of the dry weight of the medicinal material papain. Then, immerse the amplitude transformer of the ultrasonic generator 15 mm below the surface of the liquid, and perform ultrasonic enzymatic hydrolysis for 60 s in a mode of working for 3 s and intermittent for 3 s under the conditions of 40 kHz, 1800 W and 60% amplitude. Then, centrifuge the enzymatic hydrolysate at 8000 r / min for 10 min to obtain the Gastrodia elata-Lycium barbarum extract.

[0083] (4) The above-mentioned Gastrodia elata-Lycium barbarum extract was pre-frozen in a low-temperature freezer at -4°C for one day. The vacuum degree of the vacuum freeze dryer was set to 0Pa, the partition temperature was set to 45°C, and the heating rate was controlled at 5°C / h. The pre-frozen Gastrodia elata-Lycium barbarum extract was transferred to a vacuum freeze dryer and dried for 4 days to obtain Gastrodia elata-Lycium barbarum extract.

[0084] Example 3

[0085] This embodiment provides a method for preparing Gastrodia elata-Lycium barbarum extract. The difference between this method and Example 1 is that:

[0086] (1) Weigh 5g of Gastrodia elata and 5g of Lycium barbarum, mix them, crush them into powder using an ultra-micro pulverizer, and sieve them through a 300-mesh sieve to obtain the mixture powder.

[0087] (2) Add 20 times the dry weight of the medicinal materials of water, 0.05% of the dry weight of the medicinal materials of cellulase and 0.02% of the dry weight of the medicinal materials of pectin methyl esterase to the above-mentioned mixture powder, adjust the pH of the solution to 3 and the temperature to 10℃. Then, immerse the amplitude transformer of the ultrasonic generator 15 mm below the surface of the above liquid, and perform ultrasonic enzymatic hydrolysis for 60 s in a mode of working for 3 s and intermittent for 3 s under the conditions of 40 kHz, 1800 W and 60% amplitude. Then, centrifuge the enzymatic hydrolysate at 8000 r / min for 10 min to obtain the first supernatant.

[0088] (3) Adjust the pH of the first supernatant to 7.5, and add 0.02% of the dry weight of the medicinal material α-L-arabinofuranase, 0.05% of the dry weight of the medicinal material α-glucuronidase and 0.05% of the dry weight of the medicinal material papain. Then, immerse the amplitude transformer of the ultrasonic generator 15 mm below the surface of the liquid, and perform ultrasonic enzymatic hydrolysis for 180 s at 20 kHz, 900 W and 40% amplitude with a working mode of 5 s and an intermittent mode of 2 s. Then, centrifuge the hydrolysate at 8000 r / min for 10 min to obtain the Gastrodia elata-Lycium barbarum extract.

[0089] (4) The above-mentioned Gastrodia elata-Lycium barbarum extract was pre-frozen at 4°C for one day in a low-temperature freezer. The vacuum degree of the vacuum freeze dryer was set to 1.0 Pa, the partition temperature was set to 60°C, and the heating rate was controlled at 7.5°C / h. The pre-frozen Gastrodia elata-Lycium barbarum extract was transferred to the vacuum freeze dryer and dried for 2 days to obtain Gastrodia elata-Lycium barbarum extract.

[0090] Comparative Example 1

[0091] The Gastrodia elata-Lycium barbarum extract was prepared according to the method in Example 1, except that Lycium barbarum was replaced with an equal amount of Gastrodia elata, i.e., 15g of Gastrodia elata and 0g of Lycium barbarum were weighed.

[0092] Comparative Example 2

[0093] The Gastrodia elata-Lycium barbarum extract was prepared according to the method in Example 1, except that the Gastrodia elata was replaced with an equal amount of Lycium barbarum, i.e., 0g of Gastrodia elata and 15g of Lycium barbarum were weighed.

[0094] Comparative Example 3

[0095] The Gastrodia elata-Lycium barbarum extract was prepared according to the method in Example 1, except that the intermittent working mode of high-intensity ultrasound in the two enzymatic hydrolysis processes was replaced with the normal working mode, that is, uninterrupted ultrasonic enzymatic hydrolysis for 120s under the conditions of 30kHz, 1300W, and 50% amplitude.

[0096] Comparative Example 4

[0097] The Gastrodia elata-Lycium barbarum extract was prepared according to the method of Example 1, except that the high-intensity ultrasound in steps (2) and (3) was replaced with ordinary ultrasound, and the power of ordinary ultrasound was set to 400W.

[0098] Comparative Example 5

[0099] The Gastrodia elata-Lycium barbarum extract was prepared according to the method of Example 1, except that the freeze drying in step (4) was replaced by spray drying using a spray dryer with an inlet air temperature of 160°C and an outlet air temperature of 95°C.

[0100] Comparative Example 6

[0101] The Gastrodia elata-Lycium barbarum extract was prepared using the traditional water decoction method, and the specific steps are as follows:

[0102] Weigh 10g of Gastrodia elata and 5g of Lycium barbarum, mix them, and crush them into powder using an ultrafine pulverizer. Sieve the powder through a 300-mesh sieve to obtain the mixed powder. Add 20 times the dry weight of the herbs to the mixed powder, boil for 90 minutes, stirring once every 15 minutes, filter, add the same weight of water to the residue and continue boiling, repeat 3 times, combine the filtrates, and concentrate using a rotary evaporator to obtain Gastrodia elata-Lycium barbarum extract powder.

[0103] Comparative Example 7

[0104] The Gastrodia elata-Lycium barbarum extract was prepared according to the method of Example 1, except that only the enzymatic hydrolysis in step (2) was performed and not the enzymatic hydrolysis in step (3).

[0105] Comparative Example 8

[0106] The Gastrodia elata-Lycium barbarum extract was prepared according to the method in Example 1, except that the α-L-arabinofuranase in step (3) was replaced with papain in equal amounts.

[0107] Comparative Example 9

[0108] The Gastrodia elata-Lycium barbarum extract was prepared according to the method in Example 1, except that the α-glucuronidase in step (3) was replaced with papain in equal amounts.

[0109] Test case

[0110] Since angiotensin II can cause abnormal proliferation of vascular smooth muscle cells (VSMCs), angiotensin II was used to establish the model in this test case.

[0111] Weigh angiotensin II powder and dissolve it in sterile ultrapure water to prepare a 1 μM / L angiotensin II solution to induce vascular smooth muscle cells (VSMCs) for 24 h. The specific steps are as follows:

[0112] Vascular smooth muscle cells were cultured in DMEM medium (pH 7.5) containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture (100 U / ml penicillin and 0.1 mg / ml streptomycin) at 37°C and 5% CO2 saturated humidity. Vascular smooth muscle cells in the logarithmic growth phase were selected and digested with 0.25% trypsin at a concentration of 2 × 10⁶ cells / mL. 4Cell suspensions were prepared at a density of 100 μL per cell and seeded into 96-well plates (100 μL per well). The plates were then incubated at 37°C and 5% CO2 for 24 h. Angiotensin II solution at a concentration of 1 μM / L (100 μL per well) was added, and the plates were incubated at 37°C and 5% CO2 for another 24 h to obtain a solution of abnormally proliferating vascular smooth muscle cells.

[0113] Detection of the protective effect of Gastrodia elata-Lycium barbarum extract on vascular smooth muscle cells

[0114] The Gastrodia elata-Lycium barbarum extract powders from Examples 1-3 and Comparative Examples 1-8 were each prepared into Gastrodia elata-Lycium barbarum extract solutions with a concentration of 100 μg / mL. The abnormally proliferating vascular smooth muscle cell solutions were divided into 11 groups, 100 μL of each group, and 100 μL of the Gastrodia elata-Lycium barbarum extract solution from Examples 1-3 and Comparative Examples 1-8 were added to each group. After culturing in a constant temperature incubator at 37℃ and 5% CO2 for 24 h, the cell viability of each group was measured using an MTT assay kit (Sangon Biotech (Shanghai) Co., Ltd., E606334-0500) according to the instructions. The results are shown in Table 1.

[0115] Table 1 Cell Viability

[0116] Cell vitality Example 1 101.678% Example 2 104.077% Example 3 108.638% Comparative Example 1 112.692% Comparative Example 2 118.002% Comparative Example 3 115.285% Comparative Example 4 119.773% Comparative Example 5 114.802% Comparative Example 6 119.884% Comparative Example 7 116.651% Comparative Example 8 115.811% Comparative Example 9 116.371%

[0117] The test results above show that, compared to Comparative Example 1 which only contained Gastrodia elata and Comparative Example 2 which only contained Lycium barbarum, the cell viability in Examples 1-3 of this application was lower. This indicates that the mass ratio of Gastrodia elata and Lycium barbarum was reasonably set, and the two had a synergistic effect, which could further improve the abnormal proliferation of vascular smooth muscle cells. In Comparative Example 3, after replacing the intermittent working mode of high-intensity ultrasound in the two enzymatic hydrolysis processes with the normal working mode, the cell viability increased to 115.285%. This indicates that high-intensity ultrasound in the normal working mode would damage the effective active ingredients in Gastrodia elata and Lycium barbarum, while high-intensity ultrasound in the intermittent working mode is more suitable for assisting the enzymatic hydrolysis extraction of effective active ingredients in Gastrodia elata and Lycium barbarum. In Comparative Example 4, after replacing high-intensity ultrasound with normal ultrasound, the cell viability increased to 119.773%. This indicates that the high-intensity ultrasound in this application can effectively extract the effective active ingredients in Gastrodia elata and Lycium barbarum. In Comparative Example 5, after replacing freeze-drying with spray drying, the cell viability increased to 114.802%. This indicates that freeze-drying can avoid the high-temperature inactivation of effective active ingredients in Gastrodia elata and Lycium barbarum. Comparative Example 6, using the traditional water decoction method to prepare Gastrodia elata-Lycium barbarum extract, showed an increase in cell viability to 119.884%, indicating that the high-intensity ultrasound-assisted dual enzymatic hydrolysis method used in this application can further extract the effective active ingredients from Gastrodia elata and Lycium barbarum compared to the traditional water decoction method. Comparative Example 7, with only one enzymatic hydrolysis, showed an increase in cell viability to 116.651%, indicating that the two enzymatic hydrolysis processes synergistically further extract the effective active ingredients from Gastrodia elata and Lycium barbarum. In Comparative Example 8, the second enzymatic hydrolysis using only papain and α-glucuronidase increased cell viability to 115.811%, and in Comparative Example 9, the second enzymatic hydrolysis using only papain and α-L-arabinofuranylase increased cell viability to 116.371%, indicating that α-L-arabinofuranylase, α-glucuronidase, and papain have a synergistic effect, further extracting the effective active ingredients from Gastrodia elata and Lycium barbarum.

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a Gastrodia elata-Lycium barbarum extract, characterized in that, Includes the following steps: (1) Gastrodia elata and wolfberry are mixed, crushed into powder, and sieved to obtain a mixture powder; the mass ratio of Gastrodia elata to wolfberry is (1-3):

1. (2) After adding water and compound enzyme I to the mixture powder, enzymatic extraction is performed under high-intensity ultrasound at 900-1800W to obtain the first enzymatic hydrolysate. After centrifugation, the first supernatant is obtained. The compound enzyme I includes cellulase and pectin methyl esterase. The amount of cellulase added is 0.02%-0.05% of the dry weight of the medicinal material, and the amount of pectin methyl esterase added is 0.01%-0.02% of the dry weight of the medicinal material. The high-intensity ultrasound treatment mode is: working for 3-5 seconds and then pausing for 2-3 seconds under the conditions of 20-40kHz and 40%-60% amplitude. (3) After adding compound enzyme II to the first supernatant, enzymatic hydrolysis extraction was performed under high intensity ultrasound of 900-1800W to obtain the second enzymatic hydrolysate. After centrifugation, the second supernatant obtained is the Gastrodia elata-Lycium barbarum extract. The complex enzyme II comprises α-L-arabinofuranylase, α-glucuronidase, and papain; the amount of α-L-arabinofuranylase added is 0.02% to 0.05% of the dry weight of the medicinal material; the amount of α-glucuronidase added is 0.02% to 0.05% of the dry weight of the medicinal material; the amount of papain added is 0.02% to 0.05% of the dry weight of the medicinal material. The high-intensity ultrasound processing mode is as follows: under the conditions of 20-40 kHz and 40%-60% amplitude, there is a 2-3 second interval every 3-5 seconds of operation; (4) The gastrodia-goji extract was freeze-dried to obtain gastrodia-goji extract.

2. The preparation method according to claim 1, characterized in that, In step (2), the pH of the enzymatic hydrolysis is 3-5, the temperature is 4-10℃, and the time is 60-180s.

3. The preparation method according to claim 1, characterized in that, In step (3), the pH of the enzymatic hydrolysis is 6.5 to 7.5, the temperature is 4 to 10°C, and the time is 60 to 180 seconds.

4. The preparation method according to claim 1, characterized in that, The freeze-drying process includes the following steps: (1) Pre-freeze the Gastrodia elata-Lycium barbarum extract at -4 to 4°C for one day in a low-temperature freezer; (2) After pre-freezing, the Gastrodia elata-Lycium barbarum extract in the low-temperature freezer is transferred to a vacuum freeze dryer. The vacuum freeze dryer has a vacuum degree of 0-1.0 Pa, a partition temperature of 45-60℃, and a partition heating rate of 5-7.5℃ / h. (3) Dry in a vacuum freeze dryer for 2 to 4 days to obtain the Gastrodia elata-Lycium barbarum extract.

5. A Gastrodia elata-Lycium barbarum extract, prepared by the method according to any one of claims 1 to 4, characterized in that, The active ingredients in the Gastrodia elata-Lycium barbarum extract include one or more of gastrodin, p-hydroxybenzyl alcohol, barisonin A, Lycium barbarum polysaccharide, luteolin, or gallic acid.