Olive leaf extract as well as preparation process and application thereof

Abstract

The invention relates to the technical field of preparation of olive leaf extracts, in particular to an olive leaf extract and a preparation process and application thereof, and the process comprises the following steps: crushing dried olive leaves, mixing the crushed olive leaves with an ethanol aqueous solution, and carrying out multi-stage temperature control hot reflux extraction and solid-liquid separation to obtain a supernatant and a precipitate; loading the supernate to a resin column, washing with water and then eluting with ethanol, and drying the eluent to obtain an oleuropein crude product; blending the oleuropein crude product and the sample stirring silica gel, drying, filling into a chromatographic column, eluting by using an ethyl acetate-ethanol mixed solvent, and collecting a high-purity oleuropein fine product; sequentially carrying out ultrasonic dissolution, concentration, standing crystallization and filtration on the precipitate through medium-concentration and high-concentration ethanol to obtain a maslinic acid crude product and an oleanolic acid crude product; and respectively heating and dissolving with ethanol, cooling and crystallizing, and circularly recrystallizing to obtain a corresponding refined product. The invention aims to guarantee the extraction yield and quality of oleuropein by controlling the multi-stage temperature of heating reflux extraction of olive leaves.

Description

Technical Field

[0001] This application relates to the field of olive leaf extract preparation technology, specifically to an olive leaf extract, its preparation process, and its application. Background Technology

[0002] Currently, the development of olive resources both domestically and internationally mainly focuses on the fruit. A large number of olive leaves produced from pruning and harvesting olive trees are generally used as animal feed or directly discarded. Olive leaves contain a wealth of functional components such as oleuropein, oleanolic acid, hawthorn acid, hydroxyl complexol, and flavonoids, resulting in low resource utilization.

[0003] Oleuropein is the main schizocyclic ether terpenoid compound in olive leaves, which has pharmacological effects such as antioxidation, hypoglycemia, cardiovascular protection, and antibacterial activity. Oleanolic acid and scutellaric acid both belong to pentacyclic triterpenoid compounds and have similar chemical structures. Oleanolic acid has pharmacological effects such as hepatoprotection, nephropathy, and anti-inflammation, while scutellaric acid has pharmacological effects such as antitumor, antioxidant, and anti-inflammatory activity.

[0004] Reflux extraction utilizes a boiling solvent to repeatedly soak and extract the raw material, then refluxes the solvent through condensation, allowing for continuous extraction at a higher temperature. Due to its simplicity and ease of operation, it is widely used for extracting active ingredients from olive leaves. Olive leaves naturally contain endogenous enzymes such as β-glucosidase, which, under certain conditions, catalyze the hydrolysis of oleuropein. Furthermore, oleuropein is a heat-sensitive substance, highly sensitive to temperature during extraction. Prolonged high-temperature reflux can easily cause high-temperature degradation of oleuropein, directly reducing the yield of the oleuropein extract. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides an olive leaf extract, its preparation process, and its application, thereby resolving the existing issues.

[0006] The present application discloses an olive leaf extract, its preparation process, and its application, employing the following technical solution: In a first aspect, one embodiment of this application provides a process for preparing olive leaf extract, the process comprising the following steps: Pulverization and extraction: The dried olive leaves are pulverized and mixed with an ethanol-water solution. After multi-stage temperature-controlled hot reflux extraction and solid-liquid separation, the supernatant and precipitate are obtained. Purification of oleuropein: The supernatant was loaded onto a resin column, washed with water and then eluted with ethanol. The eluent was dried to obtain crude oleuropein. Silica gel column purification: The crude oleuropein was mixed with silica gel, dried, and then packed into a chromatography column. It was eluted with an ethyl acetate-ethanol mixed solvent to collect the high-purity oleuropein concentrate. Preparation of hawthorn acid and oleanolic acid: The precipitates were successively dissolved by ultrasonication in medium and high concentrations of ethanol, concentrated, and allowed to crystallize by static standing. Crude hawthorn acid and crude oleanolic acid were obtained by filtration. They were then dissolved by heating with ethanol, cooled and crystallized, and the corresponding high-quality products were obtained by repeated recrystallization. In the multi-stage temperature-controlled hot reflux extraction process, several temperature measuring points are evenly distributed circumferentially in the middle of the hot reflux extraction tank. Within each feedback cycle, the temperature record sequence of each temperature measuring point is compared with the preset temperature standard upper limit curve to screen out the over-temperature points. The proportion of over-temperature points that continuously exceed the preset number and the temperature difference between the over-temperature points and the corresponding time on the preset temperature standard upper limit curve are used to calculate the feedback temperature over-limit value of each temperature measuring point. Then, the overheating runaway assessment value of each temperature measuring point is calculated by combining the average maximum mutual information coefficient between the temperature measuring points. The normalized value of the overheat runaway assessment value is used to generate the temperature feedback ratio factor of each temperature measurement point in the next feedback cycle, and the output of the PLC controller is adjusted accordingly to control the temperature of the multi-stage heat reflow, and the heat reflow process is terminated by the real-time temperature measurement value.

[0007] Preferably, the silica gel column chromatography eluent is composed of ethyl acetate and ethanol in a volume ratio of 99:1.

[0008] Preferably, the ultrasonic dissolution temperature during the preparation of hawthorn acid is lower than that during the preparation of oleanolic acid.

[0009] Preferably, the recrystallization solvent is ethanol with a volume fraction ≥ 95%, and the recrystallization cycle is three times.

[0010] Preferably, the calculation expression for the feedback temperature exceeding the standard value is: In the formula, This is the feedback temperature exceeding the limit at the i-th temperature measuring point inside the hot reflux extraction vessel. It is the over-temperature duration weight of the temperature record sequence at the i-th temperature measurement point. It is the temperature record value corresponding to the y-th temperature exceedance point within the i-th temperature measurement point. It is the upper limit value of the curve relating the y-th overtemperature point to the upper limit of the temperature standard. It is the temperature record sequence of the i-th temperature measurement point.

[0011] Preferably, the calculation expression for the overheating runaway assessment value is: In the formula, It is the overheating runaway assessment value at the i-th temperature measurement point inside the hot reflux extraction vessel. It is the temperature exceeding the standard value reported by the i-th temperature measuring point. It is the uniformity of heat conduction at the i-th temperature measurement point. It is a non-zero positive number that is close to 0.

[0012] Preferably, the uniformity of heat conduction at the i-th temperature measuring point is determined by the average of the maximum mutual information coefficients between the i-th temperature measuring point and all remaining temperature measuring points.

[0013] Preferably, the multi-stage process divides a single reflux extraction into three consecutive time-series stages: "start-main extraction-enhancement". For each stage, the process of generating a temperature feedback scaling factor for each temperature measurement point in the next feedback cycle after normalization, and adjusting the PLC controller output accordingly, includes: The sum of the products of the temperature feedback ratio factor of each temperature measuring point in the next feedback cycle and the temperature value measured at the sampling time is used as the feedback temperature value of the heat reflux extraction tank at the sampling time. The feedback temperature value is input into the PLC controller to adjust the extraction temperature of the hot reflux extraction tank. For the enhancement phase: if the temperature value obtained from any temperature measurement point is higher than the tolerance limit for m consecutive times, the enhancement phase ends; where m is the preset number of times, and the tolerance limit is the preset start-up phase temperature.

[0014] Secondly, another embodiment of this application provides an olive leaf extract, which is prepared by the olive leaf extract preparation process described above.

[0015] Thirdly, another embodiment of this application also provides an olive leaf extract as a drug for preparing anti-inflammatory diseases.

[0016] The beneficial effects of the above scheme are as follows: This application employs a multi-stage temperature control method for olive leaf heating and reflux extraction. In the start-up stage, a rapid heating strategy and inactivation treatment are used to prevent enzymatic reactions at the source, effectively inactivating β-glucosidase, reducing the enzymatic hydrolysis of oleuropein, and further ensuring the extraction yield and quality of oleuropein.

[0017] This application constructs an overheating runaway assessment value to assess the risk of local overheating runaway within the extraction tank in real time. Through multi-stage temperature control (start-up, main extraction, and enhancement stages), the temperature feedback ratio factor is dynamically adjusted. In the start-up stage, a lower feedback temperature value is used for conditions with high risk of oleuropein degradation to increase the β-glucosidase inactivation temperature and prevent β-glucosidase from causing an enzymatic reaction with oleuropein. In the main extraction stage, a higher feedback temperature value is used for conditions with high risk of oleuropein degradation to suppress the heating function of the hot reflux extraction tank and prevent prolonged heating and reflux of oleuropein. In the enhancement stage, conditions with high risk of oleuropein degradation are terminated in a timely manner to prevent further losses. Attached Figure Description

[0018] To more clearly illustrate the technical solutions and advantages in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a flowchart illustrating the steps of a process for preparing olive leaf extract according to an embodiment of this application. Detailed Implementation

[0020] To further illustrate the technical means and effects adopted by this application to achieve the intended inventive purpose, the following, in conjunction with the accompanying drawings and preferred embodiments, details the specific implementation methods, structures, features, and effects of an olive leaf extract, its preparation process, and its application according to this application. In the following description, different "one embodiment" or "another embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The following detailed description, in conjunction with the accompanying drawings, illustrates a specific scheme for an olive leaf extract, its preparation process, and its application provided in this application.

[0023] Example 1 This application provides an embodiment of a process for preparing olive leaf extract; please refer to the flowchart for the preparation process. Figure 1 The specific preparation process is as follows: S1: Crushing and Extraction Olive leaves are pulverized to obtain dried olive leaf powder. The olive leaf powder is mixed with a 30%-70% ethanol solution at a ratio of 1g:8-12mL. A multi-stage temperature control method is used for the hot reflux extraction, dynamically adjusting the temperature of the extraction tank. Extraction is performed 2-3 times, each time lasting 2-3 hours. After extraction, the ethanol in the filtrate is removed by vacuum distillation, and the supernatant and precipitate are obtained by solid-liquid separation.

[0024] In this embodiment, olive leaf powder and 30% ethanol solution were mixed at a material-to-liquid ratio of 1g:8mL. The temperature of the hot reflux extraction tank was dynamically adjusted using a multi-stage temperature control method for olive leaf heating and reflux extraction. The extraction was performed 3 times, with each extraction lasting 3 hours.

[0025] S2: Preliminary purification of oleuropein The supernatant obtained in step S1 was loaded onto an XDA-8 adsorption resin column and eluted sequentially with ultrapure water and 45-75% ethanol solution. The ethanol eluent was collected. The eluent was dried by spray drying to obtain crude oleuropein.

[0026] For the XDA-8 adsorption resin column, the adsorption resin was immersed in a 95% ethanol solution for 24 hours to allow it to fully swell, and then rinsed with ultrapure water until no alcohol odor remained. Subsequently, it was immersed in a 4% HCl solution for 12 hours and rinsed until neutral. Finally, it was immersed in a 4% NaOH solution for 12 hours and rinsed again until neutral, completing the resin pretreatment. The adsorption resin was then packed into the column using a wet method to remove air bubbles between resin particles. The diameter-to-height ratio of the resin column was 1:4-8.

[0027] XDA-8 resin has more adsorption sites that can bind to oleuropein, and its pore size is wider and its pore volume is smaller, making it more suitable for the transfer of oleuropein. Therefore, this application uses XDA-8 as the adsorption resin.

[0028] In this embodiment, elution was performed using ultrapure water and a 45% ethanol solution.

[0029] S3: Silica gel column chromatography Dissolve crude oleuropein in 80% ethanol. Weigh out silica gel at a mass ratio of 3-5:1 (to crude oleuropein), add it to the crude oleuropein solution, and stir until homogeneous. Dry the silica gel in a vacuum dryer until it becomes powder. Separately, weigh out silica gel at a mass ratio of 20:1 (to crude oleuropein), pack it into a chromatography column, soak it in ethyl acetate to remove air, and then add the silica gel. Prepare an eluent with ethyl acetate and 80% ethanol at a volume ratio of 99:1 (total volume), and elute at a rate of 1.0-2.0 BV / h. Collect the product to obtain high-purity oleuropein.

[0030] If the ethanol content in the eluent of silica gel column chromatography is too high, other substances in the crude oleuropein will also be eluted, making it difficult to obtain high-purity oleuropein. Therefore, this application uses a ratio of ethyl acetate: 80% ethanol = 99:1.

[0031] In this embodiment, the silica gel was weighed at a mass ratio of 3:1 to crude oleuropein, and eluted at a rate of 1.0 BV / h.

[0032] S4: Preparation of hawthorn acid and oleanolic acid Add 4-6 times the volume of 45%-55% ethanol solution to the precipitate obtained in step S1, heat to 40-60℃, sonicate for 1 hour, and filter to obtain a mixed solution of hawthorn acid and filter residue. Concentrate the mixed solution to half its original volume, let it stand at room temperature for 24 hours to crystallize, and filter to obtain crude hawthorn acid.

[0033] Add 4-6 times the volume of 75%-85% ethanol solution to the above filter residue, heat to 60-80℃, sonicate for 1 hour, and filter to obtain a mixed solution of oleanolic acid. Concentrate to half the original volume, let stand at room temperature for 24 hours to crystallize, and filter to obtain crude oleanolic acid.

[0034] In this embodiment, the precipitate obtained in step S1 was added to 4 times the volume of 45% ethanol solution, heated to 40°C, and ultrasonically dissolved for 1 hour. The mixture was then filtered to obtain a hawthorn acid mixed solution and filter residue. The mixed solution was concentrated to half its original volume, allowed to stand at room temperature for 24 hours to crystallize, and then filtered to obtain crude hawthorn acid.

[0035] Add the above filter residue to 4 times the amount of 75% ethanol solution, heat to 60°C, sonicate for 1 hour, and filter to obtain a mixed solution of oleanolic acid.

[0036] Considering reagent toxicity, ethanol with a volume concentration ≥95% was used as the recrystallization solvent. The recrystallization and purification processes for crude hawthorn acid and crude oleanolic acid were consistent. To obtain crude hawthorn acid or crude oleanolic acid, the recrystallization solvent was added under heating conditions of 65-80℃. After the crude hawthorn acid or crude oleanolic acid was completely dissolved, it was cooled to room temperature and then placed in a cooling device for crystallization for 6-12 hours at a cooling temperature of 3-5℃. After cooling, the mixture was filtered, and the mother liquor was recrystallized three times to obtain refined hawthorn acid and refined oleanolic acid.

[0037] To overcome the shortcomings of the existing technology, this application proposes a multi-stage temperature control method for olive leaf heating and reflux extraction in step S1 of the olive leaf extract preparation process. This method addresses the problem that oleuropein, as a heat-sensitive substance, is highly sensitive to temperature during the extraction process. Prolonged high-temperature heating and reflux can easily cause high-temperature degradation of oleuropein, directly reducing the yield of oleuropein extract.

[0038] Step 1: Distribute at least 4 temperature measurement points evenly in the circumferential direction in the middle of the hot reflux extraction tank, and obtain the temperature record sequence of each temperature measurement point within the feedback cycle.

[0039] This application specifies that at half the volume height of the hot reflux extraction tank, the components are evenly distributed... There are 4 temperature measurement points (N) to monitor the extraction temperature during the first heating and reflux extraction process of olive leaves. The total number of temperature measurement points (N) is at least 4, and in this embodiment, it is set to 4. The temperature measuring element is a thermocouple sensor with a sampling frequency of 1Hz. A 2-minute feedback cycle is set, meaning that the temperature values ​​of all temperature measurement points are acquired every 2 minutes and arranged into a temperature record sequence according to time order.

[0040] Step 2: By monitoring the temperature at multiple temperature measurement points in the hot reflux extraction tank and comparing it with the standard temperature scale curve, the feedback temperature exceedance value and heat conduction uniformity are calculated, thereby obtaining the overheating runaway assessment value. This allows for real-time assessment and early warning of local overheating risks, preventing the degradation of oleuropein due to continuous high temperatures and ensuring extraction yield.

[0041] The products obtained from the preparation process of olive leaf extract are oleuropein, oleanolic acid, and rutin. Oleanolic acid and rutin are characteristic pentacyclic triterpenoids in olive leaves, and rutin is even a derivative of oleanolic acid; both have similar chemical structures and physicochemical properties. Oleuropein belongs to the sepsis-enriched ether glycoside class and is a polar compound. XDA-8 resin is also a polar resin; based on the principle of like attracts like, oleuropein can be separated from the extract using an XDA-8 adsorption resin column.

[0042] Oleuropein, the most abundant antioxidant active ingredient in olive leaves (approximately 1-17%), is higher than that in olive fruit, and its content in olive leaves fluctuates at different growth stages of the olive tree. As a typical heat-sensitive substance, oleuropein's chemical structure is destroyed under sustained high temperatures, leading to its inactivation. Especially in solvent environments, prolonged heating and reflux can easily trigger the decomposition and transformation of oleuropein, directly reducing the yield of oleuropein extraction.

[0043] The large volume of the reflux extraction tank results in non-linear and hysteretic temperature changes in the extract, making it easy for the extract temperature to exceed the standard temperature curve. This application obtains the start-up time period of the reflux extraction unit corresponding to the feedback cycle and extracts a local temperature curve from the standard temperature curve corresponding to the start-up time period as a local temperature scale curve. x represents any time within the feedback period.

[0044] In industrial-scale production of heat-reflux extraction, there is a certain tolerance for temperature fluctuations. This application sets the temperature tolerance to... Then the upper limit curve of the temperature standard corresponding to the local temperature scale curve can be expressed as: Among them, tolerance error One possible value range is [0.05, 0.15], with a tolerance error. The accuracy is related to the control system precision of the hot reflux extraction tank; in this embodiment, it is set to 0.1.

[0045] This application compares the temperature recording sequence at any temperature measurement point within the feedback period with a preset temperature standard upper limit curve. The sampling time where the temperature recording value exceeds the temperature standard upper limit curve is recorded as an over-temperature point. A continuous over-temperature subsequence with ≥Q consecutive over-temperature points is extracted from the temperature recording sequence. The ratio of the total number of data elements in all continuous over-temperature subsequences to the total number of data elements in the temperature recording sequence is recorded as the over-temperature persistence weight. In this embodiment, the preset value Q is 3.

[0046] This application uses the following formula to obtain the feedback temperature exceedance value at the i-th temperature measuring point inside the hot reflux extraction tank. : In the formula, It is the over-temperature duration weight of the temperature record sequence at the i-th temperature measurement point. It is the temperature record value corresponding to the y-th temperature exceedance point within the i-th temperature measurement point. It is the upper limit value of the curve relating the y-th overtemperature point to the upper limit of the temperature standard. It is the total number of all temperature-exceeding points in the temperature record sequence of the i-th temperature measurement point.

[0047] Overheating sustained weighting This indicator represents the duration of temperature exceedance in the extract within the feedback cycle. Frequent and consecutive occurrences of temperature exceedances suggest that the higher the temperature exceeds the standard at the measurement point, the more likely sensor measurement errors are to be ruled out, thus enhancing the reliability of the recorded extract temperature value. A higher weight is given to this indicator. The higher the curve indicating that the temperature exceeds the upper limit of the temperature standard at the exceedance point, the more pronounced the temperature rises. The larger the value, the more severe the temperature exceedance of the extract at the temperature measuring point, and the higher the reported temperature exceedance value. The larger.

[0048] Furthermore, the extraction temperature in the hot reflux extraction tank is easily affected by factors such as ambient temperature, system aging, and noise interference. This can cause significant nonlinear random fluctuations in the extraction temperature over time, leading to local thermal runaway and exacerbating local overheating in the hot reflux extraction tank, thereby increasing the risk of thermal degradation of oleuropein. This application obtains the temperature recording sequence at the i-th temperature measurement point. The uniformity of heat conduction at the i-th temperature measurement point inside the reflux extraction vessel is obtained using the following formula. : In the formula, Let J be the temperature record sequence of the j-th temperature measurement point, where J is the set of temperature measurement points, j ≠ i, and N is the total number of temperature measurement points. In this embodiment, N is taken as 4. It is a sequence of temperature records for calculation. , The maximum mutual information coefficient between them is a well-known technical concept and will not be elaborated further.

[0049] Used to reflect the correlation between temperature record sequences from different temperature measurement points The larger the value, the stronger the linear or nonlinear correlation between the measured temperatures at different temperature measurement points, and the higher the synergy of temperature changes in different extracts in the reflux extraction tank. This indicates that the heat from the extract can be diffused more quickly and the heat conduction is more uniform during the reflux extraction process, resulting in higher uniformity of heat conduction at the temperature measurement points. The larger.

[0050] Based on the above steps, this application uses the feedback temperature exceedance value and the uniformity of heat conduction at each temperature measurement point to determine the overheating runaway assessment value at each temperature measurement point.

[0051] This application obtains the overheating runaway assessment value at the i-th temperature measurement point inside the hot reflux extraction tank using the following formula. : In the formula, It is the temperature exceeding the standard value reported by the i-th temperature measuring point. It is the uniformity of heat conduction at the i-th temperature measurement point. It is a non-zero positive number that is close to 0, in order to prevent the denominator from being 0. In this embodiment, it is taken as 0.01.

[0052] The temperature exceeding the limit reflects the effectiveness of temperature control within the hot reflux extraction tank. Temperature measurement and heat conduction uniformity assess the uniformity of heat conduction during the heating and reflux process within the tank, directly impacting the risk of localized overheating runaway at the measurement point. Localized overheating runaway can cause the olive leaf extract temperature to consistently exceed the tolerance limit. Prolonged high-temperature heating and reflux can easily damage the chemical structure of oleuropein, deactivating it and thus reducing the extraction yield.

[0053] Step 3: This application divides the hot reflux extraction of olive leaves into three stages: initiation, main extraction, and enhancement. Through a multi-stage temperature control strategy, and based on the objectives of each stage and the real-time calculated overheating runaway assessment value, the temperature feedback ratio factor is dynamically adjusted, and the extraction temperature is regulated by a PLC controller to achieve rapid enzyme inactivation, efficient extraction, and prevention of thermal degradation of oleuropein.

[0054] Olive leaves naturally contain endogenous enzymes such as β-glucosidase. Under suitable conditions, these enzymes catalyze the hydrolysis of oleuropein, thus reducing the yield of oleuropein extraction. β-glucosidase activity does not decrease significantly in the temperature range of 4-60℃, but it is rapidly inactivated above 65℃. Therefore, reflux extraction of olive leaves not only needs to avoid thermal degradation of oleuropein due to prolonged reflux, but also requires rapid enzyme inactivation in the early stages of reflux.

[0055] This application divides the extraction time of a single heating reflux extraction into a start-up stage, a main extraction stage, and an enhancement stage. The time ratio for each stage can be set to 30%:60%:10%. If the extraction time is selected as 3 hours, then 1-54 minutes is the start-up stage, 54-162 minutes is the main extraction stage, and 162-180 minutes is the enhancement stage. Through a dynamic temperature path of "low temperature start-up - medium temperature main extraction - high temperature enhancement," multi-stage temperature control of olive leaf heating reflux extraction is achieved, and a standard temperature curve for multi-stage heating reflux extraction can be obtained through an optimized method.

[0056] (1) Start-up stage: The main functions are: first, to use preheating to break down the plant cell wall, allowing the ethanol solvent to penetrate rapidly and initially improve the extraction rate of olive leaf extract; second, to quickly and thoroughly inactivate endogenous degrading enzymes such as β-glucosidase, preventing the occurrence of enzymatic reactions from the source.

[0057] This application raises the temperature from the immersion temperature to 60-70°C at a rate of 2-5°C / min and maintains it at this temperature until the start-up phase ends; wherein, the heating rate is preferably 3°C / min and the start-up phase temperature is preferably 65°C.

[0058] If the feedback cycle is in the start-up phase, in order to avoid the enzymatic reaction of β-glucosidase, which would cause degradation of oleuropein and result in local overheating at the temperature measuring point, a lower feedback temperature value is required to promote the heating function of the hot reflux extraction tank and significantly increase the heating rate.

[0059] Temperature records at temperature measurement points with low overheat runaway assessment values ​​are low. This application obtains the overheat runaway assessment values ​​of all temperature measurement points in the hot reflux extraction tank and normalizes them using the Softmax function. The difference between the constant 1 and the normalized overheat runaway assessment value is used as the temperature feedback ratio factor of the temperature measurement point in the next feedback cycle. The temperature feedback factor of each temperature measurement point is updated once every feedback cycle.

[0060] During the heating and reflux process in the next feedback cycle, the temperature value of the temperature measuring point at the sampling time is obtained, and the sum of the product of the temperature feedback proportional factor and the temperature value of all temperature measuring points is calculated as the feedback temperature value of the hot reflux extraction tank at the sampling time. The feedback temperature value is then input into the PLC controller to adjust the extraction temperature of the hot reflux extraction tank.

[0061] (2) Main extraction stage: Further destroy the cell wall structure of olive leaves, efficiently dissolve most of the effective components such as oleuropein, oleanolic acid and hawthorn acid in the cells, and improve the solubility and diffusion rate of olive leaf extract.

[0062] This application raises the temperature to 70-80°C at a rate of 1-3°C / min and maintains it at this temperature until the end of the main extraction stage; wherein, the heating rate is preferably 2°C / min, and the start-up stage temperature is preferably 75°C.

[0063] If the feedback cycle is in the main extraction stage, in order to avoid prolonged heating and reflux of oleuropein, which would lead to thermal degradation of oleuropein, the temperature measurement point with a large overheat runaway assessment value is prone to thermal degradation of oleuropein. At this time, a higher feedback temperature value is required to suppress the heating function of the hot reflux extraction tank.

[0064] This application obtains the overheating runaway assessment values ​​of all temperature measurement points in the hot reflux extraction tank, and uses the Softmax function to normalize them. The normalized overheating runaway assessment values ​​are then used as the temperature feedback scaling factor for the temperature measurement points in the next feedback cycle.

[0065] During the heating and reflux process in the next feedback cycle, the temperature value of the temperature measuring point at the sampling time is obtained, and the sum of the product of the temperature feedback proportional factor and the temperature value of all temperature measuring points is calculated as the feedback temperature value of the hot reflux extraction tank at the sampling time. The feedback temperature value is then input into the PLC controller to adjust the extraction temperature of the hot reflux extraction tank.

[0066] (3) Enhancement stage: At the end of the heating extraction, after most of the easily extracted oleuropein has dissolved, the heating extraction temperature is briefly increased to enhance the extraction of residual components that are difficult to extract and are deeply hidden in the plant matrix, thereby increasing the extraction rate of olive leaf components.

[0067] This application raises the temperature to 80-90°C at a rate of 0.5-1°C / min and maintains this temperature until the end of the strengthening stage, at which point heating is stopped. The preferred heating rate is 1°C / min, and the preferred start-up temperature is 85°C.

[0068] If the feedback cycle is in the enhancement phase, the extraction temperature in the hot reflux extraction tank is the highest, and the heating time is the longest, resulting in an extremely high risk of thermal degradation of oleuropein. If the temperature value obtained at any temperature measurement point is higher than the tolerance limit for m consecutive times, the enhancement phase is terminated directly to prevent further thermal degradation of oleuropein. In this embodiment, the preset number of times m is 5.

[0069] Example 2 S1: Crushing and Extraction In this embodiment, olive leaf powder and 50% ethanol solution were mixed at a material-to-liquid ratio of 1g:10mL. The temperature of the hot reflux extraction tank was dynamically adjusted using a multi-stage temperature control method for olive leaf heating and reflux extraction. The extraction was performed twice, with each extraction lasting 3 hours.

[0070] S2: Preliminary purification of oleuropein In this embodiment, elution was performed using ultrapure water and a 60% ethanol solution.

[0071] S3: Silica gel column chromatography In this embodiment, the silica gel was weighed at a mass ratio of 4:1 to crude oleuropein, and eluted at a rate of 1.5 BV / h.

[0072] S4: Preparation of hawthorn acid and oleanolic acid In this embodiment, the precipitate obtained in step S1 was added to 5 times the volume of 50% ethanol solution, heated to 50°C, and ultrasonically dissolved for 1 hour. The mixture was then filtered to obtain a hawthorn acid mixed solution and filter residue. The mixed solution was concentrated to half its original volume, allowed to stand at room temperature for 24 hours to crystallize, and then filtered to obtain crude hawthorn acid.

[0073] Add 5 times the amount of 80% ethanol solution to the above filter residue, heat to 70°C, sonicate for 1 hour, and filter to obtain oleanolic acid mixed solution.

[0074] All parts not mentioned in this embodiment are the same as the corresponding parts in Embodiment 1, and will not be described in detail here.

[0075] Example 3 S1: Crushing and Extraction In this embodiment, olive leaf powder and 70% ethanol solution were mixed at a material-to-liquid ratio of 1g:12mL. The temperature of the hot reflux extraction tank was dynamically adjusted using a multi-stage temperature control method for olive leaf heating and reflux extraction. The extraction was performed twice, with each extraction lasting 2 hours.

[0076] S2: Preliminary purification of oleuropein In this embodiment, elution was performed using ultrapure water and a 75% ethanol solution.

[0077] S3: Silica gel column chromatography In this embodiment, the silica gel was weighed at a mass ratio of 5:1 (silica gel to crude oleuropein) and eluted at a rate of 2.0 BV / h.

[0078] S4: Preparation of hawthorn acid and oleanolic acid In this embodiment, the precipitate obtained in step S1 was added to 6 times the volume of 55% ethanol solution, heated to 60°C, and ultrasonically dissolved for 1 hour. The mixture was then filtered to obtain a hawthorn acid mixed solution and filter residue. The mixed solution was concentrated to half its original volume, allowed to stand at room temperature for 24 hours to crystallize, and then filtered to obtain crude hawthorn acid.

[0079] Add the above filter residue to 6 times the amount of 85% ethanol solution, heat to 80°C, sonicate for 1 hour, and filter to obtain a mixed solution of oleanolic acid.

[0080] All parts not mentioned in this embodiment are the same as the corresponding parts in Embodiment 1, and will not be described in detail here.

[0081] Test results: The olive leaf raw material of this application contains 8% oleuropein, 0.5% rutin, and 1% oleanolic acid.

[0082] The preparation process of olive leaf extract obtained by the multi-stage temperature control method of olive leaf heating and reflux extraction is used as an example. The difference between the following comparative examples and examples not mentioned is whether or not the multi-stage temperature control method of olive leaf heating and reflux extraction is used.

[0083] The HPLC conditions for oleuropein were as follows: a 250 mm × 4.6 mm, 5 μm column was used, the flow rate was 1.0 ml / min, the column temperature was 30 ℃, the wavelength was 231 nm, and the mobile phase was acetonitrile: 0.1 phosphoric acid solution = 23:77 (V / V) isocratic elution.

[0084] The HPLC conditions for hawthorn acid were as follows: a 250 mm × 4.6 mm, 5 μm column was used, the flow rate was 1.0 ml / min, the column temperature was 30 ℃, the wavelength was 215 nm, and the mobile phase was 1% acetic acid: methanol = 12:88 (V / V) isocratic elution.

[0085] The liquid chromatography conditions for oleanolic acid were as follows: a 250 mm × 4.6 mm, 5 μm column was used, the flow rate was 1.0 ml / min, the column temperature was 30 ℃, the wavelength was 205 nm, and the mobile phase was acetonitrile: 0.1 phosphoric acid solution = 80:20 (V / V) isocratic elution.

[0086] According to high performance liquid chromatography, the purity of oleuropein in Example 1 was 94.34%, and the yield was 94.58%; the purity of oleanolic acid was 98.42%-98.37%, and the yield was 90.56%; and the purity of rutin was 97.66%, and the yield was 92.42%.

[0087] According to high performance liquid chromatography, the purity of oleuropein in Comparative Example 1 was 84.13%, and the yield was 83.29%; the purity of oleanolic acid was 98.37%, and the yield was 89.76%; and the purity of ursolic acid was 96.22%, and the yield was 93.25%.

[0088] According to high performance liquid chromatography, the purity of oleuropein in Example 2 was 96.42%, and the yield was 95.77%; the purity of oleanolic acid was 98.27%, and the yield was 91.37%; and the purity of rutin was 97.56%, and the yield was 93.02%.

[0089] According to high performance liquid chromatography, the purity of oleuropein in Comparative Example 2 was 87.30%, and the yield was 86.22%; the purity of oleanolic acid was 97.51%, and the yield was 90.07%; and the purity of ursolic acid was 98.23%, and the yield was 91.77%.

[0090] According to high performance liquid chromatography, the purity of oleuropein in Example 3 was 94.73% and the yield was 94.62%; the purity of oleanolic acid was 97.74% and the yield was 92.08%; and the purity of rutin was 98.14% and the yield was 93.67%.

[0091] According to high performance liquid chromatography, the purity of oleuropein in Comparative Example 3 was 85.84%, and the yield was 84.92%; the purity of oleanolic acid was 97.15%, and the yield was 91.65%; and the purity of rutin was 98.66%, and the yield was 92.45%.

[0092] According to the test results, the multi-stage temperature control method of olive leaf heating and reflux extraction can improve the quality, purity and yield of olive bitter glycosides.

[0093] Based on the same inventive concept as the aforementioned olive leaf extract preparation process, this application also provides an olive leaf extract, which is produced by the aforementioned olive leaf extract preparation process.

[0094] Based on the aforementioned application of olive leaf extract, the olive leaf extract can be used as a drug for preparing anti-inflammatory and other diseases.

[0095] Specifically, regarding oleuropein: oleuropein has a variety of biological activities such as antioxidant, anti-inflammatory, and anti-tumor effects. It can be used to prepare drugs for treating diseases such as hypertension and arthritis, as well as cardiovascular and anti-inflammatory drugs. In addition, it can also inhibit the growth and spread of tumor cells and has a certain auxiliary effect on cancer treatment. Regarding oleanolic acid: Oleanolic acid has pharmacological effects such as antibacterial, anti-inflammatory, antiviral, and hepatoprotective properties. It can be used to treat diseases such as acute icteric hepatitis and chronic hepatitis, and it also has significant effects on improving symptoms, signs, and liver function. Regarding hawthorn acid: Hawthorn acid has a variety of pharmacological effects such as anti-inflammatory, antibacterial, antiviral, antitumor, hypoglycemic, and hypolipidemic effects. It can be used to treat indigestion, stomach pain, hyperlipidemia and other diseases, and can also be used as a drug component for the treatment of cardiovascular diseases.

[0096] The various embodiments in this application are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0097] It should be noted that, unless otherwise specified and limited, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a circuit structure, article, or device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or device. Without further limitations, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the article or device that includes said element. Furthermore, the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0098] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the invention herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not invented in this application.

[0099] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A process for preparing olive leaf extract, characterized in that, The process includes the following steps: Pulverization and extraction: The dried olive leaves are pulverized and mixed with an ethanol-water solution. After multi-stage temperature-controlled hot reflux extraction and solid-liquid separation, the supernatant and precipitate are obtained. Purification of oleuropein: The supernatant was loaded onto a resin column, washed with water and then eluted with ethanol. The eluent was dried to obtain crude oleuropein. Silica gel column purification: The crude oleuropein was mixed with silica gel, dried, and then packed into a chromatography column. It was eluted with an ethyl acetate-ethanol mixed solvent to collect the high-purity oleuropein concentrate. Preparation of hawthorn acid and oleanolic acid: The precipitates were successively dissolved by ultrasonication in medium and high concentrations of ethanol, concentrated, and allowed to crystallize by static standing. Crude hawthorn acid and crude oleanolic acid were obtained by filtration. They were then dissolved by heating with ethanol, cooled and crystallized, and the corresponding high-quality products were obtained by repeated recrystallization. In the multi-stage temperature-controlled hot reflux extraction process, several temperature measuring points are evenly distributed circumferentially in the middle of the hot reflux extraction tank. Within each feedback cycle, the temperature record sequence of each temperature measuring point is compared with the preset temperature standard upper limit curve to screen out the over-temperature points. The proportion of over-temperature points that continuously exceed the preset number and the temperature difference between the over-temperature points and the corresponding time on the preset temperature standard upper limit curve are used to calculate the feedback temperature over-limit value of each temperature measuring point. Then, the overheating runaway assessment value of each temperature measuring point is calculated by combining the average maximum mutual information coefficient between the temperature measuring points. The normalized value of the overheat runaway assessment value is used to generate the temperature feedback ratio factor of each temperature measurement point in the next feedback cycle, and the output of the PLC controller is adjusted accordingly to control the temperature of the multi-stage heat reflow, and the heat reflow process is terminated by the real-time temperature measurement value.

2. The preparation process of olive leaf extract as described in claim 1, characterized in that, The silica gel column chromatography eluent is composed of ethyl acetate and ethanol in a volume ratio of 99:

1.

3. The preparation process of olive leaf extract as described in claim 1, characterized in that, The ultrasonic dissolution temperature during the preparation of hawthorn acid is lower than that during the preparation of oleanolic acid.

4. The preparation process of olive leaf extract as described in claim 1, characterized in that, The recrystallization solvent is ethanol with a volume fraction ≥ 95%, and the recrystallization cycle is three times.

5. The preparation process of olive leaf extract as described in claim 1, characterized in that, The formula for calculating the feedback temperature exceeding the standard value is: In the formula, This is the feedback temperature exceeding the limit at the i-th temperature measuring point inside the hot reflux extraction vessel. It is the over-temperature duration weight of the temperature record sequence at the i-th temperature measurement point. It is the temperature record value corresponding to the y-th temperature exceedance point within the i-th temperature measurement point. It is the upper limit value of the curve relating the y-th overtemperature point to the upper limit of the temperature standard. It is the temperature record sequence of the i-th temperature measurement point.

6. The preparation process of olive leaf extract as described in claim 1, characterized in that, The calculation expression for the overheat runaway assessment value is as follows: In the formula, It is the overheating runaway assessment value at the i-th temperature measurement point inside the hot reflux extraction vessel. It is the temperature exceeding the standard value reported by the i-th temperature measuring point. It is the uniformity of heat conduction at the i-th temperature measurement point. It is a non-zero positive number that is close to 0.

7. The preparation process of olive leaf extract as described in claim 6, characterized in that, The uniformity of heat conduction at the i-th temperature measuring point is determined by the average of the maximum mutual information coefficients between the i-th temperature measuring point and all remaining temperature measuring points.

8. The preparation process of olive leaf extract as described in claim 1, characterized in that, The multi-stage process divides a single reflux extraction into three consecutive time-series stages: "start-up-main extraction-enhancement". For each stage, the process of generating a temperature feedback scaling factor for each temperature measurement point in the next feedback cycle after normalization, and adjusting the PLC controller output accordingly, includes: The sum of the products of the temperature feedback ratio factor of each temperature measuring point in the next feedback cycle and the temperature value measured at the sampling time is used as the feedback temperature value of the heat reflux extraction tank at the sampling time. The feedback temperature value is input into the PLC controller to adjust the extraction temperature of the hot reflux extraction tank. For the enhancement phase: if the temperature value obtained from any temperature measurement point is higher than the tolerance limit for m consecutive times, the enhancement phase ends; where m is the preset number of times, and the tolerance limit is the preset start-up phase temperature.

9. An olive leaf extract, characterized in that, The preparation process of olive leaf extract as described in claim 1 is used.

10. An olive leaf extract according to claim 9 as a drug for preparing anti-inflammatory diseases.

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