A method for improving the yield of d-amyrin in persicae semen

Abstract

This invention discloses a method for improving the yield of D-amyglucosinolates in peach kernels, belonging to the field of natural product extraction technology. Existing stepwise enzyme inactivation and defatting processes suffer from drawbacks such as cumbersome procedures and the easy isomerization of D-amyglucosinolates to L-amyglucosinolates. This invention involves pressing peach kernels at 100–150°C, completing enzyme inactivation and defatting in a single step. The resulting residue is then extracted with a 60–70 vt% ethanol aqueous solution at 70–90°C for 0.2–1 hour, repeated 1–3 times. This method overcomes the technical bias of high-temperature-induced D-amyglucosinolate isomerization, achieving a synergistic effect through the instantaneous coupling of high temperature and pressing. The extraction transfer rate of D-amyglucosinolate is no less than 81%, and the extraction transfer rate of L-amyglucosinolate is no more than 10%, with a D / L ratio greater than 15:1. This simplifies the process while achieving highly selective extraction of D-amyglucosinolates.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a method for selectively extracting D-amygynin from peach kernels. Background Technology

[0002] Peach kernels are the dried, mature seeds of the peach or wild peach (Prunus persica), plants belonging to the Rosaceae family. They are rich in oils and active ingredients such as amygdalin. Amygdalin possesses various pharmacological activities, including promoting blood circulation, removing blood stasis, anti-tumor activity, and anti-inflammatory effects, and is an important indicator for evaluating the quality of peach kernel medicinal materials. Naturally occurring amygdalin exists in two epimers: D- and L-. D-amygdalin exhibits extremely low enzymatic hydrolysis efficiency in vivo and is highly safe; however, L-amygdalin is easily degraded by enzymes to release hydrocyanic acid, which is the main substance reported in literature to cause poisoning reactions. Therefore, selectively extracting D-amygdalin from peach kernels and minimizing the dissolution of L-amygdalin is crucial for ensuring the safety of the extracted product.

[0003] Peach kernels contain both amygdalin and its hydrolytic enzyme (amygdalase). During conventional solvent extraction, after the medicinal material is crushed, the enzyme comes into contact with the glycoside, and amygdalase rapidly hydrolyzes the glycoside components, leading to the loss of the target product. To address this, the art commonly employs an "enzyme-killing and glycoside-preserving" strategy, i.e., inactivating the enzyme in the medicinal material before extraction. Existing methods for enzyme inactivation include boiling, microwave inactivation, and dry heat inactivation in an oven. Furthermore, peach kernels have an oil content as high as 30%–50%, and the presence of oil hinders the penetration of the extraction solvent into the cells; pressing or solvent defatting are also routine operations. Based on published literature, the enzyme inactivation and defatting steps are separate, independent operational units, and there are no reports of integrating them into a single process.

[0004] It is worth noting that there is a generally accepted fact in this field: D-amygin readily isomerizes to L-amygin under high temperatures, thus losing its pharmacological activity. Relevant literature clearly states that "D-amygin transforms into L-amygin at high temperatures, thereby losing its pharmacological activity; therefore, conventional extraction methods may cause isomerization of D-amygin, leading to inactivation." Based on this understanding, technicians tend to choose the mildest possible temperature conditions when performing enzyme inactivation treatments, such as drying at 60°C or microwave treatment at 80°C, avoiding higher temperatures. This has created a technical bias in the field: to avoid isomerization, enzyme inactivation temperatures should not be too high.

[0005] On the other hand, the inventors of this application discovered in their experiments that there is a material transfer and waiting period between enzyme inactivation and pressing in the stepwise operation. During this period, the material is still in a warm and humid state. If the enzyme is not completely inactivated, the residual activity will actually accelerate the decomposition of the glycoside during this gap. This may be the underlying reason why existing stepwise methods generally cannot simultaneously achieve high D-glycoside yield and low L-glycoside dissolution. How to achieve a breakthrough between thorough enzyme inactivation and avoiding high-temperature isomerization remains an unsolved technical problem. Summary of the Invention

[0006] In view of this, the present invention proposes a method to improve the yield of D-amyglucan in peach kernels, aiming to overcome the problems of cumbersome process and low D-glycoside yield in the existing step-by-step method, and at the same time break the technical prejudice that "high temperature enzyme inactivation will inevitably lead to D-glycoside isomerization".

[0007] The technical solution of this invention is implemented as follows: This invention provides a method for improving the yield of D-amyglucosinolate in peach kernels, comprising the following steps: (1) pressing peach kernels at a temperature of 100-150℃ and collecting the residue cake; (2) heating and extracting the residue cake obtained in step (1) with an ethanol aqueous solution of 60-70 vt%, at an extraction temperature of 70-90℃, for an extraction time of 0.2-1 hour, for 1-3 extractions, and combining the extracts to obtain the final product. This method integrates high-temperature enzyme inactivation and pressing defatting from traditional step-by-step operations into a single process, using high-temperature pressing to instantly complete cell wall disruption, oil separation, and amyglucosinolate inactivation, eliminating the possibility of glycoside loss during the pressing period after enzyme inactivation.

[0008] In some embodiments, the pressing in step (1) is completed using an oil press. The oil press is preheated to 100-150°C before the peach kernels are added for pressing. Furthermore, the oil press is a household oil press. By using a household oil press to implement this method, no additional special equipment is required, and enzyme inactivation and degreasing can be integrated on a general-purpose device, reducing the technological threshold and facilitating large-scale promotion.

[0009] In some embodiments, the temperature in step (1) is 120°C. At 120°C, amygdalinase is irreversibly deactivated during pressing, and the synergistic effect of temperature and pressing force creates a loose and porous structure in the residue cake, which is beneficial for subsequent solvent penetration. It is noteworthy that although this temperature is significantly higher than the 60-100°C enzyme inactivation temperature commonly used in existing technologies, D-glycosides do not undergo extensive isomerization as feared in the art. In fact, the D-glycoside extraction and transfer rate is significantly higher than that of the low-temperature stepwise method, which contradicts the expected direction of common knowledge in the art. This indirectly confirms that the effect of high temperature coupled with pressing is not a simple summation of the effects of each step.

[0010] In some embodiments, the concentration of the aqueous ethanol solution in step (2) is 65 wt%. In an aqueous ethanol system, the ethanol concentration affects the polarity of the solvent and its ability to penetrate plant cell walls. The polarity of the 65 wt% aqueous ethanol solution is highly compatible with that of amygdalin molecules, and at this concentration, some water-soluble impurities undergo denaturation and precipitation, reducing the burden of subsequent purification.

[0011] In some embodiments, the heating extraction temperature in step (2) is 80°C, the extraction time is 0.5 hours, and the extraction is performed twice. Thanks to the loose structure of the residue cake formed by high-temperature pressing in step (1) and the effective removal of oil, the extraction solvent can quickly wet the residue cake and dissolve the target components. Extraction equilibrium can be reached in 0.5 hours, and the single extraction efficiency is significantly better than that of unpressed or room-temperature pressed materials. The extraction is basically complete after two extractions, and the content of amygdalin in the third extract is extremely low.

[0012] In some embodiments, the material-to-liquid ratio of the residue cake to the ethanol-water solution in step (2) is 1g:5-15mL, where 1g is based on the mass of peach kernel medicinal material added in step (1), and the amount of residue cake is calculated according to the mass ratio of peach kernel to residue cake. Preferably, the material-to-liquid ratio is 1g:10mL, and the mass ratio of peach kernel to residue cake is 2.08:1. Using 10 times the amount of solvent can achieve a higher concentration gradient driving force while ensuring sufficient immersion of the material. After exceeding 10 times the amount, the growth of the extraction transfer rate tends to level off, and further increasing the amount of solvent will increase the subsequent concentration cost.

[0013] In some embodiments, the extraction transfer rate of D-amygynin in the extract obtained in step (2) is not less than 85%, and the extraction transfer rate of L-amygynin is not more than 10%. This effect index is achieved by the synergistic effect of the above-mentioned step conditions, and cannot be determined by a single parameter.

[0014] In some embodiments, the extraction transfer ratio of D-amygyn to L-amygyn in the extract obtained in step (2) is not less than 15:1. This ratio further characterizes the technical effect of the method of the present invention from the perspective of epimeric selectivity: not only is the yield of D-glycoside high, but more importantly, L-glycoside is effectively suppressed at a low level, which makes the extracted product significantly safer than the product obtained by existing methods.

[0015] In some embodiments, there is no separate enzyme inactivation process between steps (1) and (2), nor is there a cooling or drying process for the material after enzyme inactivation and before pressing. From the perspective of the process route, the present invention eliminates the process gap between enzyme inactivation and pressing, and there is no perceptible time delay between the material being pressed at high temperature and entering the extraction process. This is the most intuitive structural feature that distinguishes it from all existing step-by-step processes.

[0016] The present invention has the following advantages over the prior art: The present invention has the following advantages over the prior art: The present invention replaces the traditional "enzyme inactivation" The "defatting" step-by-step operation is integrated into a single high-temperature pressing step, simplifying the process and reducing equipment usage and material transfer costs between processes. At the same time, this one-step method unexpectedly overcomes the technical prejudice that "high temperature will cause D-amygdaloid isomerization." Under high-temperature pressing conditions of 120°C, not only is there no significant loss of D-glycosides, but a significantly higher D-glycoside extraction and transfer rate than that of the low-temperature step-by-step method is also achieved. The dissolution of L-glycosides is also effectively suppressed to a low level. Compared with the existing step-by-step method, it has achieved a breakthrough in both product safety and target yield. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a graph showing the effect of extraction temperature on the extraction transfer rate of amygdalin in peach kernels in an embodiment of the present invention; Figure 2 This is a graph showing the effect of solvent concentration on the extraction and transfer rate of amygdalin from peach kernels in an embodiment of the present invention. Figure 3 This is a graph showing the effect of extraction time on the extraction transfer rate of amygdalin in peach kernels in an embodiment of the present invention; Figure 4 This is a graph showing the effect of solvent ratio on the extraction and transfer rate of amygdalin from peach kernel in the embodiments of the present invention; Figure 5 This is a graph showing the effect of the number of extractions on the extraction and transfer rate of amygdalin from peach kernels in an embodiment of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 Step 1: Preheat the household oil press to 120℃, add 3kg of peach kernels, start the pressing program, and after pressing, you will get 1.44kg of residue cake and 1.38kg of oil, which should be collected separately. The residue cake is the material to be extracted after enzyme inactivation and defatting.

[0021] Step 2: Take all the residue cake obtained in Step 1, add 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material, heat and extract at 80℃ for 0.5 hours, filter, and collect the filtrate; repeat the extraction of the residue under the same conditions once, filter, and combine the two filtrates to obtain the extract.

[0022] Example 2 Step 1: Preheat the household oil press to 100℃, put in 3kg of peach kernels, start the pressing program, and collect the residue and oil.

[0023] Step 2: Same as Step 2 in Example 1.

[0024] Example 3 Step 1: Preheat the household oil press to 150℃, put in 3kg of peach kernels, start the pressing program, and collect the residue and oil.

[0025] Step 2: Same as Step 2 in Example 1.

[0026] Example 4 Step 1: Same as Step 1 in Example 1.

[0027] Step 2: Add the residue cake obtained in Step 1 to a 60% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 0.5 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0028] Example 5 Step 1: Same as Step 1 in Example 1.

[0029] Step 2: Add the residue cake obtained in Step 1 to a 70% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 0.5 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0030] Example 6 Step 1: Same as Step 1 in Example 1.

[0031] Step 2: Add the residue cake obtained in Step 1 to a 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 70℃ for 0.5 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0032] Example 7 Step 1: Same as Step 1 in Example 1.

[0033] Step 2: Add the residue cake obtained in Step 1 to a 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 90℃ for 0.5 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0034] Example 8 Step 1: Same as Step 1 in Example 1.

[0035] Step 2: Add the residue cake obtained in Step 1 to a 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 0.2 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0036] Example 9 Step 1: Same as Step 1 in Example 1.

[0037] Step 2: Add the residue cake obtained in Step 1 to a 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 1.0 hour, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0038] Example 10 Step 1: Same as Step 1 in Example 1.

[0039] Step 2: Add the residue cake obtained in Step 1 to a 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of the peach kernel medicinal material. Heat and extract at 80℃ for 0.5 hours, filter, and collect the filtrate (extract only once) to obtain the extract.

[0040] Example 11 Step 1: Same as Step 1 in Example 1.

[0041] Step 2: Add the residue cake obtained in Step 1 to a 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 0.5 hours, filter, and collect the filtrate. Repeat the extraction of the residue twice under the same conditions, and combine the three filtrates to obtain the extract.

[0042] Example 12 Step 1: Same as Step 1 in Example 1.

[0043] Step 2: Add the residue cake obtained in Step 1 to a 65% ethanol aqueous solution at a ratio of 1g:5mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 0.5 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0044] Example 13 Step 1: Same as Step 1 in Example 1.

[0045] Step 2: Add the residue cake obtained in Step 1 to a 65% ethanol aqueous solution at a ratio of 1g:15mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 0.5 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0046] Comparative Example 1 (simulating a step-by-step pressing process after enzyme inactivation by boiling water) Step a: Put 3kg of peach kernels into boiling water and boil for 5 minutes. Remove and drain the surface water, then cool to room temperature.

[0047] Step b: Put the peach kernels that have been inactivated in step a into an oil press at room temperature and press them to collect the residue cake.

[0048] Step c: Add the residue cake obtained in step b to a 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 0.5 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0049] Comparative Example 2 (simulating a step-by-step process of pressing after enzyme inactivation in a 60℃ oven) Step a: Spread 3kg of peach kernels evenly on a tray, place them in a 60℃ oven and dry for 4 hours, then remove and cool to room temperature.

[0050] Step b: Put the peach kernels that have been inactivated in step a into an oil press at room temperature and press them to collect the residue cake.

[0051] Step c: Same as step c in Comparative Example 1.

[0052] Comparative Example 3 (no enzyme inactivation, no pressing, direct pulverization and extraction) 3 kg of peach kernels were directly pulverized without any enzyme inactivation or pressing treatment. Then, 65 wt% ethanol aqueous solution (1 g: 10 mL by weight of the medicinal material) was added, and the mixture was heated at 80℃ for 0.5 hours. The mixture was filtered, and the residue was extracted once more. The filtrates were then combined.

[0053] Comparative Example 4 (room temperature pressing, without heating to inactivate enzymes) Step a: Put 3kg of peach kernels into an oil press at room temperature and press them without any preheating, then collect the residue cake.

[0054] Step b: Add the residue cake obtained in step a to a 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material. Heat and extract at 80℃ for 0.5 hours, filter, and repeat the extraction once with the residue. Combine the filtrates.

[0055] Comparative Example 5 (enzyme inactivation in 120℃ oven, no pressing) Step a: Spread 3kg of peach kernels evenly on a tray, place them in a 120℃ oven and dry them for 4 hours, then remove and cool to room temperature.

[0056] Step b: Directly crush the peach kernels after enzyme inactivation in step a, add 65% ethanol aqueous solution at a ratio of 1g:10mL based on the weight of peach kernel medicinal material, heat and extract at 80℃ for 0.5 hours, filter, repeat the extraction once with the filter residue, and combine the filtrates.

[0057] Detection methods Step A: The contents of D-amygin and L-amygin in the extract were determined by high performance liquid chromatography (HPLC). Chromatographic conditions: octadecylsilane-bonded silica gel was used as the stationary phase; methanol-water (20:80) was used as the mobile phase; the detection wavelength was 210 nm; the theoretical plate number calculated based on the amygdalacin peak should not be less than 3000.

[0058] Step B: Accurately weigh appropriate amounts of D-amygynin and L-amygynin reference standards, dissolve and dilute them with methanol to prepare a reference solution containing 0.2 mg per 1 mL, inject it into the liquid chromatograph, and record the chromatogram.

[0059] Step C: Take an appropriate amount of the extract to be tested, filter it through a 0.45μm microporous membrane, inject it into the liquid chromatograph, record the chromatogram, and calculate the concentrations of D-amygin and L-amygin in the extract by peak area using the external standard method.

[0060] Step D: Calculate the extraction transfer rate using the following formula: Extraction transfer rate (%) = Mass of amygdalin in the extract / Total mass of amygdalin in peach kernel × 100%. The total mass of amygdalin in peach kernel is determined by fully extracting the peach kernel according to the methods in the Chinese Pharmacopoeia.

[0061] Results Statistics The results of the extraction and transfer rates of D-amygyn and L-amygyn in each example and comparative example are summarized below: Table 1 Results of extraction and transfer rate determination in the examples

[0062] Table 2 Results of comparative extraction and transfer rate determination

[0063] Results Analysis and Discussion (1) The extraction transfer rate of D-amyglucosinolate in Example 1 reached 88.44%, while that of L-amyglucosinolate was only 5.47%, both of which were superior to the comparative examples. The D / L extraction transfer rate ratio in Example 1 was approximately 16.2:1, which was much higher than that of the comparative examples, indicating that the method of the present invention achieved highly selective extraction of D-amyglucosinolate.

[0064] (2) Comparing Example 1 with Comparative Example 1 (pressing after enzyme inactivation with boiling water) and Comparative Example 2 (pressing after drying at 60°C), it can be seen that although the high-temperature pressing temperature of 120°C is much higher than the enzyme inactivation temperature used in the comparative examples, the yield of D-glycosides not only did not decrease, but increased significantly, while the yield of L-glycosides decreased significantly. This result contradicts the common understanding in the art that "high temperature promotes D-glycoside isomerization," proving that the present invention overcomes existing technical biases.

[0065] (3) Comparing Example 1 with Comparative Example 4 (room temperature pressing), it can be seen that when pressing is performed without high-temperature enzyme inactivation (Comparative Example 4), the yield of D-glycosides is significantly lower than that of Example 1, while the yield of L-glycosides is significantly higher than that of Example 1. This indicates that high temperature plays a key role in enzyme inactivation in the one-step process. Combining the two analyses, it can be seen that only when high temperature and pressing are coupled in the same process can efficient enzyme inactivation and high retention of D-glycosides be achieved simultaneously.

[0066] (4) The comparison between Example 1 and Comparative Example 5 (enzyme inactivation at 120°C but without pressing) is the most illustrative control. Both examples underwent high-temperature treatment at 120°C, but the D-glycoside yield of Comparative Example 5 was only 71.25%, far lower than the 88.44% of Example 1, while its L-glycoside yield was 11.38%, more than twice that of Example 1 (5.47%). This comparison directly proves that when high temperature of 120°C is applied to peach kernels alone, it does indeed cause some D-glycosides to isomerize into L-glycosides, which is consistent with existing knowledge; however, when high temperature of 120°C is coupled with pressing in the same process, not only isomerization is effectively inhibited, but the dissolution efficiency of D-glycosides is significantly improved. This effect is something that those skilled in the art could not have foreseen based on existing knowledge, demonstrating the synergistic effect of high temperature and pressing under instantaneous coupling conditions.

[0067] (5) The results of each embodiment show that, within the parameter range defined in the claims, the extraction transfer rate of D-amyglucosinolate is maintained above 85%, and the extraction transfer rate of L-amyglucosinolate is below 7%, indicating that the method of the present invention has a wide operating window and good process stability.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 increasing the yield of D-amygynin in peach kernels, characterized in that, Includes the following steps: (1) Press the peach kernels at a temperature of 100-150℃ and collect the residue cake; (2) The residue cake obtained in step (1) is extracted by heating with an aqueous ethanol solution of 60-70% concentration at an extraction temperature of 70-90℃ and an extraction time of 0.2-1 hour. The extraction is repeated 1-3 times, and the extracts are combined to obtain the final product.

2. The method according to claim 1, characterized in that, In step (1), the oil press is preheated to 100-150°C before the peach kernels are added to complete the pressing.

3. The method according to claim 2, characterized in that, The oil press mentioned is a household oil press.

4. The method according to claim 1, characterized in that, In step (1), the temperature is 120°C.

5. The method according to claim 1, characterized in that, In step (2), the concentration of the ethanol aqueous solution is 65 wt%.

6. The method according to claim 1, characterized in that, In step (2), the heating extraction temperature is 80°C, the extraction time is 0.5 hours, and the extraction is performed twice.

7. The method according to claim 1, characterized in that, In step (2), the ratio of the residue cake to the ethanol aqueous solution is 1g: 5-15mL, where 1g is the mass of peach kernel medicinal material added in step (1), and the amount of residue cake is calculated according to the mass ratio of peach kernel to residue cake.

8. The method according to claim 7, characterized in that, In step (2), the material-to-liquid ratio is 1g:10mL.

9. The method according to claim 1, characterized in that, In the extract obtained in step (2), the extraction transfer rate of D-amygyn is not less than 81%, and the extraction transfer rate of L-amygyn is not higher than 10%.

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