Efficient biological enrichment method for alpha-eleostearic acid in bitter gourd seed oil

Abstract

The invention belongs to the technical field of natural active substance extraction, and particularly relates to a bitter gourd seed oil alpha-eleostearic acid efficient biological enrichment method and application of bitter gourd seed oil. According to the efficient biological enrichment method for alpha-eleostearic acid in bitter gourd seed oil, a superfine grinding and microwave extraction technology is innovatively adopted, the extraction process of bitter gourd seed oil is systematically optimized, and the optimal treatment conditions are determined as follows: the microwave power is 800W, the superfine grinding time is 20min, the microwave temperature is 50 DEG C and the microwave time is 20min; under the treatment, the bitter gourd seed oil is improved by 1.287 times compared with a conventional extraction mode. The balsam pear seed oil prepared by adopting the balsam pear seed oil alpha-eleostearic acid efficient biological enrichment method provided by the invention has better oxidation resistance and antibacterial effect. The method not only provides a theoretical basis for efficient and green extraction of bitter gourd seed oil, but also provides a technical reference for resource utilization of other high-oil byproducts.

Description

Technical Field

[0001] The invention belongs to the technical field of natural active substance extraction, and particularly relates to a method for efficiently bio-enriching α-eleostearic acid in bitter melon seed oil and application of bitter melon seed oil. Background Art

[0002] Bitter melon (Momordica charantia L.) is a climbing herb in the Cucurbitaceae family and genus Momordica. It is widely distributed from tropical to temperate regions. Native to eastern India, it is now cultivated throughout northern and southern China. Its fruit is named for its densely packed, warty surface and bitter taste. Both the ripe flesh and aril are edible. Bitter melon is rich in vitamin C, dietary fiber, and minerals. It contains active ingredients such as momordica charantia, insulin-like substances, oleanolic acid, cucurbitane-type triterpenes, proteins, steroids, alkaloids, saponins, flavonoids, and gallic acid. It has significant medicinal value, exhibiting antidiabetic, antibacterial, antioxidant, antimutagenic, antiulcer, anti-inflammatory, and lipid-lowering properties.

[0003] Bitter melon is a widely cultivated plant used for both medicinal and edible purposes worldwide. The pulp is primarily edible, with seeds accounting for 15% to 20% of the fruit's dry weight. Currently, bitter melon seed capsules are often discarded as processing waste or used solely for animal feed, resulting in a resource utilization rate of less than 5%, resulting in significant waste. Research has found that bitter melon seeds contain 30% to 35% oil and are rich in protein, as well as active ingredients such as momordica charantia and saponins, making them highly valuable medicinally.

[0004] Patent document CN106860502A discloses a method for preparing bitter melon seed flavonoids. The method comprises the following steps: 1) removing impurities from bitter melon seeds, washing them, drying them, and crushing them; 2) adding 6 to 10 times their weight of a solvent to the crushed bitter melon seeds, and subjecting them to ultrahigh pressure treatment at 120 to 240 MPa for 1 to 3 minutes; 3) subjecting the slurry obtained in step 2) to microwave extraction at 350 to 500 W for 8 to 16 minutes; and 4) filtering the solution obtained in step 3), concentrating it, and spray drying it to obtain bitter melon seed flavonoids. The bitter melon seed flavonoids prepared by this method exhibit antioxidant and antibacterial properties, as well as anti-aging, cerebrovascular disease treatment, and lipid-lowering effects.

[0005] Patent document CN111454342A discloses a method for extracting hypoglycemic polypeptide K from bitter melon seeds. The method comprises the following steps: (1) weighing bitter melon seed protein powder, placing it in a test tube, then adding clean water, shaking it, adjusting the pH value of the solution with NaOH solution, and then placing it in an ultrasonic extraction device for ultrasonic treatment for 10 minutes; (2) placing the test tube containing the solution in a water bath for extraction, and then centrifuging the test tube in a centrifuge for 10 minutes; (3) taking the supernatant, adjusting the pH value with 1M HCl, letting it stand for 30 minutes, and then centrifuging it in a centrifuge for 10 minutes to collect the precipitate; (4) dissolving the precipitate obtained in step (3) with 2mL of NaOH solution to obtain a hypoglycemic polypeptide K solution. The hypoglycemic polypeptide K product obtained by this method has the advantages of high purity and good activity, providing a basis for further screening bitter melon varieties with high polypeptide K content and developing bitter melon hypoglycemic functional products.

[0006] As global demand for vegetable oils continues to grow, developing new oil crops has become a research hotspot in agriculture and food science. Bitter melon seed oil, with unsaturated fatty acids accounting for over 70% and containing bioactive compounds such as polyphenols and flavonoids, offers broad development prospects and industrialization potential as a novel oil resource. Currently, there is significant waste of bitter melon seed resources. Fully developing and utilizing bitter melon seed resources could bring significant economic benefits, avoid resource waste, and alleviate the pressing needs of the oil market. Therefore, vigorous research on bitter melon seed oil is crucial. Summary of the Invention

[0007] In view of the problems of low extraction efficiency, damage to active ingredients, and residual solvents in the current traditional methods for extracting bitter melon seed oil, the present invention provides a method for efficient bio-enrichment of α-eleostearic acid in bitter melon seed oil to solve the above problems. The present invention innovatively adopts ultrafine grinding and microwave extraction technology to systematically optimize the extraction process of bitter melon seed oil, and determines the optimal processing conditions as follows: microwave power 800W, ultrafine grinding time 20min, microwave temperature 50℃, and microwave time 20min. Under this treatment, the bitter melon seed oil is increased by 1.287 times compared with the traditional extraction method. This method not only provides a theoretical basis for the efficient and green extraction of bitter melon seed oil, but also provides a technical reference for the resource utilization of other high-oil by-products.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] The present invention provides a method for efficiently bioaccumulating α-eleostearic acid from momordica charantia seed oil, comprising the following steps:

[0010] Step S1, drying the bitter melon seeds and ultrafine grinding them for 10 to 20 minutes to obtain bitter melon seed powder;

[0011] Step S2: adding the bitter melon seed powder obtained in step S1 into anhydrous ethanol for microwave extraction, filtering, collecting the extract, recovering the ethanol under reduced pressure, and drying at 80° C. to obtain bitter melon seed oil.

[0012] Furthermore, in step S1, the material-liquid ratio of bitter melon powder to anhydrous ethanol is 1 g:20 mL.

[0013] Furthermore, the conditions for the microwave extraction in step S2 are: microwave power of 600-800 W, microwave temperature of 45-50° C., and extraction time of 15-20 min.

[0014] In addition, the present invention also provides a bitter melon seed oil composition, comprising the following components and their weight parts:

[0015] 10-18 parts of bitter melon seed oil, 5-8 parts of trichosanthes seed oil, and 30-48 parts of bitter melon seed polysaccharide. The bitter melon seed oil is the bitter melon seed oil prepared by the above-mentioned bitter melon seed oil α-eleostearic acid efficient bio-enrichment method.

[0016] Furthermore, the preparation method of the Trichosanthes kirilowii seed oil is:

[0017] The seeds of Trichosanthes kirilowii are shelled, crushed, and then passed through a 20-mesh sieve to obtain Trichosanthes kirilowii seed powder. The Trichosanthes kirilowii seed powder is added to anhydrous ethanol for microwave extraction, filtered, and the extract is collected. The ethanol is recovered under reduced pressure and dried at 80° C. to obtain Trichosanthes kirilowii seed oil.

[0018] Furthermore, the material-liquid ratio of the Trichosanthes kirilowii seed powder to anhydrous ethanol is 1 g:8 mL.

[0019] Furthermore, the microwave extraction conditions are as follows: microwave power of 450-500W, microwave temperature of 45-50°C, and extraction time of 5-8 minutes.

[0020] Furthermore, the preparation method of the bitter melon seed polysaccharide is:

[0021] Step A, grinding the dried bitter melon seeds and passing them through an 80-mesh sieve, adding 4 to 6 times the volume of petroleum ether, heating and reflux for 2 to 3 hours, filtering, taking the precipitate, drying and removing the petroleum ether, adding 4 to 6 times the volume of anhydrous ethanol, heating and reflux for 2 to 3 hours, filtering, taking the precipitate, and drying to obtain bitter melon seed powder;

[0022] Step B: adding distilled water to the bitter melon seed powder prepared in step A, extracting at a temperature of 85-95° C. for 2-3 hours, extracting 2-3 times, combining the filtrates, and drying to obtain bitter melon seed polysaccharide.

[0023] Furthermore, in step B, the material-liquid ratio of bitter melon seed powder to distilled water is 1 g:25 mL.

[0024] In addition, the present invention also provides the use of the bitter melon seed oil composition in preparing a drug for preventing or treating hypoglycemic symptoms.

[0025] The present invention provides an efficient bio-enrichment method for α-eleostearic acid in bitter melon seed oil, which is an optimal process obtained through orthogonal experimental screening of four factors and four levels: microwave temperature, microwave power, microwave time, and ultrafine grinding time. The optimal treatment conditions are determined to be: microwave power 800W, ultrafine grinding time 20min, microwave temperature 50°C, and microwave time 20min. Under this treatment, bitter melon seed oil is increased by 1.287 times compared with the traditional extraction method. In addition, the present invention conducts biological activity analysis on bitter melon seed oil, and the results show that bitter melon seed oil has good antioxidant capacity and can scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals to a certain extent. It also has good antibacterial activity, especially the best antibacterial effect against Bacillus subtilis.

[0026] Furthermore, to enhance the medicinal value of bitter melon seed oil, the inventors applied bitter melon seed oil to research on its hypoglycemic efficacy. Experiments have shown that bitter melon seed oil rich in α-eleostearic acid, prepared using the present invention, exhibits excellent hypoglycemic efficacy. Furthermore, to better maintain the efficacy of bitter melon seed oil and reduce the conversion of α-eleostearic acid into catalpaic acid in the bitter melon seed oil, the inventors mixed polysaccharides extracted from bitter melon seeds with the bitter melon seed oil. Unexpectedly, the bitter melon seed polysaccharide not only improves the stability of the bitter melon seed oil but also synergizes with the bitter melon seed oil to enhance its hypoglycemic effect, further enhancing its medicinal value.

[0027] Trichosanthes kirilowii is the fruit of the Cucurbitaceae plant Trichosanthes kirilowii Maxim. It produces abundant, large, and plump seeds containing kernels, glycosides, saponins, organic acids and their salts, gums, fatty oils, and pigments. To further enhance the blood sugar-lowering efficacy of bitter melon seed oil, the inventors extracted the Trichosanthes kirilowii seeds and combined the extracted Trichosanthes kirilowii oil with bitter melon seed oil to study its hypoglycemic efficacy. During the research, the inventors unexpectedly discovered that combining Trichosanthes kirilowii oil with bitter melon seed oil significantly enhanced the hypoglycemic efficacy of the combination, achieving significantly better results than either bitter melon seed oil or Trichosanthes kirilowii oil alone. This provides a new drug for patients seeking hypoglycemic treatment. The optimal blood sugar-lowering effect was achieved when the mass ratio of bitter melon seed oil, Trichosanthes kirilowii oil, and bitter melon seed polysaccharide was 2:1:3.

[0028] In summary, compared with the prior art, the efficient bioaccumulation method of α-eleostearic acid in momordica charantia seed oil provided by the present invention has the following advantages:

[0029] (1) The method for efficiently bioaccumulating α-eleostearic acid in bitter melon seed oil provided by the present invention has a simple preparation process and a good α-eleostearic acid enrichment effect, which can effectively improve the utilization rate of bitter melon seeds and enhance the medicinal value of bitter melon seed oil.

[0030] (2) The bitter melon seed oil prepared by the bitter melon seed oil α-eleostearic acid efficient bio-enrichment method provided by the present invention has a good hypoglycemic effect and can provide a new type of hypoglycemic drug that is purely natural and has no toxic side effects for patients with hyperglycemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the fatty acid distribution diagram of bitter melon seed oil.

[0032] Figure 2 This is a graph showing the effects of ultrafine and ordinary grinding methods on the proportion of fatty acid content in bitter melon seeds.

[0033] Figure 3 This figure shows the effects of ultrafine and ordinary grinding methods on the fatty acid content of bitter melon seeds.

[0034] Figure 4 This is a graph showing the effect of different ultrafine grinding times on the proportion of fatty acid content in bitter melon seeds.

[0035] Figure 5 This is a graph showing the effect of different ultrafine grinding times on the fatty acid content of bitter melon seeds.

[0036] Figure 6 This is a graph showing the effects of ultrasound and microwave treatment on the proportion of fatty acid content in bitter melon seeds.

[0037] Figure 7 This figure shows the effects of ultrasound and microwave treatment on the fatty acid content of bitter melon seeds. DETAILED DESCRIPTION

[0038] The present invention will be further described below by way of specific embodiments, but this is not intended to limit the present invention. Those skilled in the art may make various modifications or improvements based on the basic concept of the present invention, but as long as they do not depart from the basic concept of the present invention, they are all within the scope of the present invention. The raw materials involved in the present invention can be obtained by commercially available or conventional techniques in the art.

[0039] 1. Exploration of efficient bioaccumulation method of α-eleostearic acid in bitter melon seed oil

[0040] 1. Detection of fatty acid composition of bitter melon seed oil:

[0041] (1) Preparation of bitter melon seed oil samples:

[0042] The bitter melon seeds are dried and crushed to obtain bitter melon seed powder; the bitter melon seed powder is placed in a round-bottom flask equipped with a condensation reflux device, anhydrous ethanol is added as an extraction solvent at a liquid-to-solid ratio of 20:1 (mL / g), and an extraction treatment is carried out for 30 minutes under set conditions. The extract is filtered, the extract is collected, the ethanol is recovered under reduced pressure, and the extract is dried at 80°C to obtain bitter melon seed oil.

[0043] (2) Composition analysis of bitter melon seed oil:

[0044] The extracted bitter melon seed oil sample was subjected to transmethylation, and the fatty acids in the bitter melon seed oil were converted into fatty acid methyl esters. The bitter melon seed oil was then subjected to gas chromatography component analysis using modern gas chromatography (GC) technology.

[0045] (3) Analysis results of fatty acids in bitter melon seed oil:

[0046] The fatty acid analysis results of bitter melon seed oil are as follows Figure 1 shown. Figure 1 The fatty acid distribution diagram of bitter melon seed oil is shown in Figure 2. Figure 1 It can be seen from the gas chromatogram of bitter melon seed oil that the fatty acids in bitter melon seed oil mainly include 9 fatty acids: C16:0 (palmitic acid), C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), C20:0 (arachidic acid), C20:1 (arachidonic acid), α-eleostearic acid (C18:3-9c, 11t, 13t), C22:1 erucic acid, and β-eleostearic acid (C18:3-9t, 11t, 13t).

[0047] 2. Comparison of the oil extraction effects of ultrafine grinding and ordinary grinding of bitter melon seeds:

[0048] (1) Preparation of bitter melon seed oil samples:

[0049] The bitter melon seeds were dried and then subjected to ultrafine grinding for 10 minutes and ordinary grinding for 10 minutes. The ordinary grinding was specifically grinding for 5 minutes, pausing for 2 minutes, and then grinding for another 5 minutes, for a total of 10 minutes. The ultrafine grinding was carried out using a vibrating cell-level ultrafine grinder, model XDW-6B, with an amplitude of 6 mm; the ordinary grinding was carried out using a high-speed multifunctional grinder, model 600Y, with a voltage of 220V and a power of 1400W, a grinding degree of 70-300 mesh, a rotation speed of 30,000 r / min, a working time of 5 minutes, and an interval time of 5 minutes. The bitter melon seed powders obtained by the two different grinding methods were obtained.

[0050] 1 g of bitter melon seed powder obtained by two different crushing methods was weighed respectively and placed in a round-bottom flask equipped with a condensation reflux device. Anhydrous ethanol was used as the extraction solvent and added according to a liquid-to-solid ratio of 20:1 (mL / g). The extraction was carried out for 30 minutes under the set conditions, filtered, and the extract was collected. The ethanol was recovered under reduced pressure and dried at 80°C to obtain bitter melon seed oil.

[0051] (2) Determination of the content of bitter melon seed oil:

[0052] The obtained bitter melon seed oil was transferred to an EP tube and dried to constant weight. The mass of the bitter melon seed oil was recorded and the bitter melon seed oil extraction rate was calculated. The fatty acid composition of the bitter melon seed oil samples obtained from two groups of different grinding treatments (ultrafine grinding and ordinary grinding) was analyzed by gas chromatography to determine the types and peak areas of each fatty acid.

[0053] (3) Determination results of bitter melon seed oil content:

[0054] The results of determination of oil content in bitter melon seeds are as follows Figure 2 and Figure 3 shown. Figure 2 This is a graph showing the effects of ultrafine and ordinary grinding methods on the fatty acid content of bitter melon seeds. Figure 3 The figure shows the effect of ultrafine and ordinary grinding methods on the fatty acid content of bitter melon seeds. Figure 2 It can be seen that the proportion of α-eleostearic acid in the ultrafine grinding group was significantly higher than that in the ordinary grinding treatment, increasing by 3.94%, which is the most prominent fatty acid. Figure 3 It can be seen that, except for C16:0, the fatty acid content in bitter melon seed oil after ultrafine grinding treatment is increased. For example, the content of most fatty acids such as C18:0 and α-eleostearic acid is 1.18 and 1.36 times higher than that of the ordinary powder treatment group, respectively; at the same time, the total fat content of bitter melon seed oil after ultrafine grinding treatment is significantly higher than that of the ordinary powder treatment, which is 1.34 times that of the ordinary powder treatment group.

[0055] 3. Analysis of the extraction of fatty acids from bitter melon seeds at different ultrafine grinding times:

[0056] (1) Preparation of bitter melon seed oil samples:

[0057] The bitter melon seeds are dried, and then put into an ultrafine grinder for 10 to 60 minutes, and samples are taken at 10, 20, 30, 40, 50 and 60 minutes respectively to obtain bitter melon seed powder.

[0058] 1 g of bitter melon seed powder obtained with different crushing times was weighed respectively and placed in a round-bottom flask equipped with a condensation reflux device. Anhydrous ethanol was used as the extraction solvent and added according to a liquid-to-solid ratio of 20:1 (mL / g). The extraction was carried out for 30 minutes under the set conditions, filtered, and the extract was collected. The ethanol was recovered under reduced pressure and dried at 80°C to obtain bitter melon seed oil.

[0059] (2) Fatty acid analysis of bitter melon seeds:

[0060] The obtained bitter melon seed oil was transferred to an EP tube and dried to constant weight. The mass of bitter melon seed oil was recorded and the bitter melon seed oil extraction rate was calculated. The bitter melon seed oil samples obtained at different crushing times were analyzed for fatty acid composition by gas chromatography to determine the types and peak areas of each fatty acid.

[0061] (3) Analysis results of fatty acids in bitter melon seeds:

[0062] The fatty acid analysis results of bitter melon seeds are as follows Figure 4 and Figure 5 shown. Figure 4 This is the effect of different ultrafine grinding times on the proportion of fatty acid content in bitter melon seeds. Figure 5 The figure shows the effect of different ultrafine grinding time on the fatty acid content of bitter melon seeds. Figure 4 It can be seen that different ultrasound times have a significant effect on the composition and content of each fatty acid in bitter melon seed oil. For fatty acids such as C18:0, C18:1, α-eleostearic acid, and β-eleostearic acid, the content fluctuates significantly when the ultrasound time changes. The content of α-eleostearic acid shows a trend of first increasing and then stabilizing as the ultrasound time increases. Figure 5 Ultrasound duration also affected the overall content of bitter melon seed oil and the content of individual fatty acids. The contents of C18:0, C20:1, and total lipids varied significantly with different ultrasound times. C18:0 was highest after 10 minutes of ultrasound (marked with the letter "a"); C20:1 was significantly higher after 20 minutes of ultrasound. α-Eleostearic acid and total lipids reached their highest values ​​after 20 minutes of ultrasound, with total lipid content significantly higher than at other ultrasound times.

[0063] 4. Comparison of the effects of ultrasonic treatment and microwave treatment on oil extraction from bitter melon seeds:

[0064] (1) Preparation of bitter melon seed oil samples:

[0065] The bitter melon seeds are dried and then put into an ultrafine grinder for 20 minutes to obtain bitter melon seed powder.

[0066] Ultrafinely ground bitter melon seed powder was subjected to ultrasound and microwave treatment, respectively. Four ultrasonic treatment gradients were established: 125W, 135W, 145W, and 155W, at a temperature of 50°C for 60 minutes. Four microwave treatment gradients were established: 500W, 600W, 700W, and 800W. The temperature was 50°C for 10 minutes. Each gradient was repeated three times in parallel for both experiments. One gram of bitter melon seed powder was accurately weighed and placed in a round-bottom flask equipped with a reflux system. Anhydrous ethanol was added as the extraction solvent at a liquid-to-solid ratio of 20:1 (mL / g). Extraction was performed under the specified conditions for 30 minutes. The extract was filtered, collected, and the ethanol was recovered under reduced pressure. The extract was then dried at 80°C to obtain bitter melon seed oil.

[0067] (2) Fatty acid analysis of bitter melon seeds:

[0068] The obtained bitter melon seed oil was transferred to an EP tube and dried to constant weight. The mass of the bitter melon seed oil was recorded and the bitter melon seed oil extraction rate was calculated. The fatty acid composition of the bitter melon seed oil samples obtained from the two groups of different treatments (microwave treatment and ultrasonic treatment) was analyzed by gas chromatography to determine the types and peak areas of each fatty acid.

[0069] (3) Analysis results of fatty acids in bitter melon seeds:

[0070] The fatty acid analysis results of bitter melon seeds are as follows Figure 6 and Figure 7 shown. Figure 6 This is a graph showing the effects of ultrasound and microwave treatment on the fatty acid content of bitter melon seeds. Figure 7 The figure shows the effects of ultrasonic and microwave treatment on the fatty acid content of bitter melon seeds. Figure 6 and Figure 7 It can be seen that both ultrasonic and microwave treatments increase the amount of bitter melon seed oil extracted with increasing treatment power. Among the major fatty acid components, C18:0, C20:1, α-eleostearic acid, and total lipids differed significantly between treatments. Regarding the extraction performance of the target α-eleostearic acid, the relatively high content of C20:0 was significantly higher under the 700W and 800W ultramicro-treatments than under the ultrasonic treatment group, ranking at the top of the treatment group. Meanwhile, the content of fatty acids such as C20:0 was relatively low, with minimal fluctuations across treatments. In terms of total lipid content, treatments such as the 800W ultramicro-treatment significantly increased the total lipid content of bitter melon seed oil above that of other treatments, reflecting the outstanding effectiveness of this treatment in increasing overall lipid content. Therefore, this experiment will select microwave treatment as the optimized condition for subsequent orthogonal experiments.

[0071] 2. Orthogonal experimental analysis of efficient bioaccumulation method of α-eleostearic acid in bitter melon seed oil

[0072] (1) Orthogonal experimental design:

[0073] In order to further explore the interaction of various process parameters in ultrafine grinding and microwave treatment and its effect on the oil extraction rate of bitter melon seeds, this experiment was based on the results of single factor experiment and used L16(4 5 ) An orthogonal experimental design systematically examined the optimal combination of four key factors: microwave power (A), microwave temperature (B), ultrafine grinding time (C), and microwave duration (D). By balancing the levels of each factor, orthogonal experiments can significantly reduce the number of experiments while also considering the synergistic contribution of multiple factors to oil extraction. Through range analysis and variance tests, the aim was to clarify the order of importance of each factor's impact on total fat content and determine the optimal combination of process parameters, providing a theoretical basis for subsequent verification experiments and industrial application.

[0074] Table 1 Factors and levels of the L16(45) orthogonal experiment

[0075]

[0076] Table 2L16(45) orthogonal experimental results and analysis

[0077]

[0078]

[0079] (2) Data analysis methods:

[0080] Range analysis method: calculate the average value of each factor level (K value)

[0081] Calculate the range:

[0082] Factor ranking: the greater the range, the more significant the impact

[0083] (3) Orthogonal experiment results:

[0084] From Table 2, it can be concluded that the optimal process conditions for bitter melon seed oil extraction are A4B3C2D2, namely, microwave frequency 800W, microwave temperature 50℃, ultrafine grinding time 20min, and microwave time 20min.

[0085] The contribution of factors affecting the oil extraction content of bitter melon seeds is: B>D>A>C, that is, microwave temperature>microwave time>microwave frequency>ultrafine grinding time.

[0086] Three parallel validation experiments using the A4B3C2D2 conditions yielded total lipid contents of 6.21841 mg / mL, 6.26639 mg / mL, and 6.2524 mg / mL, respectively, consistent with the orthogonal results. The optimized total lipid content of bitter melon seed oil was 1.287 times higher than the 4.8526 mg / mL obtained with conventional powder.

[0087] Example 1: A method for efficiently bioaccumulating α-eleostearic acid from bitter melon seed oil

[0088] Step S1, drying the bitter melon seeds and ultrafine grinding them for 20 minutes to obtain bitter melon seed powder;

[0089] Step S2, adding the bitter melon seed powder prepared in step S1 to anhydrous ethanol for microwave extraction, wherein the material-liquid ratio of the bitter melon powder to anhydrous ethanol is 1g:20mL, and the conditions of the microwave extraction are: microwave power of 800W, microwave temperature of 50°C, and extraction time of 20min; filtering, collecting the extract, recovering ethanol under reduced pressure, and drying at 80°C to obtain bitter melon seed oil.

[0090] Example 2: A method for preparing Trichosanthes kirilowii seed oil

[0091] The seeds of Trichosanthes kirilowii are shelled, crushed, and then passed through a 20-mesh sieve to obtain Trichosanthes kirilowii seed powder. The Trichosanthes kirilowii seed powder is added to anhydrous ethanol for microwave extraction, wherein the solid-liquid ratio of the Trichosanthes kirilowii seed powder to anhydrous ethanol is 1 g:8 mL, and the conditions of the microwave treatment are: microwave power of 500 W, microwave temperature of 45° C., extraction time of 6 min, filtering, collecting the extract, recovering ethanol under reduced pressure, and drying at 80° C. to obtain Trichosanthes kirilowii seed oil.

[0092] Example 3: Preparation method of bitter melon seed polysaccharide

[0093] Step A: Grind the dried bitter melon seeds and pass them through an 80-mesh sieve, add 6 times the volume of petroleum ether, heat and reflux for 2 hours, filter, take the precipitate, dry and remove the petroleum ether, add 6 times the volume of anhydrous ethanol, heat and reflux for 2 hours, filter, take the precipitate, and dry to obtain bitter melon seed powder;

[0094] Step B: adding the bitter melon seed powder prepared in step A to distilled water, with the material-liquid ratio of the bitter melon seed powder to distilled water being 1 g:25 mL, extracting at 90° C. for 3 h, extracting three times, combining the filtrates, and drying to obtain bitter melon seed polysaccharide.

[0095] Example 4: A bitter melon seed oil composition

[0096] The bitter melon seed oil composition is composed of the following components and their weight parts:

[0097] 10 parts of bitter melon seed oil, 5 parts of trichosanthes seed oil, and 30 parts of bitter melon seed polysaccharide, wherein the bitter melon seed oil is the bitter melon seed oil prepared in Example 1, the trichosanthes seed oil is the trichosanthes seed oil prepared in Example 2, and the bitter melon seed polysaccharide is the bitter melon seed polysaccharide prepared in Example 3.

[0098] Preparation method:

[0099] Dissolve bitter melon seed polysaccharide in distilled water, wherein the amount of distilled water added is 3 times the weight of the bitter melon seed polysaccharide, and stir evenly to obtain a mixed solution; then add bitter melon seed oil and trichosanthes seed oil into the mixed solution, stir evenly, and dry to obtain the product.

[0100] Example 5: A bitter melon seed oil composition

[0101] The bitter melon seed oil composition is composed of the following components and their weight parts:

[0102] 12 parts of bitter melon seed oil, 6 parts of trichosanthes seed oil, and 36 parts of bitter melon seed polysaccharide, wherein the bitter melon seed oil is the bitter melon seed oil prepared in Example 1, the trichosanthes seed oil is the trichosanthes seed oil prepared in Example 2, and the bitter melon seed polysaccharide is the bitter melon seed polysaccharide prepared in Example 3.

[0103] Preparation method:

[0104] Dissolve bitter melon seed polysaccharide in distilled water, wherein the amount of distilled water added is 3 times the weight of the bitter melon seed polysaccharide, and stir evenly to obtain a mixed solution; then add bitter melon seed oil and trichosanthes seed oil into the mixed solution, stir evenly, and dry to obtain the product.

[0105] Example 6: A bitter melon seed oil composition

[0106] The bitter melon seed oil composition is composed of the following components and their weight parts:

[0107] 15 parts of bitter melon seed oil, 8 parts of trichosanthes seed oil, and 45 parts of bitter melon seed polysaccharide, wherein the bitter melon seed oil is the bitter melon seed oil prepared in Example 1, the trichosanthes seed oil is the trichosanthes seed oil prepared in Example 2, and the bitter melon seed polysaccharide is the bitter melon seed polysaccharide prepared in Example 3.

[0108] Preparation method:

[0109] Dissolve bitter melon seed polysaccharide in distilled water, wherein the amount of distilled water added is 3 times the weight of the bitter melon seed polysaccharide, and stir evenly to obtain a mixed solution; then add bitter melon seed oil and trichosanthes seed oil into the mixed solution, stir evenly, and dry to obtain the product.

[0110] Comparative Example 1: A bitter melon seed oil composition

[0111] The bitter melon seed oil composition is composed of the following components and their weight parts:

[0112] 10 parts of bitter melon seed oil, 10 parts of trichosanthes seed oil, and 10 parts of bitter melon seed polysaccharide, wherein the bitter melon seed oil is the bitter melon seed oil prepared in Example 1, the trichosanthes seed oil is the trichosanthes seed oil prepared in Example 2, and the bitter melon seed polysaccharide is the bitter melon seed polysaccharide prepared in Example 3.

[0113] Preparation method:

[0114] Dissolve bitter melon seed polysaccharide in distilled water, wherein the amount of distilled water added is 3 times the weight of the bitter melon seed polysaccharide, and stir evenly to obtain a mixed solution; then add bitter melon seed oil and trichosanthes seed oil into the mixed solution, stir evenly, and dry to obtain the product.

[0115] Experimental Example 1: Antioxidant Activity of Bitter Melon Seed Oil

[0116] 1. Experimental methods:

[0117] Antioxidant activity is an important indicator for evaluating the functionality of oils and fats, directly impacting their potential applications in food, medicine, and cosmetics. This study used the DPPH free radical scavenging method to determine the in vitro antioxidant activity of the bitter melon seed oil prepared in Example 1. The DPPH free radical is a stable organic free radical, and its scavenging ability can directly reflect the antioxidant efficacy of the active ingredients in the oil (such as polyphenols and unsaturated fatty acids).

[0118] 2. Experimental results:

[0119] The experimental results are shown in Table 3.

[0120] Table 3 Free radical scavenging effect of bitter melon seed oil

[0121]

[0122] As shown in Table 3, the bitter melon seed oil prepared in Example 1 has an average DPPH free radical scavenging rate of 52.38%, which shows a certain antioxidant effect.

[0123] Experimental Example 2: Antibacterial Activity of Bitter Melon Seed Oil

[0124] 1. Experimental methods:

[0125] To evaluate the antibacterial properties and potential applications of the bitter melon seed oil prepared in Example 1, this study used the disc diffusion method to conduct antibacterial activity experiments against six common pathogenic bacteria and spoilage bacteria, including Staphylococcus aureus and Bacillus subtilis. By measuring the diameters of the inhibition zones of oil samples at different dilutions, the antibacterial effect and minimum effective concentration (MEC) were quantitatively analyzed. Chloroform was used as a blank control.

[0126] 2. Experimental results:

[0127] The experimental results are shown in Table 4.

[0128] Table 4 Bitter melon seed oil inhibition zone radius (mm) for 6 different bacterial species

[0129] Chloroform Original solution 1:10 dilution 1:100 dilution Saccharomyces cerevisiae <![CDATA[1.42±0.26 ab ]]> <![CDATA[1.55±0.48 bc ]]> <![CDATA[0.93±0.31 ab ]]> <![CDATA[1.57±0.1 a ]]> Staphylococcus aureus <![CDATA[1.53±0.21 a ]]> <![CDATA[1.88±0.2 b ]]> <![CDATA[0.23±0.03 bc ]]> <![CDATA[1.3±0.15 bc ]]> Escherichia coli <![CDATA[1.02±0.06 b ]]> <![CDATA[1.07±0.08 cd ]]> <![CDATA[0.03±0.03 c ]]> <![CDATA[0.8±0.22 d ]]> Vibrio cholerae <![CDATA[1.58±0.13 a ]]> <![CDATA[1.65±0.13 bc ]]> <![CDATA[0.4±0.31 bc ]]> <![CDATA[1.53±0.08 ab <!-- 9 -->]]> Listeria <![CDATA[0.45±0.38 c ]]> <![CDATA[0.47±0.35 d ]]> <![CDATA[0±0 c ]]> <![CDATA[0.2±0.15 e ]]> Bacillus subtilis <![CDATA[1.35±0.17 ab ]]> <![CDATA[3.4±0.57 a ]]> <![CDATA[1.57±0.86 a ]]> <![CDATA[1.27±0.06 c ]]>

[0130] Note: Different letters indicate significant differences (P>0.05).

[0131] Table 4 shows a comparison of the radii of the inhibition zones of bitter melon seed oil against six different bacterial species. The radii of the inhibition zones for different dilutions and the blank group are ranked from largest to smallest: stock solution > chloroform > 1:100 dilution > 1:10 dilution. The radius of the inhibition zone for the stock solution is larger than that for chloroform, indicating that bitter melon seed oil has a greater antibacterial effect than chloroform against the six different bacterial species. The order of the antibacterial effect of bitter melon seed oil against the six bacterial species, from strongest to weakest, is: Bacillus subtilis > Staphylococcus aureus > Saccharomyces cerevisiae > Vibrio cholerae > Escherichia coli > Listeria monocytogenes.

[0132] Experimental Example 3: Hypoglycemic Effect of Bitter Melon Seed Oil

[0133] 1. Experimental methods:

[0134] Diabetic KM mice with successfully established diabetes and blood glucose levels above 15 mmol / L were divided into five experimental groups of 15 mice each. Fifteen normal KM mice were also selected as a control group. Each experimental group received oral administration of 200 mg / kg / day of the drug, while the control group received an equivalent volume of saline. The drug was administered once daily for one month. Fasting blood glucose levels were measured 12 hours after the final dose.

[0135] The experimental groups were divided into the following groups:

[0136] Example 1 group: gavage with the bitter melon seed oil prepared in Example 1;

[0137] Group 2 of Example 2 was administered with the Trichosanthes seeds oil prepared in Example 2 by gavage;

[0138] Example 3 group: gavage with Momordica charantia seed polysaccharide prepared in Example 3;

[0139] Group 5 of Example 5: gavage with the bitter melon seed oil composition prepared in Example 5;

[0140] Comparative Example 1 group: The bitter melon seed oil composition prepared in Comparative Example 1 was administered orally.

[0141] 2. Experimental results:

[0142] The experimental results are shown in Table 5.

[0143] Table 5 Hypoglycemic effect of bitter melon seed oil

[0144]

[0145] As shown in Table 5, the bitter melon seed oil composition provided by the present invention has a significant blood sugar lowering effect.

[0146] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for efficiently bioaccumulating α-eleostearic acid from bitter melon seed oil, characterized in that: The following steps are involved: Step S1, drying the bitter melon seeds and ultrafine grinding them for 10 to 20 minutes to obtain bitter melon seed powder; Step S2: adding the bitter melon seed powder obtained in step S1 into anhydrous ethanol for microwave extraction, filtering, collecting the extract, recovering the ethanol under reduced pressure, and drying at 80° C. to obtain bitter melon seed oil.

2. The method for efficiently bioaccumulating α-eleostearic acid from momordica charantia seed oil as claimed in claim 1, wherein: In step S1, the material-liquid ratio of bitter melon powder to anhydrous ethanol is 1 g:20 mL.

3. The method for efficiently bioaccumulating α-eleostearic acid from momordica charantia seed oil as claimed in claim 1, wherein: The conditions for microwave extraction in step S2 are: microwave power of 600-800 W, microwave temperature of 45-50° C., and extraction time of 15-20 min.

4. A bitter melon seed oil composition, characterized in that It includes the following ingredients and their weight parts: 10-18 parts of bitter melon seed oil, 5-8 parts of trichosanthes seed oil, and 30-48 parts of bitter melon seed polysaccharide. The bitter melon seed oil is the bitter melon seed oil prepared by the bitter melon seed oil α-eleostearic acid efficient bioenrichment method according to any one of claims 1 to 3.

5. The bitter melon seed oil composition according to claim 4, wherein The preparation method of the Trichosanthes kirilowii seed oil is: The seeds of Trichosanthes kirilowii are shelled, crushed, and then passed through a 20-mesh sieve to obtain Trichosanthes kirilowii seed powder; the Trichosanthes kirilowii seed powder is added into anhydrous ethanol for microwave extraction, filtered, the extract is collected, the ethanol is recovered under reduced pressure, and dried at 80° C. to obtain Trichosanthes kirilowii seed oil.

6. The bitter melon seed oil composition according to claim 5, wherein The solid-liquid ratio of the Trichosanthes kirilowii seed powder to anhydrous ethanol is 1 g:8 mL.

7. The bitter melon seed oil composition according to claim 5, wherein The microwave extraction conditions are as follows: microwave power of 450-500W, microwave temperature of 45-50°C, and extraction time of 5-8 minutes.

8. The bitter melon seed oil composition according to claim 4, wherein The preparation method of the bitter melon seed polysaccharide is as follows: Step A, grinding the dried bitter melon seeds and passing them through an 80-mesh sieve, adding 4 to 6 times the volume of petroleum ether, heating and reflux for 2 to 3 hours, filtering, taking the precipitate, drying and removing the petroleum ether, adding 4 to 6 times the volume of anhydrous ethanol, heating and reflux for 2 to 3 hours, filtering, taking the precipitate, and drying to obtain bitter melon seed powder; Step B: adding distilled water to the bitter melon seed powder prepared in step A, extracting at a temperature of 85-95° C. for 2-3 hours, extracting 2-3 times, combining the filtrates, and drying to obtain bitter melon seed polysaccharide.

9. The bitter melon seed oil composition according to claim 8, wherein The material-liquid ratio of bitter melon seed powder to distilled water in step B is 1 g:25 mL.

10. Use of the bitter melon seed oil composition according to any one of claims 4 to 9 in preparing a drug for preventing or treating hypoglycemic symptoms.

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