Preparation method of total alkaloids of Pectocarpus fraxinus and application of total alkaloids in fruit preservation
Through the preparation and application of total alkaloids in potted trees, the problem of infecting fruits by Botrytis aceta, the effective preservation of fruits is achieved, and a natural food preservative is provided.
Patent Information
- Application Number
- CN202510305496.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-06-10
AI Technical Summary
The infection of Botrytis aurora on fruits, especially pears, leads to fruit rot, causing economic losses and quality problems, and it is difficult to effectively prevent and control the existing technology.
The preparation method of total alkaloids of potted trees is obtained by alcohol extraction, acid extraction, alkali precipitation, extraction and chromatography separation of dried leaves of potted trees, and total alkaloids of potted trees rich in active ingredients such as Dehydrorudgeifline and Echitamine, and used to preserve fruit freshness.
The total alkaloids of the potted tree have a significant inhibitory effect on Botrytis aceta, extend the time of fruit spoilage, and provide a natural food preservative with good application prospects.
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Figure CN120118138A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of food preservation, and particularly relates to a preparation method of total alkaloids of Calophyllum inophyllum L. and its application in fruit preservation. Background Art
[0002] Botrytis cinerea, also known as gray mold, is a plant pathogen widely distributed in nature. Its host range covers a variety of fruits, and pears are one of the more common susceptible hosts. The growth and spread of this pathogen are significantly affected by environmental factors. Under the conditions of relative humidity higher than 90% and temperature between 10 and 30 °C, its growth and reproduction are particularly active, and such temperature and humidity conditions are common in the transportation, storage, and sales of fruits after harvesting. After infection, the hyphae of Botrytis cinerea colonize on the pear epidermis and form lesions, whose color shows a gradual change from light brown to dark brown. Subsequently, the conidia produced by the pathogen spread to surrounding healthy fruits through transmission routes such as air flow and water droplet splashing, thereby triggering a larger-scale infection phenomenon. Once the pear peel is infected by Botrytis cinerea, the internal pulp tissue will gradually soften and rot, which not only seriously damages the appearance quality of the fruit but also completely loses its edible value, bringing serious economic losses and product quality hazards to the fruit industry. Therefore, actively exploring new and efficient fruit preservation drugs is of great significance for ensuring fruit quality, reducing postharvest losses, and promoting the sustainable development of the fruit industry.
[0003] As an important class of natural products, alkaloids widely exist in various biological organisms such as plants, animals, and microorganisms. They have the characteristics of diverse structural types and significant biological activities, occupying a crucial position in the field of natural products. In recent years, many alkaloid compounds with antibacterial and bactericidal activities have been reported successively, showing the great potential of alkaloid compounds in antibacterial aspects. Against the background of the current widespread application of chemical synthetic pesticides, a series of problems such as environmental pollution, pesticide residues, and enhanced resistance of harmful organisms have also emerged. Therefore, exploring and developing highly efficient and low-toxic anti-plant pathogen preparations from natural products has become a hot topic of common concern in the global scientific research field and agricultural production practice. The unique antibacterial effect exhibited by natural alkaloids has become one of the key research directions.
[0004] Alstonia rostrata is a plant of the genus Alstonia in the family Apocynaceae. It is mainly distributed in Yunnan, Guangdong, Hainan and other regions. It is a traditional medicinal plant of the Dai ethnic group. It is flat in nature and bitter in taste. Its leaves, bark and latex can all be used as medicine. The leaves and bark can be used to relieve cough and asthma, and are mainly used to treat acute and chronic bronchitis; the latex can be used externally for hemostasis. The chemical components in the leaves of Alstonia rostrata are complex, mainly including alkaloids, triterpenes, lignans and other chemical components. Among them, alkaloids are the chemical components with rich content and particularly representative research value in Alstonia rostrata, especially having significant pharmacological effects in antibacterial aspects. Summary of the Invention
[0005] The purpose of the present invention is to provide a preparation method of total alkaloids from Alstonia rostrata and its application in fruit preservation, aiming to solve the problems existing in the above-mentioned background technology. To achieve the above purpose, the technical solution adopted by the present invention is:
[0006] A preparation method of total alkaloids from Alstonia rostrata, including total alkaloids from Alstonia rostrata, and the total alkaloids from Alstonia rostrata are alcohol extracts of dried leaves of Alstonia rostrata;
[0007] The preparation method of total alkaloids from Alstonia rostrata is as follows:
[0008] S1: Take fresh leaves of mature Alstonia rostrata, dry them, pulverize them, soak them in 90% methanol-water for 3 days, then add 8 times the weight of methanol and heat under reflux for extraction four times (3 h / time), filter, combine the filtrates, concentrate by rotary evaporation, and dry to obtain the crude extract of Alstonia rostrata;
[0009] S2: The above crude extract is treated by the principle of acid extraction and alkali precipitation. Specifically, add dilute hydrochloric acid aqueous solution with pH = 2-3 to fully dissolve, filter to remove insoluble substances, that is, non-alkaloids. Add 1% ammonia water to the filtrate to adjust the pH to 8-9, and successively add organic solvents such as petroleum ether, ethyl acetate and chloroform for extraction, and combine the organic layers to obtain small-polarity alkaloid components;
[0010] S3: Secondly, extract with n-butanol solvent, collect the n-butanol layer to obtain large-polarity alkaloids. The small-polarity and large-polarity alkaloids are combined and adsorbed on a macroporous resin chromatographic column, and gradient elution is carried out with 5%-100% methanol-water as the eluent to remove pigment impurities. Guided by LC-MS detection, collect according to the gradient elution of the polarity ratio, and concentrate and evaporate to dryness to obtain the total alkaloids of Alstonia rostrata.
[0011] Preferably, the active ingredients in the total alkaloids of Alstonia rostrata include the following compounds: Dehydrorudgeifoline, Echitamine, Demethyldeformylcorymine, 17-O-acetylnorechitamine and Demethylechitamine.
[0012] Preferably, the content of Dehydrorudgeifoline in the total alkaloids of Winchia calophylla A. DC. is not less than 10.0%, the content of Echitamine is not less than 4.0%, and the content of Demethylechitamine is not less than 1.0%.
[0013] Preferably, the total alkaloids of Winchia calophylla A. DC. act on fruits after picking.
[0014] Preferably, when used for fruit preservation, the concentration of the total alkaloids of Winchia calophylla A. DC. should be greater than 92.822 μg / mL.
[0015] Preferably, the fruit is a common pear sold in the Chinese market. Further, the pear is preferably the fresh fruit of Pyrus bretschneideri Rehd. of the genus Pyrus in the Rosaceae family.
[0016] Preferably, the fruit pathogenic bacterium is Botrytis cinerea.
[0017] Advantages of the present invention:
[0018] The present invention provides a preparation method of the total alkaloids of Winchia calophylla A. DC. and its application in fruit preservation. The total alkaloids of Winchia calophylla A. DC. described in the present invention have a good inhibitory effect on the mycelia of fruit pathogenic bacteria. The experimental results show that it can extend the time of food spoilage, and thus can be used for food preservation, providing a food preservative derived from natural products, which has good application prospects in the food field. Description of the drawings
[0019] Figure 1 Shows the preservation effects of the total alkaloids of Winchia calophylla A. DC. with different concentrations on fruit pears.
[0020] Figure 2 -A shows the inhibition rate of the total alkaloids of Winchia calophylla A. DC. on Botrytis cinerea.
[0021] Figure 2 -B shows the lesion growth curves of the fruit pear groups treated with 125, 250, and 500 μg / mL of the total alkaloids of Winchia calophylla A. DC. and the blank control group.
[0022] Figure 3 Is the structural formula diagram of Dehydrorudgeifoline.
[0023] Figure 4 Is the structural formula diagram of Echitamine.
[0024] Figure 5 Is the structural formula diagram of Demethyldeformylcorymine.
[0025] Figure 6It is the structural formula diagram of 17-O-acetylnorechitamine.
[0026] Figure 7 It is the structural formula diagram of Demethylechitamine. Specific embodiments
[0027] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. The preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.
[0028] It should be noted that when an element is referred to as being "fixed to" another element, it can be directly on the other element or there can also be an intermediate element. When an element is considered to be "connected" to another element, it can be directly connected to the other element or there may be an intermediate element at the same time. On the contrary, when an element is referred to as being "directly on" another element, there is no intermediate element. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only and do not represent the only embodiments. The terms "upper end", "lower end", "left side", "right side", "front end", "rear end" and similar expressions used herein refer to the positional relationship in the drawings.
[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.
[0030] The technical solutions of this patent will be further described in detail below in conjunction with specific embodiments.
[0031] As Figures 1 to 7 shown, Example 1, this example is the first example of the present invention. This example mainly proposes a method for preparing total alkaloids from the leaves of Winchia calophylla, and the specific steps are as follows:
[0032] Take the fresh leaves of mature Winchia calophylla, dry them, pulverize them, soak them in 90% methanol-water for 3 days, then add 8 times the weight of methanol and heat under reflux for extraction four times (3 h / time), filter, combine the filtrates, concentrate by rotary evaporation, and dry to obtain the crude extract of Winchia calophylla.
[0033] The above-mentioned crude extract was treated by the principle of acid extraction and alkali precipitation. Specifically, it was fully dissolved by adding a dilute hydrochloric acid aqueous solution with a pH of 2-3, and the insoluble matter, i.e., non-alkaloids, was removed by filtration. 1% ammonia water was added to the filtrate to adjust the pH to 8-9, and then organic solvents such as petroleum ether, ethyl acetate, and chloroform were added for extraction. The organic layers were combined to obtain small-polarity alkaloid components. Secondly, n-butanol solvent was added for extraction, and the n-butanol layer was collected to obtain large-polarity alkaloids. The small-polarity and large-polarity alkaloids were combined and adsorbed on a macroporous resin chromatographic column, and gradient elution was carried out with 5%-100% methanol-water as the eluent to remove pigment impurities. Guided by LC-MS detection, collection was carried out according to the gradient elution of the polarity ratio, and evaporation to dryness after concentration gave the total alkaloids of Winchia calophylla. In this example, the target fraction was eluted with 60-75% alcohol.
[0034] The concentration of the active ingredient in the total alkaloids detection concentrate of Winchia calophylla mentioned above was measured. The detection method was as follows: 200 μL of the concentrate of the total alkaloids of Winchia calophylla was pipetted into a 1.5 mL Eppendorf centrifuge tube and centrifuged at 12,000 rpm for 15 min at 4 °C. 100 μL of the supernatant was pipetted into an injection vial containing a liner with a volume of 200 μL. Then HPLC was used for detection. 0.1% formic acid water was used as mobile phase A, and acetonitrile was used as mobile phase B, and stepwise isocratic gradient elution was carried out according to the regulations in the following table:
[0035] Table 1 Elution program
[0036]
[0037] The flow rate was 0.3 mL / min, the column temperature was 40 °C, and the injection volume was 5 μL.
[0038] After detection, the content of the compound Dehydrorudgeifoline in the total alkaloids concentrate of Winchia calophylla was 20.07%, the content of Echitamine was 7.52%, and the content of N(4)-demethylechitamine was 4.63%.
[0039] Example 2. This example is the second example of the present invention. This example provides a method for culturing Botrytis cinerea, a pathogenic bacterium of fruit pears. The specific steps are as follows:
[0040] Preparation method of PDA medium: 100 g of peeled potatoes were cut into pieces and put into distilled water and boiled for 30 min, filtered through gauze to retain the filtrate. After weighing 7.5-8.5 g of agar and 10 g of glucose, the volume was fixed to 500 mL, the pH value was natural, and sterilization was carried out at 105 °C for 60 min.
[0041] Activation of the strain: The Botrytis cinerea strain was purchased from Beina Biology - Henan Engineering and Technology Research Center for Industrial Microbial Strains. Use a sterile inoculation knife to inoculate the Botrytis cinerea stored in a glycerol tube onto a PDA plate medium and culture it in an incubator at a constant temperature of 25°C for 10 days until the mycelium covers the entire culture dish.
[0042] Example 3, this example is the third example of the present invention. This example provides a method for measuring the inhibitory effect of the total alkaloids of Winchia calophylla on the pathogenic bacterium Botrytis cinerea. The specific steps are as follows:
[0043] The mycelial growth rate method was used to detect the antibacterial activity of the total alkaloids of Winchia calophylla against Botrytis cinerea. Dissolve the total alkaloids in sterile distilled water and configure them in a PDA medium at 45°C to make plates with sample concentrations of 31.25 μg / mL, 62.50 μg / mL, 125.00 μg / mL, 250.00 μg / mL, and 500.00 μg / mL. Use the plate with only PDA medium as the blank control. Use a punch to punch out Botrytis cinerea fungal cakes (d = 9 mm) and inoculate them in the center of the plate, repeating three times. Incubate at a constant temperature of 25°C until the mycelium in the control group covers the plate. Use the cross-cross method to measure the colony diameter, calculate the inhibition rate of the total alkaloids at different concentrations on the mycelium of Botrytis cinerea, and use SPSS software to calculate the EC 50 value and obtain the virulence regression equation.
[0044]
[0045] Based on the inhibition rate data of Botrytis cinerea mycelium, perform a linear regression analysis between the probit value (y) and the logarithm of the mass concentration (x), and then determine the virulence regression equation of the total alkaloids y = a + bx, and calculate its median effective concentration (EC 50 ) and the corresponding correlation coefficient (R 2 ) to evaluate the inhibitory effect of the total alkaloids of Winchia calophylla.
[0046] Through experiments, it was found that the total alkaloids of Winchia calophylla have good antibacterial activity against Botrytis cinerea. Its EC 50 value is 92.822 μg / mL, the virulence regression equation is y = 4.415x - 8.687, and R 2 = 0.861. And as the concentration of the total alkaloids gradually increases, the diameter of the colony gradually decreases, and the antibacterial rate against Botrytis cinerea gradually increases (as shown in Table 2).
[0047] Table 2 Inhibitory effect of the total alkaloids of Winchia calophylla on Botrytis cinerea
[0048]
[0049] *Note: Mean±SD; n = 3.
[0050] Example 4. This example is the fourth example of the present invention. This example provides a method for the preservation experiment of the total alkaloids of Winchia calophylla A. DC. on fruit pears, and the specific steps are as follows:
[0051] Preparation of the spore suspension of the pathogenic fungus Botrytis cinerea: The pathogenic fungus was cultured on PDA medium for 6 - 8 days. After sporulation, the spores were scraped off and counted using a hemocytometer to prepare a spore suspension with a concentration of 1×10 6 CFU / mL.
[0052] The total alkaloids of Winchia calophylla A. DC. were dissolved in a certain concentration of Tween - 80 to prepare Winchia calophylla A. DC. extract solutions with concentrations of 125.00 μg / mL, 250.00 μg / mL, and 500.00 μg / mL.
[0053] Fresh, similarly sized, regularly shaped test pear fruits that could be infected by Botrytis cinerea were selected. Before treatment, the surfaces of the pear fruits were cleaned and disinfected. After they were naturally air - dried, a sterile stainless steel needle was used to puncture the center to form an inoculation hole of uniform size (3 mm wide and 2 mm deep).
[0054] 10 μL of the total alkaloids of Winchia calophylla A. DC. with different concentrations were placed in the holes, and the sample without total alkaloids was used as a blank control. After it was naturally air - dried, 10 μL of the above 1×10 6 CFU / mL spore suspension was added. It was placed in an incubator at 25°C with 12 - hour alternations of light and darkness for 6 - 8 days. The disease incidence of the fruits was observed, and the cross - method was used. The part with the darkest lesion color and the most severe lesion was taken as the central reference point, and two mutually perpendicular lines were drawn with a ruler to divide the lesion into four parts. These two lines should make the lesion area of each part as uniform as possible. The lengths of the four line segments (the line segments formed by the intersection of the cross - lines and the lesion edge) were measured respectively. Starting from the central reference point, the distance to the lesion edge was read along each line segment, and the diameter of the lesion on the surface of the pear fruit was measured and photographed for record.
[0055]
[0056] As shown in the Figure 1 attachment, on the surface of the pear fruits in the blank control group, rot occurred at the inoculation site on the 2nd day, and the diseased area gradually expanded in the following days. By the 8th day, the diameter of the lesion could reach 8.5 cm; while on the surface of the pear fruits treated with 125 μg / mL of the total alkaloids of Winchia calophylla A. DC., the disease symptoms were lighter. By the 8th day, the diameter of the lesion was 6.1 cm, and the control effect reached 29.27%. In addition, the groups treated with higher concentrations of 250 μg / mL and 500 μg / mL of the total alkaloids of Winchia calophylla A. DC. did not show obvious corruption until the blank group was completely spoiled.
[0057] It can be seen from the experimental results that the total alkaloids of Winchia calophylla have a good inhibitory effect on Botrytis cinerea, the pathogenic bacterium of fruit pears, prolong the spoilage time of fruit pear fruits, and the protective effect on pears becomes stronger with the increase of concentration, and it can be applied to the storage and preservation of fruits in the food field.
[0058] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those of ordinary skill in the relevant technical fields can also make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the patent protection scope of the present invention should be defined by the claims.
Claims
1. A method for preparing total alkaloids of hollyhock trees, characterized in that: The invention comprises total alkaloids of the safflower tree, wherein the total alkaloids of the safflower tree are alcohol extracts of dry leaves of the safflower tree; The preparation method of the total alkaloids of the cassia tree is as follows: S1: Take fresh leaves of mature cycads, dry them, crush them, soak them in 90% methanol water for 3 days, then add 8 times of its weight of methanol, heat and reflux to extract four times (3h / time), filter, combine the filtrate, concentrate by rotary evaporation, and dry to obtain the crude extract of cycads; S2: The crude extract is treated by the principle of acid extraction and alkali precipitation. Specifically, a dilute hydrochloric acid solution with a pH of 2 to 3 is added to fully dissolve it, and the insoluble matter, i.e., non-alkaloids, is removed by filtration. 1% ammonia water is added to the filtrate to adjust the pH to 8 to 9, and petroleum ether, ethyl acetate and chloroform organic solvents are added successively for extraction, and the organic layers are combined to obtain the low-polarity alkaloid components; S3: Then, n-butanol solvent is added for extraction, and the n-butanol layer is collected to obtain highly polar alkaloids. The low polar and highly polar alkaloids are combined and adsorbed on a macroporous resin column, and 5% to 100% methanol water is used as an eluent for gradient elution to remove pigment impurities. With LC-MS detection as a guide, the total alkaloids of the pine tree are collected by gradient elution according to the polarity ratio, and concentrated and evaporated to dryness to obtain the total alkaloids of the pine tree.
2. The method for preparing the total alkaloids of the cassia tree and its application in fruit preservation according to claim 1, characterized in that: The active ingredients in the total alkaloids of the cycad tree include the following compounds: Dehydrorudgeifoline, Echitamine, Demethyldeformylcorymine, 17-O-acetylnorechitamine and Demethylechitamine.
3. The method for preparing the total alkaloids of the cassia tree and the application thereof in fruit preservation according to claim 2, characterized in that: The total alkaloids of the cassia tree contain no less than 10.0% dehydrorudgeifoline, no less than 4.0% echitamine and no less than 1.0% demethylechitamine.
4. An application of total alkaloids of cassia bark in fruit preservation, characterized in that: The total alkaloids of the trellis tree described in any one of claims 1 to 3 are applied to the harvested fruits.
5. The method for preparing the total alkaloids of the cassia tree and the application thereof in fruit preservation according to claim 4, characterized in that: The pathogenic bacteria after the fruit is picked is Botrytis cinerea.