Method for converting quercetin glycoside into quercetin by using rhinoceros dichotoma larvae

Abstract

The invention belongs to the technical field of biotransformation, and particularly relates to a method for converting quercetin glycoside into quercetin by using rhinoceros dichotoma larvae. According to the method, high-efficiency biotransformation of quercetin is realized by utilizing a unique intestinal flora metabolism function based on natural feeding and excretion behaviors of the rhinoceros dichotomus larvae. By selecting plants rich in quercetin compounds, spraying PVP K90 aqueous solution and controlling transformation time and conditions, the survival rate of insect bodies is greater than or equal to 99%. The conversion rate of quercetin in excrement reaches 98.1%, and the subsequent purification cost is remarkably reduced. The contradiction between biotransformation efficiency and insect survival rate is solved, a new technical thought is provided for green preparation of natural products, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotransformation, and in particular relates to a method for converting quercetin glycoside into quercetin by utilizing larvae of the dimorphic rhinoceros beetle. Background Art

[0002] The larval gut microbiome of the two-horned rhinoceros beetle (Allomyrina dichotoma) possesses a unique glycoside hydrolase system. Studies have shown that its intestinal flora may secrete multiple hydrolases, including β-glucosidase (EC 3.2.1.21), which specifically degrade glycoside compounds such as quercetin-3-O-β-D-glucoside in plants. No peroxidase activity was detected in its metabolites, providing a key guarantee for maintaining the stability of the target product.

[0003] Polyvinylpyrrolidone K90 (PVP K90) is a widely used polymer renowned for its excellent film-forming properties, biocompatibility, and non-toxicity. In the pharmaceutical field, PVP K90 can be used as a tablet binder, a sustained-release agent, and a solubility aid for injectables. Its sustained-release effect prolongs drug action and reduces toxic side effects. In cosmetics, it acts as a film-former and thickener, protecting skin and hair while reducing irritation. In agricultural plant protection, PVP K90 can be used in sprays to form a protective film against pests and diseases, while exhibiting low toxicity to plants. Furthermore, it can adsorb toxic substances, reducing their toxicity, thereby playing a dual role in reducing toxicity and providing protection in a variety of fields.

[0004] Capers are rich in quercetin, with approximately 0.2 grams per 100 grams. Capers also contain quercetin glycosides, present as quercetin-3-gluco-7-rhamnoside, quercetin-3-rutinoside, and quercetin-7-rutinoside. These glycosides are not naturally free quercetin and typically require hydrolysis and other reactions to break down the glycosidic bonds and release the quercetin aglycone before they can more directly exert their biological activity.

[0005] Mulberry leaves primarily contain flavonoids such as rutin, quercetin, and isoquercetin. Rutin, quercetin, and isoquercetin are all quercetin glycosides. Rutin (5,7,3',4'-4-OH-3-O-rhamnosyl-glucosyl-flavone) is a diglycoside of quercetin, with rhamnose and glucose as glycosides. Quercetin glycosides account for approximately 10% of dried mulberry leaves, and the total flavonoid content varies between different mulberry varieties and seasonally.

[0006] Sophora japonica flowers are rich in flavonoids such as rutin, kaempferol, genistein, and isorhamnetin. Rutin is a glycoside of quercetin. Quercetin is not directly present in Sophora japonica flowers as free quercetin. Quercetin aglycone is obtained through hydrolysis of rutin and other glycosides. The rich quercetin glycosides in Sophora japonica flowers can be used to extract quercetin.

[0007] The traditional chemical method for preparing quercetin requires multiple steps such as acid hydrolysis and organic solvent extraction, which poses problems such as severe environmental pollution and low product purity (CN118084845A). In comparison, the biotransformation method has the advantages of mild conditions and strong specificity. However, the existing microbial fermentation method has the disadvantages of poor enzyme stability and long conversion cycle (CN118956994A, CN118164944A). The present invention innovatively utilizes a living insect bioreactor to achieve efficient bioconversion of quercetin glycosides into quercetin by optimizing the synergistic effect of the insect body, microbial community and substrate. Summary of the Invention

[0008] The purpose of the present invention is to provide an efficient and environmentally friendly method for extracting quercetin using a live insect bioreactor. The method is based on the natural feeding and excretion behavior of the dimorphic rhinoceros beetle. By feeding the insects with feed sprayed with a 1% PVP K90 aqueous solution and utilizing the unique metabolic function of the intestinal flora, the extraction efficiency can be significantly improved, environmental pollution can be reduced, and the method is simple to operate without the need for complex chemical treatment processes.

[0009] Specifically, the present invention is achieved through the following technical solutions:

[0010] A method for converting quercetin glycoside into quercetin using dimorphicus beetle larvae, characterized by comprising the following steps:

[0011] S1. Select the branches, leaves, flowers, fruits, seeds, etc. of plants rich in quercetin glycosides as feed, and feed them after spraying the feed with PVP K90 aqueous solution;

[0012] S2. Diplocera rhinoceros beetle larvae (third instar, weighing 8-10 g each) were starved for 24 hours at 26°C ± 1°C, 65% ± 5% humidity, 300 LUX of natural light, a light-dark cycle of 14 h:10 h, and were fed a fixed amount of food.

[0013] S3 is fed at regular intervals and quantities, and its excrement is collected every 24 hours after being processed by the intestinal flora of the Rhinoceros Beetle larvae.

[0014] Furthermore, the feed rich in quercetin glycosides in S1 is caper leaves, mulberry leaves, Sophora japonica flowers, etc.;

[0015] Furthermore, the feed processed in S3 is the feed sprayed with 10 mL of 1% PVP K90 aqueous solution;

[0016] Furthermore, the intestinal flora of the S3 rhinoceros beetle larvae has a unique glycoside hydrolase system, the optimal pH of which is 6.8-7.2 and the temperature adaptation range is 25-30°C;

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] 1. After being protected by PVP K90 aqueous solution, the insect survival rate remains ≥99% and the feed taste is improved;

[0019] 2. The quercetin aglycone in excreta reaches 98.1%, reducing the subsequent purification cost;

[0020] 3. The specific metabolism of intestinal flora avoids oxidative modification of products, and no related by-products were found in HPLC detection.

[0021] This method successfully established a live transformation system for the dimorphic rhinoceros beetle, solving the problem of balancing biotransformation efficiency and insect physiological stability, and providing a highly innovative technical solution for the green preparation of natural products. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below with reference to specific examples, but the scope of protection of the present invention is not limited thereto:

[0023] "g" represents the weight unit "gram"; "LUX" represents the light intensity unit "lux"; "mL" represents the volume unit "milliliter"; "r / min" represents the rotation speed; "℃" represents the temperature "degrees Celsius"; "min" represents the time unit "minute"; "h" represents the time unit "hour".

[0024] 1. A method for converting quercetin glycoside into quercetin using dimorphotheca larvae

[0025] A method for converting quercetin glycoside into quercetin using dimorphic rhinoceros beetle larvae, the method comprising the following steps:

[0026] S1. Select the branches, leaves, flowers, fruits, seeds, etc. of plants rich in quercetin glycosides as feed, and feed them after spraying the feed with PVP K90 aqueous solution;

[0027] S2. Diplocera rhinoceros beetle larvae (third instar, weighing 8-10 g each) were starved for 24 hours at 26°C ± 1°C, 65% ± 5% humidity, 300 LUX of natural light, a light-dark cycle of 14 h:10 h, and were fed a fixed amount of food.

[0028] S3 is fed regularly and in fixed quantities. The food is processed feed that has been treated by the intestinal flora of the dimorphic rhinoceros beetle larvae, and the excrement is collected every 24 hours.

[0029] Example 1

[0030] Starve larvae of the third instar, weighing 8-10 g each, for 24 hours. A group of 15 larvae were placed in a feeding box in an artificial intelligence atmosphere chamber at 26°C ± 1°C, 65% ± 5% humidity, 300 LUX of natural light, and a 14-hour light:10-hour dark cycle. Each day at 10:00 AM, they were fed 200 g of Capparis spinosa leaves (sprayed with 10 mL of a 1% PVP K90 aqueous solution). After 24 hours, excrement was collected.

[0031] Example 2

[0032] Starve larvae (third instar, weighing 8-10g each) for 24 hours. A group of 15 larvae were placed in a feeding box in an artificial intelligence atmosphere chamber at 26°C ± 1°C, 65% ± 5% humidity, 300 LUX of natural light, and a 14-hour light:10-hour dark cycle. Each day at 10:00 AM, they were fed 200 g of mulberry leaves (sprayed with 10 mL of a 1% PVPK90 aqueous solution). After 24 hours, excrement was collected.

[0033] Example 3

[0034] Starve larvae (third instar, weighing 8-10g each) for 24 hours. A group of 15 larvae were placed in a feeding box in an artificial intelligence atmosphere chamber at 26°C ± 1°C, 65% ± 5% humidity, 300 LUX of natural light, and a 14-hour light:10-hour dark cycle. Each day at 10:00 AM, they were fed 200 g of Sophora japonica flowers (sprayed with 10 mL of a 1% PVPK90 aqueous solution). After 24 hours, excrement was collected.

[0035] Comparative Example 1

[0036] Starve larvae of the third instar Dinomatidae rhinoceros beetle (8-10g / bird) for 24 hours. A group of 15 Dinomatidae rhinoceros beetle larvae were placed in a feeding box in an artificial intelligence atmosphere chamber at 26°C ± 1°C, 65% ± 5% humidity, 300 lux of natural light, and a 14:10 hour light:dark cycle. Each larva was fed 200g of Capparis spinosa leaves (sprayed with 10mL of purified water) at 10:00 AM daily. After 24 hours, feces were collected.

[0037] Comparative Example 2

[0038] Starve larvae of the third instar, weighing 8-10g each, for 24 hours. A group of 15 larvae were placed in a feeding box in an artificial intelligence chamber at 26°C ± 1°C, 65% ± 5% humidity, 300 LUX of natural light, and a 14-hour light:10-hour dark cycle. Each larva was fed 200g of mulberry leaves (sprayed with 10mL of purified water) at 10:00 AM daily. After 24 hours, excrement was collected.

[0039] Comparative Example 3

[0040] Starve 24 hours of Diplodocus rhinoceros beetle larvae (third instar, weighing 8-10g each). A group of 15 Diplodocus rhinoceros beetle larvae were placed in a feeding box in an artificial intelligence air chamber at 26°C ± 1°C, 65% ± 5% humidity, 300 LUX of natural light, and a light:dark cycle of 14 hours:10 hours. Each larvae was fed 200g of Sophora japonica flowers (sprayed with 10mL of purified water) at 10:00 AM daily. After 24 hours, excrement was collected.

[0041] 2. Quercetin separation and detection

[0042] Fecal Preparation: Grind 10g of fecal sample into a powder, soak in 50mL of ethanol, and ultrasonically extract for 300 minutes. Filter and discard the residue to obtain the filtrate. Repeat the extraction method three times and combine the filtrates. Analyze the filtrates using HPLC. Calculate the content of quercetin and its byproducts in the feces using a standard curve.

[0043] 3. Calculation of the conversion rate of quercetin glycoside to quercetin aglycone

[0044] Determination of total quercetin content: The fecal rate produced by feeding Sophora japonica flowers was 65.8%.

[0045] Taking Sophora japonica flower as an example, 100 mg of Sophora japonica flower sample was weighed, 10 mL of ethanol was added for ultrasonic treatment, the pH was adjusted to 2-3, the temperature was 80°C, and acid hydrolysis was performed for 10 hours. The filtrate was filtered and used as sample 1. The total content of quercetin in Sophora japonica flower was determined by HPLC using a quercetin standard as a standard sample.

[0046] Sample solution: 100 mg of feces was added to 10 mL of methanol for ultrasonic dissolution, and the filtrate was filtered to obtain the filtrate as sample 2.

[0047]

[0048] Where: C 样1 、C 样2 is the quercetin concentration of sample 1 and sample 2 obtained from the regression equation, mg·mL -1 ; V1 and V2 are the volumes of sample 1 and sample 2, mL.

[0049] When feeding other plant samples, the calculation method of quercetin aglycone conversion rate was the same as that of feeding Sophora japonica flowers.

[0050] Table 1 24-hour glycoside to aglycone conversion rate of quercetin in Examples 1-3 and Comparative Examples 1-3 (n=15)

[0051]

[0052] Table 2 Number of worms surviving for 24 hours in Examples 1-3 and Comparative Examples 1-3 (n=15)

[0053]

[0054] As shown in Table 1, the average aglycone conversion rate of Examples 1-3 and Comparative Examples 1-3 is as low as 96.4%, which shows that the leaves of plants containing quercetin glycosides, etc., in this specific biotransformation system, both in terms of the characteristics of their own components and the interaction mechanism with the glycosyltransferase in the body of the rhinoceros beetle larvae, have a unique fit, thereby promoting the large-scale and stable conversion of quercetin into quercetin aglycone, achieving the purpose of rapid and efficient separation of quercetin aglycone, and the reaction system is environmentally friendly and simple. As shown in Table 2, the number of survivors in Examples 1-3 for seven days can reach 15, with a survival rate of 100%, while the number of survivors without PVPK90 spray protection dropped to 11, with a survival rate of only 73.3%.

[0055] No other by-products were found in fecal samples by HPLC, which to some extent increased the yield of quercetin and reduced the number of by-products.

[0056] Experimental data revealed that the intestinal flora of the rhinoceros beetle exhibited remarkable quercetin glycoside metabolism. As shown in Table 1, within a 24-hour conversion period, the quercetin aglycone conversion rates of Examples 1-3 were consistently above 96.4%, with Example 1 achieving an average conversion rate of 98.1%. This conversion efficiency is over 40% higher than that of traditional chemical hydrolysis methods, and it avoids the oxidative degradation of flavonoids caused by high temperatures and strong acids, preserving the pentahedral structure of quercetin and ensuring the biological activity of the target product.

[0057] As shown in Table 2, in Examples 1-3, where the feed was pretreated with PVP solution, the 24-hour survival count remained at 15 for seven consecutive days without mortality. Spraying the feed with the PVP K90 aqueous solution significantly reduced the toxicity to the dimorphic rhinoceros beetle, while the survival count of Comparative Examples 1-3 decreased significantly over time, reaching a minimum of 11 on the seventh day. This result indicates that appropriate PVP K90 aqueous solution treatment can effectively improve feed palatability, reduce digestive system stress responses, and thus ensure the stable metabolic activity of the intestinal flora.

Claims

1. A method for converting quercetin glycoside into quercetin using the larvae of the rhinoceros beetle, characterized in that: The following steps are involved: S1. Select the branches, leaves, flowers, fruits, seeds, etc. of plants rich in quercetin glycosides as feed, and feed them after spraying the feed with PVP K90 aqueous solution; S2. Diplocera rhinoceros beetle larvae (third instar, weighing 8-10 g each) were starved for 24 hours at 26°C ± 1°C, 65% ± 5% humidity, 300 LUX of natural light, a light-dark cycle of 14 h:10 h, and were fed a fixed amount of food. S3 is fed at regular intervals and quantities, and its excrement is collected every 24 hours after being processed by the intestinal flora of the Rhinoceros Beetle larvae.

2. The method according to claim 1, characterized in that The concentration of the S1 PVP K90 aqueous solution is 5-10%, and the aqueous solution accounts for 5-10% of the mass of the feed.

3. The method according to claim 1, characterized in that The feed rich in quercetin glycosides in S1 is caper leaf, mulberry leaf, Sophora japonica flower and the like.

4. The method according to claim 1, wherein The intestinal flora of the S3 rhinoceros beetle larvae has a unique glycoside hydrolase system, the optimal pH of which is 6.8-7.2 and the temperature adaptability range is 25-30°C.

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