Radix et Rhizoma Rhei and Radix Rehmanniae Extractive Compound, Preparation Method and Application

Abstract

This invention discloses a rhubarb-Sanguisorba officinalis compound extract, its preparation method, and its applications. The compound extract is a mixture of rhubarb extract and Sanguisorba officinalis extract. This compound extract exhibits good inhibitory effects on the main pathogens causing bacterial diseases in silkworms, and shows a significant synergistic antibacterial effect compared to single herbal extracts. Experimental data shows that the rhubarb-Sanguisorba officinalis compound extract demonstrates stable control effects in multiple silkworm varieties, including p50, Jingsong, Haoyue, and 932, indicating broad applicability across various varieties. More importantly, this rhubarb-Sanguisorba officinalis compound extract is prepared using natural Chinese herbal medicines rhubarb and Sanguisorba officinalis through a water extraction process. The operation is simple, requiring no complex separation and purification steps, making it suitable for large-scale industrial production. Furthermore, it is safe and environmentally friendly, with no potential drug residue risks, contributing to the stability and sustainable development of the agricultural ecosystem and significantly reducing the economic and environmental costs of pesticide use for silkworm farmers.

Description

Technical Field

[0001] This invention relates to the field of silkworm breeding, and in particular to a rhubarb-Sanguisorba officinalis compound extract, its preparation method, and its application. Background Technology

[0002] silkworm( Bombyx mori Silkworms are important economic insects that feed on mulberry leaves and go through four developmental stages in their life cycle: egg, larva, pupa, and adult. China, as the world's leading producer of raw silk, accounts for over 75% of global cocoon and raw silk production, ranking first globally. However, bacterial diseases in silkworms have always been a major obstacle to the sustainable development of the sericulture industry. These diseases mainly include septicemia and intestinal diseases, with common pathogens including *Septicemia melanoxystrobin*, *Septicemia spp.*, and *Enterococcus*, primarily infecting silkworms through wounds and ingestion. In the early stages of infection, silkworms exhibit reduced appetite and lethargy. *Septicemia melanoxystrobin* manifests as blackish-green necrotic spots appearing on the first three segments of the silkworm's body, gradually turning the entire body black. *Septicemia spp.* initially appears as brown circular spots on the body wall, which then turn crimson, eventually leading to convulsions and death. In severe cases, the mortality rate of the silkworm colony is extremely high. This disease occurs in all seasons, but is particularly severe during the hot and humid summer and autumn.

[0003] Currently, bacterial diseases in silkworms are mainly controlled by supplementing their diet with antibiotics such as chloramphenicol and erythromycin. However, long-term use of antibiotics has brought multiple problems: the drug resistance of pathogenic bacteria is constantly increasing; in 2023, the detection rate of doxycycline-resistant strains in East China had risen to 18%; antibiotic residues remain in silkworm bodies and silk, with florfenicol having a residual half-life of up to 72 hours in silkworm cocoons; and antibiotic residues in silkworm excrement enter the farmland ecosystem, disrupting the environmental micro-ecological balance. Against this backdrop, traditional Chinese medicine (TCM) has gradually become a research hotspot for antibiotic alternatives due to its abundant sources, low likelihood of inducing drug resistance, low toxicity, and environmental friendliness. Studies have shown that various TCM herbs have varying degrees of inhibitory effects on silkworm pathogens such as *Septicemia melanogaster* and *Sterilaria lobata*. However, there is still a lack of dedicated TCM preparations targeting the main pathogens of silkworms. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a rhubarb-Sanguisorba officinalis compound extract with broad applicability for the prevention and treatment of bacterial diseases in silkworms. The second purpose is to provide a method for preparing and applying this rhubarb-Sanguisorba officinalis compound extract.

[0005] Technical solution: The rhubarb-Sanguisorba officinalis compound extract of the present invention is a mixture of rhubarb extract and Sanguisorba officinalis extract.

[0006] Preferably, the mass ratio of rhubarb extract to Sanguisorba officinalis extract, based on the amount of raw herbs, is (1~2):(1~2).

[0007] The preparation method of the rhubarb-Sanguisorba officinalis compound extract of the present invention includes the following steps: (1) Take rhubarb and raw Sanguisorba officinalis, remove impurities, crush and sieve them; (2) The pulverized rhubarb obtained in step 1 is soaked in water and decocted at 90~100℃ for 20~40 min. The filtrate is filtered and collected. The residue is decocted again with water and repeated 1~2 times. The filtrates are combined and concentrated under reduced pressure to obtain the rhubarb extract. The pulverized Sanguisorba officinalis obtained in step 1 is soaked in water and decocted at 90~100℃ for 20~40 min. The filtrate is filtered and collected. The residue is decocted again with water and repeated 1~2 times. The filtrates are combined and concentrated under reduced pressure to obtain the Sanguisorba officinalis extract. (3) The rhubarb extract obtained in step 2 is mixed with the sanguisorba officinalis extract to obtain the rhubarb-sanguisorba officinalis compound extract.

[0008] Preferably, in step 1, the rhubarb and Sanguisorba officinalis are both dried roots.

[0009] Preferably, in step 2, when the rhubarb is soaked in water, the mass-to-volume ratio of rhubarb to water is 1:(8~12); when the filter residue is boiled again with water, the mass-to-volume ratio of filter residue to water is 1:(6~10); during vacuum concentration, the final concentration, based on the amount of raw medicinal material, is 0.5–2 g / mL; when the Sanguisorba officinalis is soaked in water, the mass-to-volume ratio of Sanguisorba officinalis to water is 1:(8~12); when the filter residue is boiled again with water, the mass-to-volume ratio of filter residue to water is 1:(6~10); during vacuum concentration, the final concentration, based on the amount of raw medicinal material, is 0.5–2 g / mL.

[0010] Preferably, in step 3, when the rhubarb extract and the Sanguisorba officinalis extract are mixed, the volume ratio is (1~2):(1~2).

[0011] The pharmaceutical composition of the present invention contains the aforementioned rhubarb-Sanguisorba officinalis compound extract as an active ingredient.

[0012] The application of the rhubarb-Sanguisorba officinalis compound extract or pharmaceutical composition described in this invention in silkworm breeding.

[0013] Preferably, the application is in the preparation of a drug for the prevention and treatment of bacterial diseases in silkworms; more preferably, in the drug, the concentration of rhubarb and Sanguisorba officinalis, based on the amount of raw herbs, is 25–75 mg / mL.

[0014] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: 1. The rhubarb-Sanguisorba officinalis compound extract provided by the present invention shows good inhibitory effects on the main pathogens of bacterial diseases in silkworms. Experimental data shows that the compound extract has a significant synergistic antibacterial effect compared with single Chinese herbal extracts; 2. Experimental data shows that the rhubarb-Sanguisorba officinalis compound extract shows stable control effects in multiple silkworm varieties such as p50, Jingsong, Haoyue, and 932, and has wide applicability to various varieties; 3. The rhubarb-Sanguisorba officinalis compound extract is prepared by water extraction using natural Chinese herbal medicines rhubarb and Sanguisorba officinalis. The operation is simple, without complicated separation and purification steps, suitable for large-scale industrial production, and is safe and environmentally friendly with no drug residue risks. It is conducive to the stability and sustainable development of the agricultural ecosystem and can significantly reduce the economic and environmental costs of pesticide use for silkworm farmers. Attached Figure Description

[0015] Figure 1 Figure 1 shows the in vitro antibacterial effect of Enterococcus on single extracts of rhubarb and Sanguisorba officinalis, and the compound extract of rhubarb and Sanguisorba officinalis. Figure 2 Figure 1 shows the in vitro antibacterial effect of rhubarb, sanguisorba officinalis single herb extracts and rhubarb-sanguisorba officinalis compound extracts on Septicemia melanoma. Figure 3 Figure 1 shows the in vitro antibacterial effect of single extracts of rhubarb and Sanguisorba officinalis, and the compound extract of rhubarb and Sanguisorba officinalis, on Septicemia. Figure 4 Survival curves of single-herb extracts of rhubarb and Sanguisorba officinalis, and rhubarb-Sanguisorba officinalis compound extract for the prevention and control of bacterial diseases in p50 silkworms; Figure 5 Survival curves of single-herb extracts of rhubarb and Sanguisorba officinalis, and the compound extract of rhubarb and Sanguisorba officinalis, for the prevention and control of bacterial diseases in silkworms of the Jingsong, Haoyue, and 932 varieties. Detailed Implementation

[0016] The technical solution of the present invention will be further described below.

[0017] Example 1: Preparation of rhubarb-Sanguisorba officinalis compound extract Take dried rhubarb and raw Sanguisorba officinalis, grind them separately, and then sieve them through an 80-mesh sieve.

[0018] Take 100 g of crushed rhubarb, add 1000 mL of distilled water (material-to-liquid ratio 1:10), soak for 40 min, then decoct at 100℃ for 30 min, filter through 8 layers of gauze and collect the filtrate; add 800 mL of distilled water to the residue again, decoct at 100℃ for 30 min, filter and collect the filtrate; combine the two filtrates, continue to decoct and concentrate to 100 mL, that is, the concentration is 1 g / mL based on the amount of raw rhubarb, and store the obtained rhubarb extract at 4℃ for later use.

[0019] Take 100 g of crushed raw Sanguisorba officinalis, add 1000 mL of distilled water (material-to-liquid ratio 1:10), soak for 40 min, then decoct at 100℃ for 30 min, filter through 8 layers of gauze and collect the filtrate; add 800 mL of distilled water to the residue again, decoct at 100℃ for 30 min, filter and collect the filtrate; combine the two filtrates, continue to decoct and concentrate to 100 mL, that is, the concentration is 1 g / mL based on the raw amount of Sanguisorba officinalis, and store the obtained Sanguisorba officinalis extract at 4℃ for later use.

[0020] The above-mentioned rhubarb extract and Sanguisorba officinalis extract were mixed at a volume ratio of 1:1 to obtain a rhubarb-Sanguisorba officinalis compound extract. Before use, it was diluted with sterile water to the required concentration, calculated based on the total crude drug content.

[0021] Example 2: Evaluation of the in vitro antibacterial activity of rhubarb-Sanguisorba officinalis compound extract Using black breast bacterium ( Bacillus bombysepticus ), Serratia marcescens (Serratia marcescens, Serratia marcescens ATCC13880) and Enterococci (Enterococcus montelukast) Enterococcus mundtii The in vitro antibacterial activity of the strains (ATCC43186) was evaluated. All strains were provided by the Henan Academy of Sericulture Sciences.

[0022] After resuscitation in liquid LB medium, each bacterial strain was streaked onto LB agar plates for further culture. Single colonies were then picked and inoculated into liquid LB medium for scale-up. Cells at the same growth stage were collected, washed with sterile PBS, resuspended, and viable cell concentrations were determined spectrophotometrically using serial dilution plate counting. The concentrations were then adjusted to the required experimental concentrations with sterile PBS. All bacterial suspensions were prepared fresh before use.

[0023] The rhubarb extract, Sanguisorba officinalis extract and compound extract prepared in Example 1 were diluted with sterile water to three concentration gradients: high concentration 1 g / mL, medium concentration 0.1 g / mL and low concentration 0.01 g / mL, wherein the concentration of the compound extract was based on the total amount of raw medicinal materials.

[0024] 200 μL OD 600 Suspensions of *Septicemia melanogaster*, *Septicemia fulva*, or *Enterococcus* at concentrations of 0.25 were thoroughly mixed with equal volumes of extracts of the aforementioned different concentrations and incubated on a constant-temperature shaker for 2 hours. Subsequently, 100 μL of each bacterial suspension-extract mixture was added to 900 μL of PBS buffer and further diluted to 10⁻¹⁰ using a 10-fold serial dilution method. -5 10 -6 Or 10 -7 Concentration; take 100 μL 10 -5 10 -6 Or 10-7 The diluted solutions were evenly spread onto sterile LB agar plates and incubated at 37°C for 12 h. The number of colonies in each plate was counted. A control group (drug concentration 0) was prepared by replacing the extract with PBS buffer. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (Number of colonies in control group - Number of colonies in treatment group) / Number of colonies in control group × 100%.

[0025] Antibacterial effect against Enterococcus such as Figure 1 As shown, at high concentrations (1 g / mL), Sanguisorba officinalis, Rheum palmatum, and their combined use showed effects against 10... 5 The antibacterial rates of the diluted bacterial solutions all exceeded 90%, with 90.65% for Sanguisorba officinalis, 91.22% for Rheum palmatum, and 99.21% for the compound extract. At a medium concentration (0.1 g / mL), the antibacterial rates of Sanguisorba officinalis were 52.85%, Rheum palmatum was 67.80%, and the compound extract was 64.12%. This indicates that all three treatments have good antibacterial effects against Enterococcus.

[0026] The antibacterial effect against *Septicemia melanogaster* is as follows: Figure 2 As shown, at high concentrations (1 g / mL), Sanguisorba officinalis, Rheum palmatum, and their combined use showed effects against 10... 5 The antibacterial rate of all diluted bacterial solutions reached 100%; at a medium concentration (0.1 g / mL), the antibacterial rate of Sanguisorba officinalis was 96.43%, while the antibacterial rates of Rheum palmatum and the compound extract were both 100%; at a low concentration (0.01 g / mL), the antibacterial rate of Sanguisorba officinalis was 50.89%, that of Rheum palmatum was 61.61%, and that of the compound extract was 69.77%.

[0027] Antibacterial effect against *Septicemia* and *Clostridium perfringens*, etc. Figure 3 As shown, at high concentrations (1 g / mL), the three treatments had an effect on 10 6 The antibacterial rate of the diluted bacterial solutions all reached over 90%; at a medium concentration (0.1 g / mL), the antibacterial rate of Sanguisorba officinalis was 56.77%, that of Rheum palmatum was 79.03%, and that of the compound extract was 76.33%; at a low concentration (0.01 g / mL), the antibacterial rate of Sanguisorba officinalis was 34.46%, that of Rheum palmatum was 44.22%, and that of the compound extract was 69.08%.

[0028] Example 3: Evaluation of the efficacy of rhubarb-rehmannia glutinosa compound extract in the prevention and treatment of bacterial diseases in silkworms. 1. Validation of the therapeutic effect of bacterial diseases on individual silkworms The silkworm variety used in the experiment was p50 (provided by the Henan Academy of Sericulture Sciences), and it was conventionally raised in an environment with a temperature of 25±1℃ and a relative humidity of 70±5% RH.

[0029] Fifth instar silkworms were selected for the experiment and placed in an artificial climate chamber at a temperature of 30±1℃ and a relative humidity of 85±5% RH for 12 h to induce bacterial disease in silkworms.

[0030] Silkworms infected with bacterial disease were randomly divided into an infection control group (n=30), a rhubarb single-drug group (n=90), a raw rehmannia single-drug group (n=90), and a combined treatment group (n=90). Thirty normal 5th instar silkworms were selected as the normal group.

[0031] For the infected control group silkworms, feed them with a 500 μL / 225 cm² spray solution. 2 Sterile water and dried mulberry leaves were used; for normal silkworms, untreated mulberry leaves were fed; for silkworms treated with rhubarb alone, mulberry leaves sprayed with 25, 50, or 75 mg / mL rhubarb extract and dried were fed at a spraying rate of 500 μL / 225 cm³. 2 The concentration was 30 silkworms per dose; for silkworms treated with Sanguisorba officinalis alone, they were fed dried mulberry leaves sprayed with 25, 50, or 75 mg / mL Sanguisorba officinalis extract, at a spraying rate of 500 μL / 225 cm². 2 Each treatment concentration was for 30 silkworms; for the combined treatment group, silkworms were fed dried mulberry leaves sprayed with 25, 50, or 75 mg / mL rhubarb-Sanguisorba officinalis compound extract, at a spraying volume of 500 μL / 225 cm². 2 Each dose was administered to 30 silkworms; the silkworms were fed the medication continuously for 5 days, and their survival was observed and recorded daily.

[0032] p50 Silkworm survival curve as follows Figure 4 As shown, the number of surviving silkworms in the infected control group was 6 (survival rate 20.0%); in the single-drug group of Sanguisorba officinalis, the number of surviving silkworms treated at 50 mg / mL and 25 mg / mL concentrations was 14 (survival rate 46.7%), and the number of surviving silkworms treated at 75 mg / mL concentration was 12 (survival rate 40.0%); in the single-drug group of rhubarb, the number of surviving silkworms treated at 50 mg / mL concentration was 13 (survival rate 43.3%), and the number of surviving silkworms treated at 25 mg / mL and 75 mg / mL concentrations was 10 (survival rate 33.3%); in the combined treatment group, the number of surviving silkworms treated at 25 mg / mL concentration was 19 (survival rate 63.3%), the number of surviving silkworms treated at 50 mg / mL concentration was 12 (survival rate 40.0%), and the number of surviving silkworms treated at 75 mg / mL concentration was 11 (survival rate 36.7%).

[0033] The results showed that the in vivo protective effect of the combined treatment group was significantly better than that of the single treatment group of Sanguisorba officinalis or rhubarb, with 25 mg / mL being the optimal dosage concentration. The survival rate of silkworms in the 25 mg / mL combined treatment group was 43.3% higher than that in the control group, confirming that rhubarb and Sanguisorba officinalis played a synergistic role in the treatment.

[0034] 2. Verification of the control effects of different silkworm varieties Three silkworm varieties—Jingsong, Haoyue, and 932 (all provided by the Henan Academy of Sericulture Sciences)—were selected to further verify the applicability of the rhubarb-Sanguisorba officinalis compound extract. In vivo efficacy experiments were conducted at a concentration of 25 mg / mL. Each group consisted of 15 silkworms, and the experimental method was the same as in Example 3.

[0035] Fifth instar silkworms of various varieties were selected for the experiment and placed in an artificial climate chamber at a temperature of 30±1℃ and a relative humidity of 85±5% RH for 12 h to induce bacterial disease in silkworms.

[0036] For silkworms infected with bacterial diseases, each variety was randomly divided into an infection control group, a rhubarb single-drug group, a raw rehmannia single-drug group, and a combination group, with 15 silkworms in each group.

[0037] For silkworms in the control group, they were fed mulberry leaves sprayed with sterile water and then dried; for silkworms in the rhubarb-only group, they were fed mulberry leaves sprayed with 25 mg / mL rhubarb extract and then dried; for silkworms in the Sanguisorba officinalis-only group, they were fed mulberry leaves sprayed with 25 mg / mL Sanguisorba officinalis extract and then dried; for silkworms in the combined treatment group, they were fed mulberry leaves sprayed with 25 mg / mL rhubarb-Sanguisorba officinalis compound extract and then dried. The spraying volume of sterile water or drugs was 500 μL / 225cm. 2 Administer the medication continuously for 5 days, and observe and record the survival status of the silkworms daily.

[0038] Survival curves of various silkworm varieties are as follows Figure 5 As shown, in the Haoyue strain, all control group organisms died after infection; in the single-drug treatment group (using *Sanguisorba officinalis*), 3 organisms survived; in the single-drug treatment group (using *Rheum palmatum*), 4 organisms survived; and in the combined treatment group, 7 organisms survived. In the Jingsong strain, all control group organisms died after infection; in the single-drug treatment group (using *Sanguisorba officinalis*), 4 organisms survived; in the single-drug treatment group (using *Rheum palmatum*), 3 organisms survived; and in the combined treatment group, 6 organisms survived. In the 932 strain, all control group organisms died after infection; in the single-drug treatment group (using *Sanguisorba officinalis*), 4 organisms survived; in the single-drug treatment group (using *Rheum palmatum*), 2 organisms survived; and in the combined treatment group,

[0039] The results indicate that the rhubarb-Sanguisorba officinalis compound extract also showed significant control effects in different silkworm varieties, and the survival rate of the combined treatment was significantly higher than that of the single-drug group, confirming that the rhubarb-Sanguisorba officinalis compound extract has broad-spectrum applicability.

Claims

1. A rhubarb-Sanguisorba officinalis compound extract, characterized in that, The compound extract is a mixture of rhubarb extract and Sanguisorba officinalis extract.

2. The rhubarb-Sanguisorba officinalis compound extract according to claim 1, characterized in that, In the rhubarb extract and the Sanguisorba officinalis extract, the mass ratio of rhubarb to Sanguisorba officinalis, based on the amount of raw herbs, is (1~2):(1~2).

3. A method for preparing the rhubarb-Sanguisorba officinalis compound extract according to claim 1 or 2, characterized in that the step... include: (1) Take rhubarb and raw Sanguisorba officinalis, remove impurities, crush and sieve them; (2) The pulverized rhubarb obtained in step 1 is soaked in water and decocted at 90~100℃ for 20~40 min. The filtrate is filtered and collected. The residue is decocted again with water and repeated 1~2 times. The filtrates are combined and concentrated under reduced pressure to obtain the rhubarb extract. The pulverized Sanguisorba officinalis obtained in step 1 is soaked in water and decocted at 90~100℃ for 20~40 min. The filtrate is filtered and collected. The residue is decocted again with water and repeated 1~2 times. The filtrates are combined and concentrated under reduced pressure to obtain the Sanguisorba officinalis extract. (3) The rhubarb extract obtained in step 2 is mixed with the sanguisorba officinalis extract to obtain the rhubarb-sanguisorba officinalis compound extract.

4. The preparation method according to claim 4, characterized in that, In step 1, the rhubarb and Sanguisorba officinalis roots are both selected from dried roots.

5. The preparation method according to claim 4, characterized in that, In step 2, when the rhubarb is soaked in water, the mass-to-volume ratio of rhubarb to water is 1:(8~12); when the residue is boiled again with water, the mass-to-volume ratio of residue to water is 1:(6~10); during vacuum concentration, the final concentration, based on the amount of raw medicinal material, is 0.5–2 g / mL. When the Sanguisorba officinalis is soaked in water, the mass-to-volume ratio of Sanguisorba officinalis to water is 1:(8~12); when the residue is boiled again with water, the mass-to-volume ratio of residue to water is 1:(6~10); during vacuum concentration, the final concentration, based on the amount of raw medicinal material, is 0.5–2 g / mL.

6. The preparation method according to claim 4, characterized in that, In step 3, when the rhubarb extract and the Sanguisorba officinalis extract are mixed, the volume ratio is (1~2):(1~2).

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the rhubarb-Sanguisorba officinalis compound extract as described in claim 1 or 2 as an active ingredient.

8. The application of the rhubarb-Sanguisorba officinalis compound extract as described in claim 1 or 2, or the pharmaceutical composition as described in claim 7, in silkworm breeding.

9. The application according to claim 8, characterized in that, The application is in the preparation of drugs for the prevention and treatment of bacterial diseases in silkworms.

10. The application according to claim 9, characterized in that, In the aforementioned drugs, the concentrations of rhubarb and sanguisorba officinalis, based on the amount of raw herbs, are both 25–75 mg / mL.

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