A rhubarb-based extract for preventing and treating fish ciliate parasites, its preparation method and application

Abstract

This invention discloses a rhubarb extract for preventing and treating ciliate parasites in fish, its preparation method, and its application, relating to the field of aquaculture disease control technology. The method includes the following steps: 1) Activating rhubarb with citric acid for 1 hour, soaking in 55%~70% ethanol for 23 hours, filtering, and obtaining the first filtrate; 2) Continuing to soak the residue from step 1) in 55%~70% ethanol for 24 hours, obtaining the second filtrate; 3) Continuing to soak the residue from step 2) in 55%~70% ethanol for 24 hours, obtaining the third filtrate; 4) Combining the three filtrates from steps 1) to 3), concentrating at low temperature, and obtaining the citric acid-activated rhubarb ethanol extract. The rhubarb extract prepared by this invention, which enhances anti-ciliate parasite activity, ensures high anthraquinone content, reduces fish toxicity, and increases activity against Tetrahymena thermophila. The extract is also less prone to mold growth during storage and exhibits good control effects against Trichodina and Ichthyophthirius multifiliis diseases in fish fry.

Description

Technical Field

[0001] This invention relates to the field of aquaculture disease prevention and control technology, and more specifically to a rhubarb extract for preventing and controlling fish ciliate parasites, its preparation method, and its application. Background Technology

[0002] Fish ciliate parasites are a class of ciliate protozoa that live on or inside the bodies of fish. They belong to the subphylum Ciliophora within the phylum Protozoa. Their bodies are covered with cilia used for movement and feeding, and they have both macronuclei and micronuclei.

[0003] Common fish ciliate parasites include Ichthyophthirius multifiliis (Ichthyophthirius multifiliis). Ichthyophthirius multifiliis ), large wheel beetle ( Trichodina spp.), small wheel beetle ( Trichodinella spp.), Chilodonella ( Chilodonella spp), stimulating cryptocaryon ( Cryptocaryon irritans Among them, Ichthyophthirius multifiliis is the most serious parasite that causes white spot disease in fish and harms freshwater fish. Its developmental stages include three stages: the trophozoite that parasitizes the fish, the cyst stage that forms after leaving the fish, and the larva (predator) that is formed through asexual reproduction and released into the water.

[0004] rhubarb( Rhubarb, Rheum palmatum L.): Rhubarb is the Polygonaceae plant *Rheum tanguticum* (Rheum palmatum). Rheum tanguticum Maxim. ex Balf.), Rheum palmatum ( Rheum palmatum L.) or medicinal rhubarb ( Rheum officinale Rhubarb is the dried root and rhizome of *Rheum palmatum*. It is a plant with a rich historical background, valued for its medicinal properties for thousands of years. In Traditional Chinese Medicine, rhubarb is renowned for its purgative, heat-clearing, blood-cooling, detoxifying, blood-activating, and dampness-reducing effects. Its history as a laxative and health-restoring agent dates back to ancient China and the Mediterranean region of Europe. According to the pharmacopoeia *Yaoxing Fu*, rhubarb is used locally to clear the intestines and reduce inflammation, and systemically to clear heat and detoxify. Oral administration of rhubarb can clear heat, detoxify, strengthen the stomach, and aid digestion. Rhubarb is listed in the pharmacopoeias of China, Japan, and Europe.

[0005] Currently in China, rhubarb is an important ingredient in compound traditional Chinese medicines listed in the national standards, used to treat various diseases, including those affecting the gastrointestinal tract, liver, and kidneys. Modern research indicates that rhubarb has antibacterial and antiviral effects, and recent studies have shown that it also has antiparasitic effects. Commercially available fish medicines (rhubarb powder, three-yellow powder) prepared primarily from rhubarb have achieved certain results in the prevention and control of fish diseases. However, in the approved veterinary drug list, only powdered formulations made from rhubarb are available. Although there are reports of rhubarb extract being effective against ciliated parasites, no research products on the antiparasitic effects of rhubarb extract have been reported.

[0006] Rhubarb has antiparasitic effects: Modern research shows that rhubarb has antibacterial and antiviral effects, and recent studies have shown that rhubarb also has antiparasitic effects.

[0007] Human medical research has shown that rhubarb has a certain inhibitory effect on protozoa such as *Amoebae histolytica*, *Trichomonas vaginalis*, and *Trichomonas vaginalis*, and also has a certain inhibitory effect on the protocercariae of *Echinococcus granulosus* collected from hepatic hydatid cysts in sheep. Its effects on aquatic ciliate parasites have been extensively studied. In an experiment on the killing effect of 36 kinds of natural plant extracts on *Ichthyophthirius multifiliis* infecting freshwater fish, rhubarb extract showed the strongest effect. It also showed good anthelmintic effects on *Pseudocactus trichomonas* infecting marine fish and had a certain effect on *Cryptocactus irritans*. Commercially available veterinary drugs (rhubarb powder, *Sanhuang* powder) prepared with rhubarb as the main raw material have achieved certain effects in fish disease prevention and control. However, in the approved veterinary drug list, only powdered formulations of rhubarb are available. Although there are reports of rhubarb extract being effective against ciliate parasites, no research products on the antiparasitic effects of rhubarb extract on aquatic parasites have been reported. This may be related to the fact that existing extraction technologies, processes, or product efficiency do not meet the requirements for the prevention and control of parasitic diseases in aquatic products.

[0008] The prior art discloses various extraction methods for rhubarb, including water extraction, alcohol extraction, ammonia extraction, and ultrasonic extraction.

[0009] The water extraction method involves soaking rhubarb in 5-10 times its volume of purified water for 30 minutes to 1 hour, then placing it in a decoction pot or ceramic container. Bring to a boil over high heat, then reduce to a simmer and maintain a gentle boil for 30 minutes to 1 hour. Filter the mixture, and repeat the decoction process 1-2 times with the dregs, combining the filtrates. The ethanol extraction method uses ethanol as a solvent to extract anthraquinones (such as rhein and chrysophanic acid), tannins, polysaccharides, and other active ingredients from rhubarb. For example, the preparation of rhubarb fluid extract involves: taking 100g of rhubarb powder → adding 800mL of 70% ethanol and refluxing twice (1.5h / time) → combining the filtrates → concentrating under reduced pressure to obtain an extract → freeze-drying to obtain the extract. The ammonia extraction method utilizes the reaction of ammonia (weakly alkaline) with anthraquinone components (such as bound anthraquinone glycosides) in rhubarb, hydrolyzing them into free anthraquinones and dissolving them. Simultaneously, the alkaline conditions precipitate some acidic impurities (such as tannins). For example: 100g rhubarb powder + 1L 8% ammonia → stirring at 50℃ for 1h → filtration → adjusting the pH of the filtrate → drying the precipitate → obtaining crude anthraquinones (yield approximately 3%~5%). The ultrasonic extraction method utilizes the cavitation effect of ultrasound (high-frequency vibration generates microjets and shock waves) to disrupt the cell wall structure of rhubarb, accelerating solvent penetration (such as water and ethanol) and dissolving anthraquinones (emodin, rhein, etc.), polysaccharides, and other effective components.

[0010] Ethanol extraction: Rhubarb is extracted with ethanol, typically at a concentration of 70-95% (Wu Rui et al., 2008; Zhang Li, 2014; Hou Tinglong et al., 2023). The specific method generally involves ethanol soaking followed by hot reflux (at a relatively high temperature) (Wu Rui et al., 2008; Zhang Li, 2014; Wang Wanqian et al., 2015). Rotary evaporators are also used, which, while increasing pressure and slightly lowering the temperature, still require heating in a water bath to a certain temperature to evaporate the solvent. Wu Yuliang et al. studied the optimal alcohol extraction process for total anthraquinones from rhubarb, a traditional Chinese medicine. They set an ethanol concentration gradient of 50-90%, obtaining anthraquinone contents of 21.29-24.95 mg / L. The temperature gradients were 50℃, 60℃, 70℃, 80℃, and 90℃. The designed extraction times were 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h. The solid-liquid ratios were 1:10, 1:15, 1:20, 1:25, and 1:30. The results showed that the optimal extraction process for total anthraquinones from rhubarb was: 70% ethanol concentration, 70℃ extraction temperature, 1 h extraction time, and a solid-liquid ratio of 1:20. Sun Yunpeng et al. (2017) used orthogonal design experiments to optimize the extraction process of the Four-Ingredient Coptis chinensis Lotion containing rhubarb (the Four-Ingredient Coptis chinensis Lotion is an empirical formula for treating skin diseases, and the prescription consists of four Chinese herbs: rhubarb, Coptis chinensis, Phellodendron chinense, and Scutellaria baicalensis). The optimal extraction process was: extract with 12 times the amount of 80% ethanol three times, each time for 1.5 h.

[0011] Rhubarb used in combination with alkaline substances: It is generally believed that the advantage of using rhubarb in combination with alkaline substances is that it can reduce side effects in vivo, but in vitro experiments show that it reduces the efficacy. Rhubarb is a bitter and cold purgative, and in traditional Chinese medicine theory, it is generally not used with alkaline substances (such as preparations containing calcium carbonate or aluminum hydroxide). This is because an alkaline environment may change the solubility or chemical structure of the active ingredients in rhubarb (such as anthraquinone compounds), weakening its purgative effect. However, the combination of rhubarb with drugs containing weak alkalinity is a common clinical medication regimen. For example, the combination of rhubarb and aged lime is used in traditional Chinese medicine external application therapy, usually for treating trauma or skin diseases. Rhubarb is bitter and cold, promoting blood circulation and cooling the blood, and has the effect of stopping bleeding without leaving blood stasis; aged lime (weakly alkaline) is astringent and cold, and can astringe and stop bleeding. The combination of the two drugs enhances their hemostatic and analgesic effects. Rhubarb sodium bicarbonate tablets (also known as rhubarb sodium bicarbonate tablets) are a compound preparation of traditional Chinese and Western medicine, mainly composed of rhubarb powder and sodium bicarbonate (baking soda, a weakly alkaline substance). They have stomach-strengthening, digestive-aiding, and laxative effects. Rhubarb sodium bicarbonate tablets were previously a commonly used drug in livestock production, used in cattle, sheep, and pig farming to treat gastrointestinal diseases (Yang Juqin and Li Xingru, 2013; Zhao Junxi and Hong Aiying, 2004; Zhu Huadong, 2014). The anthraquinone components in rhubarb (such as emodin) can stimulate gastrointestinal motility and promote the secretion of digestive juices; sodium bicarbonate neutralizes gastric acid, improves the rumen or intestinal environment, alleviates acidosis (such as rumen acidosis in ruminants), and balances electrolytes. However, when used internally, it has many side effects, such as rhubarb overdose leading to diarrhea and dehydration, and long-term use of sodium bicarbonate potentially disrupting electrolyte balance. In modern terrestrial animal farming, targeted additives (such as probiotics and enzyme preparations) are increasingly used to replace rhubarb sodium bicarbonate tablets to reduce the side effects of oral administration. In aquaculture, external application reduces the side effects of oral administration. For example, in practical production applications, rhubarb can be extracted with 3% ammonia water and applied to the whole pond to prevent gill rot in fish, but the mechanism of action has not been explained.

[0012] Rhubarb is extracted using acidic substances, which may have a synergistic effect. Most of the anthraquinone components in rhubarb are bound to glucose, existing as anthraquinone glycosides. Acid hydrolysis can generate free hydroxy aglycones, which can then be extracted using organic solvents. An acid-hydrolysis followed by solvent extraction method can be used, sometimes employing sulfuric acid or hydrochloric acid. For some traditional Chinese medicines (such as Coptis chinensis and Scutellaria baicalensis), acid precipitation ensures a high transfer rate of active ingredients. However, research has not yet been conducted on which acid extraction method is most effective against parasites.

[0013] Current research indicates that all four extraction methods have some inhibitory and killing effects on ciliated parasites. However, each has the following drawbacks, which are described below: ① Water extraction: Relatively mild, however, the extract has limited efficacy against Tetrahymena thermophila and ciliated parasites, and is difficult to preserve. Even after high-temperature boiling, it is prone to mold growth after a certain period of time (e.g., 3 months). ② Ammonia extraction: Ammonia is corrosive and toxic. The storage and extraction process pose certain risks to operators and containers. Moreover, if the solvent in the extract is not completely removed, it contains molecular ammonia, which can be toxic to fish if the concentration in water is too high. ③ Ultrasonic extraction: Although it has the advantage of short extraction time, the strong cavitation effect may damage some sensitive components, resulting in lower purity of the target component. Furthermore, it requires significant equipment investment and has high requirements for process conditions, and is currently only used in small-scale laboratory environments. ④ Ethanol extraction: The effect of ethanol extracts on parasites is still unclear, and the concentration range is not well defined.

[0014] Using "cilia" and "medicine" as keywords, 12 patents were found concerning traditional Chinese medicines used to treat fish ciliate parasites. The main ingredients include: emodin, glucose solution, proanthocyanidins, combinations of traditional Chinese and Western medicines (such as Bupleurum, Gallnut, and Oxytetracycline), curcumin, 10-gingerol, cynanchine C, a mixture of seven traditional Chinese medicines including Artemisia annua and Melia toosendan bark, Sophora flavescens powder mixed with formaldehyde solution, pure plant-based traditional Chinese medicines combined with lysozyme, folic acid synthesis inhibitors, and / or folic acid activation inhibitors. These patent applications indicate that traditional Chinese medicines possess anti-ciliaate parasite properties. Specifically, emodin is an extract of rhubarb, suggesting that rhubarb extract has anti-parasitic efficacy. No patents were found that utilize rhubarb to enhance anti-parasitic effects.

[0015] Using "rhubarb" and "fish parasites" as keywords, a search for patent applications related to rhubarb extract's anti-fish parasite properties revealed that the applications mainly involve the combined use of rhubarb extract with other traditional Chinese medicine extracts, such as "A pure traditional Chinese medicine insecticide for fish, shrimp, sea cucumber, etc. and its preparation method" (application number: 201310368230.9), "A compound feed for preventing and treating loach parasitic diseases" (application number: 201510381480.5), and "A traditional Chinese medicine for treating sturgeon parasitic diseases and its preparation method." Patent applications 201710660339.8, "A Traditional Chinese Medicine Composition for Treating Internal and External Parasites in Animals" (Application No. 201610078770.7), and "A Compound Traditional Chinese Medicine Additive for Preventing and Treating Parasitic Diseases in Fish and Its Production Method" (Application No. 201510798703.8) all describe methods involving the mixing of various traditional Chinese medicine components containing rhubarb, which have anthelmintic and insecticidal functions against fish parasitic diseases. However, the methods described are based on the drying and pulverization of the traditional Chinese medicines followed by mixing in a specific ratio. "An Ethanol Extract Phase of Rhubarb Root and Its Application" (Application No. 202510308884.5) describes the application of rhubarb extract as an antibacterial agent. No patent applications have been found that describe the synergistic effect of rhubarb extract in antiparasitic treatment.

[0016] Therefore, providing a rhubarb extract for the prevention and treatment of fish ciliate parasites, its preparation method, and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0017] To achieve the above objectives, the present invention adopts the following technical solution:

[0018] A method for preparing rhubarb extract includes the following steps: 1) Rhubarb powder was activated with citric acid for 1 hour, soaked in 55%~70% volume concentration ethanol for 23 hours, filtered, and the first filtrate was obtained and concentrated at low temperature. 2) Step 1) The filter residue is soaked in 55%~70% volume concentration ethanol for 24 hours to obtain the second filtrate, which is then concentrated at low temperature; 3) The filter residue from step 2) is soaked in 55%~70% ethanol for 24 hours to obtain the third filtrate, which is then concentrated at low temperature; 4) Combine the concentrated filtrates from steps 1)-3) to obtain the citric acid-activated rhubarb ethanol extract.

[0019] Furthermore, step 1) specifically involves: Rhubarb powder was soaked in 1% citric acid for 1 hour at a mass-to-volume ratio of 1g:3mL, with stirring three times intermittently during soaking to ensure thorough mixing of the rhubarb powder and liquid and activate the antiparasitic active ingredients. Then, 95%~100% ethanol was added, with a mass-to-volume ratio of rhubarb powder to ethanol of 1g:6~7mL, so that the total ethanol volume concentration in the soaking solution was 55%~70%. Soaking was carried out for 23 hours, with stirring 3~4 times during the soaking period to ensure thorough mixing. The solution was filtered through four layers of sterile gauze, and the filtrate was collected and concentrated at low temperature until the mass-to-volume ratio of the original rhubarb powder to the concentrated filtrate was 1g:2mL. The first concentrated filtrate was stored in a pre-sterilized clean brown bottle.

[0020] Furthermore, step 2) specifically involves: Step 1) Add an appropriate amount of 55%~70% volume concentration ethanol to the obtained filter residue. The mass-volume ratio of the original rhubarb powder to ethanol is 1g:3~4mL. Continue soaking for 24 hours, stirring 3~4 times during the period to ensure thorough mixing. Filter through 4 layers of sterile gauze, take the filtrate, and concentrate it at low temperature until the mass-volume ratio of the original rhubarb powder to the concentrated filtrate is 1g:2mL. Store the second concentrated filtrate in a pre-sterilized clean brown bottle.

[0021] Furthermore, step 3) specifically involves: Step 2) Add an appropriate amount of 55%~70% volume concentration of ethanol to the obtained filter residue. The mass-volume ratio of the original rhubarb powder to ethanol is 1g:3~4mL. Continue to soak for 24 hours, stirring 3~4 times during this period to ensure thorough mixing. Filter through 4 layers of sterile gauze, take the filtrate, and concentrate it at low temperature until the mass-volume ratio of the original rhubarb powder to the concentrated filtrate is 1g:2mL to obtain the third concentrated filtrate. Store it in a pre-sterilized clean brown bottle.

[0022] Furthermore, step 4) specifically involves: Combine the concentrated filtrates obtained in steps 1)-3), mix them well, and obtain a volume ratio of 1g:6mL for the liquid extract to the original rhubarb powder, that is, 1g of rhubarb yields about 6mL of liquid. Then concentrate it to 0.5 times, that is, the final volume ratio of the liquid extract to the original rhubarb powder is 1g:3mL, that is, 1g of rhubarb yields 3mL of liquid.

[0023] A method for preparing rhubarb extract.

[0024] Application of rhubarb extract in the preparation of drugs for the prevention and treatment of fish ciliate parasites.

[0025] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes a weakly acidified hydrolysis followed by solvent extraction method, ensuring a high extraction rate of active ingredients. It overcomes the drawbacks of water extraction (low efficiency and susceptibility to mold growth), ammonia and simple alcohol extraction (low insect resistance), and ultrasonic extraction (requiring sophisticated equipment).

[0026] This invention employs a method of activating rhubarb with citric acid, followed by ethanol extraction and low-temperature concentration to prepare a rhubarb extract with enhanced anti-ciliary parasite activity. This method ensures high anthraquinone content, reduced toxicity to fish, and increased activity against Tetrahymena thermophila. The extract is also less prone to mold growth during storage and has good effects on Trichodina and Ichthyophthirius multifiliis diseases in fish fry. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 The survival rate of goldfish infected with Ichthyophthirius multifiliis in an in vivo drug treatment experiment; Figure 2 The survival rate of grass carp naturally infected with Trichodina in vivo under the influence of drugs; Figure 3 The experimental procedure is as described in this embodiment of the invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1 A method for extracting the effective components of the traditional Chinese medicine rhubarb, the extraction steps are as follows: 1) Rhubarb was activated with citric acid for 1 hour and then soaked in 55%~70% ethanol for 23 hours to obtain the first filtrate. Rhubarb powder (Guangzhou Pharmaceutical Company, rhubarb powder, 100 mesh) was soaked in 1% citric acid at a mass-to-volume ratio of 1g:3mL for 1 hour, with stirring three times intermittently during soaking to ensure thorough mixing of the rhubarb powder and liquid, thus activating the antiparasitic active ingredients. Then, 95%–100% ethanol was added at a mass-to-volume ratio of 1g:6–7mL to the original rhubarb powder, bringing the total ethanol concentration in the soaking solution to 55%–70%. Soaking continued for 23 hours, with stirring 3–4 times during this period to ensure thorough mixing. In total, acid activation for 1 hour and ethanol soaking for 23 hours resulted in a total action time of 24 hours. The solution was filtered through four layers of sterile gauze. The filtrate was then concentrated at low temperature (e.g., using a low-temperature vacuum centrifuge) to a volume twice that of the original rhubarb powder (volume / mass ratio = 1g:2mL), i.e., 1g of rhubarb yielded 2mL of filtrate. The first concentrated filtrate was stored in a pre-sterilized clean brown bottle.

[0031] 2) The filter residue from the previous step is soaked in 55%~70% ethanol for 24 hours to obtain the second filtrate; Add an appropriate volume of 55%–70% ethanol to the filter residue obtained in step 1), with the mass-to-volume ratio of ethanol to the original rhubarb powder being 1 g: 3–4 mL. Continue soaking for 24 hours, stirring 3–4 times during this period to ensure thorough mixing. Filter through four layers of sterile gauze, collect the supernatant filtrate, and concentrate it at low temperature to twice the volume of the original rhubarb powder (volume / mass ratio = 1 g: 2 mL). Store the resulting second concentrated filtrate in a pre-sterilized clean brown bottle.

[0032] 3) The filter residue from the previous step is soaked in 55%~70% ethanol for 24 hours to obtain the third filtrate. In step 2), the filter residue obtained is added again with an appropriate volume of 55%–70% ethanol. The mass-to-volume ratio of the added ethanol to the original rhubarb powder is 1 g: 3–4 mL. The mixture is then soaked for another 24 hours, stirring 3–4 times during this period to ensure thorough mixing. The solution is filtered through four layers of sterile gauze. The filtrate is then concentrated at low temperature to twice the volume of the original rhubarb powder (volume / mass ratio = 1 g: 2 mL) to obtain the third concentrated filtrate. This filtrate is then stored in a pre-sterilized clean brown bottle.

[0033] 4) Combine the three filtrates to obtain the citric acid-activated rhubarb ethanol extract. Combine the concentrated filtrates from steps 1)-3), mix well, and obtain an extract liquid with a volume 6 times the original rhubarb mass, i.e., approximately 6 mL of liquid from 1 g of rhubarb. Then concentrate the extract to 0.5 times the original volume, i.e., the final extract liquid with a volume 3 times the original rhubarb mass, i.e., 3 mL of liquid from 1 g of rhubarb. The final liquid is called citric acid-activated rhubarb ethanol extract, or EECtR for short.

[0034] Methods for quality inspection and in vitro efficacy determination of rhubarb extract against Tetrahymena thermophila Includes sterility testing, high-performance liquid chromatography (HPLC) for the detection of anthraquinone components and content, and half-maximal inhibitory concentration (IC50) against Tetrahymena thermophila. 50 ) Measurement.

[0035] Sterility test: Take the test solution and spread it on two pre-prepared sterilized solid culture media (nutrient agar and Sabouraud dextrose agar: both bacteria and fungi can grow on nutrient agar, while fungi are more likely to grow on Sabouraud dextrose agar). Incubate at 28°C for 24-48 hours and observe whether bacteria and fungi grow. Inoculate 100µL of the test solution on a plate with a diameter of 8.5 cm. Set up 3 replicates for each sample and each culture medium.

[0036] Determination of anthraquinone content: The total anthraquinone content in rhubarb extract was determined by high performance liquid chromatography (HPLC), according to the following: Chinese Veterinary Pharmacopoeia, Part II, 2020 Edition, General Chapter 0512, pp. 32-34, Appendix 54-Appendix 59, Chinese Veterinary Pharmacopoeia Commission, Beijing: China Agriculture Press.

[0037] Inhibitory effect on Tetrahymena thermophila: Rhubarb extract was diluted to different concentrations with sterile deionized water and applied to Tetrahymena thermophila in the logarithmic growth phase. The half-maximal inhibitory concentration (IC50) of rhubarb extract against Tetrahymena thermophila was calculated. 50 ).

[0038] Preparation of Tetrahymena thermophila: Tetrahymena thermophila strain B2086.2, stored at room temperature, was inoculated at a concentration of 5% (v / v) into test tubes containing 10 mL of Neff medium (0.25% peptone, 0.25% yeast extract, 0.5% glucose, and 9.0 g / L FeCl3 stock solution added at a 0.1% (v / v) ratio). The cells were cultured at 28-30°C and 100-150 rpm on a shaker until the logarithmic growth phase (24-48 h). The Tetrahymena thermophila in the logarithmic growth phase were then inoculated into fresh 10 mL of medium at an initial density of approximately 5000 cells / mL. After culturing for 24-48 h, the cells were ready for use (Tetrahymena thermophila density was 1.0 × 10⁻⁶). 5 ~1.3×10 5 (units / mL).

[0039] Add rhubarb extract: Dilute the prepared Tetrahymena thermophila cells in the logarithmic growth phase (24h~48h) with Neff medium to a 10mL system, with a concentration of 4.0×10⁻⁶. 4 ~5.0×10 4Tetrahymena thermophila cell suspensions were prepared using different concentration gradients of rhubarb extract in Neff medium to prepare a series of final drug concentrations (e.g., 0.08, 0.16, 0.32, 0.8, 1.6, 3.2, 8.0, 16.0, 32.0 g / L). Three replicates were set up for each drug concentration group, along with a blank control (medium only) and a negative control (medium, Tetrahymena thermophila, no drug). The cells were cultured and exposed for 24 hours at 28℃~30℃ and 100~150 rpm using a shaker. The Tetrahymena thermophila cell density in each experimental group was measured using a cell counter according to the method provided in the patent (A reagent and counting method for rapid fixation and counting of Tetrahymena thermophila cells. National Invention Patent, Patent No.: ZL201911291709.0).

[0040] IC 50 Calculations: A line graph was plotted with the concentration of rhubarb extract (g / L) on the x-axis and the density of Tetrahymena thermophila (10⁴ cells / mL) on the y-axis to calculate the density of Tetrahymena thermophila under different concentrations of monomer. The half-maximal inhibitory concentration (IC50) of the drug on Tetrahymena thermophila growth was calculated using R software. 50 The results were statistically analyzed, and p < 0.05 was considered statistically significant, while p ≥ 0.05 was not considered statistically significant.

[0041] A comparison of the inhibitory effects of rhubarb extracted using the method of this invention and the control method on the growth of Tetrahymena thermophila. Rhubarb was extracted using the method of this invention (ethanol extraction after citric acid activation) and control methods (boiling in water, ammonia, ethanol alone, and citric acid alone). The half-maximal inhibitory concentrations (IC50) of the obtained extracts (drug solutions) against Tetrahymena thermophila strain B2086.2 are shown in Table 1. The results showed that the drug solution obtained using the method of this invention had an IC50 of 0.333 ± 0.532 g / L against Tetrahymena thermophila, significantly stronger than that obtained by boiling in water, soaking in citric acid alone (using the citric acid concentration corresponding to that of this invention), and extraction with ammonia (p < 0.05). This was approximately 30 times stronger than boiling in water, approximately 50 times stronger than soaking in citric acid alone, and approximately 6.3 times stronger than extraction with ammonia. It was approximately 1.13 times stronger than extraction with ethanol alone, but the difference was not statistically significant (p ≥ 0.05). The results indicate that, compared with the four control methods, the drug solution extracted using the method of this invention had the strongest inhibitory effect on Tetrahymena thermophila.

[0042] Table 1. Half-maximal inhibitory concentrations (g / L, mean ± standard deviation) of rhubarb extracted using the method of this invention and the control method against Tetrahymena thermophila.

[0043] Note: Different letters in each column indicate significant differences (p<0.05), while the same letters indicate no significant differences (p≥0.05).

[0044] Comparison of the content of free anthraquinones and other components in rhubarb extracted using the method of this invention and a control method. High-performance liquid chromatography (HPLC) was used to analyze the total free anthraquinones (calculated as the total amount of aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether on the dried product) and the contents of the anthraquinone components aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether. The total anthraquinone content extracted by this method was 1.54%, which is higher than that extracted by simple ethanol extraction, ammonia extraction, and water boiling extraction. Among the free anthraquinone components, except for aloe-emodin which was lower than that extracted by ammonia extraction, the contents of other anthraquinone components were higher than those of the other three groups. This indicates that the anthraquinone components of rhubarb can be effectively extracted using this patented method. Interestingly, the content of rhein was 0 by ammonia extraction, which may be due to acid-base neutralization. See Table 2 for details.

[0045] Table 2. Content of anthraquinone components extracted from rhubarb using this method and the control method (high performance liquid chromatography).

[0046] Note: (1) Free anthraquinones are calculated as the total amount (%) of aloe-emodin, rhein, emodin, chrysophanol and emodin methyl ether on the dried product. The values ​​of the contents of each anthraquinone component are the mean ± variance. (2) Different letters in each row indicate significant differences (p<0.05), and the same letters indicate no significant differences (p≥0.05).

[0047] Sterility testing of rhubarb using the method of this invention and a control method Rhubarb extract stored at room temperature for one month was spread on nutrient agar and Sabouraud dextrose agar plates. No bacteria or fungi (mainly molds) grew on either medium. However, simple soaking in deionized water, 3% ammonia, or 0.3% citric acid resulted in mold growth on both nutrient agar and blood agar plates. When extracted with 50% ethanol, mold growth was observed on nutrient agar plates after 24 hours, but not after 48 hours; mold growth was observed on ethanol extracts at concentrations of 30%–50%. With ethanol alone, concentrations ≥55% and ≤95% showed no mold growth on either medium. Interestingly, with 100% ethanol alone, no mold growth was observed on either medium after 24 hours, but fungal growth was observed after 48 hours. This suggests that 100% ethanol has a lower efficacy in killing mold. This may be because 100% ethanol, lacking moisture, rapidly dehydrates the proteins on the surface of microorganisms, forming a hardened protective layer that hinders further penetration of ethanol into the microorganisms, thus reducing its antifungal effect.

[0048] The results show that the rhubarb extract showed no fungal growth and the test results are detailed in Table 3.

[0049] Table 3. Mold growth in rhubarb extracted using this method and the control method.

[0050] Note: "-" indicates no mold growth, and "+" indicates mold growth.

[0051] The killing effect of rhubarb extract on Ichthyophthirius multifiliis cysts in fish The assay method was used to determine the killing effect on ciliated parasites (taking Ichthyophthirius multifiliis cysts as an example) and to evaluate the effectiveness of drug control in preventing ciliated parasitic diseases in fish.

[0052] Tomont is a stage in the life cycle of Ichthyophthirius multifiliis after it leaves the fish. It is a form in which the trophozoite develops in the environment (such as the bottom of the water, the tank wall, or the surface of plants) after leaving the fish. The outer layer of the cyst is composed of mucus and a gelatinous membrane secreted by the worm. Asexual reproduction takes place inside, which can produce hundreds to thousands of larvae (predators).

[0053] Using a cell scraper, trophozoites infected with Ichthyophthirius multifiliis were scraped from the surface of goldfish and placed into a culture dish containing sterile deionized water. When the trophozoites began to swim in the water, they were transferred to another culture dish using a pipette. This elution step was repeated 2-3 times to prepare Ichthyophthirius multifiliis cysts.

[0054] One blank control group and six experimental groups were set up, with three replicates in each group. The killing experiment was conducted in a 12-well plate. Using deionized water as the solvent, 0, 0.125 g / L, 0.25 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, and 4.0 g / L concentrations of the rhubarb extract of this invention and the boiled rhubarb decoction (control group) were prepared. 3.0 mL was added to each well, and 10 worms were added to each well using a pipette. The incubator temperature was controlled at 23 ℃ ± 1.0 ℃. The 24-well plate was then placed in the incubator, and the mortality rate of the cysts in each group was recorded at 0 and 3 hours. Cysts were considered dead if they lacked a nucleus, showed no ciliary movement, or showed no division, and were observed under an inverted microscope.

[0055] Table 4 shows that the rhubarb extract of the present invention has a killing effect on the cysts of Ichthyophthirius multifiliis. The results show that 0.125 g / L extract can kill more than 50% of the cysts within 3 h, and 1.0 g / L extract can completely kill the cysts within 3 h. In contrast, the water boiling method in the control group requires a concentration of 0.25 g / L to kill more than 50% of the cysts within 3 h, and 4.0 g / L to achieve 100% killing.

[0056] Table 4. Killing rates of different concentrations of the rhubarb extract of the present invention and the water-boiling method against Ichthyophthirius multifiliis cysts.

[0057] Safety test of the rhubarb extract of the present invention on fish Drug immersion was used to determine the concentration at which all target animals (goldfish and grass carp) would die within 24 hours and the concentration at which no mortality would occur within 96 hours through preliminary experiments. Based on the experimental results, six concentration gradient groups and a control group without drug were set up. Experimental fish that had been temporarily housed for 14 days and confirmed to be healthy were placed in the group. One-third of the test solution was replaced daily in each group, and observation was conducted for 96 hours. Fish activity and the number of dead fish were recorded daily. An oxygen pump was used to provide oxygen during the experiment, and water quality indicators such as water temperature, ammonia nitrogen, and nitrite nitrogen were recorded. The 48- and 96-hour LC50 of the drug on the fish was calculated using the Kohl's method. 50 The safe concentration was calculated using the Standard Method for Aquatic Toxicology (MATC method) (as shown in Formula 1 below).

[0058] Formula 1: Safe concentration = 0.1 × average tolerance limit = 0.1 ×

[0059] Safety of goldfish Ten healthy goldfish, each weighing (2.97±0.45)g, were stocked in a 10L polyethylene white plastic bucket. Concentration groups of the drug were set up at 2.0g / L, 5.0g / L, 7.5g / L, 9.0g / L, 10g / L, and 15g / L, with three replicates for each concentration group. A control group without the drug was also included. The goldfish were immersed in the extract of this invention at the designed concentration and observed continuously for 96 hours, with the number of dead and surviving fish recorded daily. The 96-hour median lethal concentration (LC50) of the extract of this invention for goldfish was calculated. 50 The concentration was approximately 9.12 g / L, 48 h LC. 50 It is approximately 10.43 g / L. Using formula 1 for safe concentration, the safe concentration is 0.975 g / L.

[0060] Safety of grass carp Ten healthy grass carp, each weighing (7.11 ± 1.45) g, were stocked in a 10L polyethylene white plastic bucket. Concentration groups of the drug were set up at 2.0 g / L, 5.0 g / L, 7.5 g / L, 9.0 g / L, 10 g / L, and 15.0 g / L, with three replicates for each concentration group. A control group without the drug was also included. The grass carp were soaked in the extract of this invention at the designed concentration and observed continuously for 96 hours, with the number of dead and surviving fish recorded daily. The 96-hour LC50 of the extract of this invention on grass carp... 50 Approximately 7.56 g / L, 48h LC 50 The concentration was 8.21 g / L, and the safe concentration was 0.788 g / L.

[0061] The effect of rhubarb extract of the present invention on the prevention and treatment of ichthyophthirius multifiliis disease in fish Ichthyophthiriasis, also known as white spot disease, is caused by the ciliated parasite *Ichthyophthirius multifiliis*, which infects fish. Mortality rates can reach over 90% in severe cases, even resulting in total annihilation. To evaluate the efficacy of drugs in treating Ichthyophthiriasis in fish, experimental fish such as goldfish and grass carp, which are susceptible to *Ichthyophthirius multifiliis*, are used. Common methods include co-infection (healthy experimental fish and naturally infected fish are kept together in the same pond) or artificial infection (hatched *Ichthyophthirius multifiliis* larvae infect healthy experimental fish in a certain proportion). The drug solution of this invention is prepared at a specific concentration and used to immerse fish infected with *Ichthyophthirius multifiliis*. A control group infected with the parasite but without the drug solution is also included. The mortality and survival rates of the fish are recorded. The cure rate of the drug is calculated by comparing the control group (Formula 2).

[0062] Formula 2: Cure rate (effective protection rate, %)

[0063] The effect of preventing and treating goldfish Ichthyophthirius multifiliis disease Goldfish with numerous white spots on their body surface observed by the naked eye and diagnosed as infected with *Ichthyophthirius multifiliis* by microscopic examination were selected. These goldfish were placed in a 5.0L beaker of deionized water, and the mature cysts were gently detached from the skin using a sterile cell scraper. The cysts were then gently washed and transferred to a 1L glass beaker containing a small amount of sterile deionized water. They were incubated at 23℃ ± 1.0℃ for 20 hours to develop into larvae. Ten 20μL portions of the larval fluid were collected, and the number of live *Ichthyophthirius multifiliis* larvae was counted using a cell counter to determine the number for use in the goldfish infection experiment. 180 goldfish (3.95 g ± 0.79 g) that had been temporarily held for 14 days and confirmed to be healthy were randomly divided into 18 tanks (10 goldfish per tank), each containing 15L of aerated water. The quantitative infection dose was (predator:goldfish = 4000:1). Rhubarb extract was added to each breeding tank to achieve final concentrations of 0 g / L, 0.125 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, and 1.0 g / L, respectively. Each group had three replicates. During the 14-day experiment, one-third of the aerated water was replaced daily for the first four days, and the drug solution of this invention was added to maintain the same drug concentration in each experimental group since the first day. Medication was discontinued in the later stages. Every other day, bottom feces were removed, and one-third of the aerated water was replaced. Dead individuals were promptly removed to prevent water quality deterioration, and oxygenation was maintained throughout the process using an oxygen pump to ensure excellent water quality. The survival and mortality of the goldfish were observed and recorded daily. Figure 1 .

[0064] 14-day statistics: The survival rate of the goldfish control group (no medication) was 3.3%. The survival rates of the experimental groups (medication concentration groups) at 0.125 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, and 1.0 g / L were 13.3%, 46.7%, 63.3%, 73.3%, and 86.7%, respectively. The cure rate of the drug in vivo for preventing Ichthyophthirius multifiliis infection in goldfish was calculated using Formula 2. The cure rates for the 0.125 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, and 1.0 g / L concentration groups were 10.31%, 44.86%, 62.0%, 72.38%, and 86.24%, respectively. This indicates that the 0.50 g / L, 0.75 g / L, and 1.0 g / L concentration groups showed better curative effects.

[0065] The rhubarb extract of this invention is effective in preventing and treating trichodiniasis in fish. Trichodina, a ciliate parasite, can infect various freshwater fish. While the mortality rate after infection is generally lower than that of Ichthyophthirius multifiliis (white spot disease), its impact on fish fry is still more severe. Infected fish fry can be obtained through natural infection or cohabitation infection. The infected fry are then immersed in a solution prepared according to this invention at a specific concentration. A control group infected with the parasite but without the treatment solution is also established. The mortality and survival rates of the fish are recorded. The cure rate is calculated by comparing the control group with the treatment solution (Formula 2).

[0066] Formula 2: Cure rate (effective protection rate, %)

[0067] The efficacy of rhubarb extract in treating trichodiniasis in grass carp. Grass carp fry cultured in net cages at the Xilang Pearl River Experimental Base in Liwan District, Guangzhou City, spontaneously developed Trichodiniasis. The grass carp measured 18.64 g ± 1.74 g. 180 grass carp were randomly divided into 18 groups of 10 each, and cultured in white plastic buckets containing 20 L of aerated water. Starting from day 1, rhubarb extract of this invention was added to each bucket, with final concentrations of 0 g / L, 0.125 g / L, 0.25 g / L, 0.50 g / L, 0.75 g / L, and 1.0 g / L, respectively. Each concentration group had three replicates. During the 14-day experiment, medication was administered on day 1, and no medication was given on day 2 (no water change). On day 3, half of the aerated water was replaced and the drug solution of this invention was added. Medication was discontinued in the later stages. Every other day, bottom feces were removed and one-third of the aerated water was replaced. Dead individuals should be removed promptly to prevent water quality deterioration, and oxygenation should be maintained throughout the process using an oxygen pump to ensure good water quality. The survival rate of grass carp should be observed and recorded daily; results can be found in [link to relevant documentation]. Figure 2 .

[0068] 14-day statistics: The survival rate of grass carp infected with Trichodina infection in the control group (no drug treatment) was 23.3%. The survival rates of the experimental groups (drug concentration groups) at 0.125 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, and 1.0 g / L were 36.7%, 53.3%, 66.7%, 80.0%, and 86.7%, respectively. The cure rate of the drug in vivo for treating grass carp infected with Trichodina infection was calculated using Formula 2. The cure rates for the 0.125 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, and 1.0 g / L concentration groups were 17.5%, 39.2%, 56.6%, 73.9%, and 82.7%, respectively. This indicates that the 0.75 g / L and 1.0 g / L concentration groups showed better cure effects.

[0069] See the summary of the experimental procedure. Figure 3 .

[0070] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing rhubarb extract, characterized in that, Includes the following steps: 1) Rhubarb powder was activated with citric acid for 1 hour, soaked in 55%~70% volume concentration ethanol for 23 hours, filtered, and the first filtrate was obtained and concentrated at low temperature. 2) Step 1) The filter residue is soaked in 55%~70% volume concentration ethanol for 24 hours to obtain the second filtrate, which is then concentrated at low temperature; 3) The filter residue from step 2) is soaked in 55%~70% ethanol for 24 hours to obtain the third filtrate, which is then concentrated at low temperature; 4) Combine the concentrated filtrates from steps 1)-3) to obtain the citric acid-activated rhubarb ethanol extract.

2. The preparation method according to claim 1, characterized in that, Step 1) Specifically: Rhubarb powder was soaked in 1% citric acid for 1 hour at a mass-to-volume ratio of 1g:3mL, with stirring three times intermittently during soaking to ensure thorough mixing of the rhubarb powder and liquid and activate the antiparasitic active ingredients. Then, 95%~100% ethanol was added, with a mass-to-volume ratio of rhubarb powder to ethanol of 1g:6~7mL, so that the total ethanol volume concentration in the soaking solution was 55%~70%. Soaking was carried out for 23 hours, with stirring 3~4 times during the soaking period to ensure thorough mixing. The solution was filtered through four layers of sterile gauze, and the filtrate was collected and concentrated at low temperature until the mass-to-volume ratio of the original rhubarb powder to the concentrated filtrate was 1g:2mL. The first concentrated filtrate was stored in a pre-sterilized clean brown bottle.

3. The preparation method according to claim 1, characterized in that, Step 2) specifically involves: Step 1) Add an appropriate amount of 55%~70% volume concentration ethanol to the obtained filter residue. The mass-volume ratio of the original rhubarb powder to ethanol is 1g:3~4mL. Continue soaking for 24 hours, stirring 3~4 times during the period to ensure thorough mixing. Filter through 4 layers of sterile gauze, take the filtrate, and concentrate it at low temperature until the mass-volume ratio of the original rhubarb powder to the concentrated filtrate is 1g:2mL. Store the second concentrated filtrate in a pre-sterilized clean brown bottle.

4. The preparation method according to claim 1, characterized in that, Step 3) specifically involves: Step 2) Add an appropriate amount of 55%~70% volume concentration of ethanol to the obtained filter residue. The mass-volume ratio of the original rhubarb powder to ethanol is 1g:3~4mL. Continue to soak for 24 hours, stirring 3~4 times during this period to ensure thorough mixing. Filter through 4 layers of sterile gauze, take the filtrate, and concentrate it at low temperature until the mass-volume ratio of the original rhubarb powder to the concentrated filtrate is 1g:2mL to obtain the third concentrated filtrate. Store it in a pre-sterilized clean brown bottle.

5. The preparation method according to claim 1, characterized in that, Step 4) specifically involves: Combine the concentrated filtrates obtained in steps 1)-3), mix them well, and obtain a volume ratio of 1g:6mL for the liquid extract to the original rhubarb powder, that is, 1g of rhubarb yields about 6mL of liquid. Then concentrate it to 0.5 times, that is, the final volume ratio of the liquid extract to the original rhubarb powder is 1g:3mL.

6. The rhubarb extract prepared by any one of the preparation methods according to claims 1-5.

7. The use of the rhubarb extract according to claim 6 in the preparation of drugs for the prevention and treatment of fish ciliate parasites.

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