Application of gossypol in preparation of medicine for preventing and treating chicken necrotic enteritis

Abstract

The invention relates to application of gossypol in preparation of a medicine for treating chicken necrotic enteritis, and belongs to the field of medicine. In-vitro antibacterial activity tests, hemolytic activity inhibition tests, biofilm formation inhibition tests and chicken necrotic enteritis model tests prove that gossypol has a prevention and treatment effect on chicken necrotic enteritis caused by clostridium perfringens infection. The gossypol is preferably gossypol acetate, the minimum inhibitory concentration of the gossypol acetate to clostridium perfringens is 16 mu g / mL, and the minimum bactericidal concentration of the gossypol acetate to clostridium perfringens is 32 mu g / mL. It is found for the first time that gossypol can play a dual anti-infection role by inhibiting the hemolytic activity and biofilm formation of clostridium perfringens. The gossypol provided by the invention can effectively inhibit the growth of clostridium perfringens and the expression of virulence factors of the clostridium perfringens, meanwhile, the intestinal injury and growth performance of sick chickens are improved, and a new technical scheme is provided for green prevention and control of chicken necrotic enteritis.

Description

Technical Field

[0001] This invention relates to the application of gossypol in the preparation of drugs for treating necrotizing enteritis in chickens, and belongs to the pharmaceutical field. Background Technology

[0002] Poultry farming is one of the most important components of the agricultural production system. Although large-scale poultry farming and management have become increasingly sophisticated, the industry still faces threats from various pathogenic microorganisms. Among these, broiler gut health is a major factor affecting the economic benefits of broiler production. Chicken intestinal diseases caused by Eimeria parasitic protozoa or certain bacterial pathogens such as Clostridium perfringens (CP) are the most economically significant. In recent years, issues such as drug residues and drug resistance have further limited the use of anticoccidial and anti-C. perfringens drugs. Furthermore, with the implementation of the global antibiotic ban (AGP ban), the incidence and prevalence of coccidiosis and necrotic enteritis (NE) in chickens have continued to rise globally. The latest estimates from *World Poultry Journal* (October 2015) show that necrotic enteritis causes economic losses of 6.25 US cents per chicken to the global poultry industry, amounting to nearly $6 billion annually worldwide, seriously jeopardizing the healthy development of the poultry farming industry. It is well known that enteric pathogens such as Clostridium perfringens and Eimeria can disrupt the integrity and permeability of the intestinal mucosa, damaging epithelial cells, breaking down the mucin barrier, or disrupting the tight junctions between epithelial cells, thus leading to disease. Therefore, in the post-antibiotic era, regulating the intestinal microbiota and maintaining intestinal health in poultry requires the development of ideal antibiotic alternatives. Healthy, green, and highly effective antibiotic alternatives have become a hot topic in research and application both domestically and internationally.

[0003] Natural plant extracts, containing bioactive substances such as flavonoids, polyphenols, alkaloids, anthocyanins, tea polyphenols, and polysaccharides, possess various physiological functions in animals, including antibacterial, antioxidant, enhanced intestinal immunity, promotion of digestive enzyme secretion, and improvement of intestinal health. They also feature wide availability of raw materials, flexible formulation, no residues, and no drug resistance. In recent years, plant extracts have become a research hotspot in antibiotic alternatives research. In particular, the active substances in plant extracts can maintain the microecological balance of the digestive system through antibacterial activity and selective bacterial inhibition. They can also regulate poultry intestinal health by improving intestinal immunity and intestinal barrier function, promoting intestinal mucosal development, and enhancing digestive enzyme secretion. Plant extracts are widely recognized as a class of active substances for ensuring poultry intestinal health.

[0004] Gossypol is a natural polyphenolic aldehyde compound derived from cotton plants, possessing various medicinal properties, including anti-HIV, anti-cancer, anti-tumor, antiviral, antiparasitic, and antibacterial activities. Gossypol exists as a mixture of two enantiomers ((+)-gossypol and (−)-gossypol), and changes in their ratio alter its biological activity. Acetate gossypol (GAA), as a polyphenolic aldehyde, contains equimolar amounts of (+)- and (−)-enantiomers. Currently, research on the application of gossypol in livestock and poultry production focuses primarily on growth regulation, antioxidant effects, and improvement of reproductive performance. There are no reports on its application in the prevention and treatment of necrotizing enteritis in chickens, nor are there systematic studies on its inhibitory effects on Clostridium perfringens and its protective and repairing effects on the chicken intestinal mucosa. Therefore, this invention aims to provide a novel application of gossypol in the preparation of drugs for the prevention and treatment of necrotizing enteritis in chickens, providing a new technical solution and material basis for the green prevention and control of necrotizing enteritis in chickens.

[0005] Current technical problems:

[0006] Currently, antibiotics remain the primary treatment for necrotizing enteritis in chickens. However, due to long-term use, Clostridium perfringens resistance is becoming increasingly serious, with frequent emergence of multidrug-resistant strains, leading to a decline in antibiotic efficacy. Therefore, the industry is committed to developing antibiotic alternatives, such as probiotics, organic acids, and plant extracts. However, existing alternatives generally suffer from the following technical shortcomings:

[0007] (1) The antibacterial effect is unstable and slow to take effect, making it difficult to quickly control pathogens in the early stage of infection;

[0008] (2) Most plant extracts only have antibacterial effects and lack the ability to inhibit bacterial virulence factors (such as hemolysins and biofilms), and cannot effectively block the pathogenic process.

[0009] (3) It is difficult to balance antibacterial activity and intestinal mucosal protection and repair function.

[0010] Gossypol is a natural polyphenolic aldehyde compound that has been reported to possess various biological activities, including antibacterial, antiviral, and antitumor effects. However, to date, no technical solutions for the use of gossypol or its derivatives to prevent and treat necrotizing enteritis in chickens caused by Clostridium perfringens have been disclosed, nor have there been any reports of its use in preventing and treating this disease by inhibiting the hemolytic activity and biofilm formation of Clostridium perfringens. Summary of the Invention

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0012] The purpose of this invention is to provide the application of gossypol in the prevention and treatment of necrotic enteritis in chickens. Through antibacterial activity tests, hemolytic activity inhibition tests, biofilm formation inhibition tests, and necrotic enteritis model tests of gossypol against Clostridium perfringens, it is demonstrated that gossypol has a preventive and therapeutic effect on necrotic enteritis in chickens caused by Clostridium perfringens infection.

[0013] Preferably, the concentration of gossypol is greater than or equal to 16 μg / mL.

[0014] Preferably, when the concentration of gossypol is greater than or equal to 16 μg / mL, it has a good antibacterial effect against Clostridium perfringens.

[0015] Preferably, when the concentration of gossypol is greater than or equal to 32 μg / mL, it has a good bactericidal effect on Clostridium perfringens.

[0016] Preferably, the gossypol is gossypol acetate.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) This invention is the first to apply gossypol (especially gossypol acetate) to the prevention and treatment of necrotic enteritis in chickens caused by Clostridium perfringens. In vitro antibacterial experiments showed that the minimum inhibitory concentration (MIC) of gossypol against two representative Clostridium perfringens strains was 16 μg / mL and the minimum bactericidal concentration (MBC) was 32 μg / mL. The time-bactericidal curves showed that the bacteria could be completely killed within 3 hours at a concentration of 32 μg / mL, indicating rapid onset of action.

[0019] (2) This invention has found that gossypol can significantly inhibit the hemolytic activity and biofilm formation of Clostridium perfringens at sub-inhibitory concentrations (e.g., ...). Figure 2 , Figure 3 As shown in the figure, it can not only directly kill bacteria, but also weaken the pathogenicity of pathogens. This dual mechanism of action is significantly different from ordinary antibacterial agents.

[0020] (3) This invention was verified by a subclinical chicken necrotizing enteritis model. Adding 600 mg / kg of gossypol acetate to the diet can significantly reduce the jejunal and ileal injury scores of broiler chickens. Figure 4 It significantly increased average daily gain (ADG) and average daily feed intake (ADFI), and reduced feed conversion ratio (F / G), indicating that it has a protective effect on diseased chickens and improves growth performance.

[0021] (4) The gossypol used in this invention is derived from natural plant extracts, has low residues in animals, meets food safety requirements, and provides a new safe, efficient and low-drug-resistance technical solution for antibiotic replacement. Attached Figure Description

[0022] Figure 1Time-bactericidal curves of the tested Clostridium perfringens under the action of acetic acid and gossypol; a: the tested strain is ATCC13124; b: the tested strain is CVCC2030.

[0023] Figure 2 Hemolytic activity of the tested Clostridium perfringens under the action of acetic acid and gossypol; a: the tested strain was ATCC13124; b: the tested strain was CVCC2030.

[0024] Figure 3 Biofilm of Clostridium perfringens under the action of acetic acid and gossypol; a: test strain is ATCC13124; b: test strain is CVCC2030.

[0025] Figure 4 Effects of gossypol acetate on intestinal injury scores in subclinical nocturnal nephrotic syndrome (NE) broiler chickens; a: jejunal injury score; b: ileal injury score. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0028] All raw materials, reagents, instruments, and equipment used in the examples are commercially available or can be prepared using existing methods. Unless otherwise specified, all reagents used in this invention are of analytical grade.

[0029] All experimental data in this section are expressed as mean ± standard deviation (Mean ± SD) and statistical analysis was performed using SPSS 27.0 software. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0030] Example 1: Study on the in vitro antibacterial activity of gossypol acetate against Clostridium perfringens

[0031] (1) Experimental scheme

[0032] 1. Determination of MIC and MBC values

[0033] Take BHI liquid culture medium and add gossypol acetate (GAA) to it to make final GAA concentrations of 2, 4, 8, 16, 32, and 64 μg / mL. Inoculate with overnight-cultured Clostridium perfringens suspension (1×10⁻⁶). 5 CFU). Alternatively, BHI medium without GAA was selected, and 1×10⁻⁶ cells were inoculated. 5 CFU suspension was used as the positive control; the negative control was inoculated with an equal volume of autoclaved BHI liquid medium. All test tubes were incubated at 37 °C for 48 h, and the results were observed. 0.1 mL of culture medium from each group in the above MIC value determination experiment was aspirated and inoculated onto solid plates, and then incubated anaerobically at 37 °C for another 48 h, and the results were observed.

[0034] 2. Plotting the time-sterilization curve

[0035] Overnight-cultured *Clostridium perfringens* was used, and the OD value at 600 nm was adjusted to approximately 0.3. The culture was then aliquoted into test tubes, with GAA added to each tube to achieve final concentrations of 1 / 2 MIC, MIC, and 2 MIC. Separate test tubes were added to DMSO (considered the 0 μg / mL GAA group) and thoroughly mixed. All tubes were then incubated anaerobically at 37 °C. Bacterial culture was collected from each tube at 1 h, 3 h, 5 h, 7 h, and 9 h, serially diluted, and spread onto solid plates. The plates were then incubated anaerobicly at 37 °C for 24 h. *Clostridium perfringens* colonies were counted on the plates, and the effect of GAA on the growth of *Clostridium perfringens* was recorded. Time-killing curves of different concentrations of GAA on *Clostridium perfringens* were plotted with time (h) on the x-axis and the logarithm of colony count (lg) on ​​the y-axis.

[0036] 3. Determination of hemolytic activity

[0037] Clostridium perfringens was cultured in BHI containing different mass concentrations of GAA until the logarithmic growth phase, and then diluted to OD. 600The value was 0.1. The culture supernatant was collected by centrifugation and filtered through a 0.22 μm filter membrane for sterilization, with the supernatant from a single culture of *Clostridium perfringens* used as a control. 100 μL of supernatant and 100 μL of 2% sheep erythrocytes were added to a 96-well cell culture plate and gently mixed by pipetting. Equal volumes of 2% erythrocytes in sterile physiological saline and sterile water were used as controls for non-hemolysis and 100% hemolysis, respectively. The 96-well cell culture plate was incubated at 37 °C for 1 h, then placed at 4 °C overnight. After centrifugation at 800 × g for 10 min, 100 μL of supernatant was transferred from each well to a new 96-well plate, and the OD value of the solution was detected using a multi-mode microplate reader. 540 The value was used to assess the amount of heme released from sheep erythrocytes. Hemolytic activity was calculated using the formula: Hemolytic activity = (OD value of different concentrations of gossypol acetate) 540 Value - OD after treatment with physiological saline 540 Value) / (OD value of sterilized water treatment) 540 Value - OD after treatment with physiological saline 540 (Value) ×100%.

[0038] 4. Effects of gossypol acetate on biofilm formation in Clostridium perfringens

[0039] The formation of biofilms in *Clostridium perfringens* was determined using crystal violet staining. *Clostridium perfringens* in the logarithmic growth phase was diluted to OD0.05. 600 The value was 0.1, and then the bacteria were inoculated into TSB medium containing different mass concentrations of GAA at a volume ratio of 1:100. Bacteria were inoculated into TSB medium without GAA as a control. The medium was placed in 96-well plates, 200 μL per well, with 8 replicates for each mass concentration. Fresh TSB medium was used as a blank control. After anaerobic incubation for 72 h, the 96-well plates were gently washed twice with sterile PBS to remove free cells. 200 μL of 1% crystal violet solution was added for staining for 30 min. The plates were then washed three times with PBS to remove unbound dye. 200 μL of 33% acetic acid solution was added and incubated for 30 min to dissolve the bound dye. The OD of the solution was detected using a multi-mode microplate reader. 570 value.

[0040] (2) Experimental results

[0041] 1. Determination of MIC and MBC values ​​of gossypol acetate against Clostridium perfringens

[0042] This experiment selected gossypol acetate as a potential test compound against *Clostridium perfringens*. The MIC and MBC values ​​of gossypol acetate against two *Clostridium perfringens* strains (ATCC13124 and CVCC2030) were determined to investigate its antibacterial and bactericidal activities. The results showed that the MIC value of gossypol acetate against both strains was 16 μg / mL, and the MBC value was 32 μg / mL. These results preliminarily demonstrate that gossypol acetate possesses good antibacterial and bactericidal activity against the tested *Clostridium perfringens*.

[0043] Table 1. Results of determination of MIC and MBC of gossypol acetate against Clostridium perfringens.

[0044] Tested strain MIC (μg / mL) MBC (μg / mL) ATCC13124 16 32 CVCC2030 16 32

[0045] 2. Time-bactericidal curve of gossypol acetate against Clostridium perfringens

[0046] The time-sterilization curve results are as follows: Figure 1 As shown, under the treatment of 16 μg / mL and 32 μg / mL gossypol acetate, the colony counts of *Clostridium perfringens* ATCC13124 and CVCC2030 decreased significantly. Under the treatment of 32 μg / mL gossypol acetate, the colony counts fell below the minimum detectable count after 3 hours, and the lg value of CFU dropped to 0. While the growth rate of *Clostridium perfringens* CVCC2030 slowed down after 1 hour under the treatment of 8 μg / mL gossypol acetate, significant regrowth occurred after 3 hours, and the final bacterial growth was close to that of the control group without gossypol acetate, suggesting that the antibacterial activity of 8 μg / mL gossypol acetate was weak. These results further demonstrate that 16 μg / mL gossypol acetate has a good antibacterial effect against *Clostridium perfringens*, and 32 μg / mL gossypol acetate has a good bactericidal effect against *Clostridium perfringens*.

[0047] 3. Effect of Gossypol Acetate on the Hemolytic Activity of Clostridium perfringens

[0048] Hemolytic activity was assessed by detecting the release of heme from sheep erythrocytes. The results showed that treatment with 16 μg / mL, 32 μg / mL, and 64 μg / mL gossypol acetate significantly reduced the hemolytic activity of both Clostridium perfringens strains (ATCC13124 and CVCC2030) (P<0.01), indicating that gossypol acetate can inhibit the hemolytic activity of Clostridium perfringens. See Figure 2 .

[0049] 4. Effects of gossypol acetate on biofilm formation in Clostridium perfringens

[0050] The formation of biofilms in *Clostridium perfringens* was determined using crystal violet staining. The results showed that after treatment with 8 μg / mL, 16 μg / mL, and 32 μg / mL of gossypol acetate, the OD values ​​of two *Clostridium perfringens* strains (ATCC13124 and CVCC2030) were significantly reduced. 570 The values ​​were all significantly reduced (P<0.01), indicating that gossypol acetate can inhibit the formation of Clostridium perfringens biofilm. See Figure 3 .

[0051] Example 2: Study on the anti-subclinical necrotizing enteritis in chickens using gossypol acetate

[0052] (1) Experimental design:

[0053] Currently, there are two main methods for constructing necrotizing enteritis models in chickens: one is direct challenge with high doses of *Clostridium perfringens*, and the other is to damage the intestinal mucosal structure through coccidia infection, thereby providing a suitable microenvironment for *Clostridium perfringens* colonization and pathogenicity. Studies have shown that the coccidia-induced modeling method has high stability and good reproducibility, and is currently a widely used modeling method in clinical and scientific research. Based on this, this embodiment selects the coccidia-combined *Clostridium perfringens* method to establish a necrotizing enteritis model in chickens.

[0054] 1. Experimental Grouping

[0055] The experiment employed a completely randomized block design, selecting 144 healthy, one-day-old male yellow-feathered broiler chickens of similar weight (± 5 g) and randomly dividing them into 6 groups: NC (blank control group), PC (NE infection group), LCM (NE infection + 670 mg / kg lincomycin group), GAA200 (NE infection + 200 mg / kg gossypol acetate group), GAA400 (NE infection + 400 mg / kg gossypol acetate group), and GAA600 (NE infection + 600 mg / kg gossypol acetate group). Each group had 3 replicates, with 8 chickens per replicate.

[0056] 2. Subclinical chicken necrotizing enteritis model

[0057] At 13 days of age, except for the NC group (which received the same volume of physiological saline), the other groups were administered a 25-fold dose of coccidiosis vaccine. From days 17 to 21, except for the NC group (which received the same volume of physiological saline), the other groups were administered 1 mL of 5×10 [units of saline] per chick daily. 8 CFU (clostridium perfringens) was used to test Clostridium perfringens. The test period was 23 days.

[0058] 3. Growth performance testing

[0059] Record and calculate ADFI, ADG, and F / G for broilers in 13-23, using repetitions as the unit.

[0060] Calculation formula:

[0061] (1) Average daily weight gain (ADG) = (average final weight of each group - average initial weight of each group) / number of days of the trial

[0062] (2) Feed conversion ratio (F / G) = Average daily feed intake / Average daily weight gain

[0063] (3) Average daily feed intake (ADFI) = Total feed intake / (Number of experimental days × Number of experimental chickens)

[0064] 4. Intestinal Injury Score

[0065] All chickens were euthanized at 23 days of age with cervical dislocation. Necropsy was performed, and the duodenum to jejunum was harvested for gross observation of intestinal pathological changes. The jejunum and ileum of each chicken were longitudinally dissected, rinsed thoroughly with physiological saline, and intestinal damage was assessed using a 0-4 scoring system based on the criteria of Gholamiandehkordi et al. (2007) and Deshmukh et al. (2010): 0 points, no obvious lesions; 0.5 points, severe congestion of the mesentery and small intestinal wall; 1 point, thinning and fragility of the intestinal wall, with more than 5 hemorrhage points; 2 points, gas in the intestinal lumen, numerous hemorrhage points on the intestinal wall accompanied by a few necrotic or ulcerous points; 3 points, regional necrosis and ulceration of the intestinal wall; 4 points, a large amount of gas in the intestinal lumen and diffuse necrosis.

[0066] (2) Experimental results

[0067] 1. Effects of gossypol acetate on growth performance of broiler chickens with subclinical nocturnal nephrotic syndrome (NE)

[0068] As shown in Table 2, compared with the NC group, the ADG and ADFI of broiler chickens in the PC group were significantly decreased, but the F / G ratio was significantly increased. Compared with the PC group, the ADG and ADFI of broiler chickens in the GAA600 and LCM groups were significantly increased, but the F / G ratio was significantly decreased. There were no significant differences in ADG and F / G between the GAA200 and GAA400 groups. This indicates that gossypol acetate can improve the growth performance of broiler chickens suffering from necrotizing enteritis.

[0069] Table 2. Effects of gossypol acetate on growth performance of broiler chickens with subclinical NE.

[0070] Indicators Parameters NC PC GGA 200 GGA 400 GGA 600 LCM P-value Average daily feed intake (ADFI), g / animal / d <![CDATA[47.12±0.07 a ]]> <![CDATA[42.97±0.02 f ]]> <![CDATA[43.47±0.02 e ]]> <![CDATA[43.07±0.02 d ]]> <![CDATA[44.05±0.03 c ]]> <![CDATA[45.02±0.01 b ]]> <0.001 Average daily weight gain (ADG), g / animal / d <![CDATA[18.2±0.2 a ]]> <![CDATA[13.0±1.2 c ]]> <![CDATA[13.9±0.3 bc ]]> <![CDATA[14.3±0.4 bc ]]> <![CDATA[15.2±0.2 b ]]> <![CDATA[16.5±0.3 b ]]> <0.001 Material weight ratio F / G, g / g <![CDATA[2.60±0.02 b ]]> <![CDATA[3.35±0.32 a ]]> <![CDATA[3.14±0.06 a ]]> <![CDATA[3.02±0.08 ab ]]> <![CDATA[2.90±0.05 b ]]> <![CDATA[2.73±0.06 b ]]> 0.026

[0071] 2. Effects of gossypol acetate on intestinal injury scores in subclinical nocturnal nephrotic syndrome (NE) broiler chickens

[0072] like Figure 4 As shown, compared with the NC group, the jejunal and ileal injury scores of broiler chickens in the PC group were significantly increased; compared with the PC group, the addition of 600 mg / kg GAA to the diet significantly reduced the jejunal and ileal injury scores of broiler chickens. This indicates that gossypol acetate has a good protective effect against necrotizing enteritis caused by Clostridium perfringens in chickens.

[0073] Example 3: Preliminary safety observation of gossypol acetate on broiler chickens

[0074] In the experiment of Example 2, no adverse reactions such as abnormal feed intake, lethargy, or death were observed in any of the gossypol acetate supplementation groups (200, 400, and 600 mg / kg) throughout the entire experimental period. After the experiment, necropsy examinations were performed on the chickens in each group, and no obvious pathological changes were found in the heart, liver, spleen, lungs, kidneys, or other organs. This indicates that under the conditions of this experiment, gossypol acetate at a dose of 600 mg / kg did not show significant toxicity to broiler chickens.

[0075] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. The application of gossypol in the preparation of drugs for the prevention and treatment of necrotic enteritis in chickens, characterized in that: The aforementioned necrotic enteritis in chickens refers to necrotic enteritis caused by Clostridium perfringens.

2. The application according to claim 1, characterized in that, The gossypol is gossypol acetate.

3. The application according to claim 1 or 2, characterized in that, The concentration of gossypol in the drug is above 16 μg / mL.

4. As described in claim 3, characterized in that, When the concentration of gossypol in the drug is above 32 μg / mL, it has a good bactericidal effect against Clostridium perfringens.

5. The application according to claim 1, characterized in that, The gossypol is used to prevent necrotizing enteritis in chickens by inhibiting the hemolytic activity and / or biofilm formation of Clostridium perfringens.

6. A pharmaceutical composition for preventing and treating necrotic enteritis in chickens, characterized in that, It contains an effective amount of gossypol and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The gossypol is gossypol acetate, and the effective amount refers to the concentration of gossypol in the pharmaceutical composition being not less than 16 μg / mL.

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