Uses of sugarcane extract in the treatment or prevention of microbial infections and dysbiosis

Abstract

This document discloses methods and compositions for use in various therapeutic, preventive, and control measures comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane. The therapeutic, preventive, and control measures involve administering the extract to a subject, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols. These therapeutic and preventive measures include antimicrobial activity, particularly against respiratory viruses in humans, and treatment of microsporidia infections, such as EHP in shrimp and prawns. Control measures include reducing methane production in ruminants. The use of these compositions in, for example, dairy cows improves cow health by minimizing mastitis while increasing milk yield.

Description

Technical Field

[0001] This disclosure pertains to the field of inhibiting or inactivating viruses and preventing or treating microbial infections in animals. These extracts are particularly effective against viruses that cause respiratory diseases in animals.

[0002] This disclosure also relates to the use of sugarcane-derived extracts, including polyphenols, for inhibiting or inactivating microsporidian parasites and for preventing or treating microsporidian infections in animals. These extracts are particularly effective against *E. hepatocellular carcinoma* (EHP), a disease-causing parasite in aquatic animals such as shrimp and prawns.

[0003] This disclosure also relates to the use of sugarcane-derived extracts, including polyphenols, for inhibiting or inactivating protozoa and for preventing or treating protozoan infections in animals. These extracts are particularly effective against coccidia and Plasmodium species (the cause of malaria).

[0004] This disclosure also relates to the use of sugarcane-derived extracts, including polyphenols, for inhibiting or inactivating archaea and for preventing or treating archaea infections in animals. These extracts are particularly suitable for treating ecological imbalances and combating methanogens present in ruminants.

[0005] This disclosure also relates to the use of sugarcane-derived extracts, including polyphenols, for the prevention or treatment of mastitis in ruminants. These extracts are particularly suitable for the prevention or treatment of mastitis in lactating cows.

[0006] This disclosure also relates to the use of sugarcane-derived extracts, including polyphenols, for inhibiting or inactivating fungi and for preventing or treating fungal infections in animals, particularly against Ascomycota and Basidiomycota.

[0007] Mechanistically, this disclosure relates to the use of sugarcane-derived extracts, including polyphenols, for the treatment, prevention, or minimization of cytokine storms in animals, which are commonly found in all pathogen invasions of animals.

[0008] Mechanistically, this disclosure also relates to the use of sugarcane-derived extracts, including polyphenols, for treating, preventing, or minimizing at least one of inflammation, tissue, and organ damage in animals caused by an acquired immune response by modulating Toll-like receptors.

[0009] This disclosure also relates to the use of sugarcane-derived extracts, including polyphenols, alone or in combination with known adjuvants such as Freund's adjuvant, as adjuvants. Background Technology

[0010] Patients infected with SARS-CoV-2 may be asymptomatic. However, symptomatic patients may experience fever, dry cough, and shortness of breath, which are symptoms of an upper respiratory tract infection. Patients may also present with flu-like symptoms. More severe illness and breathing difficulties may occur as the infection becomes more serious and spreads deeper into the lungs. Secondary infections and / or non-pulmonary conditions (cardiac, kidney complications) may also occur. Severe illness is often characterized by pneumonia that leads to acute respiratory disease syndrome (ARDS), one of the leading causes of death from COVID-19.

[0011] There are no established treatment options for ARDS caused by SARS-CoV-2. Instead, supportive care and nonspecific treatment regimens have been used to alleviate patients' symptoms. In the absence of effective drug therapy, ventilator strategies, alone or in combination with, for example, broad-spectrum antiviral agents, antibiotics, or convalescent plasma, remain the primary available option.

[0012] Furthermore, drug candidates for ARDS may lack the required safety and activity. For example, steroids should generally not be used because they are known to reduce the immune response and may increase viral shedding. Trials of steroid treatment for patients in previous SARS or MERS epidemics have been less effective than trials with approved antiviral agents (ribavirin, lopinavir-ritonavir) and immunomodulators. Side effects can be observed with these agents. For example, anemia has been observed with ribavirin.

[0013] Recently, dexamethasone (a corticosteroid used for a variety of conditions due to its anti-inflammatory and immunosuppressive effects) has been tested in hospitalized patients with COVID-19 in the UK National Clinical Rehabilitation Trial and found to be beneficial in critically ill patients. According to preliminary findings shared with the WHO, treatment showed a reduction in mortality of approximately one-third in patients on ventilators and approximately one-fifth in patients requiring only oxygen.

[0014] Although no specific antiviral drugs have been approved yet, remdesivir has been found to reduce recovery time.

[0015] While the development of effective treatments is desired, preventing viral infection is the preferred option. Alternatively, vaccination becomes the way to combat infection.

[0016] Prevention of coronavirus infection is typically achieved through physical means and disinfection. This includes effective handwashing, the use of face shields and protective clothing, gloves, and other forms of personal protective equipment. Simply washing hands with soap and water and hand sanitizer can effectively inactivate the coronavirus. Such measures prevent individuals from spreading the virus from their hands to their mouth or nose, thereby infecting the respiratory system. Additionally, it also prevents transmission through contact with other individuals.

[0017] Once an individual is infected with the coronavirus through their respiratory system, the use of a mask minimizes the spread of the virus via aerosols. Similarly, masks worn by uninfected individuals reduce the likelihood of infection through inhaling virus-containing aerosols.

[0018] However, handwashing products are not suitable for the mouth or nose, which are often where initial infections occur.

[0019] Therefore, there remains a clear need for reagents that are effective not only in handwashing but also in the mouth and nose.

[0020] More generally, viral infections are a cause of serious disease in humans and animals. Developing appropriate vaccines and effective treatments remains an important priority, especially when the underlying virus is highly infectious.

[0021] The following are some viral families that are clinically significant to humans and / or animals:

[0022] Coronavirus

[0023] SARS-CoV-1

[0024] SARS-CoV-2

[0025] ·MERS

[0026] • Bird coronaviruses—causing significant economic losses to broilers and laying hens.

[0027] • Porcine epidemic diarrhea virus.

[0028] Bovine coronavirus—causes a disease called winter dysentery, which can cause severe diarrhea and respiratory infections in calves.

[0029] Caliciviridae

[0030] Norovirus—causing 200,000 deaths annually, with no available vaccine or specific treatment. It accounts for approximately 18% of all gastroenteritis cases.

[0031] Feline calicivirus—found in 50% of cats with upper respiratory infections.

[0032] Orthomyxoviridae

[0033] • Types A, B, C, and D influenza—all of these can infect humans.

[0034] Examples of influenza A virus strains

[0035] H1N1 caused the "Spanish flu" of 1918 and the "swine flu" of 2009.

[0036] -H2N2 caused "Asian influenza".

[0037] -H3N2 caused the "Hong Kong-type influenza".

[0038] -H5N1, also known as "bird flu" or "avian influenza".

[0039] -H7N7 has an unusual potential to cause zoonotic diseases.

[0040] -H1N2 can infect pigs and humans.

[0041] • Infectious Salmon Anemia Virus—causes anemia and losses in salmon farms

[0042] Paramyxoviridae

[0043] ·measles

[0044] mumps

[0045] Hendra virus

[0046] Canine distemper virus—kills many puppies. There is a vaccine, but full protection against the virus doesn't occur until 16 weeks of age.

[0047] Newcastle disease virus—a major disease in poultry, for which there is a vaccine, but only eye drops or sprays are available.

[0048] Pulmonary Virology

[0049] Human metapneumovirus (HMV) – Common symptoms include runny nose, congestion, sore throat, cough, headache, and fever, which can be mistaken for a common cold. People over 72 years of age are at risk of developing pneumonia.

[0050] Flaviviridae

[0051] West Nile virus

[0052] Dengue virus – 390,000,000 infections annually

[0053] Tick-borne encephalitis – No specific treatment. 10,000 to 12,000 cases annually.

[0054] Zika – No treatment available; vaccine estimated for 2028.

[0055] Hepatitis C

[0056] Theillers virus—affects horses and is a common cause of hepatitis and liver failure in equines.

[0057] Bovine viral diarrhea virus

[0058] African swine fever

[0059] Japanese encephalitis virus—68,000 cases annually

[0060] Yellow fever

[0061] Clonorchiidae

[0062] Several equine encephalitis viruses

[0063] Animal parasitic diseases impose a significant burden on animal welfare, which is linked to the economic costs of the entire agricultural industry. There are generally three main types of parasites: protozoa, worms, and arthropods.

[0064] There are many protozoan parasites that are known to cause diseases in vertebrate and invertebrate species. These parasites are pathogens of diseases including, but not limited to, malaria, dysentery, Giardia, Cryptosporidium, Chaga's disease, and coccidiosis.

[0065] While some protozoa are specific to a single host species, others can infect a range of host species. Therefore, the unchecked use of antimicrobial drugs, particularly throughout the food supply chain, is facing increasing restrictions. This is to ensure these drugs are effective in emergency situations.

[0066] Therefore, non-antibiotic alternatives are needed to help prevent the development of disease, which is an important means of improving global health and well-being outcomes without undermining economic viability.

[0067] Microsporidian parasites are economically significant in aquatic environments, particularly in shrimp and shrimp aquaculture. Enterocytozoa hepatocellularis (EHP) is known to infect only the tubular epithelial cells of the shrimp's hepatopancreas. Infection occurs due to the protrusion or growth of the microsporidian's tiny tubules, allowing them to attach to the shrimp's hepatopancreas.

[0068] Some shrimp species that are particularly susceptible to EHP are Penaeus monodon, Penaeus vannamei, and Penaeus stylirostris.

[0069] Effective prevention or treatment of infections such as EHP will have considerable benefits in the aquaculture industry.

[0070] Microbial agents pose a threat to life on Earth not only by causing disease in a wide variety of living organisms. This is because methanogenic archaea contribute the most to greenhouse gas emissions from agriculture.

[0071] Methanogens are not associated with disease states or parasitic diseases in the animals in which they were isolated. However, the production of gases such as methane during fermentation has been identified as a factor involved in increased greenhouse gas emissions and global warming. Therefore, methanogens harm the planet through their environmental impacts, rather than through disease affecting individual animals.

[0072] Ruminants produce the majority of agricultural methane, but it is also crucial for global food and textile supplies. Therefore, strategies are needed to mitigate methane production while maintaining or improving yields.

[0073] Dietary strategies that modulate the animal microbiome to suppress the proliferation of methanogens while promoting microbial species communities that support animal health will achieve these goals.

[0074] Mastitis is a common ailment in dairy ruminants. Mastitis is a bacterial infection that causes inflammation of the udder in cows. This infection is caused by a broad spectrum of pathogens and is epidemiologically classified as infectious mastitis and environmental mastitis. Mastitis is the most prevalent and damaging disease in dairy cattle worldwide.

[0075] Over the past decade, milk electrical conductivity (EC) has been introduced as an indicator of mastitis. EC is determined by the concentrations of anions and cations. If a cow has mastitis, the concentrations of Na+ and Cl- in the milk increase, which leads to increased conductivity in milk from the infected teat. Most automated milking systems incorporate EC sensors for measuring EC during milking.

[0076] In the *Journal of Dairy Science* 75 A description of the practicality of conductivity measurement can be found in 606-614 (1992).

[0077] At 25°C, the electrical conductivity measurements in milk from healthy cows typically range from 4.0 to 5.0 millisiemens (mS), while the absolute conductivity values ​​from infected teats typically range from 5.0 to 9.0 mS. In the typical EC distribution of cows with mastitis, infected teats show higher EC levels for most of the milking process, peaking at the beginning and end of milking.

[0078] Mild, uncomplicated mastitis of a single teat is usually treated with intramammary antibiotics such as β-lactams (amoxicillin, penicillin, and cephalosporins). Systemic antibiotics are used when more than one teat is infected, when udder changes are significant, or when the cow is clearly ill. The combination of systemic and intramammary antibiotics is believed to improve bacteriological cure rates and is used in severe cases of mastitis.

[0079] The main components of the economic impact of mastitis involve reduced milk production, milk processing, the cost of medications used for treatment, and labor costs associated with animal treatment and slaughter. These factors together reduce farmers' income from milk. It is estimated that Australian dairy farmers lose more than $150 million annually due to poor breast health. Furthermore, the use of antibiotics is now discouraged due to the development of bacterial resistance and the potential presence of undesirable antibiotic residues in the meat and milk of animals consumed by humans. Summary of the Invention

[0080] In one aspect of this disclosure, a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane is provided for use in inhibiting or inactivating a virus or in preventing or treating a viral infection in a subject, the extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g, and wherein the use is by means of external application, pulmonary administration, inhalation, intravenous administration or oral administration.

[0081] In another aspect of this disclosure, a method is provided for inhibiting or inactivating a virus or preventing or treating a viral infection in a subject, the method comprising administering to the subject an effective amount of a composition via topical, pulmonary, inhalation, intravenous, or oral routes, the composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0082] In another aspect of this disclosure, a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane is provided for use in the prevention or treatment of aquatic animals by inhibiting or inactivating microsporidian parasites. The extract comprises polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g, and wherein the use is by including the extract in an aquatic environment.

[0083] In another aspect of this disclosure, a method for inhibiting or inactivating microsporidian parasites in aquatic animals is provided, the method comprising administering an effective amount of a composition to the animal via an aquatic environment, the composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0084] In another aspect of this disclosure, a composition is provided comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the composition being used for the prevention or treatment of protozoan infections in animals by inhibiting or inactivating protozoa, the extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g.

[0085] In another aspect of this disclosure, a method for inhibiting or killing protozoa within an active object is provided, the method comprising administering to the animal an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0086] In another aspect of this disclosure, a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane is provided for use in preventing or treating archaeal infections in animals by inhibiting or inactivating archaea, the extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g.

[0087] In another aspect of this disclosure, a method for inhibiting or killing archaea in living organisms is provided, the method comprising administering to an animal an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0088] In another aspect of this disclosure, a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane is provided for use in treating ecological imbalances in animals by inhibiting or inactivating methanogens, the extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g.

[0089] In another aspect of this disclosure, a method for treating ecological imbalances in an animal is provided, the method comprising administering to the animal an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0090] In another aspect of this disclosure, a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane is provided for use in preventing or treating fungal infections in animals by inhibiting or inactivating fungi, the extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g.

[0091] In another aspect of this disclosure, a method for inhibiting or killing fungi in living objects is provided, the method comprising administering to an animal an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0092] In another aspect of this disclosure, a method is provided for inhibiting or inactivating the life cycle of protozoan parasites in or in the bodies of vertebrates and invertebrates, the method comprising administering to the animal an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0093] In another aspect of this disclosure, a composition is provided comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the composition being used for inhibiting or inactivating the life cycle of protozoan parasites in or in the environment in which vertebrates and invertebrates reside, the extract comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0094] In another aspect of this disclosure, a method for regulating the microbiome of an animal is provided, the method comprising administering to the animal an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0095] In another aspect of this disclosure, a composition is provided comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the composition being used for regulating the microbiome of an animal, the extract comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0096] In another aspect of this disclosure, a method for mitigating greenhouse gas emissions from animals by altering the composition of an animal-associated microbial community is provided, the method comprising administering to the animal an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of a sugarcane-derived extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or from about 100 CE mg / g to about 500 CE mg / g.

[0097] In another aspect of this disclosure, a composition is provided comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the composition being used for mitigating greenhouse gas emissions from animals by altering the composition of an animal-associated microbial community, the extract comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0098] In another aspect of this disclosure, a method for preventing or treating mastitis in ruminants is provided, the method comprising administering to the animal an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0099] In another aspect of this disclosure, a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane is provided for use in the prevention or treatment of mastitis in ruminants. The extract comprises from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0100] In another aspect of this disclosure, a method is provided to reduce methane emissions in ruminants by altering the composition of the microbial community associated with ruminants and treating or preventing mastitis in ruminants. The method comprises administering to the ruminant an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of a sugarcane-derived extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0101] In another aspect of this disclosure, a composition is provided comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the composition being used for mitigating greenhouse gas emissions from animals by altering the composition of the animal-associated microbial community and treating or preventing mastitis in ruminants, the extract comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0102] In another aspect of this disclosure, a method for increasing milk production in ruminants is provided, the method comprising administering to the ruminants an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g.

[0103] In another aspect of this disclosure, a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane is provided for use in increasing milk production in ruminants. The extract comprises from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0104] In another aspect of this disclosure, a method for mitigating methane emissions from ruminants by altering the composition of the microbial community associated with ruminants while simultaneously improving ruminant growth is provided. The method comprises administering to the ruminant an effective amount of a composition comprising from about 0.00001% by weight to about 100% by weight of a sugarcane-derived extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols.

[0105] In another aspect of this disclosure, a composition is provided comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the composition being used for mitigating methane emissions from ruminants by altering the composition of the microbial community associated with ruminants while simultaneously improving ruminant growth, the extract comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g.

[0106] In another aspect of this disclosure, a composition is provided comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the composition being used alone or in combination with a second adjuvant as an adjuvant, the extract comprising polyphenols from about 10 catechin equivalents (CE) g / L to about 50 CE g / L or polyphenols from about 100 CE mg / g to about 500 CE mg / g.

[0107] In another aspect of this disclosure, a method for improving the immune response to a vaccine is provided, the method comprising including in a vaccine composition a composition comprising from about 0.00001% by weight to about 100% by weight of an extract derived from sugarcane, the extract comprising from about 10 catechin equivalents (CE) g / L to about 50 CE g / L of polyphenols or from about 100 CE mg / g to about 500 CE mg / g of polyphenols. Attached Figure Description

[0108] Figure 1 This is a bar chart showing the results of Example 3 as described in this article.

[0109] Figure 2 This is a bar chart showing the results of Example 5 as described in this article.

[0110] Figure 3 This is a bar graph showing the results of Example 6 as described in this article. (Number of Eimeria sporozoites after incubation at 37°C for 14 hours. For all results, there was no statistically significant difference between salinomycin and the extract. For all results, the difference between the control and the salinomycin / extract was highly statistically significant (P < 0.001). For all results, the difference between the control and the extract was highly statistically significant (P < 0.001).)

[0111] Figure 4 This is a bar graph showing the results of Example 7 as described in this article. (Differences indicated by superscripts (a, b, c) are statistically significant (P < 0.05).)

[0112] Figure 5This is a bar chart showing the results of Example 8 as described in this article.

[0113] Figure 6 This is a bar chart showing the results of Example 14 as described herein.

[0114] Figure 7 This is a bar chart showing the results of Example 13 as described herein.

[0115] Figure 8 This is a graph showing the results achieved for the 0.5% composition in Example 19.

[0116] Figure 9 This is a graph showing the results achieved for the 0.5% composition in Example 19.

[0117] Figure 10 This is a bar chart showing the results of Example 20 as described herein. Detailed Implementation

[0118] Any embodiment described herein should be considered as requiring necessary modifications to be applied to any other embodiment, unless otherwise expressly stated.

[0119] This disclosure is not limited to the specific embodiments described herein, which are intended for illustrative purposes only. As described herein, functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0120] Throughout this specification, unless otherwise expressly stated or required by the context, references to a single step, substance composition, group of steps, or group of substance compositions shall be understood to cover one or more of such steps, substance compositions, groups of steps, or groups of substance compositions.

[0121] Unless otherwise expressly defined, all technical and scientific terms used herein should be considered to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., chemistry, biochemistry, cell culture, molecular biology, and pharmaceutical). Furthermore, unless the context otherwise requires, singular terms should include plural meanings and plural terms should include singular meanings. Therefore, as used in this specification and the appended claims, the singular forms “an,” “a,” and “described” include plural indicators unless the context clearly indicates otherwise. Thus, the term “subject” means “one or more subjects” unless the context clearly indicates otherwise.

[0122] As used herein, “application” should be interpreted broadly and includes administering to a subject an extract as described herein or a composition comprising the extract, as well as providing to cells an extract as described herein or a composition comprising the extract.

[0123] As used herein, the phrase “effective amount” refers to a quantity sufficient to elicit a biological or medical response in an tissue, system, animal, or human that is sought by researchers, veterinarians, physicians, or other clinicians. Undesirable effects (e.g., side effects) sometimes accompany desired effects; therefore, practitioners must weigh potential benefits against potential risks when determining the appropriate “effective amount.” The exact amount required varies from subject to subject, depending on the subject’s species, age and general condition, administration method, etc. Therefore, it may be impossible to specify an exact “effective amount.” However, the appropriate “effective amount” in any individual case can be determined by a person skilled in the art using routine laboratory methods. “Relief” includes reducing adverse symptoms, inducing a state of comfort or health, or removing or reducing biochemical, physiological, or clinical markers of disease or symptom.

[0124] As used herein, the terms “treating,” “treat,” “treatment,” “improving,” “improve,” or “improvement” include the application of an effective amount of the extract of this disclosure or a composition comprising the extract, which is sufficient to reduce or delay the onset or progression of a particular condition, or to reduce or eliminate at least one symptom of the condition. As will be understood by those skilled in the art of treating microbial infections, the term “treating” includes the cure of an infection; however, it does not necessarily mean that the infection is completely cured.

[0125] As used herein, the term "preventing" or "prevention" includes the application of an effective amount of the extract of this disclosure or a composition comprising the extract, which is sufficient to prevent the onset of a particular disease or to prevent at least one symptom of the disease. As will be understood by those skilled in the art of disease prevention, the term "prevention" includes complete prevention of the disease; however, it does not necessarily mean that the disease is completely prevented.

[0126] As used herein, "subject" refers to an animal, such as a mammal (including a human), that may benefit from the sugarcane-derived extract, the composition containing the extract, and the methods and uses described herein. There is no limitation on the types of animals that may benefit from the sugarcane-derived extract, the composition containing the extract, and the methods and uses described herein. Whether human or non-human animal, a subject may be referred to as an individual, subject, animal, host or recipient, or patient. This disclosure applies to human medicine, human cosmetics, and veterinary medicine.

[0127] The reference to "composition for use" should be understood to encompass the methods of this disclosure.

[0128] As used in this article, the term "about" refers to a range of + / - 5% of a specified value.

[0129] As used herein, the term “CE” or “catechin equivalent” is a measure of total polyphenol content, expressed as mg catechin equivalent / g raw material or g catechin equivalent / L raw material.

[0130] As used herein, the term "sugarcane-derived products" refers to the products of sugarcane milling and refining processes, including but not limited to sugar, molasses, sugar paste, bagasse, virgin juice, pulp, clarified sugarcane juice, clarified syrup, molasses waste, golden syrup, impurities, sugarcane peel, leaves, growth cones, pulp, and sugar residue, as well as combinations thereof. Sugar residue is the residue produced when products such as sugar or molasses are fermented to produce, for example, ethanol. Sugarcane sugar residue is also known as bio-sugar residue, flask distillate, or distiller's grains. As used herein, the terms "sugar residue," "bio-sugar residue," "flask distillate," and "distiller's grains" are equivalent and used interchangeably.

[0131] Throughout this specification, various aspects and components of this disclosure may be presented in a range format. The inclusion of the range format is for convenience and should not be construed as an inflexible limitation on the scope of this disclosure. Therefore, a description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range, unless specifically indicated. For example, a description of a range such as 1 to 5 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 5, 3 to 5, etc., and individual digits and decimal numbers within said range, such as 1, 2, 3, 4, 5, 5.5, and 6, unless the context requires or implies integers. This applies regardless of the width of the disclosed range. Where specific values ​​are required, they will be indicated in the specification.

[0132] Throughout this specification, the word "comprise" or its variations such as "comprises" or "comprising" should be understood to imply inclusion of the stated elements, integers, or steps, or groups of elements, integers, or steps, but does not exclude any other elements, integers, or steps, or groups of elements, integers, or steps. In one embodiment, the sugarcane-derived extract of this disclosure comprises at least about 10 CE g / L of polyphenols or at least about 150 mg CE / g of polyphenols. As mentioned above, the term "CE" or "catechin equivalent" is a measure of total polyphenol content, expressed as catechin equivalent mg / g of sugarcane-derived extract or catechin equivalent g / L of sugarcane-derived extract.

[0133] In one embodiment, the sugarcane-derived extract of this disclosure comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 CE g / L of polyphenols.

[0134] In one embodiment, the sugarcane-derived extract of this disclosure comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 250, 275, 300, 325, 350, 375, 400, 425, 450, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775 or 800 mg CE / g of polyphenols.

[0135] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 1 CE g / L to about 50 CE g / L or from about 10 CE mg / g to about 500 CE mg / g.

[0136] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 1 CE g / L to about 25 CE g / L or from about 10 CE mg / g to about 250 CE mg / g.

[0137] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 1 CE g / L to about 10 CE g / L or from about 10 CE mg / g to about 100 CE mg / g.

[0138] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 1 CE g / L to about 5 CE g / L or from about 10 CE mg / g to about 100 CE mg / g.

[0139] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 5 CE g / L to about 50 CE g / L or from about 50 CE mg / g to about 500 CE mg / g.

[0140] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 5 CE g / L to about 25 CE g / L or from about 50 CE mg / g to about 250 CE mg / g.

[0141] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 5 CE g / L to about 10 CE g / L or from about 50 CE mg / g to about 100 CE mg / g.

[0142] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 100 CE g / L or from about 100 CE mg / g to about 1000 CE mg / g.

[0143] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 90 CE g / L or from about 100 CE mg / g to about 900 CE mg / g.

[0144] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 80 CE g / L or from about 100 CE mg / g to about 800 CE mg / g.

[0145] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 70 CE g / L or from about 100 CE mg / g to about 700 CE mg / g.

[0146] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 60 CE g / L or from about 100 CE mg / g to about 600 CE mg / g.

[0147] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 50 CE g / L or from about 100 CE mg / g to about 500 CE mg / g.

[0148] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 25 CE g / L or from about 100 CE mg / g to about 250 CE mg / g.

[0149] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 15 CE g / L to about 50 CE g / L or from about 150 CE mg / g to about 500 CE mg / g.

[0150] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 15 CE g / L to about 25 CE g / L or from about 150 CE mg / g to about 250 CE mg / g.

[0151] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 70 CE g / L or from about 100 CE mg / g to about 700 CE mg / g.

[0152] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 60 CE g / L or from about 100 CE mg / g to about 600 CE mg / g.

[0153] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 10 CE g / L to about 50 CE g / L or from about 100 CE mg / g to about 500 CE mg / g.

[0154] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 15 CE g / L to about 40 CE g / L or from about 150 CE mg / g to about 400 CE mg / g.

[0155] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 20 CE g / L to about 30 CE g / L or from about 200 CE mg / g to about 300 CE mg / g.

[0156] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 20 g / L to about 27 g CE / L or from about 200 mg / g to about 270 mg / g.

[0157] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 27 CE g / L to about 35 g CE / L or from about 270 CE mg / g to about 350 CE mg / g.

[0158] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 35 CE g / L to about 40 g CE / L or from about 350 CE mg / g to about 400 CE mg / g.

[0159] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 40 g / L to about 50 g / L or from about 400 mg / g to about 500 mg / g.

[0160] In one embodiment, the sugarcane-derived extract of this disclosure comprises polyphenols ranging from about 45 CE g / L to about 50 g CE / L or from about 450 CE mg / g to about 500 CE mg / g.

[0161] The sugarcane-derived extracts of this disclosure may contain flavonoid polyphenols. The sugarcane-derived extracts may contain any amount of flavonoids. In one embodiment, the sugarcane-derived extracts of this disclosure comprise at least about 1 CE g / L of flavonoids or at least about 10 CE mg / g of flavonoids.

[0162] In one embodiment, the sugarcane-derived extract of this disclosure comprises flavonoids ranging from about 1 CE g / L to about 15 CE g / L or from about 10 CE mg / g to about 150 CE mg / g. In one embodiment, the sugarcane-derived extract of this disclosure comprises flavonoids ranging from about 3 CE g / L to about 10 CE g / L or from about 30 CE mg / g to about 100 CE mg / g. In one embodiment, the sugarcane-derived extract of this disclosure comprises flavonoids ranging from about 5 CE g / L to about 8 CE g / L or from about 50 CE mg / g to about 80 CE mg / g. In one embodiment, the sugarcane-derived extract of this disclosure comprises flavonoids ranging from about 6 CE g / L to about 8 CE g / L or from about 60 CE mg / g to about 80 CE mg / g. In one embodiment, the sugarcane-derived extract of this disclosure comprises flavonoids ranging from about 6.5 CE g / L to about 7.5 CE g / L or from about 65 CE mg / g to about 75 CE mg / g.

[0163] The sugarcane-derived extracts of this disclosure may contain proanthocyanidin polyphenols. The sugarcane-derived extracts may contain any amount of proanthocyanidins. In one embodiment, the sugarcane-derived extract of this disclosure comprises at least about 1.5 CE g / L of proanthocyanidins or at least about 15 CE mg / g of proanthocyanidins. In one embodiment, the sugarcane-derived extract of this disclosure comprises at least about 1.8 CE g / L of proanthocyanidins or at least about 18 CE mg / g of proanthocyanidins. In one embodiment, the sugarcane-derived extract of this disclosure comprises proanthocyanidins from about 1.5 CE g / L to about 2.5 CE g / L or from about 15 CE mg / g to about 25 CE mg / g of proanthocyanidins. In one embodiment, the sugarcane-derived extract of this disclosure comprises proanthocyanidins from about 1.8 CE g / L to about 2.2 CE g / L or from about 18 CE mg / g to about 22 CE mg / g of proanthocyanidins.

[0164] The polyphenols derived from sugarcane extracts disclosed herein include, but are not limited to, one or more of the following: syringic acid, chlorogenic acid, caffeic acid, vanillin, sinapic acid, p-coumaric acid, ferulic acid, gallic acid, vanillic acid, geraniol, geraniin, apigenin, vitexin, sennain, sennain, sennain, zeaxanthin, (+)-catechin, gallic acid (-)-catechin ester, (-)epicatechin, quercetin, kaempferol, myricetin, rutin, sennatin, isosennatin, luteolin, cytisine, and / or their derivatives. The polyphenols derived from sugarcane extracts disclosed herein may also include, but are not limited to, one or more of the following: hydroxycinnamic acid, isennain, sennain, neosennain, isovitexin, vitexin, and / or their derivatives.

[0165] Polyphenols derived from sugarcane extracts also include conjugates such as glycosides, glucosides, galactosides, galacturonic acid glycosides, ethers, esters, arabinosides, sulfates, phosphates, pentoaloses (xylose, arabinose) and hexanooses.

[0166] In one embodiment, the sugarcane-derived extracts of this disclosure include syringic acid, chlorogenic acid, caffeic acid, vanillin, sinapic acid, geraniol, geraniin, apigenin, vitexin, scutellarin, scutellarin, scutellarin, and zeaxanthin and / or their derivatives.

[0167] In one embodiment, the sugarcane-derived extracts of this disclosure include syringic acid, chlorogenic acid, and geraniol and / or their derivatives.

[0168] In one embodiment, the sugarcane-derived extract of this disclosure includes syringic acid. In one embodiment, the sugarcane-derived extract of this disclosure includes chlorogenic acid. In one embodiment, the sugarcane-derived extract of this disclosure includes geraniol. In one embodiment, the sugarcane-derived extract of this disclosure includes caffeic acid. In one embodiment, the sugarcane-derived extract of this disclosure includes vanillin. In one embodiment, the sugarcane-derived extract of this disclosure includes sinapic acid. In one embodiment, the sugarcane-derived extract of this disclosure includes vitexin. In one embodiment, the sugarcane-derived extract of this disclosure includes p-coumaric acid. In one embodiment, the sugarcane-derived extract of this disclosure includes ferulic acid. In one embodiment, the sugarcane-derived extract of this disclosure includes gallic acid. In one embodiment, the sugarcane-derived extract of this disclosure includes vanillic acid. In one embodiment, the sugarcane-derived extract of this disclosure includes geraniol. In one embodiment, the sugarcane-derived extract of this disclosure includes apigenin. In one embodiment, the sugarcane-derived extract of this disclosure includes sennatin. In one embodiment, the sugarcane-derived extract of this disclosure includes sennatin. In one embodiment, the sugarcane-derived extract of this disclosure includes quercetin. In one embodiment, the sugarcane-derived extract of this disclosure includes zeaxanthin. In one embodiment, the sugarcane-derived extract of this disclosure includes (+)-catechin. In one embodiment, the sugarcane-derived extract of this disclosure includes gallic acid (-)-catechin ester. In one embodiment, the sugarcane-derived extract of this disclosure includes (-)-epicatechin. In one embodiment, the sugarcane-derived extract of this disclosure includes quercetin. In one embodiment, the sugarcane-derived extract of this disclosure includes kaempferol. In one embodiment, the sugarcane-derived extract of this disclosure includes myricetin. In one embodiment, the sugarcane-derived extract of this disclosure includes rutin. In one embodiment, the sugarcane-derived extract of this disclosure includes schafodin. In one embodiment, the sugarcane-derived extract of this disclosure includes isoschafodin. In one embodiment, the sugarcane-derived extract of this disclosure includes luteolin. In one embodiment, the sugarcane-derived extract of this disclosure includes cytisine. In one embodiment, the sugarcane-derived extract of this disclosure includes hydroxycinnamic acid. In one embodiment, the sugarcane-derived extract of this disclosure includes isohexanthin. In one embodiment, the sugarcane-derived extract of this disclosure includes sennatin. In one embodiment, the sugarcane-derived extract of this disclosure includes neostigmine. In one embodiment, the sugarcane-derived extract of this disclosure includes isovitexin. In one embodiment, the sugarcane-derived extract of this disclosure includes vitexin.

[0169] In one embodiment, syringic acid, chlorogenic acid, and geraniol are the three most abundant polyphenols in the sugarcane-derived extracts of this disclosure.

[0170] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 5 to 20 μg / g dry weight of syringic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 7 to 15 μg / g dry weight of syringic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 10 to 12 μg / g dry weight of syringic acid. In one embodiment, when present, the sugarcane-derived extract of this disclosure comprises about 10.9 μg / g dry weight of syringic acid. The sugarcane-derived extract may be in syrup form.

[0171] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 50 to 200 μg / g dry weight of syringic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 90 to 130 μg / g dry weight of syringic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 100 to 120 μg / g dry weight of syringic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 107 μg / g dry weight of syringic acid. The sugarcane-derived extract may be in powder form.

[0172] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 1 to 15 μg / g dry weight of chlorogenic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 3 to 10 μg / g dry weight of chlorogenic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 5 to 8 μg / g dry weight of chlorogenic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 6.53 μg / g dry weight of chlorogenic acid. The sugarcane-derived extract may be in syrup form.

[0173] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 30 to 150 μg / g dry weight of chlorogenic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 60 to 90 μg / g dry weight of chlorogenic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 70 to 80 μg / g dry weight of chlorogenic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 74 μg / g dry weight of chlorogenic acid. The sugarcane-derived extract may be in powder form.

[0174] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 10 to 30 μg / g dry weight of geraniol. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 15 to 25 μg / g dry weight of geraniol. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 18 to 21 μg / g dry weight of geraniol. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 19 to 45 μg / g dry weight of geraniol. The sugarcane-derived extract may be in syrup form.

[0175] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 100 to 300 μg / g dry weight of geraniol. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 190 to 260 μg / g dry weight of geraniol. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 210 to 240 μg / g dry weight of geraniol. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 227 μg / g dry weight of geraniol. The sugarcane-derived extract may be in powder form.

[0176] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 7 to 15 μg / g dry weight of syringic acid, and / or about 4 to 9 μg / g dry weight of chlorogenic acid, and / or about 0.1 to 0.5 μg / g dry weight of caffeic acid, about 0.05 to 0.3 μg / g dry weight of vanillin, and / or about 0.1 to 0.3 μg / g dry weight of sinapic acid, and / or about 15 to 25 μg / g dry weight of geraniol, and / or about 0.1 to 0.4 μg / g dry weight of sennatin, and / or about 0.4 to 0.9 μg / g dry weight of sennatin, and / or about 0.05 to 0.3 μg / g dry weight of disomentin. The sugarcane-derived extract may be in syrup form.

[0177] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 10 to 12 μg / g dry weight of syringic acid, and / or about 5 to 8 μg / g dry weight of chlorogenic acid, and / or about 0.2 to 0.4 μg / g dry weight of caffeic acid, and / or about 0.1 to 0.2 μg / g dry weight of vanillin, and / or about 0.1 to 0.25 μg / g dry weight of sinapic acid, and / or about 18 to 21 μg / g dry weight of geraniol, and / or about 0.2 to 0.3 μg / g dry weight of sennatin, and / or about 0.5 to 0.8 μg / g dry weight of sennatin, and / or about 0.1 to 0.2 μg / g dry weight of disomentin. The sugarcane-derived extract may be in syrup form.

[0178] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 10.9 μg / g dry weight of syringic acid, and / or about 6.53 μg / g dry weight of chlorogenic acid, and / or about 0.29 μg / g dry weight of caffeic acid, and / or about 0.153 μg / g dry weight of vanillin, and / or about 0.18 μg / g dry weight of sinapic acid, and / or about 19.45 μg / g dry weight of geraniol, and / or about 0.245 μg / g dry weight of sennatin, and / or about 0.69 μg / g dry weight of sennatin, and / or about 0.15 μg / g dry weight of disomentin. The sugarcane-derived extract may be in syrup form.

[0179] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 90 to 130 μg / g dry weight of syringic acid, and / or about 60 to 90 μg / g dry weight of chlorogenic acid, and / or about 4 to 10 μg / g dry weight of caffeic acid, and / or about 1 to 4 μg / g dry weight of vanillin, about 1 to 3 μg / g dry weight of sinapic acid, and / or about 190 to 260 μg / g dry weight of geraniol, and / or about 3 to 7 μg / g dry weight of scutellarin, and / or 3 to 8 μg / g dry weight of scutellarin, and / or about 0.05 to 0.3 μg / g dry weight of disomentin. The sugarcane-derived extract may be in powder form.

[0180] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 100 to 120 μg / g dry weight of syringic acid, and / or about 70 to 80 μg / g dry weight of chlorogenic acid, and / or about 6 to 8 μg / g dry weight of caffeic acid, about 2 to 3 μg / g dry weight of vanillin, and / or about 1.5 to 2.5 μg / g dry weight of sinapic acid, and / or about 210 to 240 μg / g dry weight of geraniol, about 4 to 5 μg / g dry weight of sennatin, 4 to 6 μg / g dry weight of sennatin, and / or about 0.1 to 0.2 μg / g dry weight of disomentin. The sugarcane-derived extract may be in powder form.

[0181] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 107 μg / g dry weight of syringic acid, and / or about 74 μg / g dry weight of chlorogenic acid, and / or about 7.5 μg / g dry weight of caffeic acid, and / or about 2 μg / g dry weight of vanillin, and / or about 1.7 μg / g dry weight of sinapic acid, and / or about 227 μg / g dry weight of geraniol, and / or about 4.5 μg / g dry weight of scutellarin, 5.2 μg / g dry weight of scutellarin, and / or about 0.16 μg / g dry weight of disomentin. The sugarcane-derived extract may be in powder form.

[0182] The sugarcane-derived extracts disclosed herein may contain a range of organic acids naturally found in sugarcane. These organic acids may include, but are not limited to, aconitic acid (cis and trans), oxalic acid, citric acid, lactic acid, tartaric acid, glycolic acid, succinic acid, citric acid, malic acid, fumaric acid, and shikimic acid. In one embodiment, the sugarcane-derived extract contains higher levels of citric acid and malic acid than other organic acids. In another embodiment, the sugarcane-derived extract contains trace amounts of oxalic acid, citric acid, tartaric acid, glycolic acid, succinic acid, and citric acid. In yet another embodiment, the two most abundant organic acids in the sugarcane-derived extract are trans-aconitic acid and cis-aconitic acid.

[0183] The sugarcane-derived extract of this disclosure may contain trans-aconitic acid and / or cis-aconitic acid. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 10,000 to 40,000 mg / kg of trans-aconitic acid and / or about 3,000 to 7,000 mg / kg of cis-aconitic acid. In one embodiment, the sugarcane-derived extract of this disclosure contains about 17,000 to 30,000 mg / kg of trans-aconitic acid and / or about 4,000 to 6,500 mg / kg of cis-aconitic acid. In one embodiment, the sugarcane-derived extract of this disclosure may contain about 20,000 to 25,000 mg / kg of trans-aconitic acid and / or about 5,000 to 5,500 mg / kg of cis-aconitic acid.

[0184] The sugarcane-derived extracts of this disclosure may contain amino acids. In one embodiment, the total amino acid level of the sugarcane-derived extract of this disclosure is about 50,000 to 80,000 μg / g, or about 60,000 to 70,000 μg / g, or about 65,000 μg / g. In one embodiment, about 10 to 40% of these total amino acids are essential amino acids. In one embodiment, about 15 to 30% of these total amino acids are essential amino acids. In one embodiment, about 20 to 25% of these total amino acids are essential amino acids.

[0185] The sugarcane-derived extracts of this disclosure may contain free amino acids. In one embodiment, the sugarcane-derived extract of this disclosure comprises about 10,000 to 50,000 μg of free amino acids per gram. In one embodiment, the sugarcane-derived extract of this disclosure may contain about 20,000 to 35,000 μg of free amino acids per gram. The sugarcane-derived extract of this disclosure may contain about 25,000 to 30,000 μg of free amino acids per gram.

[0186] As defined above and used in this article, the term "free amino acid" refers to an amino acid that is a single molecule and is not structurally attached to peptide bonds that are attached to other amino acids.

[0187] The sugarcane-derived extracts disclosed herein may contain leucine, a branched-chain essential amino acid. In one embodiment, the concentration of leucine in the sugarcane-derived extract is about 1 to 5 mM, or about 1.5 to 4 mM, or about 2 to 3 mM. In one embodiment, the amount of leucine in the sugarcane-derived extract is about 1,000 to 20,000 μg / g, or about 1,000 to 10,000 μg / g, or about 1,000 to 5,000 μg / g, or about 1,000 to 2,000 μg / g, or about 5,000 to 10,000 μg / g, or about 10,000 to 20,000 μg / g.

[0188] The sugarcane-derived extracts disclosed herein may contain minerals. In one embodiment, the sugarcane-derived extract contains minerals naturally found in sugarcane. In another embodiment, the sugarcane-derived extract contains one or more minerals, including but not limited to potassium, sodium, calcium, magnesium, iron, zinc, selenium, and chromium.

[0189] In one embodiment, the sugarcane-derived extract contains minerals bound to polyphenols. In one embodiment, the sugarcane-derived extract contains divalent ions bound to polyphenols. In one embodiment, the sugarcane-derived extract contains calcium, magnesium, and / or iron bound to polyphenols. In one embodiment, the sugarcane-derived extract contains iron bound to polyphenols.

[0190] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 20,000 to 32,000 mg of potassium per kilogram, and / or about 300 to 600 mg of sodium per kilogram, and / or about 800 to 1,300 mg of calcium per kilogram, and / or about 3,000 to 6,000 mg of magnesium per kilogram, and / or about 40 to 90 mg of iron per kilogram, and / or about 3 to 10 mg of zinc per kilogram, and / or about 500 to 900 μg of selenium per kilogram, and / or about 1,000 to 1,600 μg of chromium per kilogram. The sugarcane-derived extract may be in syrup form.

[0191] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 25,000 to 27,000 mg of potassium per kilogram, and / or about 400 to 500 mg of sodium per kilogram, and / or about 1,000 to 1,200 mg of calcium per kilogram, and / or about 4,000 to 5,500 mg of magnesium per kilogram, and / or about 55 to 75 mg of iron per kilogram, and / or about 5.5 to 7.5 mg of zinc per kilogram, and / or about 700 to 850 μg of selenium per kilogram, and / or about 1,200 to 1,400 μg of chromium per kilogram. The sugarcane-derived extract may be in syrup form.

[0192] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 26,000 mg of potassium / kg, and / or about 450 mg of sodium / kg, and / or about 1,090 mg of calcium / kg, and / or about 4,700 mg of magnesium / kg, and / or about 65 mg of iron / kg, about 6.6 mg of zinc / kg, and / or about 786 μg of selenium / kg, and / or about 1,300 μg of chromium / kg. The sugarcane-derived extract may be in syrup form.

[0193] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 50 to 350 mg of potassium per kilogram, and / or about 5 to 70 mg of sodium per kilogram, and / or about 7,000 to 10,000 mg of calcium per kilogram, and / or about 1,000 to 3,000 mg of magnesium per kilogram, and / or about 500 to 1,300 mg of iron per kilogram. The sugarcane-derived extract may be in powder form.

[0194] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 100 to 250 mg of potassium per kilogram, and / or about 10 to 50 mg of sodium per kilogram, and / or about 8,000 to 9,000 mg of calcium per kilogram, and / or about 1,500 to 2,500 mg of magnesium per kilogram, and / or about 800 to 1,000 mg of iron per kilogram. The sugarcane-derived extract may be in powder form.

[0195] In one embodiment, the sugarcane-derived extract of this disclosure comprises about 190 mg of potassium per kilogram, and / or about 30 mg of sodium per kilogram, and / or about 8,800 mg of calcium per kilogram, and / or about 2,000 mg of magnesium per kilogram, and / or about 890 mg of iron per kilogram. The sugarcane-derived extract may be in powder form.

[0196] The sugarcane-derived extracts disclosed herein may contain monosaccharides, disaccharides, oligosaccharides, and / or polysaccharides. Examples of such sugars include, but are not limited to, sucrose, glucose, galactose, xylose, ribose, mannose, rhamnose, fructose, maltose, lactose, maltotriose, xylpyranose, raffinose, 1-sucrose trisaccharide, theanderose, 6-sucrose trisaccharide, panose, neosucrose trisaccharide, sucrose tetrasaccharide, glucan, and xylan.

[0197] In one embodiment, the composition and method comprise from about 0.00001% by weight to about 10% by weight of an extract.

[0198] In one embodiment, the composition and method comprise from about 0.00001% by weight to about 50% by weight of an extract.

[0199] In one embodiment, the composition and method comprise from about 0.00001% by weight to about 50% by weight of an extract.

[0200] In one embodiment, the composition and method comprise from about 5% by weight to about 10% by weight of an extract.

[0201] In one embodiment, the composition and method comprise from about 0.05% by weight to about 10% by weight of an extract.

[0202] In one embodiment, the composition and method comprise from about 0.05% by weight to about 5% by weight of an extract.

[0203] In one embodiment, the virus is a coronavirus.

[0204] In one embodiment, the virus is an influenza virus, including H1N1, swine flu, and avian flu.

[0205] In one embodiment, the virus is SARS-CoV-2.

[0206] In one embodiment, the virus is a flavivirus, including dengue fever, Zika, West Nile, and yellow fever.

[0207] In one embodiment, the virus is Hendra.

[0208] In one embodiment, the disease is caused by protozoa, such as malaria, dysentery, Giardia, Cryptosporidium, Chagas disease, and coccidiosis.

[0209] In one embodiment, agricultural greenhouse gas emissions are caused by ancient methanogens.

[0210] In one embodiment, the disease is caused by microsporidia such as Enterocytozoa hepatisimidae.

[0211] In one embodiment, the composition for use in inhibiting and / or treating viral infections is a topical wash, preferably a hand sanitizer.

[0212] In one embodiment, the composition for use in inhibiting and / or treating viral infections is an oral preparation, preferably a mouthwash, gargle, or drinking liquid, or a solid, such as a tablet, capsule, lozenge, or candy. Capsules are particularly preferred.

[0213] In one embodiment, the composition for oral administration may be contained in a liquid, solid, or semi-solid food.

[0214] In one embodiment, the composition may be included in non-human animal feed.

[0215] In one embodiment, the composition for use in inhibiting and / or treating viral infections is an aerosol for pulmonary application.

[0216] In one embodiment, the composition used for inhibiting and / or treating viral infections is an intranasal spray or is formulated for nasal administration.

[0217] In one embodiment, the composition for use in inhibiting and / or treating viral infections is formulated for application via a sprayer.

[0218] In one embodiment, the composition for use in inhibiting and / or treating viral infections is formulated for intravenous administration.

[0219] In one embodiment, the composition for use in inhibiting and / or treating viral infections is formulated for pulmonary administration.

[0220] In one embodiment, the composition for use in inhibiting and / or treating viral infections is formulated for sublingual or oral administration.

[0221] In one embodiment, the composition for use in inhibiting and / or treating viral infections is formulated for transdermal delivery.

[0222] The preparation of sugarcane extracts disclosed herein has been extensively described in WO 2019 / 028506. In particular, the preparation of these extracts is described on pages 17 through 40, line 22 of WO 2019 / 028506 (incorporated herein by cross-reference).

[0223] Similarly, compositions of sugarcane extracts are described on page 50, line 16, through page 51, line 24 of WO 2019 / 028506 (incorporated herein by cross-reference). Furthermore, the characteristics of sugarcane extracts are described on pages 64 through 88, line 9 of WO 2019 / 028506.

[0224] More generally, the contents of WO 2019 / 028506 are incorporated herein by cross-reference.

[0225] Detailed analysis of the sugarcane extract samples disclosed herein revealed the following components:

[0226] chlorogenic acid 2423 ng / mL

[0227] Vanillic acid 16088 ng / mL

[0228] Caffeic acid 482 ng / mL

[0229] trans-caffeic acid 273 ng / mL

[0230] p-Coumaric acid 14445 ng / mL

[0231] In addition, luteolin, trans-resveratrol, geraniol, quercetin, and rutin were found to coexist with flavonoids, including apigenin-6-C-arabinosyl-8-C glucoside, apigenin-6-C-glucosyl-8-C-arabinosyl-6,8-C-diglucoside, apigenin-6"-O-glucosyl-8-C-glucoside, schafotaside-2"-O-glucoside, and apigenin-6-C -Rhamnose-8-C-glucoside, apigenin-6,8-C-diarabinoside, methoxyluteolin-8-C-glucoside, methoxyluteolin-6"-O-rutinose-8-C-glucoside, methoxyluteolin-6"-O-glucoside-8-C-glucoside, tyrosin 7-O-neoheisteroside, tyrosin 7-O-rhamnose-glucoside and tyrosin 7-O-glycoside.

[0232] The extracts can be formulated into compositions suitable for human use by any method well known in the art of formulation. Exemplary techniques for formulating the compositions of this disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, Vol. 1000. 22 Found in version 2012.

[0233] In one embodiment, the extract of this disclosure can be spray-dried and directly incorporated into capsules for human use. Typically, such compositions do not require an additional carrier, but a carrier such as lactose can be used. For this type of product, one capsule can contain 10 to 1000 mg. Preferred capsules contain 100 to 500 mg of spray-dried material, particularly preferred are 250 mg or 500 mg capsules.

[0234] In one embodiment, the extract disclosed herein can be spray-dried and formulated into sublingual tablets for oral ingestion. For this type of product, a tablet may contain 10 to 1000 mg. Preferred tablets contain 100 to 500 mg of spray-dried material, and particularly preferred are 250 mg or 500 mg tablets of spray-dried material.

[0235] In one embodiment, the extract of this disclosure can be formulated as a liquid or semi-solid, such as a syrup or gel, for sublingual ingestion into the human mouth. For this type of product, the liquid or gel may contain 10 to 30% w / w of the extract. Preferred concentrations are 15 to 20% w / w or 20 to 30% w / w.

[0236] The spray drying of the extract disclosed herein can be prepared by known methods. Such methods may or may not include a carrier such as maltodextrin.

[0237] In one embodiment, the extract can be spray-dried at 100% concentration, with an inlet temperature of 140 to 150°C and an outlet temperature of 90 to 95°C.

[0238] In one embodiment, the extract can be spray-dried at a concentration of 80% using a maltodextrin carrier, with an inlet temperature of 150 to 160°C and an outlet temperature of 90 to 95°C.

[0239] In one embodiment, the extract of this disclosure can be formulated into a spray for nasal or oral administration in the human body. For this type of product, the spray may contain 0.01 to 10% w / v, 0.01 to 5.0% w / v, 0.1 to 2.0% w / v, or 1.0 to 2.5% w / v.

[0240] Compositions suitable for nonhuman animals have been extensively described in WO 2019 / 028506, for example in

[238] to

[251] and

[253] to

[254] , the contents of which are incorporated herein by cross-reference.

[0241] In one embodiment, in compositions suitable for non-human animals, the extracts of this disclosure may be incorporated into feed at a concentration of 0.001% to 10% w / w or w / v for liquid feed.

[0242] In one embodiment, in compositions suitable for non-human animals, the extract of this disclosure may be incorporated into feed at a concentration of 0.01% to 5% w / w or w / v for liquid feed.

[0243] In one embodiment, in compositions suitable for non-human animals, the extract of this disclosure may be incorporated into feed at a concentration of 0.1% to 2% w / w or w / v for liquid feed.

[0244] In one embodiment, in a composition suitable for ruminants, the extract of this disclosure may be incorporated into the feed at a concentration of 0.001% to 10% w / w, preferably 0.01% to 5%, most preferably 0.1% to 2% or % w / v for liquid feed to achieve a methane reduction of 3% to 80%.

[0245] In one embodiment, in a composition suitable for ruminants, the extract of this disclosure may be incorporated into the feed at a concentration of 0.001% to 10% w / w, preferably 0.01% to 5%, most preferably 0.1% to 2% w / v for liquid feed to achieve a methane reduction of 5% to 70%.

[0246] In one embodiment, in a composition suitable for ruminants, the extract of this disclosure may be incorporated into the feed at a concentration of 0.001% to 10% w / w, preferably 0.01% to 5%, most preferably 0.1% to 2% w / v for liquid feed to achieve a methane reduction of 10% to 60%.

[0247] In one embodiment, in a composition suitable for ruminants, the extract of this disclosure may be incorporated into the feed at a concentration of 0.001% to 10% w / w, preferably 0.01% to 5%, most preferably 0.1% to 2% w / v for liquid feed to achieve a methane reduction of 5% to 50%.

[0248] In one embodiment, in a composition suitable for ruminants, the extract of this disclosure may be incorporated into the feed at a concentration of 0.001% to 10% w / w, preferably 0.01% to 5%, most preferably 0.1% to 2% w / v for liquid feed to achieve a methane reduction of 5% to 40%.

[0249] In one embodiment, in a composition suitable for ruminants, the extract of this disclosure may be incorporated into the feed at a concentration of 0.001% to 10% w / w, preferably 0.01% to 5%, most preferably 0.1% to 2% w / v for liquid feed to achieve a methane reduction of 10% to 30%.

[0250] In one embodiment, in a composition suitable for ruminants, the extract of this disclosure may be incorporated into the feed at a concentration of 0.001% to 10% w / w, preferably 0.01% to 5%, most preferably 0.1% to 2% w / v for liquid feed to achieve a methane reduction of 5% to 10%.

[0251] In one embodiment, in a composition suitable for ruminants, the extract of the present disclosure may be incorporated into the feed at a concentration of 0.001% to 10% w / w, preferably 0.01% to 5%, most preferably 0.1% to 2% w / v for liquid feed, to achieve an increase in milk production of 1 to 15%, 2 to 10%, or 3 to 7%.

[0252] In one embodiment, in compositions suitable for non-human aquatic animals (such as shrimp and prawns), the extracts of this disclosure may be incorporated into the aquatic environment at a concentration of 0.1 to 2.0% v / v.

[0253] In one embodiment, in compositions suitable for non-human aquatic animals (such as shrimp and prawns), the extracts of this disclosure may be incorporated into the aquatic environment at a concentration of 0.1 to 1.0% v / v.

[0254] In one embodiment, in compositions suitable for non-human aquatic animals (such as shrimp and prawns), the extracts of this disclosure may be incorporated into the aquatic environment at a concentration of 0.1 to 0.6% v / v.

[0255] In one embodiment, in compositions suitable for non-human aquatic animals (such as shrimp and prawns), the extracts of this disclosure may be incorporated into the aquatic environment at a concentration of 0.3 to 0.6% v / v.

[0256] Example 1 of the preparation of the extract of this disclosure will now be described. The extract is named Polygain.

[0257] • The lees are stored in a reflux tank before use.

[0258] • Pass the distiller's grains through a 10-micron sieve to remove any large particles.

[0259] Then the raw materials are mixed under high shear.

[0260] • After mixing, heat it to 55°C.

[0261] • The heated material is filtered through a 0.1-micron membrane filtration system.

[0262] After filtration, it is evaporated to 60 Brix.

[0263] • If stored in liquid form, heat it to 80°C and package it.

[0264] • If spray drying of the material is required, mix the 60 Brix product with maltodextrin and blend thoroughly.

[0265] • The resulting slurry is spray-dried at an inlet temperature of 150 to 160°C and an outlet temperature of 90 to 95°C.

[0266] The polyphenol content of Polygain is determined as follows:

[0267] Polyphenols Molecular formula chlorogenic acid <![CDATA[C 16 H 18 O9]]> Trans caffeic acid & caffeic acid <![CDATA[C9H8O4]]> clove acid <![CDATA[C9H 10 O5]]> trans-coumaric acid <![CDATA[C9H8O3]]> trans-sinapic acid <![CDATA[C 11 H 12 O5]]> Vitex negundo <![CDATA[C 21 H 20 O 10 ]]> When the flavin <![CDATA[C 22 H 22 O 10 ]]> Hybridized Herbal Extract & Herbal Extract <![CDATA[C 21 H 20 O 11 ]]> Quercetin <![CDATA[C 15 H 10 O7]]> Celery <![CDATA[C 15 H 10 O5]]> Triticulin <![CDATA[C 23 H 24 O 12 ]]>

[0268] Example 2 of the preparation of the extract of this disclosure will now be described. The extract is named Virofonol.

[0269] 1. Mix 300 kg of sugarcane extract with 900 L of hot water (80 to 85 °C).

[0270] 2. Mix thoroughly and check that the Brix content is 20, then add it to a settling tank containing FPX-66 resin.

[0271] 3. Mix for 20 minutes, then allow the resin to settle for 15 to 20 minutes.

[0272] 4. Pass through a 200-micron pre-filter before entering the tank.

[0273] 5. Drain and dispose of the remaining liquid.

[0274] 6. Rinse the resin with approximately 1000L of cold water. Mix thoroughly and drain.

[0275] 7. Add 71 to 76% ethanol to the settling tank containing resin.

[0276] 8. Mix for 20 minutes, then allow the resin to settle for 15 to 20 minutes.

[0277] 9. The liquid enters the tank through a 200-micron pre-filter and is collected.

[0278] 10. Repeat steps 7 through 9.

[0279] 11. After collecting all the liquid, add 500L of warm water (35 to 40°C) to the resin.

[0280] 12. Mix for 20 minutes, then allow the product to settle for 15 to 20 minutes.

[0281] 13. Drain and collect the liquid.

[0282] 14. Add all the collected liquid to the evaporator and concentrate to 45 Brix.

[0283] 15. Spray dry 45 Brix liquid with an inlet temperature range of 140 to 150°C and an outlet temperature range of 90 to 95°C, then sieve and package.

[0284] Further examples of this disclosure, such as Example 3, will now be described.

[0285] Test Plan The study evaluated four solutions to determine if any of them could directly inactivate SARS-CoV-2. Solution 4 was the composition disclosed herein, and water was used as a negative control.

[0286] Option 1 Add 50 μL of undiluted SARS-CoV-2 stock solution to 50 μL of each solution. After 1 minute (simulating the time spent in the mouth), add 500 μL of infection culture medium to 'quench' the sterilization reaction. Then, as soon as possible, plate the samples directly onto TCID50 plates (n = 4 / dilution). Record the percentage of CPE-positive wells compared to the positive control samples.

[0287] 100 μL of SARS-CoV-2 stock solution was exposed to an equal volume (100 μL) of purified dilution solution. After 1 minute, the virus:substrate solution was serially diluted and TCID50 was performed. The change in TCID50 / mL was compared with that of the positive control sample.

[0288] 50% tissue culture infection dose determination (TCID50): In the PC2 laboratory, plates were inoculated 24 hours prior to assay to establish a Vero cell monolayer of approximately 95%. After verifying the quality / density of the monolayer, the plates were washed with infection medium (to remove any cell debris) and then transferred to the PC3 laboratory. Samples were generated, serially diluted, and a known volume was transferred to each well. n = 4 replicates / sample. The plates were incubated to enable the virus to infect the monolayer, and then MEM infection medium (containing penicillin / streptomycin, glutamine, HEPES, but without FBS) + TPCK trypsin (1 μg / mL) was added. The plates were returned to the incubator (37°C, 5% CO2) and examined for cytopathic effect (CPE) on the cells every 24 hours (up to 72 hours) under a microscope. The TCID50 / mL of infectious virus present in the original samples was then determined by the method of Reed and Meunch, 1938, “A simple method of estimating fifty percent endpoints”, *The American Journal of Hygiene*:3.

[0289] result When directly exposed to SARS-CoV-2, solution 4 exhibited fully effective antiviral activity. For both quenching and TCID50 assays, no virus-induced CPE was detected when using undiluted solution 4. In contrast, solutions 1 and 2 showed no antiviral activity. Solution 3 showed some antiviral activity. Results are illustrated in [Figure number missing]. Figure 1 middle.

[0290] In another example, 4, the compositions of this disclosure were evaluated in a SARS-CoV-2 virus yield reduction assay. A SARS-CoV-2 patient isolate (Australia / VIC01 / 2020) was used, with the composition sample resuspended at 10 mg / mL in infection medium (MEM+Abx+HEPES, serum-free). Samples were tested at 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, and 0.625 μg / mL (final concentrations after virus addition = 5, 2.5, 1.25, 0.625, and 0.313 μg / mL).

[0291] Test protocol: Vero cells were seeded into 24-well plates one day before the start of the experiment.

[0292] • The next day, the cells were infected with 100 TCID50 in 100 μL and incubated at 37°C for 1 hour.

[0293] • Use new stock solution (TCID50 / mL = 10) 5.59 ).

[0294] For this experiment, back titration in 100 μL of inoculum yielded 943 ± 233 TCID. 50 .

[0295] • Resuspend the compound in DMSO at 10 mM (from the initial 10 mg / mL stock solution), then immediately dilute to the above dose range in infection medium and use on the same day.

[0296] • After incubating with the virus for 1 hour, add the antiviral compound to 100 μl of SF medium. After another 1 hour, add 0.8 ml of infection medium containing 1.2 μg / ml trypsin (total volume in wells = 1 mL).

[0297] • Immediately after adding the culture medium, take a 125 μL sample for the t=0 titer. Replace the 125 μL sample to maintain a volume of 1 mL in the well.

[0298] • At 24 and 48 hours post-infection, aliquots of the supernatant were collected and TCID50 was performed to assess the infectious viral titer.

[0299] 50% Tissue Culture Infection Dose Assay (TCID) 50 ):

[0300] • In the PC2 laboratory, plates were inoculated 24 hours before assay to establish a Vero cell monolayer of approximately 95%.

[0301] • After verifying the quality / density of the monolayer, the plate was washed with infection medium (to remove any cell debris) and then transferred to the PC3 laboratory.

[0302] • Generate samples, serially dilute them, and transfer the known volume into each well. n = 4 copies / sample.

[0303] • Incubate the plate to allow the virus to infect the monolayer, then add MEM infection medium (containing penicillin / streptomycin, glutamine, HEPES, but without FBS) + TPCK trypsin (1ug / mL).

[0304] • Return the plate to the incubator (37°C, 5% CO2) and examine the cells for cytopathic effects (CPE) under a microscope every 24 hours (up to 72 hours).

[0305] Then the TCID of the infectious virus present in the original sample 50 / mL was determined using the Reed and Meunch method.

[0306] Results: Log-TCID at 24 hours post-infection 50 / mL value:

[0307]

[0308] Result: TCID 48 hours after infection 50 / mL value:

[0309]

[0310] In another example 5, Vero cells were infected with West Nile virus for two hours and then treated with different concentrations of the composition disclosed herein.

[0311] The results of Example 5 are shown in Figure 2 middle.

[0312] In another example, 6, six species of coccidia protozoa were cultured in 1980 μl of RPMI medium. These species were isolates at the sporozoic stage. These species included: *Eimeria giantiflora*, *Eimeria heapensis*, *Eimeria bryoniflora*, *Eimeria tenuifolia*, *Eimeria toxicae*, and *Eimeria sluggishae*. These species were then treated with 20 μl of the composition disclosed herein.

[0313] The results are shown in Figure 3 middle.

[0314] In another example, 7, a coccidiosis challenge experiment was conducted using Ross 308 male broilers. One-day-old chickens were assigned to experimental groups whose feed contained different concentrations of the composition disclosed herein, or whose feed contained the antibiotic ionocarrier salinomycin, or to a control group whose feed contained no additives. On day 14, the chickens were administered a 20-fold dose of a live vaccine formulation containing three strains of Eimeria. These strains included *Eimeria giantiformis*, *Eimeria scabra*, and *Eimeria tenella*. Seven days after the Eimeria challenge, chickens from each treatment group were euthanized, and lesions formed in the gastrointestinal tract by coccidiosis infection were scored by a qualified veterinarian.

[0315] The results are shown in Figure 4 middle.

[0316] In another example, 8, the fecal microbiota of eight thoroughbred horses was genetically sequenced for five days to determine the baseline microbiota present. 100 ml of the composition disclosed herein was administered every 24 hours for 28 days. The five-day genetic sequencing of the fecal samples was then repeated. The administration of the extract was then stopped. After another 28 days, the fecal microbiota of the eight horses was repeated again.

[0317] The results are shown in Figure 5 middle.

[0318] In another example 9, the production of infectious influenza A virus (IAV) progeny was evaluated in vitro in the presence of a series of concentrations of the composition of this disclosure (referred to in this example as "Virofonol") (added after cells were exposed to the virus). Quantitative assays were used to measure the infectious virus titer.

[0319] Influenza A virus (IAV) (a representative seasonal strain of the A / Beijing 89 strain—H3N2 subtype) was used. The final concentrations of the disclosed composition (Virofonol) added 1 hour after infection with the virus were 10, 5, and 2.5 μg / mL. Viral recovery was assessed at 2 and 24 hours post-infection (hpi). The 2-hour timepoint was used to evaluate residual introduced virus, and the 24-hour timepoint was used to evaluate replication and post-viral infectivity.

[0320] The infection protocol used is as follows:

[0321] Seed MDCK cells in 12-well plates one day before the experiment. Reserve one well for cell counting before infection.

[0322] On the second day, wash the cells twice with infection medium and count the cells. Infect with 150 μL of medium at MOI = 1.0 and incubate at 37°C for 1 hour. Gently shake the plate every 10 minutes.

[0323] • Resuspend the compound in the infection culture medium at 10 mg / mL. Filter through a 0.22 μm syringe filter.

[0324] • Dilute the compound to 10, 5 and 2.5 μg / mL in the infection culture medium.

[0325] • After incubation for 1 hour, remove the inoculum and wash all wells twice with infection culture medium.

[0326] • Immediately add the diluted antiviral compound at a volume of 1 mL per well.

[0327] • At 2 and 24 hours post-infection (hpi), the supernatant was collected and centrifuged at 13,000 rpm for 5 minutes in a microcentrifuge. (i.e., harvest the cell-free supernatant containing the virus).

[0328] • Store the clarified supernatant in two equal portions at -80°C.

[0329] • Perform plaque assays to assess infectious virus titers.

[0330] The plaque assay protocol used is as follows:

[0331] Place 2x L15 culture medium in a 45°C water bath.

[0332] • Pre-activate the viral sample with trypsin Worthington at 4 μg / ml to ensure that IAV HA is lysed.

[0333] • Perform serial 10-fold dilutions on viral samples in cold, serum-free RPMI+.

[0334] • Aspirate the culture medium from a six-well plate containing MDCK cells and replace it with 2 ml of serum-free RPMI+. Repeat.

[0335] • Aspirate RPMI+ and add 150 μl of virus dilution to each of the two wells.

[0336] Then place the plate at 37°C / 5% CO2 for 1 hour to allow the virus to infect it.

[0337] Heat the agarose in a microwave until it is completely dissolved and place it in a water bath at 45°C.

[0338] • Add an agarose capping layer (3 ml total) consisting of L15 medium containing 0.9% (w / v) agarose and 2 μg / ml trypsin Worthington.

[0339] • Allow the capping layer to harden, then return the cells to the incubator for 3 days (37°C / 5% CO2) to form viral plaques.

[0340] Hold the board up to the light and count the plaques.

[0341] The results are as follows (Table 1): Table 1

[0342]

[0343] As can be seen, when added post-infection, Virofonol exhibited antiviral activity against representative seasonal IAV strains, indicating an intracellular mechanism of action. Note that no significant changes were observed at 2 hpi, which is an assessment of the introduced virus before replication establishment. Maximum antiviral activity was observed when Virofonol was added at a concentration exceeding 5 μg / mL 1 hour post-infection and analyzed at 24 hours post-infection. This suggests that Virofonol can exert antiviral activity against IAV at a specific stage of the viral life cycle after viral entry and replication initiation.

[0344] In another example, 10, the production of infectious IAV progeny during and after infection was assessed in vitro in the presence of a range of concentrations of Virofonol. The TCID50 assay was used as a quantitative measure of infectious viral titer because it provides greater technical replication using fewer samples, and all assays can be performed in the same plate to ensure greater consistency.

[0345] Influenza A virus (IAV) (a representative seasonal strain of the A / Beijing 89 strain—H3N2 subtype) was used. Final concentrations during and after virus addition were 10, 5, and 2.5 μg / mL. Viral recovery was assessed at 2 and 24 hours post-infection (hpi).

[0346] The infection protocol used is as follows:

[0347] Seed MDCK cells in 12-well plates one day before the experiment. Reserve one well for cell counting before infection.

[0348] • On the second day, the cells were washed twice with infection culture medium and the cells were counted.

[0349] • Resuspend the compound in the infection culture medium at 10 mg / mL. Filter through a 0.22 μm syringe filter.

[0350] • Dilute the compound to 10, 5 and 2.5 μg / mL in the infection culture medium.

[0351] • Incubate the virus with each concentration of diluted compound in the infection medium at 150 μL / well at 37°C for 1 hour, MOI = 1.0.

[0352] • Infect cells with the virus incubated with the compound for 1 hour. Remove the inoculum. Wash all wells twice with infection medium.

[0353] • Add the corresponding diluted antiviral compound again at a volume of 1 mL per well.

[0354] • At 2 and 24 hpi, collect the supernatant and centrifuge at 13,000 rpm for 5 minutes in a microcentrifuge (to harvest cell-free supernatant).

[0355] • Store the clarified supernatant in two equal portions at -80°C.

[0356] • Perform TCID50 assay to assess infectious viral titer.

[0357] The TCID50 scheme used is as follows:

[0358] • Seed MDCK cells in 96-well plates the day before the experiment. Prepare 8 wells for each viral dilution. One plate per sample.

[0359] • On the second day, the cells were washed twice with infection culture medium.

[0360] • Add trypsin Worthington to the infection culture medium to a final concentration of 2 μg / ml.

[0361] • Pre-activate the viral sample with trypsin Worthington at 4 μg / ml to ensure that IAV HA is lysed.

[0362] • Dilute the virus in the infection medium at a ratio of 1:10, and transfer 100 μl of each dilution to 6 wells. Use column 7 as a positive control (virus of known titer) and column 8 as a negative control.

[0363] • Incubate at 37°C in 5% CO2 for 3 days. Monitor daily.

[0364] • The estimated PFU / mL was calculated based on the standard conversion of TCID50.

[0365] The results are as follows (Table 2):

[0366] Table 2

[0367]

[0368] As can be seen, when incubated with the virus as both a pre-infection and post-infection treatment, Virofonol exhibited antiviral activity against representative seasonal IAV strains. These findings suggest a possible virucidal effect of Virofonol directly against the virus and its cellular mechanism of action. Note that no significant changes were observed at 2 hpi, which is an assessment of the introduced virus before replication is established. The maximum effect was observed with Virofonol at concentrations greater than 5 μg / mL when viral titers were quantified at 24 hpi.

[0369] In another example, 11, the production of infectious ZIKV viral progeny was assessed in vitro during and after infection in the presence of a series of added concentrations of Virofonol. The TCID50 assay was used as a quantitative measure of infectious viral titer because it provides greater technical replication using fewer samples, and all assays can be performed in the same plate to ensure greater consistency. Using Asian Zika virus (ZIKV), the final concentrations during and after viral addition were 10, 5, and 2.5 μg / mL. Time points were 24 and 48 hours post-infection (hpi).

[0370] The infection protocol used is as follows:

[0371] Seed Vero cells in 12-well plates one day before the experiment. Reserve one well for cell counting before infection.

[0372] • On the second day, wash the cells twice with warm PBS and count the cells.

[0373] • Resuspend the compound in the infection culture medium at 10 mg / mL. Filter through a 0.22 μm syringe filter.

[0374] • Dilute the compound to 10, 5 and 2.5 μg / mL in the infection culture medium.

[0375] • Incubate the virus with each concentration of diluted compound in the infection medium at 300 μL / well at 37°C for 1 hour, MOI = 1.0.

[0376] • Infect cells with the virus incubated with the compound for 1 hour. Remove the inoculum. Wash all wells twice with warm PBS.

[0377] • Add the corresponding diluted antiviral compound again at a volume of 1 mL per well.

[0378] • At 24 and 48 hpi, collect the supernatant and centrifuge at 13,000 rpm for 5 minutes in a microcentrifuge.

[0379] • Store the clarified supernatant in two equal portions at -80°C.

[0380] • Perform TCID50 assay to assess infectious viral titer.

[0381] The TCID50 scheme used is as follows:

[0382] • Seed Vero cells in 96-well plates the day before the experiment. Prepare 8 wells for each virus dilution. One plate per sample.

[0383] • On the second day, wash the cells twice with warm PBS.

[0384] • Dilute the virus 1:10 in infection medium (serum reduced) and transfer 100 μl of each dilution to 6 wells. Use column 7 as a positive control (virus of known titer) and column 8 as a negative control.

[0385] • Incubate at 37°C in 5% CO2 for 5 days. Monitor daily.

[0386] • Calculate the estimated PFU / mL based on TCID50.

[0387] The results are as follows (Table 3):

[0388] Table 3

[0389]

[0390]

[0391] Virofonol exhibited antiviral activity against ZIKV-IAV when incubated with the virus as a pretreatment and maintained in cell culture during assays. These findings suggest both the potential extracellular virucidal activity of Virofonol (directly targeting the virus) and the intracellular mechanism of action of this compound (acting on the cell). Note that no significant changes were observed at 24 hpi, which was an assessment of the introduced virus before full replication was established at 48 hpi. The maximum effect was observed with 5 g / mL Virofonol after 48 hpi. This indicates that Virofonol affects the viral particles themselves (acting against the virus) and / or their ability to infect and replicate within cells (cellular effects). It was also noted that ZIKV has a longer replication cycle than IAV; therefore, IAV was assessed at 2 and 24 hpi, while ZIKV was assessed at 24 and 48 hpi.

[0392] In summary, Examples 9 through 11 demonstrate that Virofonol exhibits antiviral activity against both IAV and ZIKV when applied directly to the viral inoculum itself and post-infection. These results indicate that Virofonol can be used as an extracellular and intracellular antiviral agent.

[0393] In another example 12, the dose response of two compositions of this disclosure (referred to as Polynol and Polygain) against ILTV CSW1 (infectious laryngotracheitis virus) was evaluated.

[0394] In this example, chicken (Leigh Hen) liver cancer cell line (LMH cells) was seeded into 12-well plates until 90% confluence.

[0395] • Use 1×10 of 400ul 6Cells were infected with pfu / mL ILTV CSW1 virus for 1 hour.

[0396] • After 1 hour, various concentrations (Polynol: 0.5, 1 and 1.5 mg / ml, Polygain: 1, 1.5 and 2 mg / ml) were added to the corresponding wells.

[0397] • Incubate for 48 hours. After incubation, collect the cell culture medium for plaque assay.

[0398] The results for this example are as follows (Table 4):

[0399] Table 4

[0400]

[0401]

[0402] In another example 13, the ability of the composition of this disclosure (Polygain) to reduce methane emissions from non-milk-producing cows was evaluated.

[0403] Non-milk-producing cows were fed either 3 kg of a concentrate mixture (control) or a concentrate mixture with 100 g of Polygain. The methane concentration was determined at 11:00 AM and re-determined at 1:00 PM.

[0404] Figure 7 The results clearly show that, compared to the control group, Polygain significantly reduced methane emissions during the first two hours. These results demonstrate the eco-regulating effects of the compositions disclosed herein.

[0405] In another example 14, the ability of the disclosed composition (Polygain) to reduce methane formation was evaluated in vitro. In this example, rumen fluid samples were obtained and used to ferment samples of typical farm diets or *Gnaphalium affine*. In the samples evaluated, each fermentation included a 15% concentration of Polygain.

[0406] The results described below show that, for the feedlot diet, methane production was reduced by 54% per gram of digested dry matter after 24 hours. For the *Gnaphalium affine* diet, methane production was reduced by 16.5% per gram of digested dry matter after 24 hours.

[0407]

[0408] In another example 15, the ability of the composition (Polygain) of this disclosure to inhibit the electrode protrusion activity of EHP was evaluated.

[0409] Figure 6The results clearly demonstrate that Polygain is highly effective in inhibiting EHP. Note that PBS is the control. Therefore, it is evident that Polygain will prevent shrimp from being infected with EHP, and in fact, it will also prevent other aquatic species susceptible to EHP infection from becoming infected.

[0410] In another example 16, the ability of the composition (Polygain) of this disclosure to increase milk production in dairy cows was evaluated. In this example, the cows were fed their normal diet (control) or their normal diet containing 0.5% or 1.0% Polygain. Milk volume and concentrate feed were measured daily, and the results are shown in Table 5.

[0411] Table 5

[0412] sky Polygain concentration (% w / w) Milk (liters / day / dairy cow) Concentrated feed / 100kg milk 7 0 33.2 23.9 7 0.5 34.9 22.9 7 1.0 35.3 22.7 41 0 31.0 25.4 41 0.5 32.2 25.7 41 1.0 32.8 24.6

[0413] These results showed that at 7 days, milk production increased by 2.1 L / day / cow in cows supplemented with Polygain. This continued at 41 days, where milk production increased by 1.8 L / day / cow in cows supplemented with Polygain. This indicates increases of 6.3% and 5.8%, respectively.

[0414] Furthermore, the protein and fat content of all cows supplemented with Polygain remained unchanged.

[0415] Finally, cows supplemented with Polygain require lower levels of concentrate feed to achieve increased milk production.

[0416] In another example 17, the ability of the composition (Polygain) of this disclosure to improve calf growth was evaluated. In this experiment, calves were either fed their normal diet (control) or their normal diet containing 10g of Polygain.

[0417] The diet consists of 4.0 kg wheat, 1.0 kg corn, 1.0 kg rapeseed meal, 0.1 kg limestone, 0.1 kg salt, 0.05 kg dicalcium phosphate, 0.04 kg magnesium oxide, vitamins, and trace mineral premix. It is fed as pellets or loose pellet mixture.

[0418] The experimental results are listed in Table 6. These results show that the average total weight gain in the control group was 67.6 kg, compared to 70.5 kg in the experimental diet including Polygain. The average daily weight gain in the control group was 0.82 kg, compared to 0.86 kg in the other group. This corresponds to a difference of 4.9%, which is highly significant in terms of economic value per animal.

[0419] Based on examples related to reduced methane production, it is clear that the disclosed sugar extract offers a dual benefit: increased body weight while simultaneously reducing methane production.

[0420] Table 6:

[0421]

[0422]

[0423] Table 6: Continued

[0424]

[0425] Table 6 (continued)

[0426]

[0427] Table 6 (continued)

[0428]

[0429] Table 6 (continued)

[0430]

[0431] Table 6 (continued)

[0432]

[0433] In another example 18, the ability of the disclosed composition (Polygain) to improve the health of dairy cows by preventing and / or treating mastitis was evaluated. Conductivity data were collected from over 4,000 milkings of 27 cows over a 4-month period, who were divided into a control group and a group whose diet included 1% w / w Polygain (on a total dry matter basis).

[0434] As mentioned earlier, milk conductivity directly indicates the presence of mastitis in a dairy cow. Typically, conductivity increases with the occurrence of mastitis. It is important to note that it is uncommon for all four teats of a cow to be infected. Therefore, by comparing the teat with the lowest conductivity to each of the other teats to obtain a ratio, it is possible to indicate the presence of mastitis in one or more teats. Such measurements are usually collected automatically in so-called robotic milking machines. Of course, milk samples can also be obtained manually, and conductivity can be evaluated teat by teat.

[0435] In this example 18, samples were obtained in a robotic milking apparatus. As shown in Table 7, not only did the average base conductivity decrease over time, but the average ratio between teats also decreased. This strongly indicates an improvement in the health of the cow's mammary glands, which may be due to prevention, treatment, and / or a reduction in the incidence of mastitis.

[0436] Table 7

[0437]

[0438] In another example 19, the ability of the composition (Polygain) of this disclosure to reduce methane emissions from non-milk-producing cows was evaluated. Each dairy cow test group's feed included 0.5% or 1% w / w of Polygain on a dry matter basis.

[0439] At the start of the trial, methane concentrations were captured periodically over a period of approximately 42 hours. After 32 days of feeding, methane concentrations were again captured periodically over a period of approximately 42 hours.

[0440] The experimental results at 0.5% Polygain are shown in Figure 8 The results at 1% Polygain are shown in [the table / data]. Figure 9 (Chinese) Reference Figure 8 Qualitatively, it can be seen that the methane concentration in the 0.5% test group recorded only four values ​​greater than 0.2. In contrast, at the beginning, many values ​​exceeding 0.2 were recorded.

[0441] refer to Figure 9 Qualitatively, it can be seen that the methane concentration in the 1% test group did not exceed 0.2. In contrast, at the beginning, many values ​​exceeding 0.2 were recorded.

[0442] Therefore, Example 19 clearly illustrates the dysregulation effect of the compositions of this disclosure.

[0443] In another example 20, the methanogenic archaea populations in the feces of a cow fed a control diet and a second cow fed the same diet supplemented with 100g of Polygain were evaluated.

[0444] The results of Example 20 are depicted in Figure 10 These results show that the population of *Bacillus methanogenus* (Methanobacteriaceae) decreased by approximately 15%, while that of *Bacillus methanogenus* (Methanobacteriaceae) decreased by approximately 30%. Such results strongly support the findings in other examples disclosed herein that the methane reduction in Polygain is due to changes induced in the ruminant microbiota, particularly the population of methane-producing microorganisms.

[0445] Those skilled in the art will understand that various changes and / or modifications can be made to the above embodiments without departing from the broad scope of this disclosure. Therefore, the embodiments of this disclosure are to be considered illustrative rather than restrictive in all respects.

Claims

1. Use of a sugarcane-derived extract from 0.05% to 100% by weight in the preparation of a composition for the prevention or treatment of shrimp infected with Enterocytozoa hepatica by inhibiting or inactivating EHP, said extract comprising polyphenols from 10 catechin equivalents CE g / L to 100 CE g / L or from 100 CE mg / g to 1000 CE mg / g, wherein said use is by including said extract in an aquatic environment. And among them, The extract is prepared by a method comprising the following steps: filtering a supernatant of sugarcane residue, the filtration step being microfiltration or ultrafiltration; concentrating the filtered supernatant to remove water, thereby providing an extract having a Bx value of 50-70°Bx.

2. The use according to claim 1, wherein the extract comprises polyphenols ranging from 10 CE g / L to 80 CE g / L or from 100 CE mg / g to 800 CE mg / g.

3. The use according to claim 1 or 2, wherein the extract comprises polyphenols ranging from 10 CE g / L to 50 CE g / L or from 100 CE mg / g to 500 CE mg / g.

4. The use according to claim 1 or 2, wherein the extract comprises flavonoids ranging from 1 CE g / L to 15 CE g / L or from 10 CE mg / g to 150 CE mg / g.

5. The use according to claim 1 or 2, wherein the concentration of the extract in an aquatic environment is 0.1 to 2.0% v / v.

6. The use according to claim 1 or 2, wherein the concentration of the extract in an aquatic environment is 0.1 to 1.0% v / v.

7. The use according to claim 1 or 2, wherein the concentration of the extract in an aquatic environment is 0.1 to 0.6% v / v.

8. The use according to claim 1 or 2, wherein the concentration of the extract in an aquatic environment is 0.3 to 0.6% v / v.

9. The use according to claim 1 or 2, wherein the shrimp is selected from tiger prawn, Litopenaeus vannamei, and Litopenaeus vannamei.

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