Patent Application
20220257684
The present invention relates generally to phytogenic formulations for use in treatment of coccidiosis, and more specifically to anti-coccidial phytogenic formulations comprising a combination of Artemisia indica and Bidens Pilosa.
Coccidiosis is a major parasitic disease of poultry and causes a considerable economic loss in the poultry industry. The genus Eimeria, a coccidia subclass, belongs to spore-forming, unicellular protozoan parasites. They are intestinal parasites and can infect fish, reptiles, birds, mammals. Chickens are susceptible to at least 11 species of Eimeria, tenella, E. necatrix, E. brunetti and E. maxima are the more virulent species and E. acervulina, E. praecox and E. mitis are less virulent species in chickens. Eimeria infection is usually asymptomatic but shows severe clinical symptoms such as diarrhea, bloody droppings, dehydration, droopiness, listlessness, loss of appetite, paleness, ruffled feathers and huddling in young and immune-compromised animals.
The life cycle of Eimeria has intracellular, extracellular, asexual, and sexual stages. Once the chickens are infected with Eimeria, the parasites develop in the chicken and give rise to a microscopic egg (called an oocyst) which is passed out in the droppings. Under proper conditions of temperature and moisture the oocyst develops within one to two days to form a sporulated oocyst which is capable of infecting other chickens. At this stage the ooycyst contains eight bodies (called sporozoites), each of which is sable to enter cells in the chicken's intestine after the oocyst is eaten. After entering the cells, sporozoites divide many times producing offspring (merozoites). Each merozoite in turn may enter another intestinal cell. This cycle may repeat several times and cause a large number of intestinal cells being destroyed. Eventually, the cycle stops and sex cells (male and female) are produced. The male fertilizes the female to produce oocyst which ruptures from the intestinal cell and passes in the droppings. Thousands of oocysts may be passed in the droppings of an infected chicken. Therefore, poultry raised in crowded or unsanitary conditions are at great risk of becoming infected.
Plants are recognized as an excellent source for human and animal medicines. Bidens pilosa, an Asteraceae family, is claimed as an anti-coccidial herb for treating coccidiosis. U.S. Pat. No. 9,072,312 discloses Bidens Pilosa and polyacetylenic compounds for prevention and treatment of coccidiosis. Although Bidens Pilosa and its active compounds can prevent and treat coccidiosis, this prevention and treatment are sometimes ineffective in dirty chicken houses.
In one aspect, the invention relates to an anti-coccidial composition comprising: (a) an effective amount of Artemisia indica extract; (b) an effective amount of Bidens pilosa extract, and (c) a pharmaceutically acceptable vehicle, excipient, or carrier.
In another aspect, the invention relates to an anti-coccidial composition comprising: (a) an effective amount of extracts or powders of Artemisia indica; and (b) an effective amount of extracts or powders of Bidens pilosa.
In one embodiment of the invention, the anti-coccidial composition comprises: (a) an effective amount of extracts of Artemisia indica; and (b) an effective amount of extracts of Bidens pilosa; or (a) an effective amount of extracts of Artemisia indica; and (b) an effective amount of powders of Bidens pilosa; or (a) an effective amount of powders of Artemisia indica; and (b) an effective amount of extracts of Bidens pilosa; or (a) an effective amount of powders of Artemisia indica; and (b) an effective amount of powders of Bidens pilosa. The anti-coccidial composition may further comprise a pharmaceutically acceptable vehicle, excipient, or carrier.
In one embodiment, the anti-coccidial composition is an oral dosage form. The oral dosage form may be selected from the group consisting of tablets, pills, softgel capsules, hard capsules, granules, powders, concentrates, liquids, molded balls and any combinations thereof.
In another embodiment, the anti-coccidial composition further comprises an animal feed, or a non-human animal feed or food.
In another embodiment, the animal feed is selected from the group consisting of poultry feed, fish food, amphibian feed, reptile food, bird feed, and non-human mammal food.
In another embodiment, the Artemisia indica extract and the Bidens pilosa extract are present in a ratio of about from 0.0001:1.0 to 1.0:1.0.
In another embodiment, the amount of Artemisia indica extract ranges from 0.0001% to 0.01% by weight of the composition. The amount of Artemisia indica may range from about 0.00005% to about 0.005% (w/w) or from about 0.001% to about 0.01% (w/w).
In another embodiment, the amount of Bidens pilosa extract ranges from 0.01% to 0.1% by weight of the composition. The amount of Bidens pilosa may range from about 0.005% to about 0.05%.
In another embodiment, the Artemisia indica extract comprises chlorogenic acid.
The invention also relates an anti-coccidial composition consisting essentially of or consisting of: (a) an effective amount of extracts or powders of Artemisia indica; (b) an effective amount of extracts or powders of Bidens pilosa, and (c) a non-human animal feed or food.
The invention further relates to an anti-coccidial composition consisting essentially of or consisting of: (a) an effective amount of extracts or powders of Artemisia indica; and (b) an effective amount of extracts or powders of Bidens pilosa.
In one embodiment of the invention, the anti-coccidial composition is in a non-aqueous liquid, aqueous liquid, suspension or powder form.
Further in another aspect, the invention relates to use of the anti-coccidial composition of the invention in the manufacture of a medicament for killing coccidian oocysts and inhibiting coccidian oocyst sporulation.
Further in another aspect, the invention relates to use of the anti-coccidial composition of the invention in the manufacture of a medicament for killing coccidian oocysts, inhibiting oocyst sporulation, reducing sporozoite invasion, and lessening bloody stools, or for alleviating or treating coccidiosis, in a subject in need thereof.
Yet in another aspect, the invention relates to use of an anti-coccidial formulation comprising an effective amount of Artemisia indica extract, and a pharmaceutical acceptable vehicle in the manufacture of a medicament for killing coccidian oocysts and inhibiting coccidian oocyst sporulation, and sporozoite invasion.
These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.
The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.
The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
As used herein, “around”, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
By about 0.00005% to about 0.005% it meant that all hundred-thousandth, ten-thousandth, thousandth, hundredth, tenth and integer unit amounts within the range are specifically disclosed as part of the invention. Thus, 0.00005%, 0.00006%, 0.00007% . . . 0.00009%, 0.0001%, 0.0002% . . . and 0.0009%, 0.001%, 0.0011% . . . and 0.0048, 0.0049, 0.005% unit amounts are included as embodiments of this invention.
By about 0.0001% to about 0.01% it meant that all ten-thousandth, thousandth, hundredth, tenth and integer unit amounts within the range are specifically disclosed as part of the invention. Thus, 0.0001%, 0.0002%, 0.0003% . . . 0.0009%, 0.001%, 0.0011% . . . and 0.0098, 0.0099, 0.01% unit amounts are included as embodiments of this invention.
By about 0.005% to about 0.05% it meant that all thousandth, hundredth, tenth and integer unit amounts within the range are specifically disclosed as part of the invention. Thus, 0.005%, 0.006%, 0.007% . . . 0.0099, 0.01%, 0.011% . . . and 0.048%, 0.049%/a, 0.05% unit amounts are included as embodiments of this invention.
By about 0.01% to about 0.1% it meant that all hundredth, tenth and integer unit amounts within the range are specifically disclosed as part of the invention. Thus, 0.01%, 0.02%, 0.03% . . . and 0.08%, 0.09%, 0.1% unit amounts are included as embodiments of this invention.
The term “a mixture” shall generally mean any combination or blend of different kinds.
The invention relates to the anti-coccidial activity of phytogenic formulations comprising Bidens
Preparation of B. pilosa and Artemisia indica Extracts
Artemisia indica extract. Artemisia indica plants were collected from the campus of Miaoli, Taiwan. Approximately 1 kg of dried whole plants in their entirety was refluxed in 10 L of 100° C. water for two hours. After removal of aqueous phase, insoluble materials was again refluxed in 10 L of water for two hours. The combined aqueous solutions were lyophilized to yield crude extract (0.15 kg). The chlorogenic acid was used as a marker of batch consistency. Chlorogenic acid was obtained and characterized by 1H NMR and 13C NMR.
Bidens pilosa extract. B. pilosa plants were collected and processed using a similar method as described above. Approximately 1 kg of dried whole plants was refluxed in 10 L of 100° C. water for two hours and the extraction was repeated once. The combined water solutions were lyophilized to yield crude extract (0.12 kg). The cytopiloyne was used as a marker of batch consistency. Polyacetylenic compounds was obtained and characterized by 1H NMR and 13C NMR. The pure compounds thus obtained can be further derivatized to provide a number of other polyacetylenic compounds (U.S. Pat. No. 7,763,285, and Kusano et al (JP 2004083463), all of which are incorporated herein by reference in their entireties).
The extract of A. indica and B. pilosa are used in form of powder. Calculation of the percentage of the plant extract powder is as follows: the plant powder weight/the plant powder weight±basic chicken feed=% of the plant powder.
The invention relates to the discovery of anti-coccidial properties of Artemisia indica and a formulation comprising Artemisia indica and Bidens pilosa.
Chemicals. Salinomycin, PBS, and H&E stain were purchased from SIGMA-ALDRICH™. Artemisia indica and B. pilosa were extracted in boiling water and mixed at a variety of ratios for further use.
Invasion assay. Madin-Darby bovine kidney (MDBK, ATCC® CCL-22) cells, grown in DMEM containing fetal bovine serum (10%) and supplements, were seeded onto glass cover slips in 24 wells at 2×105 cells/well. One day later, the cells were pre-incubated with DMEM medium containing salinomycin, or plant extracts and phytochemicals at doses indicated for 0.5 h. Fresh sporozoites (2×105) were added to the cells for additional 4 h. Ater extensive PBS washing, the cells were fixed and stained with H&E. Photographs were taken with a microscope. Invasion percentage (%) was obtained by the formula, 100%×(the number of cells invaded by sporozoites/total cell number).
Viability test. The E. tenella sporozoites were incubated with plant extract or salinomycin for 4.5 h. Microscopy was used to distinguish life and death in sporozoites (
Sporulation assay. The E tenella oocysts were pre-treated with PBS, boiling (100° C. for 30 min) or plant extracts at indicated doses for 48 h. The oocysts were incubated with 2% potassium dichromate for 2 days before sporulation. The percentage of sporulating oocysts (%) was counted.
Propidium iodide (PI) staining of E. tenella sporozoites. The ET oocysts underwent PBS (1 h), boiling treatment (100° C. for 30 min) or incubation with plant extracts at indicated doses for 1 h. The oocysts were stained with PI. After PBS washing, the oocysts were examined using a microscope.
Birds, diets, and experimental design. One-day-old Lohmann broiler chicks hatched at Taichung Hatchery (Taiwan) were wing-banded upon arrival, weighed, and randomly placed in Petersime starter brooder units. At 1 to 2 days post-hatching, the birds were given free access to water and diets. The diets were formulated by mixing a base diet with a carrier alone (control diet) or with an indicated dose of a phytogenic formulation comprising Artemisia indica (AI), B. pilosa (BP), or both AI and BP. After grouping, birds were tube-fed with Eimeria oocysts to get infection. Growth performance (body weight and feed conversion), pathology (bloody droppings and gut morphometry) and survival rate were measured and examined (
Immunohistochemical staining. Multiple parallel sections of the ceca from chickens that had access to control diet and the diet containing Artemisia indica (AI), B. pilosa (BP), or both for 14 days were flash-frozen. The sections were stained with H&E or anti-insulin antibody, with development of diaminobenzidine tetrahydrochloride, followed by image analysis (Yang et al. Research in Veterinary Science (2015) 98:74-81).
Statistical analysis. The results from three or more independent experiments were presented as mean±S.E. Data were analyzed by ANOVA. Differences with P value less than 0.05 were considered statistically significant.
To investigate the effects of the extracts of A. indica (AI), B. pilosa (BP) and combined extracts of AI and BP, the direct killing activities of AI, BP and a combination of AI and BP in E. tenella (ET) oocysts were examined, respectively. Boiling treatment as a positive control effectively killed 30% of ET oocysts as demonstrated by propidium iodide (PI) staining (Boiling,
The effects of the plant extracts on sporulation of E. ET oocysts were tested. Seventy percent of ET oocysts were able to sporulate in the in vitro culture (PBS,
The effect of B. pilosa on the entry of ET sporozoites into MDBK cells was examined. As reported by Yang et al. (Scientific Reports (2019) 9:2896), ET sporozoites could invade into 20% of the cells (
Effects of Plant Extracts on Survival Rate, Body Weight Gain, and Feed Conversion Rate in E. tenella Challenged Chickens
The anti-coccidial effects of the extracts of A. indica (AI), B. pilosa (BP) and a combination of both extracts were examined in vivo. Chickens were randomly divided into 8 groups and given daily (Day 1 to Day 14) access to standard chicken feed (Groups 1 and 2), feed containing salinomycin (Group 3), or feed containing the plant extracts (Groups 4 to 8) at the indicated doses (
Table 1 shows the effect of plant extracts on the body weight gain in the ET challenged chickens. Body weights (BW) of the chickens from
aBody weight gain (BWG) was obtained by the formula: body weight on Day 7 or Day 14 minus body weight on Day 1. Day 7-1 stands for Day 7 BW gain, and Day 14-1 stands for Day 14 BW gain,
bStatistic comparison made between Group 1 and other groups.
cStatistic comparison made between Group 2 and other groups.
Table 2 shows the effect of plant extracts on feed conversion rate (FCR) in ET challenged chickens. On Day 14 post ET infection, the average FCR of control chickens (Group 1), infected chickens (Group 2), infected chickens fed with salinomycin (Group 3), 0.01% BP (Group 4), 0.0001% AI plus 0.01% BP (Group 5), 0.001% AI plus 0.01% BP (Group 6) 0.01% AI plus 0.01% BP (Group 7) and 0.01 AI (Group 8) were 2.24, 3.38, 3.05, 2.60, 3.01, 3.04, 2.88 and 2.96, respectively. The results indicate that AI, BP, and a combination of AI and BP all significantly ameliorated the reduction in the body weight gain and also ameliorated the increase in FCR caused by the ET infection to a greater degree than salinomycin or control feed alone. Part of this amelioration could be attributed to the weight-gaining effect of the plant extracts.
aStatistic comparison made between Group 1 and other groups.
bStatistic comparison made between Group 2 and other groups.
Effect of Plant Extracts on Fecal Oocyst Excretion in E. tenella Challenged Chickens
Table 3 shows fecal oocyst excretion in each group. Excretion of Eimeria oocysts in the feces of ET-infected chickens is an indicator of Eimeria multiplication. The oocysts per gram feces (OPG) in the chickens from
aStatistic comparison made between Group 1 and other groups.
bStatistic comparison made between Group 2 and other groups.
Effect of Plant Extracts on Intestinal Lesions in E. tenella Challenged Chickens
Table 4 shows gross cecal lesion scores on Day 7 post ET infection. Gross lesions in the ceca of the chickens from
Table 5 shows microscopic lesion scores in the ceca of the chickens from
No microscopic cecal lesions (score=0) were observed in the uninfected unmedicated control group (Group 1). The infected unmedicated animals (Group 2) showed serious microscopic lesions (score=7.2) in the cecum 7 days after the ET infection. Severe ulceration, hemorrhage and decreased villi in the cecum were also observed (data not shown). Oocysts, gametocytes and schizonts appeared inside the cecal epithelia (data not shown). The infected salinomycin-fed animals (Group 3) showed mild improvement in microscopic lesions (score=6.29) in the cecum as compared to the infected unmedicated animals (Group 2) post infection. However, the infected animals fed with B. pilosa, B. pilosa combining AI, or AI alone (Groups 4 to 8, Table 5) showed significantly reduced microscopic lesions (scores of 4.78 to 5.80) in the cecum. Consistently, B. pilosa decreased ulceration and hemorrhage and preserved more mucosae and villi in chicken ceca than control diets (data not shown). B. pilosa also decreased the number of oocysts, gametocytes and schizonts inside the cecal epithelia to a greater extent than salinomycin and control diets (data not shown). Overall, B. pilosa, combination of B. pilosa and AI, or AI alone significantly reduced gut pathology in chickens following ET infection. The data suggest that a combination of AI and BP reduced microscopic lesions in chicken guts more than either one alone.
1Microscopic lesion score is represented as a fraction, in which the numerator and denominator are indicated. The numerator and denominator in a fraction represent the sum of microscopic lesions (0 to 4) in the gut samples, 5 section slides per gut, and the number of the examined gut samples multiplied by 5 per group.
2The difference in microscopic lesion scores of the chickens between infected medicated groups and uninfected unmedicated group (Group 1) is analyzed with a chi-square test after multinomial transformation and shown by Pa value. Similarly, the difference in microscopic lesion scores of the chickens between infected medicated groups and infected unmedicated group (Group 2) is shown by Pb value.
Effect of Plant Extracts on Bloody Stools in E. tenella Challenged Chickens
All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/42692 | 7/18/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62876633 | Jul 2019 | US |
