Compositions and methods for preventing/treating metabolic syndrome

Abstract

Broth compositions prepared from poultry are disclosed. Selected poultry raw materials are processed to obtain a broth having high protein content. Certain specific amino acids and proteins are present at relatively higher concentration as compared to home-made broth. The disclosed broth compositions are effective in preventing and/or treating metabolic syndrome and may also provide other nutritional and health benefits.

Claims

1. A pharmaceutical composition comprising an effective amount of a broth prepared from poultry parts, said pharmaceutical composition comprising an enhanced quantity of chondroitin sulfate and having certain ratio between amino acids proline and histidine derived from said poultry parts, wherein the enhanced quantity of chondroitin sulfate derived from said poultry parts is at least 6% by weight of total dry solids of the pharmaceutical composition, and wherein said proline and histidine are present in said pharmaceutical composition at a ratio between proline and histidine of at least 4:1 by weight, and wherein said effective amount is an amount of the broth effective in treating a condition selected from the group consisting of joint pain and inflammation caused by the action of COX-2.

2. The pharmaceutical composition of claim 1, wherein said effective amount of the composition is an amount effective in reducing/alleviating joint pain.

3. The pharmaceutical composition of claim 1, wherein said effective amount of the composition is an amount effective in reducing COX-2 activity to treat inflammation caused by the action of COX-2 while not affecting activity of COX-1 in the individual.

4. A method of treating a condition in a subject, comprising administering to the subject an effective amount of a composition prepared from poultry parts, said composition comprising an enhanced quantity of chondroitin sulfate and having certain ratio between amino acids proline and histidine derived from said poultry parts, wherein the enhanced quantity of chondroitin sulfate derived from said poultry parts is at least 6% by weight of total dry solids of the pharmaceutical composition, and wherein said proline and histidine are present in said pharmaceutical composition at a ratio between proline and histidine of at least 4:1 by weight, and wherein said effective amount is an amount of the composition effective in treating the condition, said condition being selected from the group consisting of joint pain and inflammation caused by the action of COX-2.

5. The method of claim 4, wherein said effective amount of the composition is an amount effective in reducing/alleviating joint pain in the subject.

6. The method of claim 4, wherein said effective amount of the composition is an amount effective in reducing COX-2 activity to treat inflammation caused by the action of COX-2 while not affecting activity of COX-1 in the individual.

7. The method of claim 4, wherein at least 1 mg of said composition per pound of body weight of the subject is administered to the subject per day.

8. The method of claim 4, wherein between 1-100 mg of said composition per pound of body weight of the subject is administered to the subject per day.

9. The method of claim 4, wherein said composition is administered to the subject in a form selected from the group consisting of a medicament, a pharmaceutical composition, a food, a food ingredient, a supplement, and a nutraceutical ingredient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the protein profile by staining of proteins separated by SDS-PAGE.

(2) FIG. 2 shows the effect on triglyceride levels in Obese rats fed with the disclosed compositions as compared with controls.

(3) FIG. 3 shows the effect on cholesterol levels in Obese rats fed with the disclosed compositions as compared with controls.

(4) FIG. 4 shows that up-regulation of PKA is significantly repressed in animals fed with AAC1 broth as compared to animals fed with a commercially available product TSN.

(5) FIG. 5 shows nocifensive responses of AAC1 compared to a commercial product.

DETAILED DESCRIPTION

(6) Chicken soup and chicken broth have been available for human consumption for centuries. Many studies have been performed that show various health benefits of chicken soup or broth. According to conventional home-style method, chicken soup or broth is prepared by boiling whole chicken or large chicken parts in a pot of water for an extended period of time. The soup or broth prepared according to this method may not have maximized the health promoting effects of the soup or broth because some of the compounds may have been lost during cooking or during processing, while other compounds may not have been extracted from the chicken parts.

(7) The present disclosure provides an improved method by processing chicken parts prior to cooking and by controlling the processing temperature and cooking time to maximize the extraction of beneficial compounds while, concomitantly, minimizing loss of activity due to harsh processing conditions.

(8) Specially selected raw materials from poultry are processed according to the disclosed methods to obtain a broth having high protein content. Certain amino acids are present in relatively higher concentration as compared to a home-made broth or other commercial products. As shown in various animal studies described herein, the disclosed broth compositions may prove effective in preventing and/or treating joint diseases and the underlying pathology. The compositions may also provide other nutritional and health benefits such as decreasing inflammation.

(9) The terms “broth” and “soup” refer to a liquid composition containing at least one solute and may also be used to refer to a ready to serve form, a concentrate, a stock in either liquid or solid form.

(10) The poultry broth of the disclosure may contain a significant amount of different amino acids. Such amino acids may be present in the form of a protein or as free amino acids in the broth. Total amino acids include both amino acids present in the form of proteins and those present as free amino acids. For purpose of this disclosure, the ratio of different amino acids in a broth composition refers to the ratio between total amino acids.

(11) The term “dry solid” or “solid” as used herein refers to the components of a liquid composition that remain after all free liquid is removed from the liquid composition. In the case of an aqueous broth, the free liquid is water.

(12) The term “administer” means delivering of a material to an individual, such as by oral ingestion.

(13) The term “subject” or “individual” is used to refer to a mammal, including human being.

(14) The term “substantially” means by at least 10-20%.

(15) The following items describe some of the embodiments of the present disclosure:

(16) Item 1. A pharmaceutical composition comprising an effective amount of a broth prepared from poultry parts, said pharmaceutical composition comprising proline and histidine derived from said poultry parts, wherein said proline and histidine are present in said pharmaceutical composition at a ratio between proline and histidine of at least 4:1 by weight, and wherein said effective amount is an amount of the broth effective in preventing or treating metabolic syndrome or a symptom or condition associated with metabolic syndrome.

(17) Item 2. The pharmaceutical composition of Item 1, wherein said effective amount of the composition is an amount effective in reducing/alleviating at least one symptom of the metabolic syndrome, said at least one symptom being selected from the group consisting of a) proinflammatory or inflammation state, b) joint pain, c) diminishing cognition, d) increased BMI, e) gut dysbiosis, f) hypertension, and (g) high cholesterol.

(18) Item 3. The pharmaceutical composition of Item 1, wherein said effective amount of the composition is an amount effective in reducing/alleviating two or more symptoms of the metabolic syndrome, said two or more symptoms being selected from the group consisting of i) proinflammatory or inflammation state, ii) joint pain, iii) diminishing cognition, iv) increased BMI, v) gut dysbiosis, vi) hypertension vii) high serum lipids, viii) pain, ix) insulin resistance with or without glucose intolerance, and (x) high cholesterol.

(19) Item 4. The pharmaceutical composition of any of the preceding Items, wherein said effective amount of the composition is an amount effective in reducing COX-2 activity while not affecting activity of COX-1 in the individual.

(20) Item 5. The pharmaceutical composition of any of the preceding Items, wherein said pharmaceutical composition comprises an enhanced level of chondroitin sulfate derived from said poultry parts, said enhanced level of chondroitin sulfate being at least 4% by weight of total dry solids of the composition.

(21) Item 6. A method of preventing or treating metabolic syndrome in an individual, comprising administering to the individual an effective amount of a composition prepared from poultry parts, said composition comprising proline and histidine derived from said poultry parts, wherein said proline and histidine are present in said pharmaceutical composition at a ratio between proline and histidine of at least 4:1 by weight, and wherein said effective amount is an amount of the composition effective in preventing or treating metabolic syndrome or a symptom or condition associated with metabolic syndrome.

(22) Item 7. The method composition of Item 6, wherein said composition comprises an enhanced level of chondroitin sulfate derived from said poultry parts, said enhanced level of chondroitin sulfate being at least 4% by weight of total dry solids of the composition.

(23) Item 8. The method of Item 6, wherein said effective amount of the composition is an amount effective in reducing/alleviating at least one symptom of the metabolic syndrome, said at least one symptom being selected from the group consisting of a) proinflammatory or inflammation state, b) joint pain, c) diminishing cognition, d) increased BMI, e) gut dysbiosis, f) hypertension, and (g) high cholesterol.

(24) Item 9. The method of Item 6, wherein said effective amount of the composition is an amount effective in reducing/alleviating two or more symptoms of the metabolic syndrome, said two or more symptoms being selected from the group consisting of i) proinflammatory or inflammation state, ii) joint pain, iii) diminishing cognition, iv) increased BMI, v) gut dysbiosis, vi) hypertension vii) high serum lipids, viii) pain, ix) insulin resistance with or without glucose intolerance, and (x) high cholesterol.

(25) Item 10. The method of Item 6, wherein said effective amount of the composition is an amount effective in reducing/alleviating three or more symptoms of the metabolic syndrome, said three or more symptoms being selected from the group consisting of i) proinflammatory or inflammation state, ii) joint pain, iii) diminishing cognition, iv) increased BMI, v) gut dysbiosis, vi) hypertension vii) increased serum lipids, viii) pain, ix) insulin resistance with or without glucose intolerance, and (x) high cholesterol.

(26) Item 11. The method of any of Items 6-10, wherein said effective amount of the composition is an amount effective in reducing COX-2 activity while not affecting activity of COX-1 in the individual.

(27) Item 12. The method of any of Items 6-11, wherein said effective amount of the composition is an amount effective in reducing the level of expression or concentration of C-Reactive protein, or protein kinase A (PKA) in the individual.

(28) Item 13. The method of any of Items 6-12, wherein said effective amount of the composition is an amount effective in reducing the amount of adiponectin in the individual.

(29) Item 14. The method of any of Items 6-13, wherein said effective amount of the composition is an amount effective in reducing low density lipoproteins (LDL) in the individual.

(30) Item 15. The method of any of Items 6-14, wherein said effective amount of the composition is an amount effective in lowering ratio between low density lipoproteins (LDL) and high density lipoproteins (HDL) (LDL/HDL) in the individual.

(31) Item 16. The method of any of Items 6-15, wherein at least 1 mg of said composition per pound of body weight of the individual is administered to the individual per day.

(32) Item 17. The method of any of Items 6-15, wherein between 1-100 mg of said composition per pound of body weight of the individual is administered to the individual per day.

(33) Item 18. The method of any of Items 6-17, wherein said composition is administered to the individual in a form selected from the group consisting of a medicament, a pharmaceutical composition, a food, a food ingredient, a supplement, and a nutraceutical ingredient.

EXAMPLES

(34) The following examples are provided for purposes of illustration of the embodiments only and are not intended to be limiting. The raw materials, reagents, chemicals, and other materials are presented as exemplary components or reagents, and various modifications may be made in view of the foregoing discussion within the scope of this disclosure. Unless otherwise specified in this disclosure, components, reagents, protocol, and other methods used in the system and the assays, as described in the Examples, are for the purpose of illustration only.

Example 1 Preparation of Broth from Turkey Parts

(35) In this example, turkey parts were used to prepare a broth and the overall quality and potential health benefits of the broth were determined. Briefly, raw turkey was mechanically separated and the parts were finely comminuted to less than 2 mm in size to maximize extraction. The small-sized parts from the turkey were gently cooked to 195° F. in steam for about 15 minutes or less. The broth was then separated from the insoluble fraction by decanting. Freed fat in the broth was also removed from the broth by a centrifugal separator. The broth was concentrated in a commercial evaporator, chilled, and packaged for sale.

Example 2 Preparation of Broth from Chicken Parts

(36) In this study, chopped chicken parts containing chicken bones and cartilage were cooked in water at a temperature greater than 250° F. for more than 6 hours to maximize the extraction of certain broth fraction and/or compounds. The broth obtained from this process was designated “AAC1” for internal reference. More particularly, USDA inspected chopped raw chicken bones remaining after major muscles were removed were cooked in water in a large commercial stainless steel cooking tank. After cooking, the liquid broth portion was separated from the chicken solids by decanting. The broth was concentrated in a commercial evaporator then spray dried and packed in labeled containers.

Example 3 Comparing the Protein and Amino Acid Profiles of Different Broths

(37) Proteins are large molecules composed of one or more chains of amino acids that perform a wide array of functions in biological systems including, for example, functioning as enzymes, facilitating cell communication, and providing structural support to cells. Humans, as well as other animals, obtain essential amino acids from protein consumed as part of their diet since they lack enzymes needed to synthesize them. Ingestion of proteins leads to their break down into amino acids through the digestive process. The amino acids can then be used in protein biosynthesis in muscle production and maintenance, glucose production, serve as a dietary nitrogen source, and serve as a fuel source if necessary. The objective of this study was to determine the concentration and size range of proteins in chicken broth AAC1 as compared to a home-made product.

(38) One sample prepared according to this disclosure (AAC1) and another sample prepared according to home-style cooking methods (Homemade) were compared. The percent solid (w/v) for AAC1 was 8% solid, while the percentage (w/v) for Homemade was 1.7% solid.

(39) Prior to determining the amount of protein by the Bradford method, each sample was diluted in distilled water to a final 1% w/v solution. A standard curve was prepared using bovine serum albumin (0-3.5 μg/μL). All samples were analyzed in triplicate. The amount of total protein was determined using a plate reader at a wavelength of 595 nm. Results are shown in Table 1.

(40) TABLE-US-00001 TABLE 1 Amount of total protein in broth samples Mean Sample Values Results Results Concentration SD CV AAC1 0.446 1.558 1.691 1.691 0.132 7.8 0.461 1.693 0.474 1.821 Homemade 0.544 2.496 2.52  2.52  0.034 1.3 0.549 2.544

(41) To correct for differences in the percent solids in AAC1 (8%) and Homemade (1.7%) samples, the protein values based on a 1% solution for AAC1 and Homemade were multiplied by their respective starting % solids. The final adjusted amount of total protein for AAC1 and Homemade are shown in Table 2.

(42) TABLE-US-00002 TABLE 2 Adjusted total protein concentration in AAC1 and homemade broth Adjusted Final % of Sample Concentration AAC1 AAC1 13.6 μg/ul — Homemade 4.3 μg/ul 31.6%

(43) To determine the protein profile of each sample, equal volumes of the AAC1 and Homemade samples (15.6 μl) were each mixed with Laemmli's sample buffer and a reducing agent, heated at 95° C. for 5 minutes, and separated on a 4-12% Bis Tris gel. The relative size range of the proteins was determined by comparison to a commercially available protein standard (ranging from 4.5 kDa to 300 kDa). A constant voltage of 150V was applied to the gel for 30 minutes, allowing for separation of proteins in each sample. The proteins were visualized in the gel using SimplyBlue™ SafeStain.

(44) The results are shown in FIG. 1. Lane 1 shows the protein profile for homemade broth, lane 2 for the AAC1 broth, and lane 3 is molecular weight standard. Based on the protein profiles, AAC1 is composed of approximately 3 times more protein than a homemade product. Proteins in the homemade product displayed a wider range of molecular weight distribution, containing both large (up to about 300-500 kD) and small proteins (about 5-15kD). By contrast, the majority (i.e., greater than 50%) of proteins in AAC1 has molecular weight of between 70 kDa and 15 kDa.

(45) Individual amino acid content was also analyzed. Table 3 below shows the individual amino acid content of the broth compositions prepared according to the disclosed methods as compared to those prepared using home-style methods as well as other commercial products.

(46) TABLE-US-00003 TABLE 3 Amino acid composition of different broths Table Two: Content values above calculated to 100% Commercial solids basis: 3823 product Home- Home- — W/W% % made-1 made-2 AAC1-1 AAC1-2 AAC1-3 TAURINE 2.50 ASPARTIC ACID 3.63 2.44 3.63 3.47 7.04 5.34 5.38 THREONINE 1.44 0.76 1.52 1.42 1.75 2.13 1.79 SERINE 1.56 1.34 1.96 1.89 2.08 1.88 2.40 GLUTAMIC ACID 10.25 6.98 9.44 9.26 10.52 9.50 10.14 PROLINE 3.53 3.05 5.93 5.57 9.59 10.66 11.07 HYDROXYPROLINE 2.19 2.99 5.31 4.92 na 7.00 9.39 GLYCINE 5.97 7.38 8.79 9.47 18.02 15.41 17.68 Lanthionine 0.06 0.00 0.00 0.00 0.00 0.00 0.00 ALANINE 3.91 3.37 4.60 4.58 8.06 7.53 7.53 CYSTINE 0.44 0.29 0.34 0.40 0.00 0.25 0.17 VALINE 1.44 0.73 1.43 1.42 2.44 2.19 2.21 ISOLEUCINE 1.13 0.47 1.18 1.02 1.54 1.59 1.59 LEUCINE 2.72 1.51 2.48 2.32 3.38 3.47 3.43 METHIONINE 0.81 0.52 0.93 0.77 1.01 1.06 1.10 TYROSINE 0.88 0.44 0.81 0.65 1.09 1.00 0.90 PHENYLALANINE 1.00 0.73 9.53 7.03 2.30 2.16 1.98 HYDROXYLYSINE 0.16 0.00 0.00 0.00 0.00 0.00 0.00 HISTIDINE 2.03 2.56 2.70 1.73 1.32 1.16 1.00 ORNITHINE 2.25 0.00 0.00 0.00 0.00 0.00 0.00 LYSINE 3.75 2.67 3.07 3.00 3.96 3.66 3.05 ARGININE 3.06 2.79 4.01 3.75 8.24 6.69 6.61 TRYTOPHAN 0.16 0.06 0.12 0.09 0.22 0.15 Total 54.84 41.08 67.80 62.79 82.35 82.88 87.55

Example 4 Comparing Levels of Chondroitin Sulfate (CS) in Different Broths

(47) Chondroitin sulfate (CS) is an important structural compound found in cartilage and is implicated in joint health. CS has been shown to reduce the levels of many inflammatory mediators such as iNOS, PGE2, COX-2 (Gwendolyn, Spine 2011), and NFkB (Vallières, Osteoarthritis Cartilage, 2010). The goal of this study was to determine the amount of CS in various chicken broth samples. Chicken broths were analyzed for total CS using enzymatic digestion and LC-UV detection (Ji, Journal of AOAC International, 2007). Results were calculated as area on the curve compared to standard samples and the limit of quantification as 8 mg chondroitin sulfate/g dry material (Table 4). Among all chicken broths tested, AAC1 had the greatest amount of CS (8.797% w/w).

(48) TABLE-US-00004 TABLE 4 Amounts of chondroitin sulfate (CS) Average Dry Dry Samples (mg) mg/g % w/w Powdered Chicken 17.480 87.971 8.797 Broth AAC1 Powdered Chicken 15.443 67.553 6.755 Broth H814 Frozen 10.886 52.059 5.206 Concentrate TSN Frozen 8.627 42.104 4.210 Concentrate IDF Chicken 823 Home Style Broth 5.866 28.258 2.826 from Back Bones Frozen 5.243 26.227 2.623 Concentrate Turkey 824 Powdered Chicken 4.979 24.335 2.434 Broth HML 34511 Powdered Chicken 3.817 18.305 1.830 Broth A1004 Powdered Chicken 3.075 15.054 1.505 Broth P1301 Home Style Broth 2.185 10.99 1.09 from Chicken Parts Home Style Broth 1.937 9.361 0.936 from Chicken Necks

Example 5 Comparison of Concentration of Polyphenols in Different Chicken Broths

(49) The concentration of polyphenols in different chicken broths was determined using a modified Folin-Ciocalteau method (Slinkard, American Journal of Enology and Viticulture, 1977). Polyphenols are a class of chemical compounds known to have anti-oxidant and anti-inflammatory properties. While both AAC1 and home-style broths contained polyphenols, AAC1 contained 4828.8 μg/mL GAE of polyphenols, which is approximately 7 times more GAE of polyphenols than a homemade broth (687.7 μg/mL GAE) made from the same kind of chicken parts.

Example 6 Effect of the Broth Composition on Metabolic Syndrome in Obese Rats and Lean Healthy Rats

(50) In this test, AAC1, Homemade4 (854.5 mg/kg/day and 312.3 mg/kg/day respectively) broth, or water control were fed to Obese rats or lean healthy rats and the effects were assessed.

(51) There were many models used to study MS; however, the Zucker Fatty Rat appeared to be the preferred choice for this type of study. The Zucker Rat contains a genetic spontaneous autosomal recessive mutation on Chromosome 5. This mutation promotes the development of obesity by 5 weeks of age, and continues throughout their lives. As seen in human presentation of MS, as the obesity state develops, the animals present with hyperglycemia, mild glucose intolerance, hyperlipidemia, and moderate hypertension. In this study, we were working with a factorial design with repeated measures. Assuming that we were anticipating a medium effect size (f=0.20) were α=0.05 powered at 0.80, we anticipated the use of 48 animals (24 Zucker Lean, 24 Zucker) in order to suitably observe the effects detailed in this study. We looked for changes in 6 groups (Lean+Water, Lean+AAC1, Lean+Homemade, Obese+Water, Obese+AAC1, Obese+Homemade) allowing for an n=8 in each condition.

(52) A total of 24 Zucker lean (cat #186) and 24 Zucker Obese (Cat #185) were purchased from Charles River Laboratories at 8 weeks of age and allowed to acclimate to the facility conditions for 1 week prior to initiation of studies. During this time, investigators handled the animals for 5 minutes each day to acclimate the animals to the investigators involved in this study. Once acclimated to facility conditions, animals were acclimated to blood pressure holding devises used in this study. Animals entered the study at the age of 10 weeks old.

(53) Two chicken broths (ACC1 and Homemade 4) were used in this study. Broth ACC1 was made at a 1% (w/v) by placing 10 g of powdered broth in a clean, autoclaved bottle. The bottle was then filled to the 1 L mark with filtered water. Solutions for Homemade broth were made in a similar fashion. Broth was measured out into 125 mL increments and placed into clean bottles as previously described. The bottle was then filled to the 1 L mark with filtered water, leaving the final concentration 0.415% solid. All solutions were then placed in a warm, sonication bath for 30 minutes to allow all components of the broths to evenly go into solution. Broths were placed in the refrigerator until they were used. Animals that received broth diets were fed their assigned broth for a complete twelve weeks after initial baseline assessments prior to any additional testing. Bottles were changed three times a week in order to ensure fresh product was consumed and to avoid any bacterial contamination in the water bottles during feeding.

(54) While AAC1 or Homemade4 (H4) (854.5 mg/kg body weight/day and 312.3 mg/kg body weight/day respectively) broth did not seem to have a statistically significant effect on weight gain (Data not shown), Food Intake (Data not shown), Girth (Data not shown), Blood Pressure (Data not shown), or HbA1c (Data not shown), some trends were observed in Triglyceride (FIG. 2) and Cholesterol levels (FIG. 3). For example, Obese animals that were fed AAC1 showed reduced change in circulating Triglyceride levels as compared to both the Obese water group and Obese Homemade4 fed group. Note that considerable variations existed in these groups.

(55) Although no significant increase in HDL levels were observed, significant differences existed with respect to LDL levels in the AAC1 group as compared to the control groups fed with water or H4 broth. These findings suggested that AAC1 broth lowered LDL levels, similar to the effect of statin drugs. Importantly, an improved (protective) change in the ratio of LDL to HDL in the Obese animals on the AAC1 diet was also observed as compared to the H4 or water controls.

(56) Consumption of AAC1 or Homemade4 (883.3 mg/kg body weight/day and 327.8 mg/kg body weight/day, respectively) broth did not seem to cause a significant change in Lean healthy rats as compared to the control group fed with water alone. No differences in weight (Data not shown), food intake (Data not shown), Girth (Data not shown), Blood Pressure (Data not shown), or HbA1c (Data not shown) were observed. However, AAC1 appeared to lower the Triglycerides levels as compared to the control group fed with water alone (Data not shown). AAC1 also appeared to lower the LDL levels as compared to the control group fed with water alone (Data not shown). Note that considerable variations were observed in these groups.

Example 7 Effect of the Broth Composition on COX-2 Activity

(57) The COX family of enzymes is responsible for the synthesis of prostanoids such as prostaglandins. COX-1 is constitutively active while COX-2 is inducible and hence is upregulated during inflammation and pain. Compounds that block COX-2 activation while not effecting COX-1 are of particular interest due to their ability to block inflammation while not causing unwanted side effects mediated by blocking COX-1 activity. The level and specificity of COX inhibition by the chicken broth prepared according to the disclosed methods was investigated.

(58) Using an in vitro COX enzyme inhibitor assay (Abcam), chicken broth samples were assayed for their ability to block COX-1 and COX-2 activity according to the manufacturer's protocol. The results are shown in Table 5.

(59) TABLE-US-00005 TABLE 5 Selective inhibition of COX-1 and COX-2 Ratio of Sample Enzyme % Inhibition COX2/COX1Inhibition Powdered IDF AAC1 COX-1 −2.3 18.34 COX-2 42.4 Frozen IDF 823 COX-1 −51.5 0.43 COX-2 22.1 Home Style Broth COX-1 −17.9 1.57 from Back Bones COX-2 28.1 Home Style Broth COX-1 −32.5 0.78 from Necks COX-2 25.3 IDF Turkey 824 COX-1 −21.7 1.51 COX-2 32.7 Chicken Broth K COX-1 −25.5 1.21 COX-2 30.8 Commercial Broth COX-1 −5.1 5.16 TSN COX-2 26.3 Commercial Broth COX-1 −39.9 0.28 H814 COX-2 11.3 Commercial Broth COX-1 −9.2 4.01 A1004 COX-2 36.7 Commercial Broth COX-1 −27.0 1.01 HML COX-2 27.3 Commercial Broth COX-1 −19.9 2.36 PLNT COX-2 47.1 Home Style Broth COX-1 −5.2 0.67 from same raw parts COX-2 3.5

(60) Broth labeled “Home Style Broth from same raw parts” was a broth prepared using traditional home-style cooking method with the same raw materials as those used for preparing AAC1. As shown in Table 5 above, chicken broth AAC1 showed the greatest ratio of COX-2 inhibition to COX-1 inhibition (18.34) with greater than 40% inhibition of COX-2 enzyme activity as compared to other commercial products or “Home Style Broth from same raw parts.”

Example 8 Effect of the Disclosed Broth on Joint Diseases by Regulating Protein Kinase A

(61) Temporomandibular Joint Disorder (TMD) is a disease affecting the temporomandibular or jaw joint (TMJ), the muscles of mastication, or both. TMD is believed to be caused by activation of neurons and glia cells located in the trigeminal system (Tjakkes et al., 2010). The prevalence of TMD symptoms have been reported in up to 93% in the general population with varying incidence rates (Zhao et al., 2011). Further development of TMD may also lead to the development of a chronically sensitized state of the disorder.

(62) Protein kinase A (PKA) is a member of the family of cyclic AMP (cAMP) dependent enzymes that act as pro-inflammatory molecules in peripheral and central nervous systems. Increased levels of PKA expression by sensory nociceptive neurons has been reported during the chronic sensitized state. The objective of this study was to study the effects of commercially available broths, home-style broths, and the broths according to the instant disclosure. These various products may contain certain anti-inflammatory molecules that have some effects on TMD. These molecules may exert their effects through PKA in regulating the development and progress of TMD.

(63) Three chicken broths (AAC1, TSN, and a homemade broth) were investigated in this study. Broth AAC1 (8% solids) was prepared at a concentration of 0.5% (w/v). First, 5 g of powdered broth was placed in a clean, autoclaved bottle. The bottle was then filled to the 1 L mark with filtered water and the mixture was stirred to allow the powder to dissolve in water. A homemade style broth (1.7%) was also tested as a control. To achieve a dosage that would allow for the comparison of AAC1 and the homemade-style broth, the homemade broth was diluted 16 fold with filtered water by placing 62.5 mL of stock broth and diluting the broth to 1 L. A commercially available broth, TSN, was also tested as another control. To ensure TSN was tested at a concentration similar to that of AAC1, the solution for TSN (33.3% solid) was made in a similar manner. The TSN broth was allowed to warm until a consistency of the stock broth was non-gelatinous. Broths were measured out into 16.7 g increments and placed into clean bottles as previously described. The bottle was then filled to the 1 L mark with filtered water. All solutions were then placed in a warm, sonication bath for 30 minutes to allow all components of the broths to be evenly solubilized. Broths were stored in the refrigerator until they were administered to the animals via water bottle administration for 2 weeks prior to TMD induction.

(64) Adult Sprague-Dawley male rats (200 g-300 g) were used in this study. The rats were divided into three groups, with each group being fed with water, a commercial product (TSN), or AAC1 broth prepared according to this disclosure.

(65) Mechanical stress was applied to each group and upper spinal cord tissues containing the spinal trigeminal nucleus (STN) samples were obtained and processed for immuno-staining. Prior to immunostaining, slides were placed at room temperature and covered with 1×PBS for 5 minutes. PBS was removed, and liquid was replaced with a 5% normal donkey serum 0.1% triton solution for 20 minutes. Tissues were then washed with 5 mL 1×PBS. Working dilutions of rabbit anti-rat PKA (BD Biosciences, San Jose, Calif.; 1:500) were made using 5% normal donkey serum. Primary antibodies were allowed to incubate on tissues (100 μl/tissue) for 3 hours at room temperature. Samples were then washed with 5 mL 0.1% Tween 20-PBS and 2 mL 1×PBS. Working dilutions of donkey anti-rabbit IgG Alexa 488® was made by diluting stock antibodies 1:200 in 1×PBS. Secondary antibodies were allowed to incubate on sample slide (100 μl/tissue) for 1 hour at room temperature while protected from light. Slides were again washed with 5 mL 0.1% Tween 20-PBS and 2 mL 1×PBS, and mounted for fluorescent analysis using Vectashield fluorescent mounting media containing the fluorescent dye DAPI. Slides were cover slipped, sealed using clear nail polish, and stored at 4° C. until microscopic images were collected.

(66) A Zeiss Z1 imager with apotome was used to acquire 10× Z-Stacked images of the V3 areas of the trigeminal ganglia and medullary horn of the STN. Zen 2011 software was used to evenly balance the background of each image prior to analysis. Gray scale jpeg images were opened in ImageJ software, where 10 non-overlapping regions of interests (RIOs) with an area of equal size were placed in areas representative of protein expression in each image, and the integrated pixel densities were measured. Background intensities were also acquired through similar procedure, and averaged. The average background intensity acquired from each image was then subtracted from each integrated density values from areas of interest. Subtracted integrated densities were averaged and fold changes were calculated as the average change±SEM from TMD control levels. Statistical differences were determined using the Mann-Whitney U test in SPSS software, and were considered to be different when p<0.05.

(67) The data in FIG. 4 provide evidence that elevated PKA levels caused by prolonged jaw opening are greatly repressed in AAC1 fed animals when compared to levels in animals consuming either water or a commercial product (TSN). In Table 6, the results of several experiments are summarized as the average fold change in the intensity of immunostaining when compared to water whose mean intensity was made equal to one.

(68) TABLE-US-00006 TABLE 6 AAC1 repression of elevated PKA levels in response to jaw stress caused by prolonged jaw opening Average Fold P Value P Value Broth Change (vs. TMD) (vs. TSN) Water 1.00 ± 0.06 1.000 0.239 (TMD) AAC1 0.75 ± 0.02 0.001 0.000 TSN 0.94 ± 0.04 0.239 1.000

(69) The AAC1 broth disclosed herein showed a greater ability to modulate the levels of PKA in the STN following joint stress when compared to a commercially available product. Based on the difference in composition of AAC1 and a homemade broth, we would predict that AAC1 would be significantly better at repressing stress-induced elevations in PKA levels in the STN.

Example 9 Nocifensive Responses of AAC1 as Compared to Other Broth Products

(70) Temporomandibular Joint Disorder (TMD), is characterized by the continuation of pain behavior and sensation despite a decrease in nociceptive inputs (Herb et al., 2006). The increased sensitivity can be attributed to the development of a chronically sensitized state of the nerves that provide sensory innervation of the joint, muscles, ligaments, and tendons. TMD patients often report that their symptoms negatively affect other aspects of their life (Sessle, 2008). Some TMD patients develop protective behavior modifications that may limit or minimize their everyday activities. This protective behavior is termed “nocifensive” behavior. The objective of this study was to determine if AAC1 chicken broth could reduce TMJ stress-induced nocifensive behaviors in response to mechanical stimulation, and how its performance compares to homemade broth and commercially available products.

(71) Three chicken broths (AAC1, TSN, and a home-style broth) were investigated in this study. Broth AAC1 (8% solids) was made at a 0.5% (w/v) while a homemade style broth (1.7%) was tested at a dose that would allow for an equal comparison of the ratio of percent solids between AAC1 and a homemade broth. Thus, the homemade broth was diluted 8 fold with filtered water by placing 62.5 mL of stock broth, and diluting the broth to 1 L. To test a competing commercially available broth at a similar concentration, a solution for TSN (33.3% solids) was made in a similar manner with the modification that the broth was allowed to warm until a consistency of the stock broth was non-gelatinous.

(72) Broths were measured out into 16.7 g increments and placed into clean bottles as previously described. The bottle was then filled to the 1 L mark with filtered water. All solutions were then placed in a warm, sonication bath for 30 minutes to allow all components of the broths to evenly solubilize. Broths were placed in the refrigerator until they were administered to the animals via water bottle administration for 2 weeks prior to TMD induction.

(73) Adult Sprague-Dawley male rats (200 g-300 g) were housed separately in clean, standard plastic rat cages (VWR, West Chester, Pa.) with non-restricted access to both food and water in a room with 12 hour/light dark cycles. Three consecutive days prior to testing, animals were allowed to enter the Ugo Basile Durham animal holding device (Ugo Basile, Collegeville, Pa.) for 5 min to acclimate to testing conditions. During this acclimation period, stimulation of hair follicles and epidermis located in the masseter and TMJ region of the face occurred by gently rubbing the area with a pipette tip. This stimulation was used to improve the condition of the animal to the testing procedure, thus reducing the number of false reactions to testing filaments. Baseline nocifensive behaviors were assessed by utilizing a modified version of the well-established von Frey method. A series of calibrated von Frey filaments were applied in increasing force to the cutaneous area over the masseter muscle. Prior to application, the muscle was palpated with the tip of the testing filament to insure proper placement. Once placement was established, a scientist blinded to the experimental conditions placed enough force on the location to accomplish a bend in the filament. Reactions observed after initiation of force to the area and prior to the bend of the filament, were verified by one other scientist and recorded. Each filament was applied 5 times, and recorded as the number of reactions obtained from 5 applications of each specific calibrated filament. Measurements were collected over both the right and left masseter muscles of each animal, which were averaged together to obtain a combined average number of reactions out of 5. Baseline readings were prior to 2 week feeding of all chicken broth products and again 24 hours prior to TMD induction.

(74) The number of nocifensive responses after 2 hr, 3 days, or 7 days in animals experiencing a mechanically induced TMD pathology was measured. The results of this study, which are shown in FIG. 5, demonstrate that AAC1 significantly reduces the number of nocifensive responses after 2 hr, 3 days, or 7 days in animals experiencing a mechanically induced TMD pathology. AAC1 significantly decreases the number of nocifensive responses as compared to TMD animals that consumed only water. The TSN product did not cause a decrease in nocifensive responses at any of the measured time points. Thus, while product AAC1 reduces nocifensive behaviors in animals experiencing chronic inflammation resulting from the prolonged jaw opening TMD model, this ability is not exhibited by a commercially available product. Based on results obtained from previous examples, as well as data presented in this example, we would predict that a homemade broth product would not significantly reduce TMJ stress induced nocifensive responses.

(75) All animal studies described herein were performed using approved protocols in compliance with government rules and regulations. Changes may be made in the above methods and systems without departing from the scope hereof. It should thus be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover generic and specific features described herein, as well as statements of the scope of the present method and system, which, as a matter of language, might be said to fall there between.

(76) Although each of the embodiments described above has been illustrated with various components having particular respective orientations, it should be understood that the system and methods as described in the present disclosure may take on a variety of specific configurations with the various components being located in a variety of positions and mutual orientations and still remain within the spirit and scope of the present disclosure. Furthermore, suitable equivalents may be used in place of or in addition to the various components, the function and use of such substitute or additional components being held to be familiar to those skilled in the art and are therefore regarded as falling within the scope of the present disclosure. Therefore, the present examples are to be considered as illustrative and not restrictive, and the present disclosure is not to be limited to the details given herein but may be modified within the scope of the appended claims.

(77) All references cited in this disclosure, including patents, patent applications, scientific papers and other publications, are hereby incorporated by reference into this application.