Claims:We Claim:

1. A method of reducing interleukin-6 levels in an obese human comprising administering to the obese human an effective amount of a fructo-oligosaccharide composition.

2. The method as claimed in claim 1, wherein the fructo-oligosaccharide composition comprises about 50 to about 99%fructo-oligosaccharides.

3. The method as claimed in claim 1, wherein the fructo-oligosaccharide composition comprises about 90 to 95% fructo-oligosaccharides.

4. The method as claimed in claim 2 or 3, wherein the fructo-oligosaccharide comprises about 40-50% 1-kestose(GF2), 40-50% nystose (GF3) and 5-10% fructofuranosylnystose (GF4).

5. The method as claimed in claim 1, wherein the fructo-oligosaccharide composition is in the form of a liquid or a solid dosage form for oral administration.

6. The method as claimed in claim 1, wherein the fructo-oligosaccharide composition is in the form of an oral syrup.

7. The method as claimed in claim 6, comprising administering 2- 20 ml of the fructo-oligosaccharide composition.

8. The method as claimed in claim 1, comprising reducing C-reactive protein levels in the obese human.

Dated 26th day of April, 2017

Essenese Obhan
Of Obhan & Associates
Agent for the Applicant
Patent Agent No. 864 , Description:FIELD OF THE INVENTION
The present disclosure relates to a method of reducing interleukin-6 (IL-6) levels in an obese human.

BACKGROUND
Obesity in humans is associated with chronic activation of certain cells such as monocytes, macrophages, and neutrophils in the immune system, as well as adipocytes in fat tissue, which results in an abnormal production of cytokines such as tumor necrosis factor type-alpha (TNF-a), interleukins 1 and 6 (IL-1a, ß and IL-6), interferon-gamma (IFN-?), monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and leptin. IL-6 is one of the principal cytokines mediating immune response in humans. The increased levels of these cytokines, particularly IL-6, leads to the development of metabolic disorders and complications such as insulin resistance, hyperinsulinemia, hypertension, lipid disorders, type 2 diabetes, cardiovascular diseases, cancer, osteoarthritis, gallstones and mental health problems. Therefore, the modulation of cytokines, specifically IL-6 is critical to avoid the development of these metabolic disorders and the associated complications in obese humans.
Use of peptides (such as lunasin, a soy peptide) and antibodies (such as tocilizumab) that antagonize IL-6 in humans is an established therapy in many inflammatory diseases and many cancers. However, these interventions are expensive. Thus, there is a need for a cost-effective method of regulating IL-6 and associated immune response in obese human population which is effective in prevention as well as treatment of metabolic disorders such as insulin resistance, hyperinsulinemia, hypertension, lipid disorders, type 2 diabetes, cardiovascular diseases, cancer, osteoarthritis, gallstones and mental health problems.

SUMMARY OF THE INVENTION
A method of reducing interleukin-6 levels in an obese human is disclosed. The method comprises of administering to the obese human an effective amount of a fructo-oligosaccharide composition.

BRIEF DESCRPTION OF DRAWINGS
Figure 1A depicts a diagram showing IL-6 level in subjects (test and control) before the beginning of a study carried out in accordance with examples of the present disclosure.
Figure 1B depicts a diagram showing IL-6 level in subjects before and after the study comprising administering 10 ml of fructo-oligosaccharide composition as supplement (FOS intervention) carried out in accordance with examples of the present disclosure.
Figure 1C depicts a diagram showing IL-6 level in subjects before and after the study comprising administering 10 ml of sugar solution (sugar intervention) carried out in accordance with examples of the present disclosure.
Figure 2 depicts a diagram showing IL-6 distribution before and after FOS intervention carried out in accordance with examples of the present disclosure.
Figure 3 depicts a diagram showing IL-6 distribution before and after sugar intervention carried out in accordance with examples of the present disclosure.
Figures 4 depicts a density plot of IL-6 level of the subjects (moderate active obese) after FOS intervention is carried out in accordance with examples of the present disclosure.
Figure 5 depicts a density plot of IL-6 level of the subjects (moderate active obese) after sugar intervention is carried out in accordance with examples of the present disclosure.
Figures 6 depicts a diagram showing IL-6 level of the subjects (consolidated) after FOS intervention is carried out in accordance with examples of the present disclosure.
Figure 7 depicts a diagram showing IL-6 level in subjects (consolidated) after sugar intervention is carried out in accordance with examples of the present disclosure.

DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the disclosed product and process, and such further applications of the principles of the invention therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to be restrictive.
Reference throughout this specification to “one embodiment” “an embodiment” or similar language means that a particular feature, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The present disclosure relates to a method of reducing interleukin- 6 (hereinafter referred to as IL-6) levels in an obese human. The method comprises administering to the obese human an effective amount of a fructo-oligosaccharide composition (hereinafter referred to as FOS Composition).
The obese human is a human having a Body-Mass Index (BMI) of more than 25 kg/m2. The obese human may belong to any age group and includes both men and women.
In accordance with an embodiment, the FOS composition comprises about 50 to about 99% fructo-oligosaccharide. In accordance with a specific embodiment, the FOS composition comprises about 90 to about 95% fructo-oligosaccharide.
In accordance with an aspect, the fructo-oligosaccharide in the FOS composition is a mixture of oligosaccharides consisting of a sucrose molecule (glucose -fructose disaccharide, GF1) linked to one (GF2), or two (GF3) or three (GF4) additional fructose units added by ß-2-l glycosidic linkages to the fructose unit of the sucrose. The GF2, GF3, and GF4 oligosaccharides, are also called 1-kestose, nystose and fructofuranosylnystose, respectively.
In accordance with an embodiment, the fructo-oligosaccharide in the FOS composition comprises about 40-50% 1-kestose (GF2), 40-50% nystose (GF3) and 5-10% fructofuranosylnystose (GF4).
In accordance with an embodiment, the FOS composition is a composition for oral administration. In accordance with an embodiment, the FOS composition is in the form of a solid dosage form or a liquid dosage form. In an example, the FOS composition is in the form of an oral syrup.
In accordance with an embodiment, the FOS composition is administered to the obese human as a medicament. In accordance with an alternative embodiment, the FOS composition is administered to the obese human as a nutraceutical supplement. The FOS composition may be administered to the obese human along with water, food or a drink.
In accordance with an embodiment, the FOS composition in the form of the oral syrup is administered to the obese human in a dose of 2 to 20 ml. In an example, 10 ml of the oral syrup is administered.
The present disclosure also provides a method of reducing C-reactive protein (CRP) levels in the obese human.CRP is a blood test marker for inflammation in the body. CRP is produced in the liver and its level is measured by testing the blood. It has been found that by administrating the FOS composition as disclosed, the CRP levels are reduced in the obese human.

Examples- Effect of FOS on IL-6 levels in Obese Humans

Subject selection
A randomized double blind trial was conducted on 55 obese subjects having a Body Mass Index of more than 25. Specific inclusion and exclusion criteria were followed to consider a subject for the trial. Inclusion criteria for the subject were based on age (20-50 yrs), body mass index (BMI >25) and willingness to consume FOS supplement. Subjects with reported diabetes, hypertension, CVD, celiac disease, HIV, cancer, renal disorder and liver disorder, infections and inflammatory disorders and hospitalization history in the last one month were excluded. Underweight subjects, highly active subjects and subjects already on different supplements were also excluded. Subjects reporting a low expression of IL-6 (0.1 pg/mL) were also excluded from the study. Each of the subject was randomly assigned to either a control group or a test group. For assessment of results, the subjects were also divided into two groups: sedentary obese group and moderately active group, based on Metabolic Equivalent (MET) of the subjects. ‘MET’ is defined as the ratio of the work metabolic rate to the resting metabolic rate. In other words, one MET is equal to 1 kcal/kg/hour and is roughly equivalent to the rate of energy expenditure while at rest.
The subjects with an activity of less than 600 MET minutes per week were classified as sedentary, those with activity level between 600-3000 MET minutes per week were classified as moderately active and above 3000 MET minutes per week were classified as vigorously active.

The Table 1, below provides that distribution of subjects for the present study:
Test group (FOS composition given as supplement) Control group (Sugar given as supplement)
Sedentary 21 11
Moderate 9 14
Consolidate 30 25

Trial and monitoring
The trial comprised of a daily intervention of 10 ml of either FOS composition or sugar (sucrose) as supplement for 30 days. Depending on the group (control or test group), each subject was provided with the appropriate supplement. The supplement was consumed along with either meals or fluids.
Monitoring of the daily supplement intake was carried out using a compliance card and daily SMS reminders were sent to each of the subjects to ensure compliance. Subjects were advised not to alter their usual calorie intakes and were asked to document any unusual symptoms or side effects and to keep a diary of illness and medications. Spot observations were carried out to validate the compliance in the subjects. Subjects were required to provide blood samples before and at the end of each intervention. Blood was drawn from each subject on the 0thday of the study i.e., on the day before the subjects were to take the supplement and on the 30thday of the study.

Biochemical evaluation and assay
Venous blood sample was collected in clean, sterilized vacuum containers and allowed to stand at room temperature for 15 minutes. Serum was immediately separated and stored at -80°C until analysis. The sample was then analyzed for IL-6 using standardized IL-6 kits.IL-6 was determined using HMHMAG-34K- MILLIPLEX MAP Metabolic Hormone Magnetic Bead Panel.

Statistical Assessment:
Statistical assessment of the data was performed using the R framework and associated standard R packages. Distribution of the samples was assumed to be normal and t-test was used to report difference between distributions. In addition, cumulative distribution function was plotted to visualize the difference between the distributions.

Results

Sedentary obese data
Figure 1A provides the IL-6 level in subjects (test and control) before the beginning of the study. The mean distribution of IL-6 level in test and control subjects are similar as observed in Figure 1A. Figure 1B provides the IL-6 levels before and after study in subjects given 10 ml of FOS composition as supplement and Figure 1C provides IL-6 level before and after study in subjects given 10 ml of sugar (sucrose) solution. Cumulative distribution function was also plotted to visualize the difference between the distributions. Figure 2 provides the data for IL-6 distribution before and after FOS intervention and Figure 3 provides the data for IL-6 distribution before and after sugar intervention
For subjects given10 ml FOS composition as a supplement, significant fall in the IL-6 levels was observed (Figure 1B). However, similar results were not observed when sugar solution was given a supplement (Figure 1C).

Moderately active obese
Few subjects reported an IL-6 level below 0.01 in pre-FOS intervention stage. These subjects did not qualify for the objective and design of the current study. Data was refined by dropping all subjects reporting IL-6 level’s below 0.01 at initial stage.
Following data refinement, assessment suggested significant reduction in IL-6 levels on FOS intervention in subjects as is evident for the Table 2, below:
Table 2: IL-6 levels in subjects (moderately active obese) pre- and post-FOS and sugar intervention.

* Statistically significant value
Figures 4 provides a density plot of the IL-6 level of the subjects (moderate active obese) who were given 10 ml of FOS composition and Figure 5 provides a density plot of the IL-6 level of the subjects (moderate active obese) who were given 10 ml of sugar solution. For subjects given 10 ml FOS composition as a supplement, significant fall in the IL-6 levels was observed (Figure 2). However, similar results were not observed when sugar solution was given a supplement (Figure 3).

Consolidated obese data
Data comprised of sedentary and moderately active obese subjects. Assessment was performed on refined dataset as described previously. Table 3 provides the consolidated data.

Table 3: IL-6 levels in subjects (consolidate) pre- and post-FOS and sugar intervention.

Figures 6 provides the IL-6 level in subjects (consolidated) who were given 10 ml of FOS composition and Figure 7 provides the IL-6 level in subjects (consolidated) who were given 10 ml of sugar solution. It is observed that intervention using FOS in obese subjects (sedentary or moderately active) reduces IL-6 levels.

Objective: Assess change in C-reactive protein(CRP) levels on FOS interventions
The CRP level was also measured for the subjects. Figure7 shows the result of FOS intervention on CRP levels. On intervention with FOS there is a fall in the CRP levels in the subjects. Similar drop is not observed in subjects intervened with sugar.

INDUSTRIAL APPLICABILITY
The process as disclosed provides a unique dietary intervention method for controlling and regulating expression of IL-6 (and immune response) in obese humans. The method disclosed is thus effective in prevention as well as treatment of metabolic disorders such as insulin resistance, hyperinsulinemia, hypertension, lipid disorders, type 2 diabetes, cardiovascular diseases, cancer, osteoarthritis, gallstones and mental health problems associated with obesity in humans.