DESC:TECHNICAL FIELD
The present disclosure relates to DINI Axis links diet to NCDs thus opens an avenue where only alteration in diet could help to curb the emergence of non-communicable diseases (NCDs) like Diabetes, Stroke, some kinds of Cancers, etc. More specifically, it relates to Diet-Infection-Nutrition-Immunity (DINI) Axis, providing a formulation and process for preparing the formulation”

BACKGROUND
The existing state-of-art mentions that Nutrition is a fundamental aspect of life, and it is linked to multiple components, systems, and processes, including those occurring during diseases, such as infection. It describes the relationship between nutrition and infection, with a focus on how malnutrition affects susceptibility to infection and the burden that infections, especially foodborne infections. A foodborne disease occurs as the result of the ingestion of contaminated food products, and it includes a huge number of illnesses caused by different pathogens. The most severe cases tend to occur in the very old, in the very young, in those who have compromised immune system function, and in healthy people exposed to a very high dose of an organism.
It mentions the ingestion of pathogens in quantities that causes symptomatic infections and how it adversely effects the body. The role of nutrition at such conditions to bring equilibrium in the disturbed immunity system of the body invaded by these pathogens in a toxic amount.

The drawback of the existing concept is that it fails to take into account the pathogens we are exposed every day that are ingested by an individual from the food we eat or the environment on a daily basis. They do not necessarily cause diseases however it does cause changes in the body. It affects the infection status whenever a foreign particle enters the body. That's how diet and infections are related, which is failed to be considered in the existing system.
Even though the infections are setting up, these pathogens can remain asymptomatic maybe due to the quantity in which they are ingested or in their level of pathogenicity, but they have an impact on the body. There is always a mild infection that is related to diet.

This alter in the infection status also alters the body's nutrient, specially the micronutrient status. That is why there is always either depletion or changes in the nutritional status because of the low profile infections which is acquired by the diet. Micronutrients play a major role in our immunity and though we are able to fight with diseases there's always a negative impact on the immunity structure happening every time because of diet or food. At any stage the body is having an infection stage; there are changes in the nutrients which hence affects the immunity system of the body. This axis is not considered in the existing system.

An infectious (communicable) disease is defined as a malady caused by a specific infectious agent or its toxic products that results from transmission of that agent or its products from an infected person, animal or reservoir to a susceptible host. This agent can be transmitted either directly or indirectly through an intermediate plant or animal host, vector or inanimate environment. Contrastingly, NCD is defined as an indication having no known causative agents to be transmitted from one affected individual to another. As time rolled on, scrupulous scientific observation has stumbled upon with an increasing number of NCDs being associated with an infectious risk factor, blurring the distinction between an infectious disease and NCD. It is, therefore, imperative to bring to the fore their linkages and interactions (1).
The human microbiota consists of a panoply of microbes (including bacteria, fungi, and viruses) living in the body and plays crucial roles in many physiological functions, including digestion, immune responses, metabolism, etc. Although microbes reside on many body sites, the majority are in the gut, with bacteria being the most widely studied. However, the extent to which microbial dispersal between humans contributes to NCDs remains unclear and subject to intense investigation. It turns out that gut microbiota is transmissible within both family and social networks. Since families share similar diets and environments, their microbiota is expected to be similar. Interestingly, spousal relationships can be determined on the basis of gut bacterial analysis. Nonetheless, it is assiduously arduous to decouple environment from microbiota, which renders the investigation of the transmissibility of NCDs a bit challenging (2). Robert Koch’s postulates (published in 1890) have been ingrained into generations of microbiologists as requirements to be filled in order to infer that a specific bacterium causes disease. While these postulates are known to have limitations, they can still be seen as a framework to understand microbial causes of ailments: (i) the microorganism must be present in all cases of the disease (ii) it can be isolated from the diseased host and grown in pure culture (iii) the one from the pure culture must cause the disease when inoculated into a healthy, susceptible laboratory animal (iv) the microorganism can be re-isolated from the new host and shown to be the same as the originally inoculated microorganism (3).
A way to be able to establish some causal links beyond mere correlations between gut dysbiosis and occurrence of NCDs is therefore needed. For instance, in case of cardiovascular diseases (CVD), the most prevalent NCD worldwide, there are strong correlations with the prevalence of particular gut microbiota that encode the enzyme choline trimethylamine (TMA)–lyase (CutC) that metabolizes phosphocholine and carnitine (from red meat) into TMA, which then undergoes hepatic oxidation into trimethylamine oxide (TMAO). To our intrigue, transmissible dysbiotic microbiota are known to play some role in some cancers (3). The inventors are now exploring the field to establish molecular pathways linking gut dysbiosis and NCDs. At the same time, the inventors are looking for immunomodulatory therapeutic nutrition to prevent and/or manage NCDs. Together, the inventors have termed it “Diet-infection-nutrition-immunity (DINI) Axis” and intend to investigate the phenomenon and provide a proof-of-concept study for colorectal cancer (CRC) in a murine model.

CRC is one of the most common cancers and carries a major health burden. Globally, among all cancers, CRC ranked third in incidence (with over 1.8 million new cases) and second in mortality (with over 860,000 deaths) in 2018. It had been predicted that in 2020, approximately 147,950 individuals would be diagnosed with CRC and 53,200 would die from the disease in the US (4, 5). As with many diseases, tumour formation in the large bowel is multifactorial and various genetic and environmental factors contribute to disease development (6). Among environmental factors, the role of microorganisms in cancer biology has been increasingly recognized. Of new cancer cases in 2012, infectious agents have been estimated to account for more than 15% of all cancers (7), including the vast majority of gastric cancers (Helicobacter pylori), hepatocellular carcinomas (hepatitis B and C viruses) and cervical cancers (human papillomavirus). Although the oncogenic mechanisms of specific infectious agents have been studied, researchers have also begun to investigate the collective microbial community in the tumour environment. This population of microorganisms, also known as a microbiota, has emerged as an important environmental factor for some cancers, including those of the colorectum, liver, biliary tract, and even the breast (8). Harbouring approximately 3 × 1013 bacteria, the colorectum interacts with a large number of microorganisms with which the intestinal epithelium has a constant crosstalk (9). These microorganisms are important for gastrointestinal physiology such as energy harvest (10) and immune maturation (11), and changes in their relative abundance can alter the equilibrium, leading to intestinal and extraintestinal diseases.
A number of human shotgun or 16S ribosomal RNA (rRNA) sequencing studies have been performed to depict the CRC microbiota, in both faecal and mucosal samples (12-15). Overall, the CRC microbiota exhibits a global compositional shift (commonly termed dysbiosis) compared with the microbiota of healthy individuals, reflecting a different ecological microenvironment in patients with CRC. Besides specific bacteria, including strains of Bacteroides fragilis, Escherichia coli, Enterococcus faecalis and Streptococcus gallolyticus, being individually linked to CRC in various association and mechanistic studies, metagenomic studies in humans have identified novel associations with other bacteria (16, 17). These bacteria include Fusobacterium nucleatum (18, 19) as well as bacteria from the Parvimonas, Peptostreptococcus, Porphyromonas and Prevotella genera that showed increased abundance in faecal and tumour samples from patients with CRC (20, 21). Quantifying and detecting the fold-changes in relative abundance of these bacteria forms the principle of using the microbiota as a CRC biomarker.

OBJECTIVES
The main objective of the present disclosure is to develop therapeutic immunomodulatory nutraceutical formulations based on DINI Axis hypothesis for use in NCDs like Diabetes, Stroke, and certain types of cancers among others like colorectal cancer (CRC). In yet another embodiment, DINI Axis links diet to NCDs thus opens an avenue where only alteration in diet could help to curb the emergence of non-communicable diseases (NCDs) like Diabetes, Stroke, some kinds of Cancers, etc.
SUMMARY
This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention. This summary is not intended to identify key or essential inventive concepts of the claimed subject matter, nor is it intended for determining the scope of the claimed subject matter.
In accordance with the present subject matter, the novel approach is that the DINI Axis takes in to account the minute changes happening to the immunity system due to the daily diet we take.
It considers the effect of the environment (such as microbiome and dysbiosis) in the pathogenesis of the non-communicable diseases (NCDs) and some cancers and provides therapeutic immunomodulatory nutraceutical intervention towards the management of the diseases.
In yet another embodiment, DINI Axis links diet to NCDs thus opens an avenue where only alteration in diet could help to curb the emergence of non-communicable diseases (NCDs) like Diabetes, Stroke, some kinds of Cancers, etc.
To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof in the detailed description.
BRIEF DESCRIPTION OF DRAWINGS:
To further clarify advantages and aspects of the invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that the drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope.

Figure 1A&B illustrate CT26 Cancer cell selective cytotoxicity of formulation F3A-D in 24h. The results shown are average values for triplicate experiments performed on the same day in accordance with an embodiment of the present invention.
Figure 2A&B illustrate CT26 Cancer cell selective cytotoxicity of formulation F3A-D in 48h. The results shown are average values for triplicate experiments performed on the same day in accordance with an embodiment of the present invention. Figure 3A&B illustrate CT26 Cancer cell selective cytotoxicity of formulation F3A-D in 72h. The results shown are average values for triplicate experiments performed on the same day in accordance with an embodiment of the present invention.
Figure 4 illustrate that Formulation F3A-D are non-toxic to normal cell (48h data shown). The results shown are average values for triplicate experiments performed on the same day in accordance with an embodiment of the present invention.
Figure 5 illustrate that Formulations F3A-D significantly down regulates expression of Bcl-2 protein and upregulates the expression of Bax protein (markers for apoptosis) in CT26 colon cancer cells in accordance with an embodiment of the present invention.
Figure 6 illustrate that Formulations F3A-D induce apoptosis in CT26 colon cancer cells. The horizontal and vertical axes represent cells labelled with FITC-Annexin V and PI, respectively, in the dot plots. Dots in the upper right quadrant in each quadrant represent late apoptotic cells (positive for both Annexin V and PI). Data shown here are representative of one experiments in accordance with an embodiment of the present invention.
Figure 7A-C illustrate Growth inhibition of E. coli upon treatment of secondary metabolites (Black label) of (A) Lactobacillus casei, (B) Lactobacillus plantarum and (C) Bifidobacterium lactis. Untreated E. coli grew spontaneously (blue label) in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION:

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the 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 illustrated system, and such further applications of the principles of the invention as illustrated 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 explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, 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 an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.

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 belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Unless otherwise defined, all terms, and especially any technical and/or scientific terms, used herein may be taken to have the same meaning as commonly understood by one having an ordinary skill in the art.
In one of the embodiment of the present disclosure, composition or formulation comprising of phyto-ingredients Silybum Marianum extract (10-40 weight %), Grape seed extract (10-40 weight %), Green Tea extract (10-40 weight %), Pomegranate extract (10-40 weight %); and optionally further comprising one or more selected from the group comprising of Parsley extract (Apigenin %), Apple extract (1-10 weight %), Allium Sativum extract (1-10 weight %), Withania somnifera extract (1-10 weight %) and Curcumin (1-10 weight %).

In one of the embodiment of the present disclosure, composition or formulation comprising of phyto-ingredients Silybum Marianum extract (Silymarin 50-80 %), Grape seed extract (Oligomeric Proanthocyanidin 70-90 %), Green Tea extract (Epigallocatechin gallate, EGCG 25-50%), Pomegranate extract (Ellagic acid 40-60%); and optionally further comprising one or more selected from the group comprising of Parsley extract (Apigenin 60-80%), Apple extract (Quercetin 2.5-5.0 %), Allium Sativum extract (Allicin 2.5-5.0 %), Withania somnifera extract (Withaniolide 2.5-5.0% weight %) and Curcumin (95%), optionally further comprising one or more selected from the group comprising of Vitamin D3 (400IU with 30 % over range), Zinc (up to 0.05 weight %), Sodium Selenite (up to 0.0005 weight %), Inulin (1-10 weight %), Fructo-oligosaccharides (1-10 weight %), Medium chain triglycerides (1-15 weight %) and Docosahexaenoic acid (1-10 weight %) powder. In yet another embodiment, it further comprises a probiotic blend along with prebiotics and other micronutrients.

In yet another embodiment the present disclosure provides a composition or formulation comprising of phyto-ingredients comprising Parsley extract (Apigenin), Apple extract (Quercetin), Silybum Marianum extract (Silymarin), Grape seed extract (Oligmoeric Pranthocyanidins), Allium Sativum extract (Allicin), Green Tea extract (Epigallocatechin gallate, EGCG), Pomegranate extract (Ellagic acid), Withania somnifera extract (Withaniolides), Curcumin (95%), and a probiotic blend along with prebiotics and other micronutrients. In one of the embodiment of the present disclosure, the composition or formulation, wherein the probiotic blend comprising of one or more selected from the group comprising Lactobacillus casei (0.5 – 5 billion cfu), Bifidobacterium lactis Bb 12 (0.5 – 5 billion cfu), Lactobacillus rhamnosus GG (0.5 – 5 billion cfu) and Lactobacillus plantarum (0.5 – 5 billion cfu).
In yet another embodiment the present disclosure provides a composition or formulation, optionally further comprising one or more selected from the group comprising of Vitamin D3 (400IU with 30 % over range), Zinc (up to 0.05 weight %), Sodium Selenite (up to 0.0005 weight %), Inulin (1-10 weight %), Fructo-oligosaccharides (1-10 weight %), Medium chain triglycerides (1-15 weight %) and Docosahexaenoic acid (1-10 weight %) powder.

In one of the embodiment of the present disclosure, the composition or formulation as and when used inhibits the growth of colorectal cancer cells and remain non-toxic to the normal cell, induces apoptosis to the colorectal cancer cells via upregulating pro-apoptotic Bax protein and downregulating anti-apoptotic Bcl-2 protein.

In yet another embodiment the present disclosure provides a composition or formulation, wherein the secondary metabolites isolated from the probiotics blend present in the composition or formulation are efficacious in complete growth inhibition of E. coli and other infectious bacterial strains responsible for Colorectal cancer (CRC) pathogenesis such as – Fusobacterium nucleatum, Bacteroides fragilis, etc.

In one of the embodiment of the present disclosure, the composition or formulation as and when used for screening and treatment of Colorectal cancer (CRC) patients having various genetic mutations comprising KRAS, BRAF and others, and environmental aberrations comprising dysbiotic microbiota with cancer causing bacteria and others.

In yet another embodiment the present disclosure provides a process for preparing formulation, said process comprising the steps of:
a) mixing the phyto -ingredients comprising Parsley extract (Apigenin), Apple extract (Quercetin), Silybum Marianum extract (Silymarin), Grape seed extract (Anthocyanidins), Allium Sativum extract (Allicin), Green Tea extract (EGCG), Pomegranate extract (Ellagic acid), Withania somnifera extract (Withanolides), Curcumin (95%), and a probiotic blend along with prebiotics and other micronutrients using mortar and pestle;
b) blending the mixture as obtained in step (a) using a grinder for 5 to 10 minutes;
c) sieving the blended mixture using sieve size 60# to obtain amorphous powder; and
d) filling and packing the amorphous powder as obtained in step (c) in sachets.

Use of the composition or formulation for screening and treatment of advanced cancers including Colorectal cancer (CRC) by Diet-Infection-Nutrition-Immunity (DINI) Axis and having anti-cancer activity and anti-bacterial activity against the infection which can cause Colorectal cancer.

In yet another embodiment, a method of treatment or prevention of Colorectal cancer (CRC), the method comprising using the Diet-Infection-Nutrition-Immunity (DINI) Axis and taking the composition or formulation in accordance with the present disclosure by a person in need thereof, wherein the composition or formulation comprises of phyto-ingredients having anti-cancer activity and probiotics having anti-bacterial activity against the infection which can cause Colorectal cancer).

In one of the embodiment of the present disclosure, a method to (i) screen or prevent or inhibit or treat cancers including Colorectal cancer, cervical cancer and breast cancer, (ii) screen or prevent or inhibit or treat non-communicable diseases including Cardiovascular diseases, Diabetes, Stroke, Autoimmune diseases, (iii) screen or prevent or inhibit or treat Polycystic ovarian syndrome or disease (PCOS or PCOD), Osteoporosis, non-alcoholic fatty liver disease (NAFLD), (iv) provides dietary interventions including phyto-ingredients and probiotics to address the sub-optimally treated infections to trigger the immunomodulation, (v) provide proper nutrition, (vi) treats and help in preventing a range of medical conditions by leveraging nutrition, (vii) provide cancer-nutraceuticals for preventive Oncology to Cancer Disease Management for cancer patients at specific stages and specific regions of the cancer in the patient, the method comprising the step of using the Diet-Infection-Nutrition-Immunity (DINI) Axis and taking the composition or formulation in accordance with the present disclosure by a person in need thereof.

It is now accepted that our nutritional status influences our health and risk of both infectious and noncommunicable diseases. But it is also accepted that billions of people in both developed and developing countries suffer from one or more forms of malnutrition, contributing to the global burden of disease. Mankind has an inherent preference for palatable, sugary, salty, fatty, and refined foods. These foods are mostly energy-dense and low in micronutrients. Food production, processing, manufacturing, marketing, and promotion have responded to this preference by making high energy-dense foods available at increasingly affordable prices. This has led to changes in food consumption patterns which unfortunately coincided with more sedentary, less active lifestyles. The resultant over nutrition of especially macronutrients is the major cause of obesity and also, together with obesity, a risk factor for many of the non-communicable disease (NCDs) such as type 2 diabetes, coronary heart disease, stroke, hypertension, dental disease, osteoporosis, and some forms of cancer.

The pathogens we are exposed every day that are ingested by an individual from the food we eat or the environment daily. They do not necessarily cause diseases however it does cause changes in the body. It affects the infection status whenever a foreign particle enters the body. That’s how diet and infections are related. Even though the infections are setting up, these pathogens can remain asymptomatic maybe due to the quantity in which they are ingested or in their level of pathogenicity, they have an impact on the body. There is always a mild infection that is related to diet.

This alter in the infection status also alters the body’s nutrient, specially the micronutrient status. That is why there is always either a depletion or changes in the nutritional status because of the low-profile infections which is acquired by the diet. Micronutrients plays a major role in our immunity and though we are able to fight with diseases there’s always a negative impact on the immunity structure happening every time because of diet or food. At any stage the body is having an infection stage, there are changes in the nutrients which hence affects the immunity system of the body.

This axis happening 24/7 in the body is prompting us to non-communicable diseases (NCDs) like Diabetes, Stroke, some kinds of Cancers, etc. This axis is named the DINI Axis.

“The rise of infectious diseases and drug-resistant pathogens demands innovative approaches to decrease mortality. Strengthening nutritional status is one such approach.”

In accordance with an embodiment of the present disclosure, the invention provides relationships between Diet Infections nutrition Immunity and NCDs which is referred as "DINI AXIS" in the present disclosure. The DINI AXIS research is categorized in to four steps, first, from ecological meta-analysis which compared different populations, the effects of migration of populations, food availability during economic development, and differences in dietary and nutrient intakes. Second, numerous epidemiological/pandemic studies have triggered the associations between diet and biological risk factors of NCDs. Third, interventions with specific nutrients and foods in placebocontrolled trials using both healthy and diseased subjects confirmed the relationships of drug-nutrient interactions and its outcomes. And fourth, molecular and genetic intervention has taken under research projects through which diet and nutrients affect genetic mutation and expression, adding to our knowledge of how nutrition influences immunity and improve quality of life.

In accordance with an embodiment of the present disclosure, the novel approach is that the DINI Axis takes in to account the minute changes happening to the immunity system due to the daily diet we take.

In yet another embodiment of the present disclosure, it considers the mild low-profile infections caused by the diet, the alteration or depletion of the micronutrient we take thus hampering the immune responses of the body as well as its major role in the NCD's which is a one of it's kind concept since it is all related to the food we take.

In accordance with an embodiment of the present disclosure, DINI Axis links diet to NCDs thus opens an avenue where only alteration in diet could help to curb the emergence of non-communicable diseases (NCDs) like Diabetes, Stroke, some kinds of Cancers, etc.

In yet another embodiment of the present disclosure, it treats & help prevent a range of medical conditions by leveraging nutrition and life sciences. The invention address specific health conditions with special emphasis on Onco Nutrition & intend to provide a 360-degree solution to global cancer-nutraceuticals portfolio right from Preventive Oncology to Cancer Disease Management using Nutrition as complementary therapy to stage & region specific cancer patients.

The present disclosure provides dietary interventions (mainly with phyto-ingredients and probiotics) to address the sub-optimally treated infections (as discussed above) to trigger the immunomodulation along with providing proper nutrition to the subject under investigation. In one of the embodiment, the inventors have integrated probiotics and prebiotics along with the therapeutic nutritional intervention to trigger immunity to combat the chronic inflammatory state caused by the pathogenic infection. Our initial experiments with involve cell studies to screen the formulations. Thereafter, the inventors have taken the optimized formulation to the next stage of investigation, i.e., murine model of CRC with subsequent set of experiments. Our goal is to provide a proof-of-concept of the DINI Axis proposal for CRC and a successful clinical translation of the standardized formulation for CRC management in humans.

Formulation and set of experiments designed for bioactivity evaluation are given below:

EXAMPLES:
The present invention illustrated with the help of following examples, which are not intended to limit the scope of the invention and any such modification therein falls within the scope of this invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the subject matter. Those skilled in the art will appreciate that many modifications may be made in the invention without changing the essence of the invention.

Example 1:
Experimental Methods:

Formulation development

Formulation design
S. No. Ingredients Batch No. Quantity (g) Quantity in 100 g
1 Parsley Extract (Apigenin 7-80%) VH/VHPE-107 0.25 1.25
2 Apple extract (Quercetin 2.5 -5%) VH/VHQE-P8 0.25 1.25
3 Silybum Marianum extract (Silymarin 60-80%) VH/VHSE-105 0.25 1.25
4 Grape seed extract Oligomeric Pronthocyanidins 60-80%) 002-2010074-28 0.35 1.75
5 Allium Sativum extract (Allicin 2.5-5.0%) VH/VHAE-106 0.035 0.175
6 Green Tea extract (Epigallectocatechins gallate, EGCG 60-80%) TA191203-02 0.2 1
7 Pomegranate extract (Ellagic acid 40-60%) PFE-020/2106/S-11 0.15 0.75
8 Withania somnifera extract (Withanolide 2.5-5%) JNL/WS/072101 0.2 1
9 Curcumin (95%) CPW-0501216015 0.35 1.75
10 Vitamin D3 400IU (with 30 % over range) CLC0421023 0.000013 0.000065
11 Zinc 0.01 0.05
12 Sodium Selenite 201113 0.000045 0.000225
13 Inulin 2019231901 2 10
14 FOS 2019150698 1.4 7
15 MCT MCT-D-210215 2.55 12.75
16 Maltodextrin 222 10 50
17 DHA powder PB20055L-S40 1 5
18 Lactobacillus casei HTBS-RM3/21010 (10 billion cfu/g) 0.25 1.25
19 Bifidobacterium lactis Bb 12 HTBS-RM21/21007 (10 billion cfu/g) 0.25 1.25
20 Lactobacillus rhamnosus GG HTBS-RM2/21018 (10 billion cfu/g) 0.25 1.25
21 Lactobacillus plantarum HTBS-RM11/21006 (10 billion cfu/g) 0.25 1.25
Total 20 100

Example 2: (see next page)

S. No. Ingredients Batch No. F3A F3B F3C F3D
1 Parsley extract (Apigenin) VH/VHPE-107 5 mg 2.5 mg 2.5 mg 2.5 mg
2 Apple extract (Quercetin) VH/VHQE-P8 5 mg 2.5 mg 7.5 mg 5 mg
3 Silybum Marianum extract (Silymarin) VH/VHSE-105 15 mg 20 mg 25 mg 20 mg
4 Grape seed extract (Anthocyanidins) 002-2010074-28 20 mg 25 mg 15 mg 20 mg
5 Allium Sativum extract (Allicin) VH/VHAE-106 5 mg 10 mg 5 mg 7.5 mg
6 Green Tea extract (EGCG) TA191203-02 25 mg 20 mg 20 mg 15 mg
7 Pomegranate extract (Ellagic acid) PFE020/2106/S11 20 mg 15 mg 20 mg 25 mg
8 Withania somnifera extract (Withanolides) JNL/WS/072101 2.5 mg 2.5 mg 2.5 mg 2.5 mg
9 Curcumin (95%) CPW-501216015 2.5 mg 2.5 mg 2.5 mg 2.5 mg
Total 100 100 100 100

Example 3

Procedure:

Appropriate amounts of ingredients were weighed as per the formula. Apigenin (Parsley Extract) (0.25 g), Quercetin (Apple Extract) (0.25g), Silymarin (Silybum Marianum Extract) (0.25g), Anthocyanidins (Grape seed extract) (0.35g), Allicin (Allium Sativum extract) (0.035g), Green tea extract (0.2g), Ellagic acid (Pomegranate Extract) (0.15g), Withania Somnifera extract (0.2g), Curcumin 95% (0.35g), The ingredients were then properly mixed using mortar and pestle. After complete mixing, blending of mixture was done using grinder (5 - 10 min). Further, blended mixture was sieved using sieve size 60# to yield amorphous powder. Separate sachets were filled with 2 gm of powder. Precise sealing and labelling of sachet were done at the end.

Procedure in Flow Chart:

Instruments used

Sr. No. Name of instrument Identification number
1 Weight balance BAL/RD/001
2 Hand Blender HBL/RD/008
3 60# sieve 60#
4 Mortar pestle ---
5 Sealing Machine SLM/RD/007

Example 4:

Experimental Methods:

Bioactivity Evaluation

Cell culture
In this study, the inventors have selected mouse colon carcinoma cell line CT26 for CRC model. The cells were purchased from NCCS, Pune and collected from Department of Biology, BITS Pilani, Hyderabad, India. Further the cells are cultured in T25 flask along with RPMI media and 10% fetal bovine serum (FBS) and found that these cells are adherent and have a fibroblast morphology. They grew the CT-26 cells in RPMI media and incubate the flask in incubator with specified conditions of 95 % air, 5 % CO2 and temperature 37°C ± 1 °C. after confluent the flask, they sub-cultured the cells from T25 to T75 flask.

Media preparation
RPMI media has been selected for cell culture studies along with 10% FBS (Fetal bovine serum) and subsequently added penicillin and streptomycin 0.5 to 1 % ratio in culture media which protect the bacterial contamination, either gram positive or gram-negative bacteria.

Cell plating
A sterile 96 well plate in cell culture lab was taken and 6 to 7 thousand cells were transferred to each well for treatment. Before the treatment, the inventors incubated the cells for 12 hr maintaining the temp 37 ± 2 °C. Thereafter, the inventors treated the CT-26 (CRC cell lines) by different formulations in 4 various concentrations i.e., 0.25 %, 0.125%, 0.063% and 0.0312 %. The inventors kept the plate in incubator for 48 hr at 37°C with continuous 5 % supply of CO2 gas. Before seeding the cells, culture media was removed from flask and the cells were washed two to three times with 5 to 10 ml phosphate buffer (PBS). Thereafter, the PBS buffer was completely removed and added 2 to 5 ml of trypsin and PBS solution in ratio (1:1). While treating concentration of trypsin solution should be 1X. The flask was tapped slowly or kept in incubator for 2 to 10 minutes. The floated cells were transferred in 50 ml sterile tube containing calculated amount of RPMI media. Subsequently, the tubes were centrifuged at 1000 RPM for 5 minute and supernatant solution was discarded. Finally, fresh media was added, checked the concentration of cells by hemocytometer and seeded for experiment.

Preparation of formulation sample for treatment
As mentioned above, the inventors prepared different DINI-AXIS-CRC Formulations-3, 3A, 3B, 3C and 3D. They accurately weighed 50 mg powder and transferred in 15 ml centrifuge tube and added 5 ml of phosphate buffer (PBS) and vortex for 5 minutes and further made up to the 10 ml with PBS. The resulting tube was sonicated for 10 minutes with continuous shaking while maintaining temperature 30 ± 2° C. Thereafter, they filtered the resulting solution through 0.22 µm PVDF sterile filter and finally diluted in four different concentrations i.e., 0.25 %, 0.125%, 0.063% and 0.0312 %. with (RPMI) culture media. All the experimentation works were performed in aseptic condition.

MTT Assay
The cultured cells were revived from T75 flask to 5 ml sterile Eppendorf tube and was subsequently seeded the cells in each well of a 96 well plate (approx. 6000 to 8000 cells per well) and incubated for 24 hr under proper CO2 and oxygen supply. After 24 hr, the resulting plate was examined under confocal microscope and treated the cells with various optimized formulation i.e., EON/CRC/F3A-D. Each formulation was prepared in 4 different concentrations for treatment. The formulations were added in triplicate for applying statistics. Assay was performed after three different time points: 24hr, 48hr and 72hr. For assay, the media was removed and 200 µl MMT reagent was added having concentration 1mg/ml and incubated for 4 hr with proper condition. Next, MMT media was removed and DMSO (cell culture grade) was added. The plate was wrapped with aluminium foil with shaking for 10 minutes. Finely, reading was collected from MMT plate reader and the IC50 value was calculated by GraphPad Prism software. The data is given below:

96 well plate
Treatment for 24/48/72 hr
Concentration in % (w/v) for Formulation

0.250% 0.125% 0.0625% 0.0312 Formulations
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 F3
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 FA
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 FB
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 FC
T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3 FD
C C C C C C C C C C C C C
C C C C C C C C C C C C C
C C C C C C C C C C C C C

Proteomics study: Western blot analysis
CT26 cells were seeded at a density of 1 × 105 cells in a 6 well plate for 18-24 h before treatment. Cells were treated for 24 h with: Formulation 3A-D respectively at IC50 concentration (0.1% w/v) as achieved from MTT, and untreated cells were used as a control. After 4 h of incubation at 37 ºC, the media was replaced with 4 mL fresh complete media (RPMI-1640 containing 10% FBS, v/v) and incubated for additional 20 h. cells were detached from the flask using a cell scrapper and the cells were lysed with RIPA buffer (Sigma) supplemented with protein cocktail inhibitor (Thermo Scientific) at 4 ºC. Total protein contents of the cell lysates were quantified by BCA protein estimation ki (Thermo Scientific, USA). 15 µg of total proteins were dissolved in SDS-PAGE sample buffer prior to separation by 12% SDS-PAGE. Proteins were transferred to PVDF membrane (Millipore) using wet blotting. Membranes were blocked for 2 h at rt with 5% BSA in PBS containing 0.05% Tween-20 (PBS-T). Blots were then incubated with rabbit Bax and Bcl-2 antibody and anti-ß-actin (as loading control) primary antibodies (at 1:1000 dilutions in 10 mL 0.05% PBS-T) for 3 h at rt. After washing with PBS-T (3 × 10 mL, 10 min each), the membranes were incubated with goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (in 10 mL 0.05% PBS-T with 1:5000 dilution) for 2 h. Protein bands were developed using BCIP/NBT chromogen solution (Sigma).

Apoptosis induction study by Flow Cytometry
CT26 cells were seeded at a density of 1 × 105 cells in a 6 well plate for 18-24 h before treatment. Cells were treated for 24 h with: Formulation 3A-D respectively at IC20 concentration (0.1% w/v) as achieved from MTT, and untreated cells were used as a control. Here, the inventors decreased the concentration (IC20) of the formulation for treatment as they observed rapid cancer cell killing at IC50 in the previous proteomics study. Also, here the inventors intended to see the induction of apoptosis in the cancer cell by DINI Axis formulation. After 4 h of incubation at 37 ºC, the media was replaced with 4 mL fresh complete media (RPMI-1640 containing 10% FBS, v/v) and incubated for additional 20 h. After 4 h of incubation at 37ºC, the media was replaced with 2 mL fresh complete media and incubated for additional 20 h. Next, cells were then trypsinized, washed with 1× PBS, centrifuged and the pellet was resuspended in 200 µL binding buffer containing Annexin-V FITC (0.25 µg) and Propidium iodide (PI) (1.0 µg). The mixture was then incubated for 15 min in dark and analyzed by flow cytometer (BD FACS Canto II).

E. coli growth inhibition study with the metabolites of Probiotics

Next, to investigate the role of the probiotics in the amelioration of the gut dysbiosis associated with the pathogenesis of colorectal cancer, the secondary metabolites were isolated from all different probiotic strains namely, Lactobacillus casei, Lactobacillus plantarum and Bifidobacterium lactis. Bacterial cells were arrested in the stationary phase, followed by centrifugation of cell suspension and further filtration through a nitrocellulose membrane. All the experiments were performed under complete sterile conditions. The resultant filtered supernatant was concentrated to get secondary metabolite content which was used to treat E. coli (taken as a model organism) to test the effect of these compounds on the bacterial cell growth. Moreover, the phyto-formulation were also mixed with it at various concentration ratio and treated on E.coli.

Example 5:

Results

In vitro Cellular Cytotoxicity
Before evaluating the efficacies of the presently described formulations of phytochemicals containing the ingredients in the prescribed amount as listed in Table 1 & 2 in promoting apoptosis, silencing Bax and Bcl-2 gene in CT26 carcinoma cells, first the inventors evaluated the cellular cytotoxicities of the all formulations in CT26 cells along with normal HEK cells by standard MTT assay after treatment for 24h, 48h and 72h. CT26 cells were treated for 24h, 48h and 72h with the formulations in varying amount of chosen phytochemicals. From these chosen ingredients, they initially made six different formulations and screened them in MTT assay. Formulation 3 with the phyto-ingredients demonstrated highest efficacy in inhibiting CT26 colon cancer cell growth. This prompted us to develop few variants of the same – Formulations 3A, 3B, 3C, 3D which showed even better efficacy in the biological experiments such as MTT, Western Blot, Flow cytometry. As shown in Figure 1-3, formulation F3A-D exhibited significant cytotoxicity in CT26 cells in all the time points 24h, 48h and 72h. Importantly, all the formulation F3A-D did not show any cellular cytotoxicity in healthy HEK cells (Figure 4). This is remarkable (Figure 4) as the phyto-formulations are helping the normal HEK cells to proliferate further. Collectively, the findings summarized in Figures 1 - 4 are consistent with the supposition that formulations (F3A-D) are efficient as anti-cancer agents selectively to cancer cells (non-toxic to normal cells).

Figure 1A&B: CT26 Cancer cell selective cytotoxicity of formulation F3A-D in 24h. The results shown are average values for triplicate experiments performed on the same day. Figure 2A&B: CT26 Cancer cell selective cytotoxicity of formulation F3A-D in 48h. The results shown are average values for triplicate experiments performed on the same day. Figure 3A&B: CT26 Cancer cell selective cytotoxicity of formulation F3A-D in 72h. The results shown are average values for triplicate experiments performed on the same day. Figure 4: Formulation F3A-D are non-toxic to normal cell (48h data shown). The results shown are average values for triplicate experiments performed on the same day.

In vitro proteomics analysis:

Next, the inventors evaluated the in vitro efficacies of the formulations F3A-D in modulation of B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X (Bax) protein levels in CT26 cancer cells in order to study the apoptotic process. It has well known that the Bcl-2 family members regulate the intrinsic (mitochondrial mediated) apoptotic pathway; particularly, pro-apoptotic Bax and anti-apoptotic Bcl-2 are important for the cytochrome C release and the subsequent downstream activation of caspase protein. Findings in western blot analysis revealed that pro-apoptotic Bax is highly upregulated in treatment group F3B and F3D and moderately upregulated in F3A and F3C compared to the untreated control cells. It also demonstrated a significant downregulation in anti-apoptotic Bcl-2 protein levels in all the treatment groups F3A-D compared to the untreated control cells. Representative bands and quantified protein band intensities are presented in Figure 5A-C.
Figure 5: Formulations F3A-D significantly down regulates expression of Bcl-2 protein and upregulates the expression of Bax protein (markers for apoptosis) in CT26 colon cancer cells.

Apoptosis studies using Flow cytometry
After the inventors confirmed the promising results of cellular cytotoxicity profiles and proteomics analysis by western blots of the formulation 3A-D, with a view to examine whether a lower concentration of the formulation incubated for a shorter period of time is capable of inducing apoptosis of CT26 cancer cells, the inventors performed Annexin-V binding based flow cytometric assays. The findings summarized in Figure 6 revealed a clear evidence of higher population of apoptotic cells (upper right quadrants, Figure 6) when treated with formulation 3A-D. Importantly, the untreated cells showed presence of significantly less population of apoptotic cells. The highest apoptosis inducing property exhibited by the F3B and F3D are consistent with highest degree of Bax upregulation in the proteomics study (Figure 5). Together, the formulations comprised of the chosen phyto-nutrients are well capable of inducing apoptosis in CT26 carcinoma cells. Figure 6: Formulations F3A-D induce apoptosis in CT26 colon cancer cells. The horizontal and vertical axes represent cells labelled with FITC-Annexin V and PI, respectively, in the dot plots. Dots in the upper right quadrant in each quadrant represent late apoptotic cells (positive for both Annexin V and PI). Data shown here are representative of one experiments.

Investigating the effect of probiotic secondary metabolites over E. Coli

Next, to investigate the role of the probiotics in the amelioration of the gut dysbiosis associated with the pathogenesis of colorectal cancer, the secondary metabolites were isolated from all different probiotic strains namely, Lactobacillus casei, Lactobacillus plantarum and Bifidobacterium lactis. These secondary metabolites were mixed with the phyto-formulation (F3B) at various concentration ratio and treated on E. coli. Importantly, the results revealed complete inhibition of E. coli growth upon treatment of the secondary metabolites of the Lactobacillus casei, Lactobacillus plantarum and Bifidobacterium lactis. The untreated E. coli grew spontaneously. It was interesting to see that the phyto-ingredients, added in different ratios, did not interfere with the growth of E. coli. Figure 7A-C: Growth inhibition of E. coli upon treatment of secondary metabolites (Black label) of (A) Lactobacillus casei, (B) Lactobacillus plantarum and (C) Bifidobacterium lactis. Untreated E. coli grew spontaneously (blue label).

It has become increasingly important to device efficient strategy to combat a bleak malady like cancer. Here, the inventors provide a holistic approach to address the daunting issues related to Colorectal cancer (CRC), to be specific. There is no dearth of evidence that that the tumour-associated inflammatory response had the unforeseen, ironical effect of enhancing tumorigenesis and progression, in turn, helping incipient neoplasias to obtain salient features of cancer and fostering their eventual development into full-blown cancers. Inflammation contributes to neoplastic growth by providing a growing list of signalling molecules: tumour growth factor EGF, the angiogenic growth factor VEGF, other proangiogenic factors such as FGF2, chemokines, and cytokines promoting the inflammatory state, proangiogenic and/or pro-invasive matrix-degrading enzymes, including MMP-9 and other matrix metalloproteinases, cysteine cathepsin proteases, heparanase, and inductive signals that lead to activation of EMT, etc. Additionally, inflammatory cells can release chemicals, notably reactive oxygen species (ROS), which are proactively mutagenic for neighbouring cancer cells, accelerating their genetic evolution toward a state of heightened malignancy. It has also been shown that the CRC microbiota exhibits a global compositional shift (commonly termed dysbiosis) compared with the microbiota of healthy individuals, reflecting a different ecological microenvironment in patients with CRC. Besides specific bacteria, including strains of Bacteroides fragilis, Escherichia coli, Enterococcus faecalis and Streptococcus gallolyticus, being individually linked to CRC in various association and mechanistic studies, metagenomic studies in humans have identified novel associations with other bacteria like fusobacterium nucleatum etc. They are responsible for the chronic inflammatory state which is a trigger for carcinogenesis.

Towards generating a nutraceutical formulation having therapeutic intervention capability, the inventors have chosen phytochemicals and probiotics to reduce the infection born the chronic inflammatory state of the CRC. Importantly, our formulation comprising of anti-inflammatory phytochemicals is showing remarkable CT26 colon cancer cell growth inhibition in MTT assay in a concentration dependent manner. Upon further investigation, it revealed to be inducing apoptosis in colon cancer cells via upregulating pro-apoptotic Bax protein and anti-apoptotic Bcl-2 protein. Probiotics present in the formulation (L. casei, L. plantarum, B. lactis) are showing strong evidence in inhibition of the growth of E. coli. In summary, this nutritional intervention has the potential to combat bacterial infections responsible for chronic inflammation causing tumorigenesis and to inhibit the growth of colon cancer cells. The present disclosure provides formulation, proven to be non-toxic to normal cells, has a dual effect: inhibition of cancer cell proliferation via apoptosis (effect of phytochemicals) and inhibition of infection crucial for chronic inflammation (effect of probiotics) and thereby tumorigenesis. For sure, there will be lot more experiments conducted in animals and humans in near future. It will pave the way for many future studies to develop microbiota oriented personalized nutraceuticals for various NCDs and cancers.

ADVANTAGES:

The following are the technical advantages of the invention:

• In the present disclosure DINI Axis links diet to NCDs thus opens an avenue where only alteration in diet could help to curb the emergence of NCDs.
• The present disclosure describes therapeutic immunomodulatory nutraceutical intervention for use in colorectal cancer management.
• The nutritional intervention has the potential to combat bacterial infections responsible for chronic inflammation promoting tumorigenesis and to inhibit the growth of colon cancer cells via inducing apoptosis.
• The formulation, proven to be non-toxic to normal cells, has a dual effect: inhibition of cancer cell proliferation via apoptosis (effect of phytochemicals) and inhibition of infection crucial for chronic inflammation (effect of probiotics) and thereby tumorigenesis.

While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention. For example, effective dosages other than the particular dosages as set forth herein above may be applicable as a consequence of variations in responsiveness of the mammal being treated for any of the indications with the compounds of the invention indicated above. The specific pharmacological responses observed may vary according to and depending upon the particular active compounds selected or whether there are present pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the res tills arc contemplated in accordance with the objects and practices of the present invention. It is intended, therefore, that the invention be defined by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.

While specific language has been used to describe the present subject matter, any limitations arising on account thereto, are not intended. As would be apparent to a person in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein.

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1. Composition or formulation comprising of phyto-ingredients Silybum Marianum extract (Silymarin 10-40 weight %), Grape seed extract (Anthocyanidins 10-40 weight %), Green Tea extract (EGCG 10-40 weight %), Pomegranate extract (Ellagic acid 10-40 weight %); and optionally further comprising one or more selected from the group comprising of Parsley extract (Apigenin 1-10 weight %), Apple extract (Quercetin 1-10 weight %), Allium Sativum extract (Allicin 1-10 weight %), Withania somnifera extract (Withanolides 1-5 weight %) and Curcumin (Curcuminoids 1-5 weight %).

2. Composition or formulation comprising of phyto-ingredients comprising Parsley extract (Apigenin), Apple extract (Quercetin), Silybum Marianum extract (Silymarin), Grape seed extract (Anthocyanidins), Allium Sativum extract (Allicin), Green Tea extract (EGCG), Pomegranate extract (Ellagic acid), Withania somnifera extract (Withanolides), Curcumin (95%), and a probiotic blend along with prebiotics and other micronutrients.

3. The composition or formulation as claimed in claim 2, wherein the probiotic blend comprising of one or more selected from the group comprising Lactobacillus casei (0.5 – 5 billion cfu), Bifidobacterium lactis Bb 12 (0.5 – 5 billion cfu), Lactobacillus rhamnosus GG (0.5 – 5 billion cfu) and Lactobacillus plantarum (0.5 – 5 billion cfu).

4. The composition or formulation as claimed in preceding claims, optionally further comprising one or more selected from the group comprising of Vitamin D3 (400IU with 30 % over range), Zinc (up to 0.05 weight %), Sodium Selenite (up to 0.0005 weight %), Inulin (1-10 weight %), Fructo-oligosaccharides (1-10 weight %), Medium chain triglycerides (1-15 weight %) and Docosahexaenoic acid (1-10 weight %) powder.

5. The composition or formulation as claimed in preceding claims, wherein the composition as and when used inhibits the growth of colorectal cancer cells and remain non-toxic to the normal cell, induces apoptosis to the colorectal cancer cells via upregulating pro-apoptotic Bax protein and downregulating anti-apoptotic Bcl-2 protein.

6. The composition or formulation as claimed in preceding claims, wherein the secondary metabolites isolated from the probiotics blend present in the composition or formulation are efficacious in complete growth inhibition of E. coli and other infectious bacterial strains responsible for Colorectal cancer (CRC) pathogenesis such as – Fusobacterium nucleatum, Bacteroides fragilis, etc.

7. The composition or formulation as claimed in preceding claims, wherein the composition as and when used for screening and treatment of Colorectal cancer (CRC) patients having various genetic mutations comprising KRAS, BRAF and others, and environmental aberrations comprising dysbiotic microbiota with cancer causing bacteria and others.

8. Process for preparing formulation, said process comprising the steps of:
e) mixing the phyto -ingredients comprising Parsley extract (Apigenin), Apple extract (Quercetin), Silybum Marianum extract (Silymarin), Grape seed extract (Anthocyanidins), Allium Sativum extract (Allicin), Green Tea extract (EGCG), Pomegranate extract (Ellagic acid), Withania somnifera extract (Withanolides), Curcumin (95%), and a probiotic blend along with prebiotics and other micronutrients using mortar and pestle;
f) blending the mixture as obtained in step (a) using a grinder for 5 to 10 minutes;
g) sieving the blended mixture using sieve size 60# to obtain amorphous powder; and
h) filling and packing the amorphous powder as obtained in step (c) in sachets.

9. Use of the composition or formulation as claimed in preceding claims for screening and treatment of advanced cancers including Colorectal cancer (CRC) by Diet-Infection-Nutrition-Immunity (DINI) Axis and having anti-cancer activity and anti-bacterial activity against the infection which can cause Colorectal cancer.

10. A method of treatment or prevention of Colorectal cancer (CRC), the method comprising using the Diet-Infection-Nutrition-Immunity (DINI) Axis and taking the composition or formulation according to claim 2 by a person in need thereof, wherein the composition or formulation comprises of phyto-ingredients having anti-cancer activity and probiotics having anti-bacterial activity against the infection which can cause Colorectal cancer).

11. A method to (i) screen or prevent or inhibit or treat cancers including Colorectal cancer, cervical cancer and breast cancer, (ii) screen or prevent or inhibit or treat non-communicable diseases including Cardiovascular diseases, Diabetes, Stroke, Autoimmune diseases, (iii) screen or prevent or inhibit or treat Polycystic ovarian syndrome or disease (PCOS or PCOD), Osteoporosis, non-alcoholic fatty liver disease (NAFLD), (iv) provides dietary interventions including phyto-ingredients and probiotics to address the sub-optimally treated infections to trigger the immunomodulation, (v) provide proper nutrition, (vi) treats and help in preventing a range of medical conditions by leveraging nutrition, (vii) provide cancer-nutraceuticals for preventive Oncology to Cancer Disease Management for cancer patients at specific stages and specific regions of the cancer in the patient, the method comprising the step of using the Diet-Infection-Nutrition-Immunity (DINI) Axis and taking the composition or formulation according to claim 1 or 2 by a person in need thereof.