Description:FIELD OF INVENTION
The invention relates to field of biotechnology particularly to a down-regulating pro-inflammatory cytokines.
BACKGROUND OF THE INVENTION
Water is elixir of life; all life forms have their origin in water. About 71% of the earth is covered by water. Ganga is a scared river of India; the origin of Ganga River is in the Himalaya Mountains at Gomukh at the terminus of the Gongotri Glacier. When the ice of this glacier melts, it forms the clear waters of the Bhagirathi which joins the Alaknanda River forming the Ganga River. There are a number of reports of River Ganga having therapeutic properties due to high levels of bacteriophage present in Ganga water. Bacteriophages or phages are viruses that infect and kill bacteria. Bacteriophages (BPs) are viruses that can infect and kill bacteria without any negative effect on human or animal cells. Bacteriophages consist of a nucleic acid molecule surrounded by a specific protein coat (capsid). Because of this particular property of bacteriophages, phage therapy is considered as an good option, alone or in combination with antibiotics, to treat bacterial infections. In a recent study by Mishra and Nath, 2020 (Raman Mishra, Raghvendra & Nath, Gopal. (2020). Detection of Bacteriophages against ESKAPE Group of Nosocomial Pathogens from Ganga River Water During Community Bath at Various Rituals Since 2013–2019. Journal of Applied Pharmaceutical Sciences and Research. 17-21. 10.31069/japsr. v3i1.5.), bacteriophages of resistant microbes such as the ESKAPE group of nosocomial and S. Typhi were detected in Ganga water samples collected on different rituals. Ganga water sample from different places (Prayagraj, Mirzapur, and Varanasi) and sites were collected. A total 210 strains of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Escherichia coli (Called as ESKAPE group) and additionally S. typhi were identified from 500 clinical samples and confirmed ESKAPE and S. typhi were used in the study. Bacteriophages were observed in the form of plaques on the bacterial lawn culture. Among 210 strains (30 each) of E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and S. Typhi total 52 phages were detected in the form of plaques on the bacterial lawn culture. The authors concluded that the Ganga water can be used as phage bank and used in phage therapy. It has been reported that at Gomukh, which is the origin of River Ganga, loads of sediment gushes out in force along with water due to the melting permafrost The Himalayan permafrost melts and forms the origin of Ganges, the researchers believe that bacteriophages trapped at a much earlier time scale in the Himalayan permafrost as abiotic particles are being released gradually with the melting permafrost, thereby making a seed source of bacteriophage at Gomukh.
The National Environmental Engineering Research Institute has reported that the Ganga contains approximately 1,100 types of bacteriophages. In addition, Ganga water exhibits high alkalinity, and its self-purificatory properties contribute to the growth of bacteriophages.
In a recent study, metagenomic DNA from the pooled sediment of 16 sample of the river Ganges to explore the abundance and diversity of phages, sediment i.e., the upper layer of soil base, around 3-4 cm (~2 inch) below the river water, was collected from these sites. (Abundance and diversity of phages, microbial taxa and antibiotic resistance genes in the sediments of the river Ganges through metagenomic approach. Narender Kumar, et al, doi: https://doi.org/10.1101/2020.04.29.067819). The study identified 285 different DNA viruses largely dominated by the 19 group of 260 distinctive phages namely Microcystis phage Ma-LMM01, Haemophilus phage HP2 264, Microcystis phage MaMV-DC, Bacillus phage G, Salmonella phage SJ46, Pseudomonas phage phiKZ, Synechococcus phage S-IOM18, Synechococcus phage S-RIM8 A.HR1, Pseudomonas phage phi3, Ralstonia phage RSL1, Synechococcus phage S-PM2 and Synechococcus phage S-SM2, members of the Myoviridae family; likewise, Caulobacter phage CcrColossus, Rhodococcus phage REQ1, Enterococcus phage phiFL4A, Enterobacteria phage lambda, Enterococcus phage EFC-1, Erwinia phage phiEaH2, Mycobacterium phage Bipper, Propionibacterium phage PFR2 belongs to the family Siphoviridae. Correspondingly, Choristoneura fumiferana granulovirus member of family Baculoviridae and BeAn58058 virus from Poxviridae family from Ganga water.
Environmental (e)DNA is the genetic material which forms a reservoir of DNA or RNA from all diverse life forms into aquatic habitat. Environmental DNA (eDNA) is defined as genetic material obtained directly from environmental samples (soil, sediment, water, etc.) without any obvious signs of biological source material. (e)DNA is directly collected from the environment and is not sampled from an individual organism. As various organisms interact with the environment, DNA is expelled and accumulates in their surroundings from various sources. The source of the environmental DNA can be urine, waste, mucus, or disrupted cells. (e)DNA collected from aquatic habitats is often used for management and assessment of a species’ distribution and entire community compositions and has become a powerful tool for improving species detection.
The genetic material which can be isolated from the environment depends on the type of genetic material, stability of its structure and resistance to various hydrolases present in the microenvironment. eDNA are largely short fragments and is composed of both nuclear (nu-DNA) and mitochondrial (mt-DNA) genomes. In general, mt-DNA has high density in the eDNA samples as compared to nu-DNA (10–1000 s of mitochondria to a single nucleus per cell), and is more stable than nu-DNA due to circular structure. eRNA or environmental RNA due its conformation as a single-stranded structure and also due to the presence of hydroxyl groups is considered less stable than its DNA counterpart, as RNA degrades rapidly after cell death. In a eukaryotic cell, ribosomal (r)RNAs comprise>80% of the total RNAs within a cell and thus are predicted to be found in greater concentrations than that of messenger (m)RNA. Additionally, rRNA is hypothesized to be less susceptible to degradation compared to mRNA due to structure stability. Degradation of eDNA and eRNA depends on abiotic factors, such as water temperature, pH, salinity, and ultraviolet (UV) radiation, and biotic factors including microbes and activity of extracellular enzymes. An accurate interpretation of environmental genetic signal is dependent upon knowledge of the shedding and degradation rates between the nu- and mt-genomes, between eDNA and eRNA, and between the RNA types within eRNA.
Extracellular RNA (exRNA) can be broadly defined as RNA species present outside of the cells in which they were transcribed. The ex-RNA is well- known to be secreted within extracellular vesicles and therefore are protected from ubiquitous RNA-degrading enzymes. exRNAs may be found in the environment or, in multicellular organisms, within the tissues or biological fluid. Therefore, ex-RNA can be environmental RNA which are secreted by the bacteria into an aquatic environment and which may be secreted encapsulated in an extracellular vesicle, thus protected from the hydrolase and extracellular RNAse.
Archaea, Bacteria and Eukarya, cells from all three domains of life, produce extracellular vesicles (EVs). Extracellular vesicles (EVs) from eukaryotic cells include exosomes that are less than 150 μm in size, microvesicles that are about 200–500μm in size, and oncosomes that are smallest in size about 1–10μm. Exosomes are shed through the multivesicular bodies of the endosomal pathway, by budding off from the plasma membrane while oncosomes are released directly from tumor cell membranes. EVs secreted by the eukaryotic cells are abundant in microRNAs (miRNAs), other types of RNAs are also detected, including small nucleolar RNAs (snoRNAs), PlWI-interacting RNAs (piRNAs), long noncoding RNAs (lncRNAs), transfer RNAs (tRNAs) and tRNA fragments, YRNAs, ribosomal RNAs (rRNAs), mitochondrial RNAs, and protein-coding RNAs. exRNA-containing vesicles are stable and can be detected in biofluids such as blood, breast milk, follicular fluid, saliva, and urine. Thus, exRNA can be used as potential biomarkers as indicators of normal biological processes or identifying the patients at risk. Tumor-derived exRNA have been detected in blood of prostate cancer patients, correlating with tumor size and malignancy. Similarly, exRNA in saliva is reported to be associated with pancreatic and esophageal cancer, circulating miRNAs are significantly elevated in advanced heart failure, and lncRNA is considered as an independent predictors of pathological cardiac re-modelling and diastolic dysfunction in patients diagnosed with diabetes type 2.
Both gram -negative and gram -positive bacteria are known to secrete EVs. Bacterial EVs play a significant role in competing with other bacteria for resources survival of microbe, material exchange, host immune modulation, transfer of nutrients within microbial communities, delivery of virulence factors and toxins, horizontal gene transfer, and modulation of host immunity, and infection. In gram-negative bacteria vesicle formation. involves blebbing of the outer membrane of the bacterial envelope, generating outer-membrane vesicles (OMVs); and also by explosive cell lysis forming outer-inner membrane vesicles (OIMVs) and explosive outer-membrane vesicles (EOMVs). In gram-positive bacteria cytoplasmic membrane vesicles (CMVs) are produced through endolysin-triggered bubbling cell death. The content of bacterial extracellular vesicles (EVs) are messenger RNA (mRNA), small noncoding (ncRNAs), DNA, proteins, and lipids among others. The host–microbe interspecies communication between human and microorganisms is presumed to be aided by microbial EVs. The bacterial EVs also modulate certain human host functions. It is also believed that EVs are employed as a novel secretory mechanism by bacteria to deliver various cargo into the host cells without the need for the bacteria to contact the host cells. The membrane composition of the EVs in gram positive and gram-negative bacteria differs depending upon the bacterial cell wall of gram positive and gram-negative bacteria. The gram-positive bacteria do not have an outer membrane but have a much thicker peptidoglycan cell wall, which is linked to the underlying cytoplasmic membrane via lipoteichoic acids (LTA). Cell surface members of the TLR family, namely TLR2 and TLR4, recognize extraluminal of the bacterial EV’s ligands such as LPS and LTA molecules, peptidoglycan, and lipoarabinomannan. The EV of the gram-positive bacteria show LTA on their surface that can engage the Toll-like receptor 2 (TLR2). Gram-negative EVs consist of an outer membrane with an interior leaflet of phospholipids and an exterior leaflet of LPS and engage Toll-like receptor 4 (TLR4).
Considering the above, it is well-established that extracellular-vesicles are well implicated in the cell-signalling, horizontal gene transfer, modulating host cell response, as biomarkers and in number of other biological processes. Thus e-RNA either as environmental RNA or as an extra-cellular RNA encapsulated within EVs can play important role in biological process and may contribute to the therapeutic properties of the Ganga Jal.
A few of the prior art discloses use of ganga water in therapy, Ranjana et al, 2021; Volume: 7, Page: 186-190; Indian Journal of Clinical and Experimental Dermatology (https://doi.org/10.18231/j.ijced.2021.037) discloses use of the naturally available cocktail of phages in the Ganga water as a treatment for chronic Psoriasis. Naturally available cocktail of phages in the water of the Ganga River was administered to the psoriasis patients without first identifying the target bacteria and isolating specific phages that may be active against them. In doing so, authors tested the hypothesis that phages present in Ganga Jal would self-identify the bacteria that are present and act against them. Patients who took Ganga water for only two weeks showed benefit but the benefit did not sustain after stoppage of the treatment and the disease relapsed to the pre-treatment levels. The same patients showed sustained benefit after they took Ganga water for four weeks.
The present study therefore aims to elucidate the mechanism by which the ganga water down regulates the proinflammatory cytokines and develop a method for down-regulation of the pro-inflammatory cytokines that are up-regulated in number of auto-immune disease using the therapeutic properties of ganga water.
OBJECT OF THE INVENTION
Accordingly, the inventors of the present inventors keeping the therapeutics property of Ganga water and the presence of environmental RNA (eRNA) in mind the object of the present invention is to develop a method to downregulate pro-inflammatory condition using therapeutic properties of the ganga water.
Another objective of the present invention is to develop a method to downregulate the pro-inflammatory cytokines by the micro-RNA (miRNA) present in the Ganga water.
Another object of the present invention is to elucidate the method by which the downregulation of the inflammatory genes is carried out by Ganga Jal.
SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts in a simplified format that is further described in the detailed description of the invention. This summary is neither intended to identify key or essential inventive concepts of the invention and nor is it intended for determining the scope of the invention.

The present invention discloses a method of down-regulating pro-inflammatory cytokines comprising; obtaining a sample of ganga water; filtering the sample of ganga water and concentrating the same; and reconstituting the sample in a suitable buffer, wherein the down regulation of the pro-inflammatory cytokines is carried out by extracellular (exRNA) contained in/attached to the extra cellular vesicle present in ganga water.
In still another embodiment a composition of the extracellular (exRNA) contained in/attached to the extra cellular vesicle obtained from Ganga Jal is disclosed.
BRIEF DESCRIPTION OF FIGURES:
The above-mentioned objectives and descriptions, features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:

Figure 1 depicts broad view of upregulation of cytokines and transcription factors in inflammation;
Figure 2 depicts the using microRNA quantity as estimated in qubit fluorophore analysis in Ganga Jal (A & B);
Figure 3 depicts the cell culture plates of mammalian A549 cell line exposed to PBS (Control), PBS+RNAse (negative control), Ganga Jal, Ganga Jal+ RNAse (positive control);
Figure 4 depicts on effect of treatment with Ganga Jal on IL-1Beta, IL-6, TNFα, IFN-gamma, IL-23, and NFĸB gene expression as compared to cell culture supernatant in the mammalian A549 cell line; and
Figure 5 depicts effect of reverse of effect on IL-17, IL-1Beta, IL-6, and TNFα genes on treatment with Ganga Jal in presence of RNase as compared to cell culture supernatant in the mammalian A549 cell line;
Figure 6 depicts particle size distribution of the extracellular vesicle isolated from the Ganga Jal;
Figure 7 depicts zeta potential of the distribution of the extracellular vesicle isolated from the Ganga Jal;
Figure 8 depicts morphology of the extracellular vesicle isolated from the Ganga Jal as observed in TEM in one embodiment of the invention (A-D);
Figure 9 depicts morphology of the extracellular vesicle isolated from the Ganga Jal as observed in TEM in one embodiment of the invention (A-D);
Figure 10 depicts (a) and (b) morphology of the extracellular vesicle isolated from the Ganga Jal as observed in TEM in one embodiment of the invention;
Figure 11 depicts (a) and (b) morphology of the extracellular vesicle isolated from the Ganga Jal as observed in SEM in one embodiment of the invention; and
Figure 12 depicts (a) and (b) morphology of the extracellular vesicle isolated from the Ganga Jal as observed in SEM in one embodiment of the invention
Further, skilled artisans will appreciate that elements in the figures are illustrated for simplicity and may not have been necessarily drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of the aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures 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 exemplary and 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.
Extracellular vesicles (EVs) are lipid bilayer enclosed packages of biomolecules released from cells into their surrounding environment, and include particles described as exosomes, ectosomes, microvesicles, oncosomes, and apoptotic bodies,
exRNA may be selected from group of microRNAs (miRNAs), other types of RNAs are also detected, including small nucleolar RNAs (snoRNAs), PlWI-interacting RNAs (piRNAs), long noncoding RNAs (lncRNAs), transfer RNAs (tRNAs) and tRNA fragments, YRNAs, ribosomal RNAs (rRNAs), mitochondrial RNAs, and protein-coding RNAs
Ganga water has a natural microflora bacteria Staphylococcus spp., E. coli, Vibrio spp., Bacillus subtilis, Campylobacter spp., Helicobacter spp., Brucella spp., Micrococcus spp., Corynebacterium spp., Pseudomonas spp., Hemophilus spp., and Clostridium spp. Several multi drug resistant bacteria are also found in the ganga water, however still ganga water is found to be self-cleansing probably due to the presence of bacteriophages in ganga water.
The present invention a method of down-regulating pro-inflammatory cytokines comprising; obtaining a sample of ganga water; filtering the sample of ganga water and concentrating the same; and reconstituting the sample in a suitable buffer, wherein the down regulation of the pro-inflammatory cytokines is carried out by extracellular (exRNA) present in ganga water.
The sample of Ganga water was procured from NIT Ghat Patna, Purnia and Begusarai. The collected sample may be filtered twice using filter paper of suitable pore size. The filtration may be carried out using filter paper of 0.45µm pore size and filter paper- 0.22µm pore size. The filtered Ganga water may be subjected to lyophilization/ freeze drying technique to concentrate the Ganga water. In an embodiment the present invention wherein the ganga water is concentrated 200-500x.
The buffer selected for dissolving the lyophilized content of ganga water may be phosphate buffer saline in the range of 0.4-1%, pH -7.4; sodium chloride 0.9%; and glycerin in the range of 1-5%.
Since the Ganga water has varied life forms in it, it is a source of a large reservoir of genetic material. Ganga water has a huge aquatic life and is a source of the extracellular DNA (eDNA) and extracellular RNA (eRNA) in the Ganga water. The present invention is directed towards the concentrating the ganga water to increase content of the eRNA and devise a method to downregulate proinflammatory cytokines. Cytokines and chemokines are small cell-signalling proteins. The receptors of these cytokines are expressed on immune cells. Cytokines play critical role in immune cell differentiation, migration, and maturation of these immune cell into functional subtypes and ultimately exerting their biological functions. T cells proliferate on antigen recognition, into specific types and in the presence of polarizing cytokines, differentiate into effector cells, which produce distinct patterns of cytokines. CD4 T cells differentiate into Th1 cells, which express T-bet and selectively produce IFN-Ƴ; Th2 cells, which express Gata3 and produce IL-4; IL-17 and the transcription factor retinoic acid receptor-related orphan receptor ƴt; Th9; Th22; Tfh and Treg among others. Among cytokines TGF-, IL-1, IL-2,IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17, IL-22, IL-23 and IFN-Ƴ play major role in the development of number of autoimmune disease.
Nuclear factor-κB (NF-κB) represents a family of inducible transcription factors. NF-κB regulate a large array of genes involved in the immune and inflammatory responses. The family have five structurally related members, including NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB and c-Rel. Theses transcription factor target genes by binding to a specific DNA element.
NF-κB proteins are normally sequestered in the cytoplasm by a family of inhibitory proteins, such as IκB. The canonical and noncanonical (or alternative) pathways results in activation of NF-κB. The canonical NF-κB pathway responds to diverse stimuli, including ligands of various cytokine receptors, pattern-recognition receptors (PRRs), TNF receptor (TNFR) superfamily members, as well as T-cell receptor (TCR) and B-cell receptor Innate immune cells such as macrophages, dendritic cells, and neutrophils, express PRRs that detect various microbial components, including pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs). Mammalian cells express five families of PRRs, including toll-like receptors (TLRs), RIG-I-like receptors, NOD-like receptors (NLRs), C-type lectin-like receptors and cytosolic DNA sensors. The primary mechanism for canonical NF-κB activation is the inducible degradation of IκBα triggered through its site-specific phosphorylation by a multi-subunit IκB kinase (IKK) complex resulting in nuclear translocation of canonical NF-κB members predominantly the p50/RelA and p50/c-Rel dimers. The noncanonical NF-κB pathway is activated ligands of a subset of TNFR superfamily members such as LTβR, BAFFR, CD40 and RANK. A central signalling molecule for this pathway is NF-κB-inducing kinase (NIK), which activates and functionally cooperates with IKKα to mediate p100 phosphorylation, inducing p100 ubiquitination and processing. The processing of p100 involves degradation of its C-terminal IκB-like structure, results in generation of mature NF-κB2 p52 and nuclear translocation of the noncanonical NF-κB complex p52/RelB. The PRRs results in activation of the canonical NF-κB pathway, which is responsible for transcriptional induction of pro-inflammatory cytokines, chemokines and additional inflammatory mediators in different types of innate immune cells. These inflammatory mediators engage in the induction of inflammation and also promotes the differentiation of inflammatory T cells. A signaling component that integrates the different PRR pathways for NF-κB activation is transforming growth factor-β-activated kinase 1 (TAK1). In response to diverse PAMPs and DAMPs, macrophages are activated and secrete a large array of cytokines and chemokines. Macrophages differentiate into phenotypically into classically activated (M1) and the alternatively activated (M2) macrophages. M1 macrophages are characterized by the production of pro-inflammatory cytokines, such as IL-1, IL-6, IL-12, TNF-α and chemokines, involved in various inflammatory processes. Signal transduction after TNF-a stimulation occurs most often via the canonical pathway. M1 macrophages also promote the differentiation of inflammatory T cells, including Th1 and Th17 cells. M2 macrophages produce anti-inflammatory cytokines, such as IL-10 and IL-13, and are important for resolution of inflammation and mediating wound healing. TLR signals have an important role in regulating macrophage polarization. TLR4 ligand lipopolysaccharide (LPS) promotes macrophage differentiation toward M1 phenotype. LPS stimulates macrophage signalling, which in turn results in activation of a ubiquitin-dependent kinase, TAK1. Upon activation, TAK1 activates the downstream kinase IKK, which in turn phosphorylate the NF-κB inhibitor IκBα, leading to ubiquitin-dependent IκBα degradation and NF-κB activation. NF-κB is a key transcription factor of M1 macrophages and is required for induction of a large number of inflammatory genes, including those encoding TNF-α, IL-1β, IL-6, IL-12p40 and cyclooxygenase.
Canonical NF-κB members, RelA and c-Rel, also mediates TCR signalling and naive T-cell activation. Deregulated NF-κB activation can cause aberrant T-cell activation, which is associated with autoimmune and inflammatory responses. NF-κB regulates T-cell differentiation and effector function. Upon activation, CD4+ T cells differentiate into different subsets of effector T cells, including Th1, Th2, Th17 and T follicular (Tfh) cells, which secrete distinct cytokines and mediate different aspects of immune responses. Th1 and Th17 cells are generally considered as inflammatory T cells, as these cells are involved in inflammatory responses against both infections and self-triggers, and are associated with various autoimmune and inflammatory conditions.
TNF-alpha is produced by macrophages and T-cells, B cells, NK-cells, neutrophils, mast cells, endothelial cells, smooth muscle cells, cardiomyocytes, fibroblasts, osteoclasts, osteoblasts, astrocytes etc including bacterial lipopolysaccharide (LPS, endotoxin) is also a stimulant triggering TNF-a production. TNF is produced as a 26 kDa 233-amino-acid transmembrane protein (mTNF) that is expressed on the cell surface, which when actively cleaved by TNF-converting enzyme to produce a 17 kDa 157-amino-acid soluble TNF (sTNF) form. mTNF and sTNF both perform cellular functions mediated by either of its two receptors: TNFR1, expressed across all human tissues, and TNFR2, expressed primarily in immune cells, neurons, and endothelial cells biological effects of TNF-a is mediated by binding on two different receptors—TNFR1 (CD120a, p55) and TNFR2 (CD120b, p75). TNFR1 and 2 signalling pathways may lead to the activation of nuclear factor-kappa B (NF-κB). The signalling pathway by sTNF via TNFR1 is known to trigger pro-inflammatory pathways, and mTNF binding to TNFR2 usually initiates immune modulation and tissue regeneration.
(e)RNA present in Ganga Jal through antisense mechanism may significantly down regulate the inflammatory response caused in the autoimmune disease. For instance, in psoriasis i.e., the use of a microRNA which encapsulates in liposome have been made to target Interleukin 17 which is a central driver in psoriasis pathogenesis.
In one embodiment the extracellular ribonucleic acid (exRNA) may be selected from a group comprising micro-Ribonucleic acid (miRNA), small interfering Ribonucleic acid (siRNA), guide Ribonucleic acid (gRNA) cRNA (circular Ribonucleic acid), piwi Ribonucleic acid (piRNA). In a preferred embodiment the extracellular (exRNA) may be microRNA (mi RNA).
In the present invention the inventors successfully isolated RNA from the Ganga Jal as presented in Table 1. The Ganga Jal was concentrated 200 times and RNA isolated and estimated to be 47.617ng /µl. In one embodiment of the present invention, the microRNA Quantification was done using qubit fluorophore The isolated RNA in a tube were further examined using qubit fluorophore analysis. The obtained concentration of micro RNAs was 4.6ng/µl (Figure 2). Therefore, the micro-RNA present in Ganga Jal downregulates proinflammatory cytokines by antisense mechanism.
The down regulation of the pro-inflammatory is carried out inhibition of the messenger-ribonucleic acid (m-RNA) of the pro-inflammatory cytokines by antisense/ribozymal cleavage by the microRNA (mi RNA) present in Ganga water.
This was further demonstrated in the mammalian A549 cell line. The cell line was treated with Ganga Jal, phosphate buffer saline for control, as a negative control, cell line was treated with Ganga Jal + RNAse, as positive control, the cell line was treated with PBS + RNase. This was followed by RNA isolation from the treated cell line and both positive and negative control, C-DNA was prepared and expression of expression of different pro-inflammatory cytokines associated with different autoimmune disorder i.e., IL-1Beta, IL-6, TNFα, IFN-gamma, IL-23, and NFĸB genes were estimated using specific primers. As clear in figure 4, all the genes were IL-23, IL-6, IFN-gamma, TNF-α downregulated when treated with Ganga Jal. However, IL-1β and NF-kb were not downregulated by Ganga Jal, showing that Ganga Jal may not be downregulating the expression the transcription factor NF-kb and IL-1β. The treatment of Ganga Jal with enzyme RNase reversed this effect (figure 5) showing that the micro-RNA present in Ganga Jal inhibits the proinflammatory cytokines except for IL-1β, which was not downregulated when RNAse was mixed with Ganga Jal. The Ganga Jal did not downregulate the IL-1β without RNAse too. NF-kb gene was not tested after treatment of Ganga Jal with enzyme RNase.
The Ganga Jal has shown gene repressions activity against the inflammatory cytokines such as IL-17, IL-6, TNF-α genes. TNFα can stimulate the generation of numerous pro-inflammatory cytokines including IL-6, IL-8, self-activation -TNFα, adhesive molecules, chemokines, and metalloproteinases, pleading to a TNFα-mediated pro-inflammatory autocrine loop. TNF-a as an inflammatory cytokine is known to mediate vasodilatation, edema, facilitation of the adhesion of leukocytes, regulation of blood coagulation during inflammation, changes in the production of reactive oxygen species etc all important processes in inflammation.
IL-17 is an inflammatory cytokine which exerts its function on myeloid cells and mesenchymal cells to induce the expression of granulocyte colony-stimulating factor (G-CSF), IL-6 and chemokines. This increase granulopoiesis and recruit neutrophils to the infection site. Human IL-17 is a homodimeric glycoprotein consisting of 155 amino acids with a molecular weight of about 35 kDa. Additionally, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F cytokines have been identified. The IL-17 receptors also constitute a distinct family of transmembrane protein cytokine receptors namely IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17R. Among these IL-17RA have been reported to be ubiquitously expressed in hematopoietic tissues, various myeloid cells, epithelial cells, fibroblasts, endothelial cells, epithelial cells, and osteoblasts.
A conserved ‘SEFIR’ (short for SEF/IL-17R) domain in the cytoplasmic tail of all IL-17Rs has been identified. ACT1 (also known as CIKS), an activator of NF-κB, was found to contain a SEFIR domain. ACT1 contains a TRAF6-binding motif and thus can bind TRAF6 and TGF-β-activated kinase 1 to deliver downstream signals, resulting in activation of the canonical NF-κB pathway. After activation, the intracellular IL-17 signalling includes ACT1-dependent and -independent downstream pathways. In the ACT1-dependent pathway- IL-17RA engages its SEFIR domain to recruit the adaptor protein ACT1. ACT1 contains a Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) binding motif and that can bind TRAF6, TRAF3 and Transforming growth factor-β-activated kinase 1 (TAK1), which subsequently leads to activation of the canonical NF-κB pathway.
IL-6 induces an extensive range of acute phase proteins such as C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen, haptoglobin, and α1-antichymotrypsin in initial stages of inflammation. High-level concentrations of SAA lead to a serious complication of several chronic inflammatory diseases through the generation of amyloid A amyloidosis and amyloid fibril deposition, which causes progressive deterioration in various organs. IL-6 is also involved in the regulation of serum iron and zinc levels via control of their transporters. IL-6 induces hepcidin production, which blocks the action of iron transporter ferroprotein 1 on gut and reduces serum iron levels. The IL-6-hepcidin axis is thus responsible for hypoferremia and anaemia associated with chronic inflammation. IL-6 also enhances zinc importer ZIP14 expression on hepatocytes and so induces hypozincaemia seen in inflammation IL-6 promotes megakaryocyte maturation, thus leading to the release of platelets in bone marrow.
IL-6 induces differentiation of naïve CD4+ T cells. IL-6 with transforming growth factor (TGF)-β, is required for Th17 differentiation from naïve CD4+ T. IL-6 also inhibits TGF-β-induced Treg differentiation. Up-regulation of the Th17/Treg balance results in disruption of immunological tolerance, and involved in the development of autoimmune and chronic inflammatory diseases. IL-6 also promotes T-follicular helper-cell differentiation and production of IL-21, which regulates immunoglobulin (Ig) synthesis and IgG4 production. IL-6 also induces the differentiation of CD8+ T cells into cytotoxic T cell. IL-6 also induce the differentiation of activated B cells into Ab-producing plasma cells, so that continuous over synthesis of IL-6 results in hypergammaglobulinemia and autoantibody production.
IL-6 in bone marrow stromal cells stimulates the RANKL, which results in differentiation and activation of osteoclasts leading to bone resorption and osteoporosis. IL-6 also induces excess production of VEGF, leading to enhanced angiogenesis and increased vascular permeability.
In still another embodiment, the method of the present invention can downregulate the pro-inflammatory gene selected that may be selected from a group comprising APOE (Apolipoprotein E), CTSB(Cathepsin-B), IL-1β (Interleukin -1 beta), IL-6(Interleukin-6), Presenilin 1 (PSEN-1), TNF-α (Tumour necrosis factor -α),, IL-4 (Interleukin-4), IL-5 (Interleukin-5), IL-6 (Interleukin-6), IL-9 (Interleukin-9), IL-12(Interleukin-12), IL-13(Interleukin-13), IL-15(Interleukin-15), IL-17(Interleukin-17), IL-18 (Interleukin-18), IL-23 (Interleukin-23), IL-33(Interleukin-33) (Interleukin-6), TGF-β (Transforming growth factor-β), TNF-α (Tumour necrosis factor -α), IL-17, IL-23, IL-6, NfκB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), TLR-9 (Toll like receptor-9), TNF-α, TGF-β, IFN-ϒ(Interferon-Ƴ), IRS-1(Insulin receptor substrate 1), CXCL-10(C-X-C motif chemokine ligand 10), ESR-1 (Estrogen Receptor 1), KISS-1, KLK-7(Kallikrein-1), CCL-11(C-C motif chemokine ligand-11), CCL-2(C-C motif chemokine down-regulating pro-inflammatory cytokines).
In one embodiment, the downregulation is carried out by the ex-RNA molecule encapsulated in extra cellular vesicle present in Ganga Jal. The extra cellular vesicle may be 40nm -1200nm in size with mean size of 410nm.
Zeta potential (ZP), as an indicator of colloidal stability which is influenced by the surface charge. The ZP is measured by electrophoretic mobility in a suspension. The cell plasma membrane possesses a negative surface charge when suspended in a neutral medium. The EV also carry a negative charge in a neutral medium. The surface charge of EVs depends on a multitude of factors such as ionization of the membrane surface groups, protonated states, inter- and intramolecular bonding, presence of H-bonds, and ion adsorption from the electrolytes present in solution. The EVs also isolated from the Ganga Jal showed a net negative charge of -14.3mV.in the PBS buffer at pH 7.4.
In one embodiment, a composition of the extra cellular vesicle comprising extracellular ribonucleic acid (exRNA) wherein the concertation of extracellular ribonucleic acid (exRNA) may be in the range of 4-12µg/ml, along with suitable pharmaceutical components is disclosed. The excipients used in the composition may be admixed with non-toxic pharmaceutically acceptable excipients. These include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, glucose, mannitol, cellulose, starch, calcium phosphate, sodium phosphate, kaolin and the like. Binding agents, buffering agents, and/or lubricating agents may also be used. Tablets and pills can additionally be prepared with enteric coatings. The composition may optionally contain sweetening, flavoring, coloring, perfuming, and preserving agents in order to provide a more palatable preparation.
Examples
The following examples are for illustration purposes and are not to be construed as limiting the invention disclosed in this document to only the embodiments disclosed in these examples.
Example 1
RNA estimation in Ganga Water
Sample Collection
The sample was collected using a sterile screw capped container from various sites of Bihar Ghats safely. The various sites are NIT Ghat Patna, Purnia and Begusarai. All the samples are collected from Eastern region, Bihar, India.
RNA isolation
50ml Ganga Jal sample was taken in autoclaved falcon tube. The Ganga Jal sample was then centrifuged for 10 min at 2000g in a cooling centrifuge. The pellet was discarded and supernatant was taken in another tube. The supernatant was centrifuged for 20 min at 14000g in a cooling centrifuge, pellet was discarded and supernatant was collected in another tube. The supernatant was centrifuged for 30 min at 20000g in a cooling centrifuge. The pellet was discarded and supernatant was a lyophilized/freeze dried for 12 hours. After 12 hours the lyophilised sample was further used for RNA isolation.
Method of RNA Isolation
The lyophilized sample was reconstituted with 200µl of nuclease free water of Ganga Jal and taken in an empty 1.5ml lysis tube. 220µl of lysis solution supplemented with 5µl carrier RNA was added and mixed thoroughly by vortex of sample The sample was incubated for 15 min at 56°C in a water bath. The sample was then centrifuged for 3-5 sec at full speed to collect drops of the sample accumulated inside of the lid. 300 µl of ethanol was added to the sample and mixed by vortex of sample. The sample was incubated at room temperature for 3 min and centrifuged for 3-5 sec at full speed to collect drops from the inside of the lid. The lysate was transferred to a prepared spin column preassembled within the wash tube. The column was centrifuged for 1min at 6000g. The wash tube containing flow-through was discarded and the spin column was placed into a new 2ml wash tube. 700 µl of wash buffer VWB supplemented with ethanol was added to the spin column. The column was centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and placed the spin column into 2ml wash tube. 500 µl of wash buffer VWB3 supplemented with ethanol was added to the spin column and the column was centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and placed the spin column into 2ml wash tube. 500 µl of wash buffer VWB3 supplemented with ethanol was added to the spin column and centrifuged for 1 min at 6000g. The wash tube containing flow through was discarded and the spin column was placed into 2ml wash tube. The column was centrifuged for 3 min at 12000g. The wash tube containing flow through was discarded. The spin column was placed into a new 1.5 ml elution tube. 50 µl of RNase free water was added, preheated to 56°C to the centre of spin column membrane. The column was incubated for 2 min at room temperature. The column was centrifuged for 1 min at 12000g. The spin column was discarded and the purified nucleic acids was stored at -20°C. The nucleic acids were estimated in the sample by taking reading epoch2 and fluorophore reading. The isolated RNA in a tube were further examined using fluorometry analysis. It was done to measure the purity and quantity of RNAs isolated from Ganga Jal sample. The instrument for the above-mentioned process was of Biotek, Model- Epoch Microplate Spectrophotometer.
Table 1: Estimation of RNA in Ganga Jal
Sample-Ganga Jal,
Sample volume- 1 µl Measurement
OD at 260λ 0.06
OD at 280λ 0.028
260/280 ratio 2.129
Quantity RNA 47.617 ng /µl

The microRNA Quantification was done using qubit fluorophore The isolated RNA in a tube were further examined using qubit fluorophore analysis. It was done to measure the quantity of microRNAs from Ganga Jal sample. The obtained concentration of micro RNAs was 4.6 ng /µl (Figure 2).
Example 2
In-vitro experiment of Ganga Jal in the mammalian A549 cell line
Culture of A549 cell line
Cell-seeding of equal amount of cell in a six well plate was done and the cells were incubated for 24 hours at 30°C in a 5% CO2 incubator for allow to reach 70-80% confluency. After 24 hours 100 µl PBS in first well, 100 µl PBS+RNAse in second well, 100 µl Ganga Jal(GJ) in third well, 100 µl Ganga Jal(GJ)+ RNAse was added in fourth well. After 24 hours RNA isolation was carried out.
RNA ISOLATION
Six 1.5ml lysis tube was taken and marked as Phosphate Buffer Saline (PBS), Phosphate Buffer Saline + enzyme RNase (PBS+RNase), Ganga Jal (GJ), Ganga Jal+RNase (GJ+RNase), samples were accordingly taken in each tube, The samples were centrifuged for 10 minutes at 2000g in a cooling centrifuge. The supernatant discarded and pellets taken. 500 µl lysis buffer (Biorad Aurum Total RNA Mini Kit) was added in each sample, mix well and vortexed. The lysate was transferred to a prepared spin column preassembled within the wash tube. 500 µl of Diethyl Pyrocarbonate plus ethanol (DEPC+ethanol) was solution in each sample, mix well and vortexed and centrifuged for 1 minute at 13000g. The supernatant was discarded. 700 µl total RNA low stringency wash solution (Biorad Aurum Total RNA Mini Kit) was added in each sample and centrifuged for 1 minute at 13000g. The supernatant was discarded and 80 µl solution of 75 µl DNAse buffer + 5 µl DNAse (Biorad Aurum Total RNA Mini Kit) was added to each sample. The samples were incubated for 20 minutes in room temperature. 700 µl total RNA high stringency wash solution (Biorad Aurum Total RNA Mini Kit) was added in each sample and centrifuged for 1 minute at 13000g in a cooling centrifuge. 700 µl total RNA low stringency wash solution (Biorad Aurum Total RNA Mini Kit) in each sample was added again and centrifuged for 1 minute at 13000g in a cooling centrifuge. The supernatant was discarded pellet was air dried centrifuge for 2 minutes at 13000g. The spin column was placed into a new 1.5 ml elution tube and 40 µl of elution was added in each tube and incubated for 2 min at room temperature. The column was centrifuged for 1 min at 12000g. The spin column was discarded and the purified nucleic acids was stored at -20°C for epoch2 and fluorophore reading.

Table 2: Estimation of RNA in Ganga Jal
Sample
OD at 260λ OD at 280λ 260/280 ratio RNA (ng /µl)
Culture supernatant 0.056 0.026 2.164 44.704
Culture supernatant + RNase 0,043 0.020 2.164 04.163
Ganga Jal 0.022 0.011 2 17.696
Ganga Jal + RNase 0.033 0.016 2.062 40.313

The isolated RNA in a tube were further examined using fluorometry analysis. It was done to measure the purity and quantity of RNAs isolated from Ganga Jal sample. The instrument for the above-mentioned process was of Biotek, Model- Epoch Microplate Spectrophotometer.
C-DNA preparation
The isolated RNAs were used to prepare cDNA. The cDNA stands for complementary DNA, which the complementary RNA sequences. Total RNA is routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR, whereas specific types of RNAs (e.g., messenger RNA (mRNA) and small RNAs such as miRNA) may be enriched for certain applications like cDNA library construction and miRNA profiling. All the eluted RNA sample were normalised (volume equalization) of all six samples i.e. PBS, PBS+RNAse, Ganga Jal (GJ), Ganga Jal(GJ)+ RNAse. Pipetting was done for proper mixing and subsequently PCR tube was run on the PCR machine for 37 minutes. cDNA was prepared and further used for RT qPCR.

RT-PCR Analysis
The expression of different pro-inflammatory cytokines associated with different autoimmune disorder i.e,. IL-1Beta, IL-6, TNFα, IFN-gamma, IL-23, and NFĸB. CDNA 2 µl, primers 5 µl (2.5 µl forward and 2.5 µl reverse) and SYBR green 20 µl was added in a PCR tube and volume makeup was done with nuclease free water. Pipetting was done for proper mixing and then each sample duplet was loaded in 96 well plates. Plate sealer was applied and subsequently, the plate was put on the rotor for proper mixing. Plate was transferred to the PCR machine and according to desired primers temperatures was and PCR was run for 1 hour 52 minutes. Cq value of desired genes was analysed at 80-93°C, multi curved were checked. The gene expression was analysed for IL-1Beta, IL-6, TNFα, IFN-gamma, IL-23, and NFĸB genes.
EXAMPLE 3
Isolation of Extracellular vesicles from Ganga Jal
The extracellular vesicles were isolated from the sample by using the ultracentrifugation technique. The sample was filtered with a 0.22 µm syringe filter and ultracentrifuged at 1,25,000 xg for 90 min at 4 °C. After completion of the centrifugation process, the tubes were removed and the supernatant was carefully collected. The EV pellet (may/may not be visible) was resuspended in 1 ml of PBS (filtered through a 0.1 µm syringe filter). The EV pellet and supernatant were stored at -20 °C for further analyses.
Dynamic Light Scattering
Sample were prepared to attain a protein concentration of 1 – 2 mg/ml. Sample buffer (PBS, pH 7.4), 100% EtOH, and MQ H2O through 0.1 µm filter. Photon Correlation Spectroscope was first calibrated with MQ H2O, followed by filtered buffer (reading should be 15,000 and 20,000 cts/s.). This is followed by taking the reading of the sample in the same cuvette, which has been calibrated with MQ H2O and sample buffer.
Weighting model Peak 1 [nm] Peak 2 [nm]
Intensity 844.7 165.52
Volume 964.9 144.05
Number 123.92 676.5

Size distribution D₁₀ [nm] D₅₀ [nm] D₉₀ [nm] Undersize span (D₉₀- D₁₀)/D₅₀
Volume 406.5 891.6 1279.6 0.979
Intensity 178.17 729.8 1194.4 1.393
Number 64.77 113.89 162.27 0.856

Two peaks of ~150 nm and ~900 nm was observed. A total of 800 particles were detected. 90 % (D90) particles were <1194.4 nm (number-162), 50 % were <729.8 nm (number-114), and 10 % were <178.17 nm (number-65). The transmittance of 84.8%, filter optical density of 0.4996 suggest the presence of particles in the sample. The mean intensity of 130.5 kcounts/s (>20 kcounts/s) was also in line with the type of sample. The value of Intercept g1² (0.8267) was slightly less than 0.85. The baseline was 1.083. The Polydispersity index of 30 % indicate heterogeneity of the sample but is less than negative control (MQ H2O).
The data suggest that the sample contains EVs (VLPs) of sizes 150 to 1200 nm. Though, the sample was filtered with 0.22µm syringe filter before analysis the presence of EVs of size around 1200nm is probably due to the aggregation of EVs.
As is clear from the figure 6, the size range is 40-1200nm in size. While the mean value of the size of the exosomes were 150nm.
Mean zeta potential -14.3 mV
Distribution peak value -8.6 mV
Processed runs 420
Adjusted voltage 55.8 V
Mean intensity 591.6 kcounts/s
Electrophoretic mobility -1.1111 µm*cm/Vs
Filter optical density 0.0000
Conductivity 16.996 mS/cm
+/- Standard deviation 0.7 mV
Transmittance 94.6 %

Preparation of specimen for TEM imaging
10 µl of EV sample was pipetted on a carbon grid coated with copper and allowed to stand at RT for 2 min. The excess liquid was removed by blotting the grids. The grid was briefly placed on 10 µL of 2% uranyl acetate followed by blotting to remove excess liquid. The above step was repeated once.The grids were air-dried at room temperature. Finally, the samples were observed under TEM, and images were captured.
A heterogeneous population of extracellular vesicles having different sizes and shapes (circular, oval, elongated) was observed in the TEM images. The size of EVs ranged from ~40 nm to 410 nm. Maximum EVs were present in the range of 100 to 200 nm. Most of the EVs demonstrated circular shapes, however, few oval and elongated shapes were also detected. The bilayered structure of EVs is clearly visible in the images.
SEM analysis
After 24 h fermentation, cells were pelleted from all samples and pre-fix in 50 μL of formaldehyde (37% v/v) and incubate for 15 min at room temperature. The fixed cells were washed three times in PBS (pH 7.4) and dehydrated in a gradient series of ethanol (50, 70, 90%, 95% and 3 × 100% for 10 min), followed by critical point drying with liquid CO2. The gold–palladium (Au–Pd) were coated on to samples with and examined in a Zeiss Evo 50 EP scanning electron microscope (Carl Zeiss AG, Oberkochen, Germany)
Second method
Cells were fixed in 5% glutaraldehyde, 0.2 M sodium Cacodylate, 0.4 M Sucrose, 10mM MgCl2 pH 7.4 and mixed 1:1 with suspension. Postfix the biofilm samples treated for 30 min with 1% osmium tetroxide, 0.7% potassium ferrocyanide, 0.1 M sodium cacodylate, 0.2 M sucrose, and 5 mM MgCl2 (pH 7.4). The samples were dehydrated through a graded series of ethanol and critical point dry using liquid carbon dioxide in a Tousimis Samdri 795 Critical Point Drier (Rockville, MD). The samples were sputter coated with gold-palladium in a Denton Vacuum Desk-2 Sputter Coater (Cherry Hill, NJ) and examined in a Zeiss Supra Field Emission Scanning Electron Microscope (Carl Zeiss Microscopy, LLC North America), using an accelerating voltage of 5 KV (doi:10.1111/mmi.12650; doi, 10.3390/microorganisms8070983)
SEM analysis of the Ganga Jal revealed extracellular vesicles (Figure 11 and 12). The size range of the EVs are of 100 to 200 nm.
While specific language has been used to describe the disclosure, any limitations arising on account of the same 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. The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.
, C , C , Claims:We Claim,
1. A method of down-regulating pro-inflammatory cytokines comprising;
i. obtaining a sample of ganga water;
ii. filtering the sample of ganga water and concentrating the same; and
iii. reconstituting the sample in a suitable buffer.
wherein the down regulation of the pro-inflammatory cytokines is carried out by extracellular (exRNA) present in ganga water; and
wherein the ex-RNA molecule encapsulated in extra cellular vesicle present in Ganga Jal.
2. The method as claimed in claim 1, wherein ganga water is concentrated 200-500x.
3. The method as claimed in claim 1, wherein the extracellular ribonucleic acid (exRNA) is selected from a group comprising micro-Ribonucleic acid (miRNA), small interfering Ribonucleic acid (siRNA), guide Ribonucleic acid (gRNA) cRNA (circular Ribonucleic acid), piwi Ribonucleic acid (piRNA).
4. The method as claimed in claim 1, wherein the pro-inflammatory gene selected from a group comprising APOE (Apolipoprotein E), CTSB(Cathepsin-B), IL-1β (Interleukin -1 beta), IL-6(Interleukin-6), Presenilin 1 (PSEN-1), TNF-α (Tumour necrosis factor -α),, IL-4 (Interleukin-4), IL-5 (Interleukin-5), IL-6 (Interleukin-6), IL-9 (Interleukin-9), IL-12(Interleukin-12), IL-13(Interleukin-13), IL-15(Interleukin-15), IL-17(Interleukin-17), IL-18 (Interleukin-18), IL-23 (Interleukin-23), IL-33(Interleukin-33) (Interleukin-6), TGF-β (Transforming growth factor-β), TNF-α (Tumour necrosis factor -α), IL-17, IL-23, IL-6, NfκB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), TLR-9 (Toll like receptor-9), TNF-α, TGF-β, IFN-ϒ(Interferon-Ƴ), IRS-1(Insulin receptor substrate 1), CXCL-10(C-X-C motif chemokine ligand 10), ESR-1 (Estrogen Receptor 1), KISS-1, KLK-7(Kallikrein-1), CCL-11(C-C motif chemokine ligand-11), CCL-2(C-C motif chemokine ligand-2).
5. The method as claimed in claim 1, wherein in the buffer is phosphate buffer saline in the range of 0.4-1%, pH -7.4; sodium chloride 0.9%; and glycerin in the range of 1-5%.
6. The method as claimed in claim 1, wherein the downregulation is carried out by the ex-RNA molecule encapsulated in extra cellular vesicle present in Ganga Jal.
7. The method as claimed in claim 1 and 8, wherein the extra cellular vesicle is 40nm -1200nm in size preferably 150nm in size and has a zeta potential of -14.3mV.
8. A composition of the extra cellular vesicle as claimed in claim 1-7 comprising extracellular ribonucleic acid (exRNA) and suitable pharmaceutical components.