A PLANT BASED EDIBLE EXOSOMES “LIFEX 1.0 (LIVER FIT EXOSOMES)” FORMULATION AND METHOD FOR PREPARATION THEREOF

Abstract

The present invention discloses harnesses the potential of Extracellular Vesicles (ExVs) from plant edible sources i.e. edible exosomes for mitigating Hepatic encephalopathy. The present invention includes a developed formulation of an oral supplement composite ‘LiFEx’ to improve liver fitness, comprises edible exosomes sourced from black carrot, ginger, garlic, turmeric and lemon after specifically isolated and characterised, offer an affordable solution to treatment of Hepatic encephalopathy. The LiFEx composite significantly reduces the urease positive taxa Escherichia-shigella, from family Enterobacteriaceae and improves colonisation of various probiotic taxa in gut of acute HE rats. LiFEx composite exerts bifidogenic effect, improves the colonisation of Bifidobacterium and other taxa like butyricoccus, ruminococcus and lactobacillus helps in restoring gut homeostasis. LiFEx composite enhance the proliferation of Dubosiella, an emerged probiotic taxa known for its hepato-protective and neuro-protective effects.

Specification

Description:[001] The present invention relates to a plant based edible exosomes formulation and method of preparation thereof. More specifically, the present invention concerns edible exosomes that possess hepato-protective roles.
BACKGROUND OF THE INVENTION
[002] Traditional Indian food components, renowned for their hepatoprotective properties, have garnered attention in mitigating Hepatic encephalopathy (HE). Nevertheless, the challenge persists in harnessing the benefits effectively without resorting to consuming large quantities, which may not be practical or sustainable.
[003] WO2022152771A1 (Abstract): discloses a composition comprising non-immunogenic, plant-derived edible exosomes for as an oral prebiotic supplement product. These exosomes are naturally occurring with their own unique endogenous cargoes.
[004] WO2019229271A1 (Abstract): Provided are edible exosomes isolated from edible products i.e. black carrot, ginger, garlic, turmeric and lemon. The edible exosomes are characterized by a unique size distribution and are particularly suitable for use in health- promoting products, food supplements and drugs.
[005] Edible exosomes have emerged as promising vehicles for therapeutic intervention due to their innate ability to facilitate intercellular communication and deliver bioactive payloads. Plant-derived edible exosomes offer a cost-effective and potentially impactful avenue for addressing HE. Despite their promise, challenges in isolation methodologies have hindered their widespread adoption, necessitating novel approaches to overcome technical hurdles and enhance their utility.

SUMMARY OF THE INVENTION
[006] In the view of the foregoing disadvantages inherent in the known types of conventional derivatives, methods and techniques, now present in the prior art, the present invention provides formulation and a method for preparation of plant based edible exosomes, which has all the advantages of the prior art and none of the disadvantages.
[007] In an aspect of the present invention, a plant-based formulation comprising, edible exosomes in a concentration of approximately 9.31x10^8 to 2.68 x 10^9 particles/mL, wherein the edible exosomes have a size distribution primarily in the range of 50 nm to 300 nm, and wherein the edible exosomes are isolated from plant-based sources selected from the group consisting of turmeric, ginger, garlic, lemon, and black carrot.
[008] In another aspect of the present invention, a method for preparation of plant-based edible exosomes oral supplement method comprising, obtaining juice from a plant-based source selected from the group consisting of turmeric, ginger, garlic, lemon, and carrot, wherein the juice is obtained, isolating edible exosomes from the obtained juice using a combination of filtration and centrifugation, wherein the edible exosomes are isolated using a centrifugal force ranging from 10,000 x g to 100,000 x g; and characterizing the edible exosomes for size, stability, viability and bioactive compounds.
[009] In this respect, before explaining at least one object of the invention in detail, it is to be understood that the invention is not limited in its application to the details set of rules and to the arrangements of the various models set forth in the following description or illustrated in the drawings. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.
[0010] These together with other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be made to the accompanying drawings and descriptive matter in which there are illustrated preferred embodiments of the invention.
OBJECTIVES OF THE PRESENT INVENTION
[0011] The objective of the present invention is to overcome the objections of the prior art.
[0012} Another objective of the present invention is to provide a formulation of plant based edible exosomes.
[0013] Another objective of the present invention is to provide a method of preparation of plant based edible exosomes.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The following sections of this article will provide various embodiments of the current invention with references to the accompanying drawings, whereby the reference numbers utilized in the picture correspond to like elements throughout the description. However, this invention is not limited to the embodiment described here and may be embodied in several other ways. Instead, the embodiment is included to ensure that this disclosure is extensive and complete and that individuals of ordinary skill in the art are properly informed of the extent of the invention.
[0015] Numerical values and ranges are given for many parts of the implementations discussed in the following thorough discussion. These numbers and ranges are merely to be used as examples and are not meant to restrict the claims' applicability. A variety of materials are also recognized as fitting for certain aspects of the implementations. These materials should only be used as examples and are not meant to restrict the application of the innovation.
[0016] The present invention provides a plant based edible exosomes formulation as oral supplemented product for liver fitness and method for preparation thereof.
[0017] In accordance with the embodiment of the present invention, a composition of plant-based edible exosomes which can be taken orally and improve the liver fitness, herein referred to as 'LiFEx 1.0' (Liver Fit Exosomes). The novelty of this invention lies in its multifaceted approach, integrating optimized concentrations of edible exosomes sourced from black Carrot, Ginger, Garlic, Turmeric, and Lemon. The composition of LiFEx 1.0 is meticulously tailored to ensure efficacy, efficiency, biological stability, and viability of the respective exosomes from each plant source.
[0018] LiFEx 1.0 is developed with the aim to improve gut health which influences liver metabolic capacity, specifically targeting inflammation and hyperammonemia. Our formulation is specially prepared to target the multifaceted targets. By harnessing the synergistic properties of the combined exosomes, LiFEx 1.0 seeks to improve gut microbiota balance and function, offering a comprehensive solution for gut and liver health both and integrity maintenance, particularly in hepatic encephalopathy. Additionally, the diverse bioactive compounds present in the LiFEx 1.0formulation are leveraged to address multiple aspects of the gut-liver axis.
[0019] The size and concentration of edible exosomes in LiFEx 1.0 range from 50nm to 300nm, with approximately 9.31x10^8 to 2.65x10^9 ± 0.65 particles/ml with the concentration varies for all the edible sources i.e. carrot, ginger, garlic, turmeric and lemon with the zeta potential ranging from -7 to -16.0 ± 2.4mV. Metabolic cargo analysis reveals that exosomes enriched with organic acids, polyphenols, and their derivatives exhibit potent therapeutic properties. Carrot Exosomes (CarEx) containing Tangeretin enhance gap junctional intercellular communication, while Ginger Exosomes (GinEx) with gingerol and ellagic acids offer hepatoprotective benefits. Garlic Exosomes (GarEx) featuring Licoisoflavone A demonstrate anti-hypertrophic effects, whereas Turmeric Exosomes (TurEx) containing Curcumin and amygladin exhibits anti-inflammatory and neuroprotection. Lemone Exosomes (LemEx) enriched with stachydrine exhibit anti-fibrotic properties, complemented by pyridines acting as vasodilators. In-vitro digestion assay performed using stomach/intestine like solutions to assess the biological stability and viability of edible exosomes resulted in reduction of particle size with intact zeta potentials. To optimize the composition of LiFEx, edible exosomes co-cultured with hepatocytes, Carrot Exosomes (CarEx) enhance the expression of ornithine and arginase genes linked to the urea cycle for detoxifying ammonia, while also boosting antioxidant activity (SOD). Ginger Exosomes (GinEx) and Turmeric Exosomes (TurEx) collectively enhance anti-inflammatory gene expression (IL-10). Garlic Exosomes (GarEx) induce a restrictive growth pattern in hepatocytes, characterized by reduced expression of the PCNA gene. Lemone Exosomes (LemEx) potentially induce cell death, possibly by altering the pH of the surrounding media. Meanwhile, when co-cultured with pro-biotic microbial model organism Lactobacillus rhamnassus GG (LGG) CarEx, GinEx and GarEx showed a significant upregulation in the growth of LGG. Based on the above data, the formulation 'LiFEx 1.0' comprises the optimised concentrations i.e. 4 parts of CarEx, 2 parts of GinEx, 2 parts of GarEx, and 1 part of each TurEx and LemEx, yielded significant outcomes, demonstrating effectiveness in reducing inflammation, mitigating cellular injury, and enhancing the urea cycle in hepatocytes more efficiently compared to the standard medical treatment lactulose.
[0020] Novelty of this study includes-
• Formulate LiFEx 1.0, as an oral supplement product integrating optimized concentrations of edible exosomes sourced from Carrot, Ginger, Garlic, Turmeric, and Lemon to improve liver fitness.
• Tailor the composition of LiFEx to ensure efficacy, efficiency, biological stability, and viability of the respective exosomes from each plant source.
• Develop LiFEx 1.0 with a multifaceted approach aimed at enhancing gut health, specifically targeting inflammation and hyperammonemia.
• Harness the synergistic properties of the combined exosomes to improve gut microbiota balance and function, offering a comprehensive solution for gut health and integrity maintenance in CLD.
• Leverage the diverse bioactive compounds present in the LiFEx 1.0 formulation to address multiple aspects of gut-liver axis.
[0021] Oral administration of LiFEx 1.0 to Thioacetamide (TAA) induced acute HE rats prophylactically results in significant reductions in plasma ammonia levels and serum liver enzymes, indicative of improved liver condition. Enhanced cognitive and sensory motor activities are also observed in rodents receiving LiFEx 1.0, alongside reductions in inflammation and cellular injury, as evidenced by histopathological and gene expression studies of liver tissues efficiently than the rats administered with lactulose.
[0022] Metagenomics analysis of rodent stools further validates the efficacy of LiFEx 1.0 composite, demonstrating enhanced colonization of beneficial probiotic bacteria and reductions in dysbiotic gut microbes associated with Non-autochthonous flora. Notably, LiFEx 1.0 composite outperforms standard pre-biotic medical treatments like lactulose, offering a safer and more effective alternative.
[0023] Hepatic encephalopathy is a neuropsychiatric disorder that develops in individuals with liver disease. Symptoms are related to progressive dysfunction of the brain and may include personality changes, intellectual impairment, impaired memory and loss of consciousness (coma). Hepatic encephalopathy can occur in individuals with acute or chronic liver disease or in individuals whose liver is bypassed by a portosystemic shunt (with no liver disease present). A portosystemic shunt is an abnormal passageway that allows blood from the gastrointestinal tract to bypass the liver. HE is more prevalent in patients with chronic liver disease, approximately 30-50% patients with cirrhosis develope this hyperammonemia. In chronic liver disease, there exist an impaired entero-hepatic circulation leading to dysbiotic gut with enhanced colonisation of urease positive bacteria majorly belongs to the family, Enterobactereacae. The urease produced by these non-autochtonous taxa hydrolyse urea in to ammonia and carbon dioxide in gut which majorly contributes to the systemic hyperammonemia. Meanwhile, the inefficiency of liver to detoxify ammonia exacerbate the disease condition, and the gaseaous ammonia crosses the blood brain barrier causing hepatic encephalopathy (HE). Without adequate diagnosis and treatment, HE exacerbates in to hepatic coma, eventually causing mortality of the patient. Many of the symptoms of hepatic encephalopathy are reversible when promptly detected and treated.
[0024] Extracellular Vesicles (ExVs) emerge as a promising solution due to their nano size and biological origin, making them ideal for translational research in diagnostics and therapeutics. Plant-derived ExVs, particularly from edible portions, offer a cost-effective means of isolation. Edible exosomes contain natural bioactive compounds and non-coding RNAs. Their small size, biocompatibility, stability, low toxicity, and non-immunogenicity position them as superior therapeutic candidates compared to mammalian ExVs. However, challenges in edible exosomes isolation include time consumption, producibility, and size uniformity.
[0025] To this end, we have synthesized in-house methodology yields approximately 9.31x10^8 to 2.68 x 10^9 particles/mL from juices of mentioned edible foods. Characterization reveals uniform particle sizes ranging from 50 nm to 300 nm, ensuring biological stability and viability. Zeta potential measurements confirm stability, and mass spectrometry identifies bioactive metabolites with therapeutic potential. Notably, the methodology addresses drawbacks associated with other techniques such as electrophoresis, enzyme-based methods, and commercially available kits, mitigating issues related to purity and interference with vesicle properties.
[0026] The present method capitalizes on the unique characteristics of edible exosomes for potential applications in translational research, diagnostics, and therapeutics. These small membrane vesicles, with a nano size and biological origin, facilitate intercellular communication by carrying various biomolecules as cargoes. In plants, natural occurrences of this nano-delivery system play a crucial role in cell-to-cell communication and immune regulation against pathogenic attacks. The focus on plant-derived ExVs, particularly from the edible portions, presents an affordable and significant means of isolating edible exosomes, offering a prospective solution to Hepatic encephalopathy.
[0027] Edible exosomes boast natural bioactive compounds and non-coding RNAs, designed to enhance stability. They have demonstrated efficient and safe delivery of both naturally occurring cargoes and artificially loaded molecules such as miRNAs and small synthetic drugs, establishing themselves as promising therapeutic candidates. The small size, biocompatibility, stability, low toxicity, and non-immunogenicity of edible exosomes position them favorably over other mammalian ExVs. However, challenges in isolation persist, including time consumption, producibility, and achieving uniformity in size, as no specific marker for Edible exosomes has been reported to date.
[0028] The in-house methodology developed for edible exosomes isolation demonstrates enhanced producibility within a short timeframe, yielding approximately 9.31x10^8 to 2.68 x 10^9 particles/mL from juices of specific edible foods. Morphological characterization and size distribution analysis were conducted using Nanoparticle Tracking Assay (NTA) and Transmission Electron Microscopy (TEM).
[0029] Despite various methods reported to date, such as gradient ultracentrifugation, electrophoresis, enzyme-based methods, and commercially available kits, challenges persist. These include contamination issues from chemicals used in gradient development affecting purity, potential interference with zeta potential in electrophoresis methods, and potential alterations to particle size or stability in enzymatic action-based methods.
[0030] The paramount advantage of Edible exosomes lies in their innocuous nature and specific uptake by host cells, whether microbes or humans, depending on their membrane composition of lipids. This characteristic enables not only intercellular but also inter-kingdom communication. Edible exosomes' potential as a non-invasive drug carrier, loaded with targeted hydrophobic drug molecules capable of oral intervention and reaching the brain by crossing the Blood-Brain Barrier (BBB), positions them as a promising candidate in nanomedicine. The ease of isolation from reproducible sources such as carrots and ginger enhance their appeal, suggesting a dynamic role for edible exosomes in the gut-liver axis by emphasizing the connection between a healthy gut and a healthy liver.
[0031] Orally administered LiFEx to acute HE rodents prophylactically resulted in a significant reduction of plasma ammonia levels and serum liver enzymes such as AST, ALT and bilirubin, indicating an improved liver condition more efficiently that lactulose. Rodents receiving LiFEx also exhibited enhanced cognitive and sensory motor activities. Histopathological analysis revealed a reduction in inflammation and cellular injury, evidenced by decreased necrosis and cholestasis, which was further validated through gene expression studies of liver tissues. Notably, LiFEx improved the expression of genes associated with urea cycle enzymes.
Metagenomics analysis of rodent stools indicated that LiFEx significantly enhanced the colonization of probiotic bacteria, including Lactobacillus, Ruminococcus and exerts bifidogenic effects by improving the colonisation of Bifidobacterium, renowned for mitigating intestinal immunopathology in a Treg-dependent manner. Moreover, the enhanced abundance of Dubosiella, a well-known probiotic associated with hepatoprotective and neuroprotective effects. Additionally, LiFEx promoted the proliferation of Butyricoccus, a butyratic acid (a well-known short-chain fatty acid-SCFA) producing microbe and restoring intestinal immune homeostasis. Conversely, the abundance of non-autochtonous microbes such as Escherichia-shigella (family Enterobactereacae) and quinella (family Veillonellaceae) positively associated to HE, was decreased by LiFEx. Abundance of Muribaculum that positively associated with inflammation and Prevotella positively associated with NAFLD, lowered upon LiFEx administration. These findings also suggests LiFEx is more effective than the ongoing pre-biotic standard medical treatment lactulose.

Fig: Differential abundance of gut microbiota improved upon oral administration of LiFEx composite compared to Thioacetamide (TAA) induced acute HE rats.
[0032] Therefore, this study demonstrates that orally administered LiFEx has the potential to improve the colonization of probiotic bacteria, leading to the restoration of gut homeostasis by mitigating intestinal immunopathology. Additionally, LiFEx mitigates the colonization of microbes positively associated with HE or inflammation, all without any observed side effects. Meanwhile, LiFEx aids in restoring hepatic homeostasis by reducing cellular injury and enhancing the expression of urea cycle enzymes.

Fig: LiFEx composite improves liver fitness. a. Plasma ammonia levels, b. Histopathology of rat liver, denoting the liver injuries as
(A. Inflammatory Infiltration, B. Confluent Necrosis, C. Cellular Cholestasis, D. Portal Expansion)
[0033]Transmission Electron Microscopy: The exosomes were morphologically characterized using Transmission Electron Microscopy (TEM). Freshly isolated exosomes were appropriately diluted and loaded onto Formvar carbon-coated copper grids. After an incubation period of 5 minutes, excess samples were washed off with decarbonated water, and the grids were dried at room temperature for 3 minutes. Subsequently, they were examined under a Talos F200C TEM (Thermo Scientific).

Fig: Identification and characterisation of Edible Exosomes. A. Representative Transmission Electron Microscopy image of edible exosomes, Physiochemical Characterization of Edible Exosomes B. Nanoparticle Tracking Assay concentration and C. Surface zeta potential.
[0034]Physiochemical Characterization of Edible Exosomes:
The hydrodynamic size and number of exosomes were analyzed using Nanoparticle Tracking Analysis (NTA) with a Nanosight LM20 equipped with NTA 3.1 software. The mass distribution of particle size was obtained and is reported as the mean diameter in nanometers (nm). Surface charge measurements of these exosomes were acquired using Zetaviewer V.7.12 from Malvern Instruments, with results reported as mean values in millivolts (mV).
[0035] Novelty of this study includes-
• Formulation of LiFEx, an oral supplement product to improve liver fitness developed by integrating optimized concentrations of edible exosomes sourced from Carrot, Ginger, Garlic, Turmeric, and Lemon.
• Tailor the composition of LiFEx to ensure efficacy, efficiency, biological stability, and viability of the respective exosomes from each plant source.
• Develop LiFEx with a multifaceted approach aimed at enhancing gut health, specifically targeting inflammation and hyperammonemia.
• Harness the synergistic properties of the combined exosomes to improve gut microbiota balance and function, offering a comprehensive solution for gut health and integrity maintenance in CLD.
• Leverage the diverse bioactive compounds present in the LiFEx formulation to address multiple aspects of gut-liver axis.
[0036] The benefits and advantages which may be provided by the present invention have been described above about specific embodiments. These benefits and advantages, and any elements or limitations that may cause them to occur or to become more pronounced are not to be construed as critical, required, or essential features of any or all the embodiments.
[0037] While the present invention has been described with reference to embodiments, the embodiments are illustrative, and the scope of the invention is not limited to these embodiments.
[0038] Many variations, modifications, additions and improvements to the embodiments described above are possible. It is contemplated that these variations, modifications, additions and improvements fall within the scope of the invention.

, Claims:1. A plant-based edible exosomes formulation comprising:
a) Edible exosomes in a concentration in a range of 9.31x10^8 to 2.68 x 10^9 particles/ml wherein the edible exosomes contains naturally occurring therapeutic cargoes, and wherein the edible exosomes are isolated from plant-based sources selected such as turmeric, ginger, garlic, lemon, and blact carrot were used to prepare the formulation to improve liver fitness, named as 'LiFEx'.
2. A method for preparation of plant-based edible exosomes, the method comprising:
a) obtaining juice from a plant-based source selected from the group consisting of turmeric, ginger, garlic, lemon, and carrot, wherein the juice is obtained by pressing the plant-based source;
b) isolating edible exosomes from the obtained juice by centrifugation performed at 1000g for 10 minutes, followed by a subsequent centrifugation step at 3000g for 20 minutes to eliminate extracts to obtain a supernatant.
c) processing the obtained supernatant through a differential ultracentrifugation step at 10000g for 40 minutes to eliminate cellular components to obtain a clear supernatant, wherein the obtained clear supernatant was ultra centrifuged at 100000g for 90 minutes at 4°C, to obtain a pellet that was washed and subsequently re-suspended in PBS.
3. The formulation as claimed in claim 1, wherein the edible exosomes have a size distribution primarily in the range of 50 nm to 300 nm.
4. The formulation as claimed in claim 1, wherein the formulation comprises the concentrations of 4 parts of CarEx (carrot exosomes), 2 parts of GinEx (ginger exosome), 2 parts of GarEx (garlic exosome), and 1 part of each TurEx (turmeric exosome) and LemEx (lemon exosome).
5. The formulation as claimed in claim 1, wherein the formulation comprises the exosomes having zeta potential ranging from -7 to -16.0 ± 2.4mV.
6. The formulation as claimed in claim 1, wherein the formulation induced a reduction of plasma ammonia levels and serum liver enzymes such as AST, ALT and bilirubin in a HE model of rodent.
7. The formulation as claimed in claim 1, wherein the formulation enhanced the colonization of probiotic bacteria, including Lactobacillus, Ruminococcus and exerts bifidogenic effects by improving the colonisation of Bifidobacterium.
8. The formulation as claimed in claim 1, wherein the formulation promoted the proliferation of Butyricoccus, a butyratic acid (a well-known short-chain fatty acid-SCFA) producing microbe and restoring intestinal immune homeostasis

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