Enhanced polyherbal NANO-admixture to retain and regain metabolic health with balanced detoxification, synergistic effect and preparation method thereof

Abstract

The Invention provides an enhanced Polyherbal Nano-admixture to retain and regain metabolic health with balanced detoxification, synergistic effect and preparation method thereof. The present invention discloses a polyherbal composition comprising a Andrographis paniculata herb extract; a Boerhavia diffusa herb extract; a Abelia triflora herb extract; a Terminalia bellerica herb extract; a Picrorhiza kurroa herb extract; a Tinospora cordifolia herb extract; a Phyllanthus emblica herb extract; a Acalypha fructicosa herb extract; a Phyllanthus niruri herb extract; a Terminalia chebula herb extract; and a Abutilon persicum herb extract.

Description

[0001] ENHANCED POLYHERBAL NANO-ADMIXTURE TO RETAIN AND REGAIN METABOLIC HEALTH WITH BALANCED DETOXIFICATION, SYNERGISTIC EFFECT AND PREPARATION METHOD THEREOF

[0002] FIELD OF THE INVENTION:

[0003] The invention, in general, relates to the technical field of herbal and nanotechnology compositions. More particularly, the present invention relates to a polyherbal nano -admixture composition to retain and regain metabolic health with balanced Detoxification and synergistic effect, and method of preparation thereof.

[0004] BACKGROUND OF THE INVENTION:

[0005] The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006] Liver ailments represent a significant global health burden, with conditions such as hepatitis, fatty liver disease, and cirrhosis contributing to morbidity and mortality worldwide. While conventional treatments exist, there is a growing recognition of the limitations and adverse effects associated with these approaches. Liver ailments encompass a spectrum of disorders ranging from acute hepatitis to chronic conditions like cirrhosis and hepatocellular carcinoma. These ailments pose a significant public health challenge globally, with factors such as viral infections, alcohol abuse, obesity, and metabolic syndrome contributing to their increasing prevalence. While conventional treatments such as antiviral medications, corticosteroids, and immune suppressants are available, they often exhibit limited efficacy, more adverse effects, and may not address the multifactorial nature of liver ailments. In recent years, there has been growing interest in exploring alternative and complementary approaches, particularly those derived from traditional medicine systems and natural products.

[0007] Liver function tests or liver enzymes, referred to as liver markers, are vital for assessing the health and functioning of the liver. These indicators offer valuable information about the overall condition of the liver and can assist in identifying liver diseases, tracking disease progression, evaluating response to treatment, and assessing liver function prior to surgical interventions. The significance of these markers lies in their capacity to identify irregularities in both the structure and function of the liver even when there are no noticeable symptoms. Liver markers play a critical role in diagnosing various hepatic conditions such as hepatitis, fatty liver disease, cirrhosis, and hepatocellular carcinoma. Elevated levels of specific enzymes like alanine aminotransferase and aspartate aminotransferase may signify damage or inflammation within the liver; meanwhile deviations observed in other indicators such as alkaline phosphatase, bilirubin, and albumin levels can indicate different types of disorders affecting the organ. Further, NAFLD and NASH are becoming leading causes of liver fibrosis and cirrhosis worldwide, but effective pharmacological treatments specifically targeting these conditions are still limited. Drugs like corticosteroids or immune suppressants used to reduce inflammation in liver conditions can increase the risk of infections or other complications

[0008] Oral medications for liver disorders may have side effects like addiction and drowsiness. Phototherapy requires day and night usage, with a slow start to its effectiveness. Exchange transfusions may lead to infection or bleeding problems, as well as the potential formation of air bubbles or blood clots causing heart, breathing, and bowel issues in infants. Additionally, herbal supplements like Silymarin are not regulated as strictly as pharmaceutical drugs in many countries. As a result, the quality, potency, and purity of these products can vary significantly between brands and batches. Inconsistent product quality makes it challenging to assess the true efficacy and safety of herbal remedies for liver disease. Thus, there is no specific medication for liver ailment in modem medicine system.

[0009] For example, US7250181B2 relates to pharmaceutical or veterinary or nutritional compositions of polyherbal extracts useful as anti-viral or immune- supporting agents. Particularly, the present invention of polyherbal composition comprises of extracts of Withania somnifera, Mangifera indica and purified Shilajit. This cost effective immune- supporting agent is ideal for use during the maintenance phase of the treatment, following an initial viral load reduction phase in which it is used as an adjuvant to conventional anti-viral drug therapy. The anti-viral and immune- supporting composition of this invention can perhaps be the sole basis of treatment where affordability is an issue. Additionally, this composition is used for the treatment, prevention or management of immune-supporting system in primates in need, especially humans.

[0010] Another application US9376369B2 discloses a method for treating inflammation in a subject comprising identifying a subject suffering from inflammation; and administering to the subject a composition consisting of an effective amount of two or more gallic acid derivatives isolated from Emblica officinalis, wherein the two or more gallic acid derivatives are selected from the group consisting of: glucose-6-gallate (GG6), glycerol- 1 -gallate, glucose- 1 -gallate (GG1), glucose- 1,6-digallate (DGG16), mucic acid-2-gallate, 1-methyl mucate-2-gallate, mucic acid 1,4-lactone 5-gallate, corilagin, chebulic acid, and m-digallic acid with minor p-digallic acid, thereby treating inflammation in the subject.

[0011] Therefore, there is a pressing need for safe, effective, and multi-targeted interventions for liver health and assess the true efficacy and safety of herbal remedies for liver ailments. Hence, the present invention provides a novel polyherbal formulation / s for their potential synergistic effects, enhanced bioavailability, and ability to target multiple aspects of liver dysfunction simultaneously.

[0012] OBJECTIVE OF THE INVENTION:

[0013] The primary object of the present invention is to overcome the drawbacks associated with prior art.

[0014] Another object of the present invention is to provide a polyherbal formulation for management of liver ailments.

[0015] Another object of the present invention is to provide a polyherbal formulation for their potential to create synergistic effects, applicability in the management of liver dysfunction.

[0016] Another object of the present invention is to provide a new herbal preparation that can be affordable and readily accessible for managing or preventing liver ailments.

[0017] Another object of the present invention is to provide an herbal preparation with minimal or no adverse effects on an individual's health and well-being.

[0018] Another object of the present invention is to normalize / regulate / control levels of liver markers such as Alanine Transaminase (ALT), Aminotransferase (AST), Alkaline phosphatase (ALP), Bilirubin markers using natural interventions.

[0019] SUMMARY OF THE INVENTION:

[0020] In an aspect of the present invention, there is provided a polyherbal nano-admixture composition to retain and regain metabolic health with balanced Detoxification and synergistic effect, comprising: a. Andrographis paniculata herb extract in an amount of 0.5-4.0 parts by weight; b. Boerhavia diffusa herb extract in an amount of 0.3-3.5 parts by weight; c. Abelia triflora herb extract in an amount of 0.2-4.0 parts by weight; d. Terminalia bellerica herb extract in an amount of 0.2-4.5 parts by weight; e. Picrorhiza kurroa herb extract in an amount of 0.7-5.0 parts by weight; f. Tinospora cordifolia herb extract in an amount of 0.5-3.7 parts by weight; g. Phyllanthus emblica herb extract in an amount of O.3-3.O parts by weight; h. Acalypha fructicosa herb extract in an amount of 0.5-5.0 parts by weight; i. Phyllanthus niruri herb extract in an amount of 0.5-2.5 parts by weight; j. Terminalia chebula herb extract in an amount of 0.3-2.4 parts by weight; k. Abutilon persicum herb extract in an amount of 0.7-3.8 parts by weight.

[0021] In an embodiment of the present invention, the formulation comprises pharmaceutically acceptable carriers or excipients.

[0022] In an embodiment of the present invention, the composition of the herbs contains different parts of the plant, but not limited to whole plants, fruits, leaves, and stems, etc.

[0023] In an embodiment of the present invention, the herb extracts ingredients at said concentration are both in wet and dry admixture to obtain a uniform admixture of the formulation.

[0024] In an embodiment of the present invention, the formulation is suitable for consumption in the form of supplements, snack bar or a drink in addition to routine formulations such as tablets, capsule or liquid oral formulation.

[0025] In an embodiment of the present invention, the Formula 24115 is effective in its inhibitory activity at inhibiting free radicals and Xanthine Oxidase with a low IC50 value.

[0026] In an aspect of the present invention, there is provided a method of preparing polyherbal nanoadmixture composition to retain and regain metabolic health with balanced Detoxification and synergistic effect as disclosed above, comprising the steps of: a. cleaning of collected plant materials to remove dirt and debris, wherein the plant materials are selected from Andrographis paniculata, Boerhavia diffusa, Abelia triflora, Terminalia bellerica, Picrorhiza kurroa, Tinospora cordifolia, Phyllanthus emblica, Acalypha fructicosa, Phyllanthus niruri, Terminalia chebula, and Abutilon persicum; b. drying the plant materials under shade; c. pulverizing weighed quantity of the dried plant materials using suitable methods to obtain a moderately coarse powder (sieve 22 / 40); d. placing the weighed quantity of powdered plant materials in a clean, dry vessel with a shaft agitator; e. adding a sufficient volume of cold solvent (20-25°C) alcohol to cover the plant materials completely; f. keeping the plant materials for 12-24 hours with a stirring at 5-15 rpm; g. filtering the mixture through a sieve (60-100 mesh) to separate the liquid extract from the solid residue; h. transferring the solid residue to another clean container filled with a warm mixture of alcohol and water; i. heating the mixture at 50-70°C with constant stirring at 25-55 rpm for 8-24 hours; j. filtering the mixture again through a sieve (60-100 mesh) to separate the liquid extract from the solid residue; and k. combining the liquid extracts and drying them under reduced pressure conditions ( 1 GO- 235 mBar) with temperatures maintained within the range of 50-75°C.

[0027] In an embodiment of the present invention, solvent used for cold processing is Alcohol and mixture of Hydro-Alcohol for warm processing.

[0028] In an embodiment of the present invention, the final dried extract is formulated into a form suitable for oral administration, such as tablets, capsules, or liquid formulations.

[0029] DETAILED DESCRIPTION OF DRAWINGS:

[0030] To further clarify the advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting in their scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:

[0031] Fig. 1: Illustrate the Schematic diagram of extraction process of the present invention. Fig. 2: Illustrate the Graphical representation of Comparison of Theoretical and Experimental Anti-oxidative Potential of the present invention.

[0032] Fig. 3: Illustrate the graphical representation of Xanthine Oxidase inhibition activity of the present invention.

[0033] Fig. 4: Illustrate the Size distribution obtained in formula 24115 after adopting process A of the present invention.

[0034] Fig. 5: Illustrate the Size distribution obtained in formula 24115 after adopting process B of the present invention.

[0035] Fig. 6: Illustrate graphical representation of the in Vitro Permeability of the present invention.

[0036] Fig. 7: Illustrate the A, B and C are Fiver function markers such as AST, ALT and ALP that are elevated in Disease Group (G2) which significantly reduced by developed formulation of the present invention.

[0037] Fig. 8: Illustrate the A, B and C are Liver function markers such as AST, ALT and ALP that are elevated in Disease Group (G2) which significantly reduced by developed formulation of the present invention.

[0038] Fig. 9: Illustrate the Graphical representation A and B of Changes in Bilirubin Level of the present invention.

[0039] DETAILED DESCRIPTION:

[0040] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings 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.

[0041] 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. In an embodiment, the present invention provides selection of Herbs used to make herbal composition. The composition of the herbs contains different parts of the herbs, such below but not limited to

[0042] 1. Whole plants

[0043] 2. Fruits

[0044] 3. Leaves

[0045] 4. Stems, etc.

[0046] In an embodiment of the present invention, there is provided an innovative process of preparation of novel compositions through following steps:

[0047] 1. Collected plant materials are cleaned to remove dirt and debris.

[0048] 2. Drying is carried out under shade.

[0049] 3. The weighed quantity of plant materials are pulverized using suitable methods to obtain moderately coarse powder (sieve 22 / 40).

[0050] 4. Weighed quantity of powdered plant materials are placed in clean, dry vessel having a shaft agitator.

[0051] 5. Sufficient volume of cold solvent (20-25°C) alcohol to cover the plant materials completely is added.

[0052] 6. Plant materials are kept for 12-24 hours with stirring at 5-15 rpm.

[0053] 7. After completion of time, the mixture is filtered through sieve (60-100 mesh) to separate the liquid extract (Extract 11) from the solid residue.

[0054] 8. Residue is transferred to another clean container filled with warm mixture of alcohol and water.

[0055] 9. Mixture is heated at 50-70°C with constant stirring at 25-55 rpm for 8-24 hours.

[0056] 10. After completion of time, the mixture is filtered through sieve (60-100 mesh) to separate the liquid extract (Extract 24) from the solid residue.

[0057] 11. The extracts are combined and dried under reduced pressure conditions (100-235 mBar) with temperatures maintained within the range of (50-75°C). This combination is termed as Extract 2411. IN AN EMBODIMENT, THE PRESENT INVENTION IS DEFINED BY NONLIMITING EXAMPLES:

[0058] 1. SCREENING FOR ACTIVITY:

[0059] The respective extracts are screened for antioxidant potential using DPPH based screening assay. Where, the free radical scavenging activity is measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH. 200 pM solution of DPPH in methanol is prepared and 150pl of this solution is added to 50 pl of different test compounds at different concentrations. After 30 minutes, the absorbance is measured at 517 nm.

[0060] % Scavenging = (Absorbance of control - Absorbance of test) / (Absorbance of control) X 100

[0061] Based on the result of % inhibition of DPPH activity only those herb extracts are selected which showed more than 35% inhibition at Img / ml concentration. Different combinations of extracts are utilized for exploration of synergistic and enhanced activity. One of the important findings of such combinations is enhancement of free radical scavenging activity. Binary combination of extracts shows synergism when compared to individual extract. Afterwards different combination is prepared by mixing different herb extracts as per details below. The ingredients at said concentration are dry blended to obtain a uniform admixture of the formulation. The formulation is suitable for consumption in the form of supplements, snack bar or a drink in addition to routine formulations such as tablets, capsule or liquid oral formulation. Composition 1 (Formula 24111)

[0062] Composition 2 (Formula 24112) Composition 3 (Formula 24113)

[0063] Composition 4 (Formula 24114) Composition 5 (Formula 24115)

[0064] Prepared compositions are evaluated for Anti-oxidative potential using DPPH assay and the comparative results are given below:

[0065] Formula 24115 is the most effective in scavenging DPPH free radicals, followed by Composition 4, Composition 2, Composition 3, and Composition 1. The very low IC50 value and standard deviation of Formula 24115 suggest it is both highly potent and consistent in its antioxidant activity. 2. COMPARATIVE XANTHINE OXIDASE ACTIVITY:

[0066] In an embodiment, the present invention provides a comparative Xanthine Oxidase Activity. Xanthine oxidase enzyme solution is prepared by diluting a stock solution of xanthine oxidase (0.2 U / ml) with phosphate buffer (0.05 M pH 7.5-7.6). Additionally, a xanthine substrate solution is prepared at a concentration of 0.15 mM in distilled water. Different dilutions of the test sample are prepared in phosphate buffer, ranging from 100 to 10 pl. For the reaction mix, 2 pl of enzyme solution was added to each well of a microplate. Then, 20 pl of the test sample (or phosphate buffer for the control reaction) is added to the respective wells. Afterward, 120 pl of phosphate buffer is added to each well, and the plate is incubated for 5 minutes at room temperature.

[0067] Following the incubation, 60 pl of the xanthine substrate solution is added to each well, and the absorbance is measured using a microplate reader at 290 nm for duration of 10 minutes.

[0068] The percentage inhibition was calculated using the formula

[0069] A

[0070] Percentage Inhibition = (1 — — )X 100 B

[0071] A is the average change in absorbance of the test sample (AbslO - AbsO), representing the absorbance difference at 10 minutes and at the start of the reaction. B is the average change in absorbance of the control reaction, representing the absorbance difference in wells where only phosphate buffer was added.

[0072] IC50 values of Xanthine oxidase assay also shows the synergistic action of developed formulation. Formula 24115 is the most effective at inhibiting Xanthine Oxidase, followed by Composition 3, Composition 1, Composition 2, and finally Composition 4. The low IC50 value and standard deviation of Formula 24115 suggest it is both highly potent and consistent in its inhibitory activity.

[0073] 3. DEVELOPMENT OF NANO-FORMULATION:

[0074] In an embodiment, the present invention, the development of nano-formulation include:

[0075] In Process A (a) Solubility and Compatibility of the Formula 24115 are screened using various excipients such as PVPVA, PND in water to ensure optimal performance and water with PVPVA is finalized for

[0076] (b) Sonicating the admixture of step (a) for 25 to 30 minutes in a probe sonicator to result in a nano-sized admixture; and

[0077] (c) Homogenizing the nano-sized admixture for 5-10 min at 9000 to 10000 RPM to thereby form the synergistic nano herbal composition being ready for use.

[0078] In Process B

[0079] (a) Solubility and Compatibility of the Formula 24115 are screened using various excipients such as PVPVA, PND, Polyethylene glycol, Lecithin, TPGS 1000, Brij in water to ensure optimal performance and water with Polyethylene glycol, Lecithin, TPGS 1000, Brij is finalized for;

[0080] (b) Sonicating the admixture of step (a) for 25 to 30 minutes in a probe sonicator at Amplitude ranging between 20 to 45% and temperature ranging between 25 to 45°C to result in a nanosized admixture; and

[0081] (c) Homogenizing the nano-sized admixture for 5-10 min at 10000 to 15000 RPM to thereby form the synergistic nano herbal composition being ready for use.

[0082] Further, the size distribution obtained in formula 24115 after adopting process B. The particle size range produced by Process A was 463.9 to 507.7 nm, while Process B yielded an average size between 151.1 to 217.9 nm. Based on these findings, Process B is selected for further formulation development as it resulted in improved nanoparticle characteristics. The developed nano-formulation can be formulated and used in the form of supplements, snack bar or a drink in addition to routine formulations such as freeze-dried product, spray dried powder, tablets, capsule or liquid oral formulation etc.

[0083] 4. COMPARATIVE IMPROVEMENT IN PERMEABILITY:

[0084] In an embodiment, the present invention, a comparative improvement in permeability is disclosed. Permeability studies are performed using Everted Gut Sac method. Ileum is cut into 10-12 cm long pieces and gently everted. Gut Sac were prepared and filled with 5 ml of ringer solution. Prepared Gut Sacs are placed in beaker containing 50 ml of 1000 ppm Formula 24115 under constant agitation and aeration at 35-37°C for 90 min. After 90 minutes gut sacs are removed, and outer surface is washed thoroughly. Sacs are opened and ringer solution is collected in pre-labelled tubes. HPTLC based Densitometry quantification is carried out.

[0085] 5. IMPROVEMENT IN STABILITY PARAMETERS:

[0086] Further, improvement in stability parameters include Stability studies for the Formula 24115 are performed as per ICH guidelines. The observations for the stability of particle size, solubility, odour, colour, etc. are carried out.

[0087] In an embodiment, the present invention, In silico efficacy evaluation was carried out using various Bio-Informatic tools. Screening is carried out using various ligands which are involved in fat metabolism and targets for the NAFLD / NASH. The ligands are Acetyl CoA carboxylase, Fatty acid synthase, HMG-COA, Stearoyl-CoA desaturase 1 (SCD1) and PPAR-a. Three- dimensional structures of these proteins are obtained from the PDB database which are then processed to generate 3D structure with least energy after removal of heteroatoms etc. Active site identification is carried out using online server Deepsite. ADMET and Drug likeness studies were carried out using pkCSM tool. The ligand and receptor interaction are done by using the Autodock4.5.6 tool which are summarized in table below:

[0088] * nM#mM

[0089] Docking with multiple ligands predicts synergistic or antagonistic effects between ligands that can bind to the same receptor. This approach is helpful in deciphering the rational design of combination therapies or multicomponent formulations, to achieve synergistic effects or target multiple pathways involved in complex diseases.

[0090] Table below is showing the fold change in inhibition constant after multiple ligands docking

[0091] The results indicate that certain combinations of Phyto extracts / Phyto molecules present in Formula 24115 can significantly enhance the inhibition of specific target proteins, offering promising therapeutic applications for liver health. An example of synergy may be observed with the combination of Gallic acid, Andrographolide, and P -sitosterol on ACC1. The results indicate that Formula 24115 ingredients / Phyto molecules are having significant synergy which indicates a promising liver protection potential of developed formula. Target enzymes mentioned in this invention are integral to liver metabolism, and their dysregulation can lead to liver diseases such as NAFLD, NASH, and hypercholesterolemia.

[0092] Targeting these enzymes with specific inhibitors can help to manage and treat conditions by:

[0093] • Reducing cholesterol and lipid synthesis.

[0094] • Improving lipid metabolism.

[0095] • Decreasing lipid accumulation in the liver.

[0096] • Reducing oxidative stress and inflammation.

[0097] 7. QUALITY PARAMETERS OF DEVELOPED FORMULATION:

[0098] In an embodiment, the present invention, quality parameters of developed formulation include Quantification of secondary metabolites is conducted for the extracts and Formula 24115 to ensure quality standards, thus aiding in meeting the quality criteria.

[0099] Further, determination of total phenolic content:

[0100] 1 mg / ml of extracts sample were prepared and then 0.1 ml of sample, 1.9 ml distilled water and 1.0 ml of Folin Ciocalteau reagent were added in a tube, and then 1.0 ml of 20% NaiCCh was added. The reaction mixture was incubated at room temperature in dark for 30 min and the absorbance of the blue colour sample was recorded at 765 nm on UV- Visible spectrophotometer. The blank consists of all reagents and solvents but no sample.

[0101] The sample was tested in triplicate and a calibration curve for gallic acid is obtained. The results are compared to gallic acid calibration curve and the total phenolic content of extracts is expressed as mg of gallic acid equivalents (GAE) per g of dry extract. Drug used Gallic acid from (Merck, India).

[0102] Further, determination of total flavonoids content:

[0103] 1 mg / ml of extracts sample were prepared and then extract or standard solutions (0.25ml) were mixed with 1.25 ml distilled water and 75 pL 5% NaNOi. After 6 min. 75 pL of 10% AlCh was added. After 5 min, 0.5 ml of 1.0 M NaOH was added to the mixture. Immediately, the absorbance of the mixture was determined at 510 nm on UV-Visible spectrophotometer. The blank consists of all reagents and solvents but no sample.

[0104] Further, Determination of glycoside: Five gram of the sample was added to 2 ml of 20% solution of dinitro salicylic acid in methanol and boiled for 2 minutes. The mixture was filtered and the residue which contains glycosides was dried, cooled and weighed. Percentage glycoside was calculated as follows:

[0105] Weiqht of Residue

[0106] % Glycosides = — — - —X 100

[0107] Weight of Sample

[0108] Further, determination of Bitters:

[0109] Three gram of the sample was added to 50 ml methanol and reflux for one hour then allowed to cool. After filtration residue was again subjected to reflux using 50 ml Methanol for one hour, filtrate was obtained, and residue was subjected to reflux using methanol third time and again filtered. All the filtrate was combined. Residue was dissolved in water. Solution was transferred to separating funnel and extracted using Ethyl acetate (25 ml). Layers were separated and ethyl acetate extraction was repeated for four times. Ethyl acetate fractions were pooled and transferred to tare dish. Evaporation was carried out on water bath followed by drying at 105°C in hot air oven. Percentage of Bitter was calculated as

[0110] Weight of Residue

[0111] % Bitter = X 100

[0112] Weight of Sample

[0113] Moreover, determination of total Alkaloid content:

[0114] Weigh 5g of the sample. Dissolve in 40ml of 10% ethanolic acetic acid; allow standing for 4 hours at room temperature. Filter with Whatman’s fdter paper (No. 42). Concentrate by evaporation over a steam bath to *4 of the original volume. Precipitate the alkaloid with cone. Ammonia solution in drops until in excess. The resulting alkaloid precipitate is recovered by filtration using previously weighed filter paper (Wl). The precipitate is washed with 9% ammonia solution, dried in the oven at 60°C for 30 minutes and cooled in a desiccator, then reweighed (W2). The concentration is determined and expressed as a percentage thus:

[0115] W2 - Wl

[0116] % Alkaloids = - - - - - — X 100

[0117] Weight of Sample

[0118] NLT-Not Less Than

[0119] 8. QUALITY CONTROL PARAMETERS FOR FORMULA 24115

[0120] In an embodiment, the present invention, the quality control parameters for formula 24115 include: Table 1 Quality Parameter for Formula 24115

[0121] Effect of developed Formula 24115 on animals with liver diseases: The effect of the Formula

[0122] 24115 on the Liver functions is measured in two different animal models i.e. Paracetamol induced liver damage and MCD Diet induced liver fibrosis. All experiments were carried out in accordance with the guidelines of Committee for the Purpose of Control and Supervision of

[0123] Experiments on Animals (CPCSEA) and with the approval of Institutional Animal Ethics Committee (IAEC). In an embodiment of the present invention, a first review of PARACETAMOL INDUCED LIVER DAMAGE:

[0124] This include Thirty-Two male Wistar rats which are divided in to four groups of eight animals. The rats from groups 1 and 2 received water once a day orally at a dose of 10 ml / kg b.w. and labelled as normal and disease controls respectively. Rats from groups 3 received positive control at a dose of 50 mg / kg, b.w. while the animals of Group 4 received Formula 24115 (3 ml / kg b.w.). All the rats received the respective assigned treatment for 14 days. On day 13, one hour after the respective assigned treatment all the rats, except group I, were administered with Paracetamol at a dose of 2g / kg, b.w. via oral gavage. On 15th day, the blood was collected from the retro-orbital plexus under mild anaesthesia and subjected for the estimation of markers of liver function such as AST, ALT and ALP. The rats were sacrificed, Liver was weighed, and histopathological changes are analysed.

[0125] N=8 animals per group

[0126] In an embodiment of the present invention, after administration of PCM levels of Liver function markers such as AST, ALT and ALP were elevated in Disease Group (G2) which is significantly reduced by developed formulation.

[0127] In an embodiment, the present invention, a second review of MCD Diet induced liver fibrosis include:

[0128] Thirty-Two male Wistar rats were divided in to four groups of eight each. The rats from groups 1 and 2 received water once a day orally at a dose of 10 ml / kg b.w. and were termed as normal and disease groups respectively. Group 1 was kept on normal diet while group 2 - 4 were kept on MCD diet for 28 days. Rats from groups 3 received Positive control at a dose of 200 mg / kg, b.w. While the animals of Group 4 received Formula 24115 (3 ml / kg b.w.). All the rats received the respective assigned treatment for 28 days. On 29th day, the blood was collected from the retro-orbital plexus under mild anaesthesia and subjected for the estimation of markers of liver function such as AST, ALT and ALP. The rats were sacrificed, Liver was weighed, and histopathological changes are analysed.

[0129] N=8 animals per group

[0130] After administration of MCD Diet of Liver function markers such as AST, ALT and ALP are elevated in Disease Group (G2) which is significantly reduced by developed formulation.

[0131] 8. CASE STUDIES OF EFFECT OF FORMULATION ON LIVER FUNCTION:

[0132] In an embodiment, the present invention effect of formulation on liver function is presented through case 1 and case 2. In Case 1: A 39-year-old, male diagnosed with Hepatitis shows the gradual change in Bilirubin levels after treatment with formulation. In Case 2: A 49-Year-old male having hepatocellular carcinoma showed elevated levels of liver function markers which was normalized after 15 days of treatment with formulation.

[0133] In an embodiment of the present invention, a novel poly-herbal nano-formulation, Formula 24115, is developed by combining various plant extracts based on their synergistic antioxidant and xanthine oxidase inhibitory potential. The efficacy of this formula is then validated using well-established animal models for liver disease, including a methionine-choline-deficient diet- induced model and a drug-induced model of liver damage. The effects demonstrated that the Formula 24115 exhibited beneficial effects in both animal models, showing promising therapeutic potential for the treatment of liver ailments.

[0134] The formula 24115 is useful in following:

[0135] • Improvement of liver functions

[0136] • Control of liver fibrosis arising due to xenobiotics, Impairment of Liver

[0137] • Improvement in liver health in conditions such as hepatitis and hepatocellular carcinoma

[0138] • Regulation of fat metabolism and cholesterol levels

Claims

We Claim:

1. A polyherbal nano-admixture composition to retain and regain metabolic health with balanced Detoxification and synergistic effect, comprising: a. Andrographis paniculata herb extract in an amount of 0.5-4.0 parts by weight; b. Boerhavia diffusa herb extract in an amount of 0.3-3.5 parts by weight; c. Abelia triflora herb extract in an amount of 0.2-4.0 parts by weight; d. Terminalia bellerica herb extract in an amount of 0.2-4.5 parts by weight; e. Picrorhiza kurroa herb extract in an amount of 0.7-5.0 parts by weight; f. Tinospora cordifolia herb extract in an amount of 0.5-3.7 parts by weight; g. Phyllanthus emblica herb extract in an amount of 0.3-3.0 parts by weight; h. Acalypha fructicosa herb extract in an amount of 0.5-5.0 parts by weight; i. Phyllanthus niruri herb extract in an amount of 0.5-2.5 parts by weight; j. Terminalia chebula herb extract in an amount of 0.3-2.4 parts by weight; k. Abutilon persicum herb extract in an amount of 0.7-3.8 parts by weight.

2. The polyherbal composition as claimed in claim 1 , wherein the formulation is combined with pharmaceutically acceptable carriers or excipients.

3. The polyherbal composition as claimed in claim 1 , wherein the composition of the herbs contains different parts of the herbs, but not limited to whole plants, fruits, leaves, and stems, etc.

4. The polyherbal composition as claimed in claim 1, wherein the herb extracts / ingredients at said concentration are dry / wet blended to obtain a uniform admixture of the formulation / extracts.

5. The polyherbal composition as claimed in claim 1, wherein the composition is suitable for consumption in the form of supplements, snack bar or a drink in addition to routine formulations such as tablets, capsule or liquid oral formulation.

6. The polyherbal composition as claimed in claim 1, wherein the composition is Formula 24115, effective in its inhibitory activity at inhibiting free radicals and Xanthine Oxidase with a low IC50 value.

7. A method of preparing a polyherbal nano admixture composition to retain and regain metabolic health with balanced Detoxification and synergistic effect as claimed in claim 1, comprising the steps of: a. cleaning collected plant materials to remove dirt and debris, wherein the plant materials comprise Andrographis paniculata, Boerhavia diffusa, Abelia triflora, Terminalia bellerica, Picrorhiza kurroa, Tinospora cordifolia, Phyllanthus emblica, Acalypha fructicosa, Phyllanthus niruri, Terminalia chebula, and Abutilon persicum; b. drying the plant materials under shade; c. pulverizing weighted quantity of the dried plant materials using suitable methods to obtain a moderately coarse powder (sieve 22 / 40); d. placing the weighed quantity of powdered plant materials in a clean, dry vessel with a shaft agitator; e. adding a sufficient volume of cold solvent (20-25°C) alcohol to cover the plant materials completely; f. keeping the plant materials for 12-24 hours with a stirring at 5-15 rpm; g. filtering the mixture through a sieve (60-100 mesh) to separate the liquid extract from the solid residue; h. transferring the solid residue to another clean container filled with a warm mixture of alcohol and water; i. heating the mixture at 50-70°C with constant stirring at 25-55 rpm for 8-24 hours; j. filtering the mixture again through a sieve (60-100 mesh) to separate the liquid extract from the solid residue; and k. combining the liquid extract obtained in step (g) and step (j) followed by drying them under reduced pressure conditions (100-235 mBar) with temperatures maintained within the range of 50-75 °C.

8. The method as claimed in claim 7, wherein the cold solvent used is alcohol and warm solvent is mixture of alcohol and Water.

9. The method as claimed in claim 7, wherein the final dried extract is formulated into a form suitable for oral administration, such as in the form of supplements, snack bar or a drink in addition to routine formulations such as tablets, capsules, or liquid formulations.

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