DESC:FIELD OF THE INVENTION
The present invention provides a phyto-pharmaceutical active polymolecular formulation. More particularly, the present invention provides pharmaceutical composition comprising combination of herbal powder and standardised solvent extract(s) of the herbal mixture useful for the treatment of liver disorders. The invention also provides a process for the preparation of such compositions and extraction process to enhance the active ingredients from the herbs.

BACKGROUND OF THE INVENTION
Liver is the largest organ in the human body. It is the only organ that is capable of regeneration of its cells, when damaged. Liver is involved in the process of removing all toxins that enter the body through food, water and air. Whatever is absorbed from the gastro intestinal route is first passed through the liver. Certain toxins that enter directly to the circulation system or through the lungs also reach the liver. It receives 25% of the blood pumped by the heart and acts like a filter that tests anything we eat.
The liver cells can be damaged through certain toxins. Regular consumption of alcohol, can damage the liver as it directly affects liver cells called hepatocytes. Even certain medication used to treat infections like tuberculosis can damage hepatocytes. The liver is capable of regeneration of damaged cells. This process of damage and regeneration can be detected through certain biochemistry tests done using serum, derived from blood drawn from the person. Alanine Liver Specific Transaminase (ALT), is one such enzyme that is specific to injury of liver cells or hepatic injury.
Hepatic injury can be transient, chronic or permanent. Transient happens whenever there is a toxin introduced to the body; drugs, alcohol, certain food toxins and environmental toxins. This is detected as a 1 – 2-fold increase in the ALT. the levels do come back to baseline in about 24 – 48 hours after stopping the ingestion of the toxin. If the toxin is consumed, like alcohol, for longer durations in moderate to large quantities then there is a shift in the baseline of the ALT and the enzymes can be elevated to about 2 – 3-fold. It also takes a longer duration, weeks to months, for it to recover to baseline levels. But in the case of a viral insult to the liver, like viral hepatitis, then there is massive destruction of the cells and the enzymes are increased nearly 100-fold. This will also take months to recover but still considered as transient as the viral cycles are transient. This may be different in the case of a chronic viral infection but again it depends on the virulence of the virus.
Non-alcoholic fatty liver disease (NAFLD) is when excess fat builds up in the liver due to causes other than alcohol use. There are two types: non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Non-alcoholic fatty liver usually does not progress to liver damage or NASH. NASH includes both a fatty liver and liver inflammation.
Sometimes the above insults remain for longer durations or at times the toxins are consumed in large quantities for very long periods of time, then this injury becomes chronic and leads to liver cirrhosis, liver cancer, liver failure, or cardiovascular disease.
Stages of NAFLD – pathophysiology

Stages of NAFLD Patient perspective Laboratory evidence Therapeutic potential
1st Stage
Healthy hepatocyte with normal fat deposits microvesicles of fat non-symptomatic Difficult diagnosis (only biopsy) currently no treatment
2nd Stage –
Healthy hepatocyte with excessive fat deposits – macrovesicles of fat or steatosis Non-symptomatic Diagnosis with ultrasound Currently no treatment
3rd Stage –
Hepatitis picture – elevated liver enzymes without fibrosis - cell death leading to rise in ALT / AST ratio Non-symptomatic to jaundice Elevated liver enzymes – AST/ALT ratio Trials of treatment with
1. SGLT 2 inhibitors
2. Metformin
3. Statins
4th Stage –
Hepatitis picture with fibrosis – steatohepatitis or NASH – Fibroscan grade >2 Symptomatic – jaundice, pain, loss of appetite Elevated liver enzymes, Fibro scan, CT Guided biopsy No established treatment for reversal
5th Stage –
Cirrhosis Symptomatic – signs of portal hypertension Elevated liver enzymes, fibroscan, CT biopsy Considered for transplant or palliative management
6th Stage – hepatic failure / hepatocellular carcinoma
Portal hypertension with or without renal involvement
Death frequency high Hepatic failure and portal hypertension picture with electrolyte imbalance Transplant is only option for patient to survive

Lifestyle is only effective in the early stages of the disease as evidenced from various guidelines prevalent at this stage. There is no consensus on the evidence from the ongoing and completed clinical trial with existing therapeutics. This gives an opportunity to bring together a novel concept “Interceptive medicine” that can support the global efforts to unifying the diagnosis criteria, prognosis criteria and treatment criteria.
There are certain herbal based products in the market currently which claims to be liver protective and/or kidney protective compositions. Such compositions comprise either multiple number of herbal components examples being LIV®-52 tablets, Bioliv syrup, Biolive syrup or compositions that is herbal powder mixture which does not constitute extract based components example being Liver Kidney CareTM capsules.
Compositions that have lower number of herbal components with optimized mixture of whole herb constituents and extract based constituents which could be effective against NAFLD is desirable.
Compositions of the present invention are an optimized mixture of whole herb constituents and extract based constituents and is a whole new genre of interceptive management for this chronic condition. Particularly, in the early stages of the disease – first to third that render a standard of care for intercepting the onset and progression to various stages of Non-alcoholic fatty liver disease (NAFLD).
Compositions of the present invention are an effective combination of whole herbal powder and extract based constituents that delivers standard doses of three herbs that is able to reduce fatty infiltration of hepatocytes (liver cells), promotes faster healing and reduce rate of cell death and thus capable of improving liver recovery from an acute or a chronic insult by liver toxins.
The present inventors have developed a unique method to combine the whole herbs and extracts of the same that enhances the active ingredients from the herbs. This not only retains the safety of the product, increases the efficacy and also ensures compliance to regulatory requirements for drug development as a botanical drug. The inventors of the present invention have identified a polymolecular cluster of 4 compounds responsible for the efficacy from the extracts.
The instant invention thus provides a composition that aids in liver recovery from acute and sub-acute insults from known liver toxins, faster than the natural recover phase seen when the insult is stopped. Further the instant composition has hepato-protective ability to prevent liver cells to go into permanent structural changes like fibrosis and death. The instant composition in humans, when consumed in the right dose for a given period of time, fasten the healing process and protect the liver cells from death and fibrosis. Further the instant extraction process enhances the active ingredients from the herbs and provides efficacy to the composition.
SUMMARY OF THE INVENTION
The present invention provides a Phyto-pharmaceutical active polymolecular formulation. More particularly, the present invention provides a formulation of herbs, Phyllanthus niruri (Bhumyamalaki), Boerhavia diffusa (Punarnava) and Picrorhiza kurroa (katuki).
The present invention provides a pharmaceutical composition comprising combination of herbal powder and standardised solvent extract(s) of the herbal mixture useful for the treatment of liver disorders. More particularly, the present invention provides a pharmaceutical composition comprising combination of herbal powder and standardised solvent extract(s) of herbs, Phyllanthus niruri (Bhumyamalaki), Boerhavia diffusa (Punarnava) and Picrorhiza kurroa (katuki). The invention also provides the extraction process and the process for the preparation of such compositions.
In one aspect, the present invention provides a phyto-pharmaceutical active polymolecular comprising:
a) herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
b) standardised extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
c) one or more pharmaceutical excipients.
In another aspect, the present invention provides a phyto-pharmaceutical active polymolecular comprising:
a) herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
b) standardised alcoholic extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
c) one or more pharmaceutical excipients.
In another aspect, the present invention provides a phyto-pharmaceutical active polymolecular comprising:
a) herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
b) standardised hydroalcoholic extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
c) one or more pharmaceutical excipients.
In another aspect, the present invention provides a phyto-pharmaceutical active polymolecular comprising:
a) herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
b) standardised alcoholic extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs,
c) standardised hydroalcoholic extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
d) one or more pharmaceutical excipients.
In another aspect, the present invention also provides process of preparing the above formulation including the steps of:
(i) Weighing and sifting (through desired sized mesh) herbal powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs and blending the same,
(ii) weighing and sifting (through desired sized mesh) standardised extract (s) of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
(iii) weighing and sifting desired excipients,
(iv) blending together the material of steps (i), (ii) and (iii),
(v) granulating the resulted blend of step (iv) using suitable granulating aid,
(vi) drying the wet mass obtained from step (v),
(vii) adding weighed and sifted extra granular excipients to dried granules of step (vi) and blending,
(viii) compressing the blend of step (vii) into tablets using desired tooling set, and
(ix) film coating the resulted tablets from step (viii).
In further aspect, the invention provides a composition to treat subjects with elevated levels of Alanine Liver Specific Transaminase (ALT), due to an acute or sub- acute consumption of known liver toxins, such as drugs, alcohol and certain kinds of micronutrient deficient diet.
In further aspect, the invention provides a composition to prevent permanent cell death in patients undergoing alcohol withdrawal therapies or sessions.
In another aspect, the present invention provides a pharmaceutical composition comprising 100-1000mg of active ingredients in the form of mixture of herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa and standardised extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs.
In further aspect, the present invention provides a composition such as a prophylactic (preventive or protective) agent to subjects who have to take medications that have known effects on the liver.
In further aspect, the present invention provides usage of the composition as per present invention as a liver tonic, for the treatment or prevention of Non-alcoholic fatty liver disease (NAFLD).
In further aspect, the present invention provides usage of the composition as per present invention as a liver tonic, for the treatment or prevention of Non-alcoholic fatty liver (NAFL).
In further aspect, the present invention provides usage of the composition as per present invention as a liver tonic, for the treatment or prevention of Non-alcoholic steatohepatitis (NASH).
BRIEF DESCRIPTION OF FIGURES:
FIG. 1: Effect of treatment on Bio chemical parameters (Day 16) in In vivo study.
FIG. 2: Effect of treatment on Liver MDA in In vivo study.

DESCRIPTION OF THE INVENTION
The present invention provides a phyto-pharmaceutical active polymolecular formulation. More particularly, the present invention provide a formulation of herbs, Phyllanthus niruri (Bhumyamalaki), Boerhavia diffusa (Punarnava) and Picrorhiza kurroa (katuki).
The present invention provides pharmaceutical compositions comprising combination of herbal powder and standardised solvent extract(s) of the herbal mixture useful for the treatment of liver and kidney disorders. More particularly, the present invention provides a pharmaceutical composition comprising combination of herbal powder and standardised solvent extract(s) of herbs, Phyllanthus niruri (Bhumyamalaki), Boerhavia diffusa (Punarnava) and Picrorhiza kurroa (katuki). The invention also provides the extraction process and the process for the preparation of such compositions.
The term "pharmaceutical composition" or "pharmaceutical formulation" as used herein refers to oral dosage forms preferably in the form of solid dosage forms such as tablets, bilayer tablets, capsules, powder, sachets and the like, liquid dosage forms such as oral solutions, suspensions and the like. The oral dosage forms can be in the form of immediate release, extended release, sustained release or biphasic release systems and are generally prepared by using one or more suitable pharmaceutically acceptable excipients. The term composition or formulation are used herein interchangeably.
The term “phyto-pharmaceutical active polymolecular formulation” and “phyto-pharmaceutical active polymolecular composition” as used herein interchangeably, provides composition comprising mixture of active moieties from the herbs Phyllanthus niruri, Boerhaavia diffusa, Picrorhiza kurroa especially Phyllanthin, Boeravinone B, Picroside I and Picroside II.
The term “standardization” or “standardized” as used herein, provides optimization of extraction process using varied process parameters and conditions such that optimal isolation of actives is achieved that was in conformation with pure analytical standards.
The term "extraction” as used herein, is defined as the process that involves the separation of medicinally active portions of plant tissues from the inactive components through the use of selective solvents.
The term "extract” as used herein, is defined as the final component achieved though the extraction process performed using the desired solvents under optimum process parameters and is selected from alcoholic extract, aqueous extract, hydroalcoholic extract or mixtures thereof.
The term "alcoholic extract” as used herein, is the extract collected using alcohol as a solvent in the extraction process under optimum process parameters.
The term “hydroalcoholic extract” as used herein, is the extract collected using mixture of water and alcohol as solvent in the extraction process under optimum process parameters.
The term "aqueous extract” as used herein, is the extract collected using water as solvent in the extraction process under optimum process parameters.
The term “standardized extract” as used herein, provides the extracts with optimal amount of actives achieved through the desired extraction process using desired solvent under optimum process parameters.
The solvent used for carrying out the extraction is selected from water, methanol (about 10% to 100%), ethanol (about 10% to 100%), chloroform, acetone, hexane or mixtures thereof. The extraction process can also be performed using supercritical fluid extraction (SFE) method using Carbon Dioxide (CO2).
When a mixture of two solvents is used for extraction, the ratio of solvents can range from about 90:10 to about 10:90 or from about 95:5 to about 5:95. The ratio of solvents used for extraction includes, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90 and 5:95.
Desired extraction processes is established by carrying out extractions with most polar solvent to non-polar solvent to identify active ingredients and qualitative and quantitative analysis is provided by LCMS, a chemical signature is provided to set standards using surrogate markers for batch to batch consistency.
The term “herbal powder” or “whole herb” as used herein, is defined as powder resulted when herbal material is dried and grounded to a powder. The material used to prepare powder can be a whole herb or specific parts of the herb such as aerial parts, leaves, branches, roots, rhizomes and certain other parts of the herb and mixtures thereof.
In one aspect, the present invention provides a pharmaceutical composition comprising:
a) herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
b) standardised extract (s) of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
c) one or more pharmaceutical excipients.
In one embodiment, the herbs used as per the present invention are as below:
1. Phyllanthus niruri
2. Boerhaavia diffusa
3. Picrorhiza kurroa
The source and geographical origin of the plant source used in the instant formulation is Northern India. Bhumyamalaki was sourced from Uttar Pradesh, Punarnava from Madhya Pradesh and Katuki was procured from Himachal Pradesh.
The details of herbs, the plant parts used (for both powder and extraction) and the corresponding major active or the marker identified and obtained in the extract are as below:
Table 1: Details of herbs and plant parts used
Name of the herb Plant Part Used Active moiety/ Marker Identified Reference
Traditional name: Bhumyamalaki

Botanical name:
Phyllanthus niruri Whole herb (Aerial parts) Phyllanthin Bhav Prakash Nikhandu, page 460-461
Traditional name:
Punarnava

Botanical name:
Boerhaavia diffusa Root Boeravinone B Bhav Prakash Nikhandu, page 70
Traditional name: Katuki

Botanical name:
Picrorhiza kurroa Rhizome with root Picroside I
Picroside II Bhav Prakash Nikhandu, page 424

Mechanism of action of the active moieties identified is explained below:
Table 2: Mechanism of action of the active moieties
Active Moiety/ moieties Mechanism of action
Phyllanthin • Regulates lipogenesis and decrease the synthesis of FFAs in liver via the modulation of ChREBP
• Decrease the production of cytokines (TGF-B1) which cause inflammation and fibrosis
• Modulate the CASP7 decreasing apotosis and hepatocyte injury
Boeravinone B • Regulates the hepatocyte insulin resistance sub-pathway and influences insulin suppression of free fatty acids (FFAs) disposal via the modulation of PI3K, Akt and GSK-3 protein kinases
• Regulates lipogenesis and decrease the synthesis of FFAs in liver via the modulation of ChREBP
• Regulates the production of reactive oxygen species (ROS) via the modulation of PERK and decrease the production of cytokines (IL-8 and TGF-B1) which causes inflammation and fibrosis.
Picroside I
Picroside II • Regulates lipogenesis and decrease the synthesis of FAAs in liver via the modulation of ChREBP
• Decrease the production of cytokines (TGF-B1) which cause inflammation and fibrosis
• Modulate the CASP7 decreasing apotosis and hepatocyte injury

In one embodiment, the present invention provides a stable pharmaceutical composition comprising;
d) herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
e) standardised alcoholic extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
f) one or more pharmaceutical excipients.
In another embodiment, the present invention provides a stable pharmaceutical composition comprising:
a) herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
b) standardised hydroalcoholic extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
c) one or more pharmaceutical excipients.
In yet another embodiment, the present invention provides a stable pharmaceutical composition comprising;
a) herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
b) standardised alcoholic extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs,
c) standardised hydroalcoholic extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
d) one or more pharmaceutical excipients.
In one embodiment, the present invention provides a significant proof for efficacy and hepatoprotective properties in in-vivo studies in a rat disease model. The instant formulation reduces ALT levels after oral administration of formulation in CCl4 induced rats.
In another embodiment the present invention relates to extraction methods to enhance the active ingredients from the herbs and providing desired efficacy to the formulation. The instant invention teaches two extraction processes i.e. Hydro-alcoholic extraction and Super Critical Fluid extraction.
Hydro-alcoholic extraction process utilizes mixing of herbal powder in 70% ethanol water mixture and subjecting to centrifugation at specific conditions of temperature time and rpm. This is followed by filtering the supernatant and concentrating the filtrate using rotary evaporator to remove the ethanol. The concentrate is then lyophilized and the moisture is removed completely to obtain a hydro alcoholic extract.
Super Critical Fluid extraction process utilizes packing the herbal powder in Super Critical Fluid extractor. This is followed by purging with CO2 gas and increasing the flow rate at a desired pressure in the presence of ethanol as a co-solvent at desired flowrate. Ethanol is removed on a rotary evaporator and vacuum dried to obtain Super Critical Fluid extract.
In an embodiment, the herbal powder in the composition of the present invention comprises Phyllanthus niruri and Boerhaavia diffusa in a ratio of about 0.01:1 to 1:0.01, preferably in a ratio of from about 0.1:1 to 1:0.1. The herbal powder as per the present invention comprises Phyllanthus niruri and Boerhaavia diffusa in a ratio of about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1:0.5, about 1:0.6, about 1:0.7, about 1:0.8 or about 1:0.9. The herbal powder as per the present invention comprises Phyllanthus niruri and Boerhaavia diffusa preferably in a ratio of about 1:0.3 to about 1:0.8.
In an embodiment, the herbal powder in the composition of the present invention comprises Phyllanthus niruri and Picrorhiza kurroa in a ratio of about 0.01:1 to 1:0.01, preferably in a ratio of from about 0.1:1 to 1:0.1. The herbal powder as per the present invention comprises Phyllanthus niruri and Picrorhiza kurroa in a ratio of about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1:0.5, about 1:0.6, about 1:0.7, about 1:0.8 or about 1:0.9. The herbal powder as per the present invention comprises Phyllanthus niruri and Picrorhiza kurroa preferably in a ratio of about 1:0.1 to about 1:0.7.
In an embodiment, the herbal powder in the composition of the present invention comprises Boerhaavia diffusa and Picrorhiza kurroa in a ratio of about 0.01:1 to 1:0.01, preferably in a ratio of from about 0.1:1 to 1:0.1. The herbal powder as per the present invention comprises Boerhaavia diffusa and Picrorhiza kurroa in a ratio of about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1:0.5, about 1:0.6, about 1:0.7, about 1:0.8 or about 1:0.9. The herbal powder as per the present invention comprises Boerhaavia diffusa and Picrorhiza kurroa preferably in a ratio of about 1:0.3 to about 1:0.9.
In one another embodiment, the herbal powder in the composition of the present invention comprises Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in a ratio of 1:1:0.1, 1:0.1:1, 0.1:1:1, 0.01:1:1, 1:1:0.01 or 1:0.01:1.
In one another embodiment, the herbal powder in the composition of the present invention comprises Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in a ratio of 1:0.1:0.1, 0.1:0.1:1, 0.1:1:0.1, 1:0.01:0.01, 0.01:0.01:1 or 0.01:1:0.01.
In another embodiment, the herbal powder in the composition of the present invention comprises Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in a ratio of 1:0.1:0.01, 1:0.01:0.1, 0.1:0.01:1, 0.01:0.1:1, 0.1:1:0.01 or 0.01:1:0.1.
In one embodiment, the composition of the present invention comprises herbal powder and the extract in a ratio from about 0.05:1 to 1:0.05. The composition of the present invention comprises herbal powder and the extract in a ratio of about 0.05:1, about 0.06:1, about 0.07:1, about 0.08:1, about 0.09:1, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1:0.9, about 1:0.8, about 1:0.7, about 1:0.6, about 1:0.5, about 1:0.4, about 1:0.3, about 1:0.2, about 1:0.1, about 1:0.09, about 1:0.08, about 1:0.07, about 1:0.06 or about 1:0.05.
In one embodiment, the present invention comprises multiple extracts in the composition for example, 2 or more extracts, preferably 2 or 3. The extracts used in the present invention are standardized extracts and are selected from alcoholic extract, hydro alcoholic extract, aqueous extract and mixtures thereof.
In another embodiment, the extracts used in the composition as per the present invention is selected from methanol extract, ethanol extract, ethanol-water extract, methanol-water extract, chloroform extract or water extract and mixtures thereof. The extracts used in the composition as per the present invention is preferably selected from ethanol extract, ethanol-water extract or water extract and mixtures thereof.
In another embodiment, when multiple extracts are used in the composition as per the present invention, the ratio of one extract to another extract ranges from about 1:0.5 to about 1:10. The ratio of one extract to another extract is selected from about 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or about 1:10. The ratio of one extract to another extract is preferably selected from about 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or about 1:5.
In another embodiment, the ethanol-water extract or methanol-water extract when used, ethanol or methanol to water ratio ranges from 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90 and 5:95. The ratio of ethanol to water is preferably selected from 90:10, 80:20, 70:30, 60:40 or 50:50. The ratio of methanol to water is preferably selected from 90:10, 80:20, 70:30, 60:40 or 50:50.
In an embodiment, the composition as per the present invention comprises herbal powder of Phyllanthus niruri in an amount of from about 10%w/w to about 50%w/w weight based on the total weight of the composition. The composition of the present invention comprises herbal powder of Phyllanthus niruri in an amount of about 10%w/w, about 15% w/w, about 20% w/w, about 25% w/w, about 30% w/w, about 35% w/w, about 40% w/w, about 45% w/w, or about 50% w/w weight based on the total weight of the composition. The composition of the present invention comprises herbal powder of Phyllanthus niruri in an amount of about 25%w/w, about 26% w/w, about 27% w/w, about 28% w/w, about 29% w/w, about 30% w/w, about 31% w/w, about 32% w/w, about 33% w/w, about 34% w/w, about 35% w/w, about 36% w/w, about 37% w/w, about 38% w/w, about 39% w/w, or about 40% w/w weight based on the total weight of the composition.
In an embodiment, the composition as per the present invention comprises herbal powder of Boerhaavia diffusa in an amount of from about 10%w/w to about 40%w/w weight based on the total weight of the composition. The composition of the present invention comprises herbal powder of Boerhaavia diffusa in an amount of about 10%w/w, about 15% w/w, about 20% w/w, about 25% w/w, about 30% w/w, about 35% w/w, or about 40% w/w weight based on the total weight of the composition. The composition of the present invention comprises herbal powder of Boerhaavia diffusa in an amount of about 15%w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, about 20% w/w, about 21% w/w, about 22% w/w, about 23% w/w, about 24% w/w, about 25% w/w, about 26% w/w, about 27% w/w, about 28% w/w, about 29% w/w, or about 30% w/w weight based on the total weight of the composition.
In an embodiment, the composition as per the present invention comprises herbal powder of Picrorhiza kurroa in an amount of from about 1%w/w to about 30%w/w weight based on the total weight of the composition. The composition of the present invention comprises herbal powder of Picrorhiza kurroa in an amount of about 1%w/w, about 5% w/w, about 10% w/w, about 15% w/w, about 20% w/w, about 25% w/w, or about 30% w/w weight based on the total weight of the composition. The composition of the present invention comprises herbal powder of Picrorhiza kurroa in an amount of about 5%w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, or about 20% w/w weight based on the total weight of the composition.
In an embodiment, the composition as per the present invention comprises herbal powder mixture of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of from about 20%w/w to about 80%w/w weight based on the total weight of the composition. The composition of the present invention comprises herbal powder mixture of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 20%w/w, about 25% w/w, about 30% w/w, about 35% w/w, about 40% w/w, about 45% w/w, about 50% w/w, about 55% w/w, about 60% w/w, about 65% w/w, about 70% w/w, about 75% w/w, or about 80% w/w weight based on the total weight of the composition.
In an embodiment, the composition as per the present invention comprises standardized extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of from about 1%w/w to about 30%w/w weight based on the total weight of the composition. The composition as per the present invention comprises standardized extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 1%w/w, about 5% w/w, about 10% w/w, about 15% w/w, about 20% w/w, about 25% w/w, or about 30% w/w weight based on the total weight of the composition. The composition of the present invention comprises standardized extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 5%w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, or about 20% w/w weight based on the total weight of the composition.
In an embodiment, the composition as per the present invention comprises alcoholic or ethanol extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of from about 1%w/w to about 30%w/w weight based on the total weight of the composition. The composition as per the present invention comprises alcoholic or ethanol extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 1%w/w, about 5% w/w, about 10% w/w, about 15% w/w, about 20% w/w, about 25% w/w, or about 30% w/w weight based on the total weight of the composition. The composition of the present invention comprises alcoholic or ethanol extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 5%w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, or about 20% w/w weight based on the total weight of the composition.
In an embodiment, the composition as per the present invention comprises aqueous or water extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of from about 1%w/w to about 30%w/w weight based on the total weight of the composition. The composition as per the present invention comprises aqueous or water extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 1%w/w, about 5% w/w, about 10% w/w, about 15% w/w, about 20% w/w, about 25% w/w, or about 30% w/w weight based on the total weight of the composition. The composition of the present invention comprises aqueous or water extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 5%w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, or about 20% w/w weight based on the total weight of the composition.
In an embodiment, the composition as per the present invention comprises hydro-alcoholic or ethanol-water extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of from about 1%w/w to about 30%w/w weight based on the total weight of the composition. The composition as per the present invention comprises hydro-alcoholic or ethanol-water extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 1%w/w, about 5% w/w, about 10% w/w, about 15% w/w, about 20% w/w, about 25% w/w, or about 30% w/w weight based on the total weight of the composition. The composition of the present invention comprises hydro-alcoholic or ethanol-water of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 5%w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, or about 20% w/w weight based on the total weight of the composition.
In an embodiment, the composition as per the present invention comprises supercritical fluid extraction extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of from about 1%w/w to about 30%w/w weight based on the total weight of the composition. The composition as per the present invention comprises supercritical fluid extraction extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 1%w/w, about 5% w/w, about 10% w/w, about 15% w/w, about 20% w/w, about 25% w/w, or about 30% w/w weight based on the total weight of the composition. The composition of the present invention comprises supercritical fluid extraction of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa in an amount of about 5%w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, or about 20% w/w weight based on the total weight of the composition.
Pharmaceutically acceptable excipients
The pharmaceutical formulations or compositions of the present invention comprise one or more pharmaceutically acceptable excipients. The term “pharmaceutically acceptable excipients” include, but not limited to, diluents, disintegrants, binders, lubricants, glidants, acidifying agent, alkalizing agent, stabilizers, surfactants, film coating materials, plasticizers, pigments, opacifiers, coloring agents and the like.
Suitable diluents, may be selected from, but not limited to the group consisting of different grades of starches or modified starches, such as maize starch, potato starch, rice starch, wheat starch, pregelatinised starch, fully pregelatinised starch, dextrin, maltodextin; cellulose derivatives, such as microcrystalline cellulose or silicified microcrystalline cellulose; sugar alcohols such as mannitol, erythritol, sorbitol, xylitol; monosaccharides like glucose; oligosaccharides like sucrose and lactose such as lactose monohydrate, lactose anhydrous, spray dried lactose or anhydrous lactose; calcium salts, such as calcium hydrogen phosphate; particularly preferably the fillers are selected from the group consisting of, maltodextrin, microcrystalline cellulose, lactose monohydrate, spray dried lactose, anhydrous lactose and calcium hydrogen phosphate and the like.
Suitable disintegrants may be selected from, but not limited to the group consisting of carmellose calcium, carboxymethylstarch sodium, croscarmellose sodium (cellulose carboxymethylether sodium salt, crosslinked), starch, modified starch such as pregelatinized starch, starch derivatives such as sodium starch glycolate, crosslinked polyvinylpyrrolidone (crospovidone), and low-substituted hydroxypropylcellulose, and disintegrating aids such as magnesium alumino-metasilicate and ion exchange resins like polacrilin potassium; particularly preferably the disintegrants are selected from the group consisting of sodium starch glycolate, croscarmellose sodium and crospovidone and the like.
Suitable binders may be selected from, but not limited to the group consisting of polyvinyl pyrrolidone (Povidone), polyvinyl alcohol, copolymers of vinylpyrrolidone with other vinyl derivatives (Copovidone), hydroxypropyl methylcellulose, methylcellulose, hydroxypropylcellulose, powdered acacia, gelatin, guar gum, carbomer such as carbopol, polymethacrylates and pregelatinized starch and the like.
Suitable lubricants may be selected from, but not limited to the group consisting of stearic acid, talc, glyceryl behenate, sodium stearyl fumarate and magnesium stearate; particularly preferably the lubricant is magnesium stearate and sodium stearyl fumarate and the like.
Suitable glidants may be selected from, but not limited to the group consisting of colloidal silica, hydrophobic colloidal silica and magnesium trisilicate, such as talc and the like.
Suitable lubricants may be selected from but are not limited to, stearic acid, magnesium stearate, calcium stearate, sodium lauryl sulphate, hydrogenated vegetable oil, hydrogenated castor oil, sodium stearyl fumarate, talc, glyceryl behenate, glyceryl monostearates, palmitic acid, carnauba wax, calcium soaps, zinc stearate, polyoxyethylene monostearates, calcium silicate, silicon dioxide, macrogols, and mixtures thereof.
Composition may contain acidifying and alkalinizing agent. Acidifying agents can be selected from, but not limited to citric acid, succinic acid, fumaric acid, malic acid, maleic acid, glutaric acid, lactic acid and mixtures thereof and the like. Alkalizing agent can be selected from, but not limited to magnesium oxide, aluminium oxide, ammonium hydroxide, magaldrate, an alkali metal salt or alkaline earth metal salt, such as sodium bicarbonate, calcium carbonate or sodium citrate, an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide, or an alkaline earth metal hydroxide such as calcium hydroxide or magnesium hydroxide, with magnesium oxide or calcium carbonate and meglumine and the like.
Composition may contain surfactants. Suitable surfactants as component can be selected from, but not limited to the group consisting of anionic surfactants, preferably sodium lauryl sulphate; polyethylene glycols (PEGs), preferably those PEGs having molecular weight in the range of about 2000 to 10000, more preferably PEG 3350, PEG 4000, PEG 6000, PEG 8000; Polysorbates, preferably Tween 20, Tween 80 or Span 80; fatty acid esters, preferably propylene glycol caprylates such as Capmul PG-8, Capryol 90; esters of glycerol and fatty acids, preferably glycerol oleates and caprylates (Capmul MCM); esters of polyethylene glycol and fatty acids, such as Labrasol and Solutol; castor oil ethoxylate (glycerol polyethylene glycol ricinoleate) such as Cremophor EL and Cremophor RH 40. More preferably the surfactant is selected from the group consisting of sodium lauryl sulphate; PEG 3350, PEG 4000, PEG 600 or, PEG 8000 and preferably PEG 6000; Tween 20 or Tween 80; and esters of polyethylene glycol and fatty acids, most preferably sodium lauryl sulphate and PEG 6000 and in particular sodium lauryl sulphate and the like.
Suitable examples of coating agents that can be used in the present application include, but are not limited to, pre-formulated film-coating materials such as Opadry® products, other hydrophilic or hydrophobic substances, and mixtures thereof. Useful components for coating include, but are not limited to, film formers, plasticizers, antiadherents, opacifiers, solvents, and optionally colorants, lubricants, pigments, antifoam agents, and polishing agents.
Suitable film-forming agents and coating materials, may include, but are not limited to hydroxypropyl methylcellulose (Hypromellose), hydroxypropyl cellulose, polyvinylalcohol, , methylcellulose, ethylcellulose, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose acetate succinate, shellac, liquid glucose, hydroxyethyl cellulose, polyvinylpyrrolidone, copolymers of vinylpyrrolidone and vinylacetate such as Kollidon® VA64 BASF, copolymers of acrylic and/or methacrylic acid esters with trimethylammoniummethylacrylate, copolymers of dimethylaminomethacrylic acid and neutral methacrylic acid esters, polymers of methacrylic acid or methacrylic acid esters, copolymers of acrylic acid ethylester and methacrylic acid methyl ester, and copolymers of acrylic acid and acrylic acid methylester and the like.
Suitable plasticizers, if used, may include, but are not limited to polyethylene glycol, diethyl phthalate and glycerol, acetyl tributyl citrate. Preference is given to polyethylene glycol and the like.
In an aspect of the above embodiments, said composition of the present invention is formulated into oral dosage form of tablets, capsules, caplets, pills, wafers, films, powders, granules or sachets.
In another aspect, the present invention provides a pharmaceutical composition comprising 100-1000mg of active ingredients in the form of mixture of herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa and standardised extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs.
In an embodiment, the present invention provides a pharmaceutical composition comprising 100-1000mg of active ingredients ((e.g., 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or 1000 mg) in the form of mixture of herbal powder comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa and standardised extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs.
In another embodiment, the present invention provides usage of pharmaceutical composition as per the invention as a liver tonic as a prophylactic (preventive or protective) or for the treatment or prevention of Non-alcoholic fatty liver disease (NAFLD) or for the treatment or prevention of Non-alcoholic fatty liver (NAFL) by administering the composition to a subject in need (human) in a frequency of once daily, twice daily, thrice daily, once two days, once three days or once in a month, preferably once daily or twice daily.
In one embodiment, said composition of the present invention is prepared by the processes including any methods known to a person skilled in the art such as, but not limited to, direct compression, dry granulation, wet granulation, roller compaction, slugging-deslugging, fluidized bed granulation by spraying a suspension or dispersion of drug in a conventional coating pan or fluidized bed equipment (such as a Wurster or Glatt) to prepare drug containing granules, followed by drying of the granules when desired granule size is achieved. Such prepared drug containing granules can be mixed with extragranular materials to prepare desired final pharmaceutical composition followed by compression into tablets and by film coating if desired.
In another embodiment, said process comprises dry granulation method, comprising at least one step selected from dry mixing, direct compression, or dry granulation such as compaction, slugging or combinations thereof. The dry process can be carried out by compacting/decompacting or slugging/deslugging a powder mixture or direct compression, in which an active agent and excipients are mixed together and granulated. These prepared granules can optionally be mixed with extragranular materials to prepare desired final pharmaceutical composition such as compressing into tablets.
In another embodiment, said process comprises wet granulation method, wherein said process comprises preparing a mixture comprising an active agent and one or more pharmaceutically acceptable excipients, and mixing with a granulating liquid, wherein said solvent is an aqueous solvent or a non-aqueous solvent or a combination of aqueous and non-aqueous solvents. The granulation liquid may be either used alone or, more usually, as a solvent containing a dissolved binder or as a suspension containing a dispersed binder. These prepared granules can optionally be mixed with extragranular materials to prepare desired final pharmaceutical composition such as compressing into tablets.
In wet granulation process, there are no particular limitations on solvents used for granulation. Suitable examples of solvents include, but are not limited to, aqueous or any of organic solvents, like water, lower alcohols such as methanol and ethanol, ketones such as acetone and methyl ethyl ketone, methylene chloride, and the like or any mixtures thereof.
In another embodiment, said process comprises melt granulation methods, in which a substance that melts on heating is mixed with an active agent, melted together and granulated and dried. These prepared granules can optionally be mixed with extra granular materials to prepare desired final pharmaceutical composition.
Suitable equipment used for preparing granules include, but are not limited to, planetary mixer, a screw mixer and the like; high-speed mixing granulation methods using a Henschel mixer, a Super mixer and the like; extruding granulation methods using a cylindrical granulator, a rotary granulator, a screw extruding granulator, a pellet mill type granulator and the like; wet high-shear granulation methods; fluidized-bed granulation methods; compression granulation methods; crushing granulation methods; and spraying granulation methods can be used.
After granulation, the granules can be dried using an oven dryer, a fluidized bed dryer, and the like, crushing, and sieving can be carried out to obtain granules or fine granules for use. Moreover, a granulation solvent may be used when preparing the composition according to the present application.
In another aspect, the present invention also provides a process of preparing the above composition including the steps of:
(i) weighing and sifting (through desired sized mesh) herbal powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs and blending the same,
(ii) weighing and sifting (through desired sized mesh) standardised extract (s) of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
(iii) weighing and sifting desired excipients,
(iv) blending together the material of steps (i), (ii) and (iii),
(v) adding weighed and sifted extra granular excipients to dried granules of step (iv) and blending,
(vi) compressing the blend of step (v) into tablets using desired tooling set, and
(vii) optionally, film coating the resulted tablets of step (vi)
In another aspect, the present invention also provides a process of preparing the above composition including the steps of:
(i) Weighing and sifting (through desired sized mesh) herbal powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs and blending the same,
(ii) weighing and sifting (through desired sized mesh) standardised extract (s) of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
(iii) weighing and sifting desired excipients,
(iv) blending together the material of steps (i), (ii) and (iii),
(v) granulating the resulted blend of step (iv) using suitable granulating aid,
(vi) drying the wet mass obtained from step (v),
(vii) adding weighed and sifted extra granular excipients to dried granules of step (vi) and blending,
(viii) compressing the blend of step (vii) into tablets using desired tooling set, and
(ix) film coating the resulted tablets from step (viii).
In further aspect, the invention provides a composition to treat subjects with elevated levels of Alanine Liver Specific Transaminase (ALT), due to an acute or sub- acute consumption of known liver toxins, such as drugs, alcohol and certain kinds of micronutrient deficient diet.
In further aspect, the invention provides a composition to prevent permanent cell death in patients undergoing alcohol withdrawal therapies or sessions.
In further aspect, the invention provides a composition such as a prophylactic (preventive or protective) agent to subjects who have to take medications that have known effects on the liver.
In further aspect, the invention also provides usage of the composition as per present invention as a liver tonic, for the treatment or prevention of Non-alcoholic fatty liver disease (NAFLD).
In further embodiment, the invention also provides usage of the composition as per present invention as a liver tonic, for the treatment or prevention of Non-alcoholic fatty liver (NAFL).
In further embodiment, the invention also provides usage of the composition as per present invention as a liver tonic, for the treatment or prevention of Non-alcoholic steatohepatitis (NASH).

EXAMPLES
Embodiments provided herein may be more fully understood by reference to the following examples. These examples are meant to be illustrative of pharmaceutical compositions and dosage forms provided herein, but are not in any way limiting.
Example 1
Preparation of ethanol extract of herbs
Below described is the extraction process of ethanol extract
(i) Raw coarse powders of herbs Phyllanthus niruri (Bhumyamalaki), Boerhavia diffusa (Punarnava) and Picrorhiza kurroa (katuki) are taken in a ratio of 2:1:1 are taken into a reactor,
(ii) ethanol is added to the reactor of step (i) in sufficient quantity till contents are dipped and is heated to 600C,
(iii) filter the contents of step (ii) and collect the filtrate and concentrate,
(iv) the filtrate of step (iii) is dried and is grinded to get the extract in powder form to get the ethanol extract.
Example 2
Preparation of 70% ethanol- water extract of herbs
Below described is the extraction process of ethanol extract
(i) Raw coarse powders of herbs Phyllanthus niruri (Bhumyamalaki), Boerhavia diffusa (Punarnava) and Picrorhiza kurroa (katuki) are taken in a ratio of 2:1:1 are taken into a reactor,
(ii) ethanol is added to the reactor of step (i) in sufficient quantity till contents are dipped and is heated to 600C,
(iii) filter the contents of step (ii) and collect the residue,
(iv) 70% ethanol in water is added to the residue and is heated to 600C,
(v) filter the contents of step (iv) and collect the filtrate and concentrate,
(vi) the filtrate of step (v) is dried and is grinded to get the 70% ethanol- water extract in powder form.
Example 3
Super Critical Fluid extraction
Dry powder of the herbal blend i.e. mixture of Bhumyamalaki, Punarnava and Katuki in 1.25:1:1 was packed in the sample vessel in Super Critical Fluid extractor. CO2 gas was purged and the flow rate was gradually increased from 1g/min up to 15g/min and the pressure was maintained at 150 psi. To facilitate optimum extraction and increase the yield, ethanol was used as a co-solvent, with a maximum of 2ml/min flowrate. The conditions were maintained for a period of 2hours. The collected extract was retrieved from the collection vessel, ethanol was removed completely on a rotary evaporator and further dried under vacuum. The dried extract was used in the formulation.
The results of dried extract obtained in examples 1, 2 and 3 were analyzed using Liquid chromatography mass spectrometry (LCMS) to identify the individual components from the extracts. Details of the retention time, molecular weight and plant source are provided in the table below.
Table 3: Retention time, molecular weight and plant source of individual components from the extract
Compound Name Retention Time
(in minutes) MW(g/mol) Plant Source
Picroside I 7.44 492.47 Picrorrhiza kurroa
Picroside II 6.34 512.46 Picrorrhiza kurroa
Boeravinone B 12.41 312.27 Boerhaavia diffusa
Phyllanthin 14.13 418.53 Phylanthus niruri

Example 4
Table 4: Film coated tablet composition
S. No. Ingredient Quantity (in %)
Intra granular portion
1. Phyllanthus niruri powder 30
2. Boerhaavia diffusa powder 20
3. Picrorhiza kurroa powder 12
4. 100% ethanol extract of blend powder of
Phyllanthus niruri,
Boerhaavia diffusa,
Picrorhiza kurroa 6
5. 70% ethanol-water extract of blend powder of
Phyllanthus niruri,
Boerhaavia diffusa,
Picrorhiza kurroa 4
6. Maltodextrin 5
7. Microcrystalline cellulose powder 15
8. Di-calcium phosphate 5
9. Purified water q. s.
Extra granular portion
10. Croscarmellose sodium 2
11. Magnesium stearate 0.5
12. Colloidal Silica 0.5
Total weight of core tablet 100.00
13. Film coating material Q.S.

Brief Manufacturing Process of tablets
(i) Weighing and sifting (through desired sized mesh) herbal powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs and blending the same,
(ii) weighing and sifting (through desired sized mesh) 100% ethanol extract,
(iii) weighing and sifting (through desired sized mesh) 70% ethanol-water extract,
(iv) weighing and sifting maltodextrin, microcrystalline cellulose powder and di-calcium phosphate,
(v) blending together the material of steps (i), (ii), (iii) and (iv),
(vi) granulating the resulted blend of step (v) using purified water,
(vii) drying the wet mass obtained from step (vi),
(viii) adding weighed and sifted extra granular excipients croscarmellose sodium
(ix) magnesium stearate and colloidal silica to dried granules of step (viii) and blending the same,
(x) compressing the blend of step (ix) into tablets using desired tooling set, and
(xi) film coating the resulted tablets from step (x) using the film coating material.
Example 5
Table 5: Film coated tablet composition
S. No. Ingredient Quantity (in %)
Intra granular portion
1. Phyllanthus niruri powder 25.0
2. Boerhaavia diffusa powder 19.5
3. Picrorhiza kurroa powder 19.5
4. 70% ethanol-water extract of blend powder of
Phyllanthus niruri
Boerhaavia diffusa
Picrorhiza kurroa
11.5
5. Supercritical fluid extraction extract of blend powder of
Phyllanthus niruri
Boerhaavia diffusa
Picrorhiza kurroa
1.5

6. Maltodextrin 5
7. Microcrystalline cellulose powder 10
8. Di-calcium phosphate 5
9. Purified water q.s.
Extra granular portion
10. Croscarmellose sodium 2
11. Magnesium stearate 0.5
12. Colloidal Silica 0.5
Total weight of core tablet 100
13. Film coating material Q.S

Brief Manufacturing Process
The composition is prepared in the process similar to that of example 4.

Example 6
In vivo animal study for testing efficacy

CCl4 induced acute liver injury model
The study focused on determining the hepatoprotective effect of the formulation in an acute liver damage model in combination with a 2-week pre-treatment along with testing the most effective composition. Alpha tocopherol treatment was added as a positive control in this study. A single dose of CCl4 (Carbon tetrachloride) administered on day 14 induced liver damage in rats as evidenced by increased ALT, AST levels (Table 7). Table 6 shows the dosing and drug compositions used for the study.
Animals used: Male Sprague Dawley Rats
Study groups and Design: 8 groups, Each group have 8 animals
Group 1: Vehicle control- Day 0 to 16 treatment with vehicle then Olive oil on day 14 at 3 ml/kg dose administered intraperitonially
Group 2: Path control - Day 0 to 16 treatment with vehicle then with carbon tetrachloride (CCL4) and olive oil mixture (1:1) on day 14 at 3 ml/kg dose administered intraperitonially
Group 3: Alpha-tocopherol- Day 0 to 14 pretreatment with alpha tocopherol at 50mg/ml dose adminstered per orally, then CCL4 on day 14 at 3 ml/kg dose adminstered intraperitonially
Group 4: Herb 1- Day 0 to 14 pretreatment with Herb 1 at dose 202 mg/kg administered b.i.d perorally, then CCL4 on day 14 at 3 ml/kg dose administered intraperitonially
Group 5: Herb 1- Day 14 CCL4, followed by Herb 1 at dose 403 mg/kg administered perorally at 2, 6, 24 and 48 hours
Group 6: Herb 2- Day 0 to 16 treatment with Herb 2 at dose 152 mg/kg administered b.i.d perorally, then CCL4 on day 14 at 3 ml/kg dose administered intraperitonially
Group 7: Herb 3- Day 0 to 16 treatment with Herb 3 at dose 75 mg/kg administered b.i.d perorally, then CCL4 on day 14 at 3 ml/kg dose administered intraperitonially
Group 8: Herb 4- Day 0 to 16 treatment with Herb 4 at dose 315 mg/kg administered perorally, then CCL4 on day 14 at 3 ml/kg dose administered intraperitonially
Several groups as above using combinations of active compounds are studied to Carbon Tetrachloride (CCl4) induces acute hepatic toxicity, marked as rising titers of serum ALT (alanine aminotransferase) which indicates liver damage.
End points studied:
1. Serum ALT, ALP (Alkaline phosphatase), Total bilirubin, Total Cholesterol (TC) and Thyroglobulin (TG)
2. MDA (Malondialdehyde) in liver tissue (Oxidative stress marker)
3. Liver gross and histopathology
Composition of herbs used in the animal studies:
Herb 1: Experimental formulation (50:50 whole herb and extract) Dose 1
Herb 2: 100% extract, combination 1
Herb 3: 100% extract, combination 2
Herb 4: Experimental formulation (50:50 whole herb and extract) Dose 2

Table 6: Dosing regimen and duration

Table 7: Liver function parameters and lipid profile in treated and untreated mice at Day 16. Values in green are statistically significant.
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
Vehicle Pathological control Alpha Tocopherol HERB1 HERB1 HERB2 HERB3 HERB4
ALT 55± 2 1418± 586 1030± 303 825± 342 1583± 523 1519± 447 1043± 206 792± 72
AST 95± 5 3538± 1019 3143± 1027 3563± 1186 4192± 1161 5041± 1198 4462± 1174 2372± 326
TC 87.36± 1.68 95.45± 6.99 105.06± 8.5 82.61± 7.19 91.81± 5.96 88.65± 3.74 92.71± 6.75 99.95± 5.78
TG 120.38± 12.07 117.14± 16.76 149.93± 28.55 129.03± 24.23 108.42± 14.24 103.16± 10.87 96.87± 11.44 98.33± 5.54
MDA-L 0.258± 0.016 0.276± 0.013 0.233± 0.014 0.221± 0.037 0.206± 0.022 0.14± 0.013 0.095± 0.007 0.144± 0.022

Inference: 50% Whole Herb + 50% Extract (Herb4) shows greater improvements in the liver function evidenced by ALT, AST than 100% extract. The best responses are being achieved in terms of liver function and levels of oxidative stress in the liver and the treatment is most effective when it is administered for a prolonged duration. There was also a reduction in liver malondialdehyde (MDA) levels, an indicator of cellular toxicity due to CCl4 in all animals that were pretreated with the whole herb and extract combination.
Example 7
Human studies
A randomized double blind placebo-controlled trial to establish efficacy and safety of compositions as per the present invention among patients with moderate to severe Nonalcoholic Steatohepatitis is being conducted. Below are some of the outcome measures to be evaluated.
Primary Efficacy Endpoint: Change in Hepatic Fat
Secondary Efficacy Endpoints: Change in Fatty liver grading, Change in ALT, AST and GGT levels, Change in HbA1C, Fasting glucose, Fasting Insulin, HOMA-IR, Change in cholesterol, LDL, HDL, triglycerides and FFA, Change in CRP, Change in fatty liver grading using Fibroscan and Change in FIB-4 score and fatty liver index score
Safety Assessment: Safety evaluations (physical examination, vital signs, laboratory investigation, electrocardiogram, adverse events and serious adverse events) will be done during the study period. Adverse events will be documented if observed, mentioned during open questioning, or when spontaneously reported.
Exploratory Endpoints: Change in serum metabolomic profile.
,CLAIMS:We claim:

1. A phyto-pharmaceutical active polymolecular formulation comprising:
(i) a herbal powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
(ii) standardised extract of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs, and
(iii) one or more pharmaceutical excipients.

2. The formulation as claimed in claim 1, wherein the ratio of herbal powder and the standardized extract are in a ratio from about 0.05:1 to 1:0.05.

3. The formulation as claimed in claim 1, wherein the standardized extract is selected from alcoholic extract, hydroalcoholic extract, supercritical fluid extract or aqueous extract and mixtures thereof.

4. The formulation as claimed in claim 1, wherein standardized extract is a mixture of alcoholic extract and hydroalcoholic extract present in a ratio of from about 1:0.5 to about 1:5.

5. The formulation as claimed in claim 1, wherein standardized extract is a mixture of alcoholic extract and supercritical fluid extract present in a ratio of from about 1:0.5 to about 1:5.

6. The formulation as claimed in claim 1, wherein the composition comprises active ingredients comprising Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa, in the form of mixture of herbal powder and standardised extract of blend powder in an amount ranging from about 100mg to1000mg.

7. The formulation as claimed in claim 1, wherein the pharmaceutical excipients are selected from the group consisting of diluents, disintegrants, binders, lubricants, glidants, acidifying agent, alkalizing agent, stabilizers, surfactants, film coating materials, plasticizers, pigments, opacifiers, coloring agents and combinations thereof.

8. The formulation as claimed in claim 1, wherein the composition is in the form of tablets, capsules, caplets, pills, wafers, films, powders, granules or sachets.

9. The formulation as claimed in claim 1, wherein the composition is suitable for administration as a liver tonic, for the treatment and/or prevention of non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) and other liver disorders.

10. A process for preparation of the formulation as claimed in claim 1, wherein the process comprises the steps of:

(i) Weighing and sifting (through desired sized mesh) herbal powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa herbs and blending the same,
(ii) weighing and sifting standardised extract (s) of blend powder of Phyllanthus niruri, Boerhaavia diffusa and Picrorhiza kurroa,
(iii) weighing and sifting desired excipients,
(iv) blending together the materials obtained in steps (i), (ii) and (iii),
(v) granulating the blend obtained in step (iv) using suitable granulating aid,
(vi) drying the wet mass obtained in step (v),
(vii) adding weighed and sifted extra granular excipients to dried granules of step (vi) and blending,
(viii) compressing the blend of step (vii) into tablets using desired tooling set, and
(ix) film coating the resulted tablets from step (viii).