DESC:FIELD OF THE INVENTION:
The present invention relates to a polyherbal composition for treatment of osteoarthritis and associated inflammation.

BACKGROUND OF THE INVENTION:
Osteoarthritis (OA) is a degenerative joint disease, wherein tissues in the joint break down over time, resulting in degradation of articular cartilage and damage in subchondral bone (Grynpas MD, Alpert B, Katz I, Lieberman I, Pritzker KP: Subchondral bone in osteoarthritis. Calcif Tissue Int. 1991, 49: 20-26. 10.1007/BF02555898.) OA affects all areas of the joint including cartilage, tendons and ligaments, bones, synovium and the meniscus in the knee. OA usually occurs after the age of 40 and the prevalence increases with age. As the damage in the soft tissues of the joint progresses with age, chronic pain, swelling, inflammation, stiffness and loss of mobility of the joints develops. Several risk factors are involved in the development of OA, such as obesity, age, oxidative stress, nutritional deficiencies, inflammation, genetic factors and trauma. (Yao, Q., Wu, X., Tao, C. et al. Osteoarthritis: pathogenic signalling pathways and therapeutic targets. Sig Transduct Target Ther 8, 56 (2023). https://doi.org/10.1038/s41392-023-01330-w).
As per a recent study published in The Lancet Rheumatology Journal (Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021; Vol 5, Issue 9 September 2023), by 2050 nearly one billion people have been projected to be living with osteoarthritis. The study revealed that 15% of the world’s population, aged 30 and above, experiences osteoarthritis after analysing osteoarthritic data from 1990-2020 for more than 200 countries.
Currently, over-the-counter pain relievers such as non-steroidal anti-inflammatory drugs (NSAIDs) are used to ameliorate the OA related pain. However, these lines of medication are involved in only short-term symptomatic relief. NSAIDs are often related with significant cardiovascular, renal and gastrointestinal adverse events (Domper Arnal M-J, Hijos-Mallada G, Lanas A. Gastrointestinal and cardiovascular adverse events associated with NSAIDs. Expert Opin Drug Saf. 2022;21(3):373–384. doi:10.1080/14740338.2021.1965988).
OA was particularly considered as a degenerative joint disease, however, over the years with the progress in molecular biology, it appears that the inflammation is often associated as a driving factor in OA. Recent studies provide evidence that inflammation is an important factor in developing synovitis, as well as is involved in progression of cartilage and bone destruction in OA. Cytokines, adipokines, abnormal metabolites all appear to play a key role in OA pathophysiology. Immune-mediated inflammation involving T- and B-cells as well as macrophages is now considered a common finding in OA synovial tissue. (Malemud CJ. Biologic basis of osteoarthritis: state of the evidence. Curr Opin Rheumatol. 2015 May;27(3):289-94. doi: 10.1097/BOR.0000000000000162. PMID: 25784380; PMCID: PMC4492522.)
Therefore, there is a need in the art for an alternative effective medication that can prevent and treat OA, specifically OA induced inflammation, without causing side effects associated with the currently available therapies.
Significant clinical research has been conducted worldwide to assess the therapeutic efficacy of natural phytocomponents in treatment of skeletal disorders (Zhou Dongyang, Zhang Hao, Xue Xu, Tao Yali, Wang Sicheng, Ren Xiaoxiang, Su Jiacan, “Safety Evaluation of Natural Drugs in Chronic Skeletal Disorders: A Literature Review of Clinical Trials in the Past 20 years”, Frontiers in Pharmacology, Vol 12, 2022,10.3389/fphar.2021.801287). For example, US 6395772 (now expired) disclosed the use of berberine or its salts, extracted from Berberis aristata in the mechanism of blocking attachment of leukocytes to endothelial cells. While the document mentions use in treating arthritis, there is no specific mention of use in osteoarthritis. Indian patent application 2509/MUM/2014 discloses a composition comprising root powder of Sida cordifolia L. and Rhizome powder of Zingiber oflicinale Rose for treatment of osteoarthritis. Further, 1830/MUM/2013 discloses a herbal mineral composition for treatment of musculoskeletal disorder.
Prior art also claims combination of Piper nigrum with other agents. Indian application 3689/MUM/2014 claims a combination of Piper nigrum and Tribulus terrestris with other agents. WO2020031108 claims lipophilic extracts of Piper nigrum, Zingiber officinale and Boswellia serrata, which are combinations different from those disclosed and claimed here. WO2009041787 claims Piper nigrum extract and the amide derived from it, for use in inflammation, infectious diseases and autoimmune diseases. KR20120056243 discloses pharmaceutical composition for the prevention and treatment of inflammatory diseases, comprising piperine or a pharmaceutically acceptable salt.
US20180311279A1 discloses a complex formulation containing extracts from over 20 different herbs. This complexity increases time, cost, and overall formulation challenges. Clearly, all of the prior art cited above discloses different combinations and not the specific polyherbal extract being disclosed and claimed herein. There is no disclosure of use of combinations of any kind as disclosed in the present disclosure.

OBJECTIVE OF THE INVENTION:
It is an object of the invention to provide a composition based on herbal extracts for use as an anti-inflammatory agent. The compositions of the present invention are useful in treating osteoarthritis.
A further object of the invention is to provide a herbal composition for use in inflammation that is safe and does not suffer from the limitations of known compositions based on synthetic compounds, which cause several side effects, such as gastrointestinal, renal and cardiovascular adverse effects.
Another object of the invention is to provide a herbal composition which is a polyherbal extract that is effective at a concentration lower than that required for each individual component of the polyherbal extract to provide anti-inflammatory effect.
A still further object of the invention is to provide a herbal composition which is effective in the treatment and management of arthritis, especially osteoarthritis.
These and other objects and advantages if the invention will be apparent from the ensuing description.
SUMMARY OF THE INVENTION:
The present invention provides a herbal composition comprising a mixture of about 50% to about 70%w/w of Phellodendron amurense or Berberis aristata, about 10% to about 30%w/w of Tribulus terrestris, about 0.5% to about 5%w/w of Piper nigrum, and about 5% to about 25%w/w of Tinospora cordifolia.
In one embodiment of the present invention, the composition comprises a hydroalcoholic extract of (i) Phellodendron amurense or Berberis aristata, (ii) Tribulus terrestris, (iii) Piper nigrum, and (iv) Tinospora cordifolia.
In another embodiment of the present invention, the composition comprises an aqueous extract of (i) Phellodendron amurense or Berberis aristata, (ii) Tribulus terrestris, (iii) Piper nigrum and (iv) Tinospora cordifolia.
In yet another embodiment of the present invention, the composition comprises an alcoholic extract of (i) Phellodendron amurense or Berberis aristata, (ii) Tribulus terrestris, (iii) Piper nigrum and (iv) Tinospora cordifolia.
In another embodiment the composition is in the form of a dosage selected from the group comprising tablets, capsules, solutions, syrups, suspension, emulsions, lozenges, solid dispersion, oral films, granules, powders filled in sachet, and dry powders for reconstitution.
In yet another embodiment of the present invention, the composition is a tablet.
In further embodiment of the present invention, the composition is a capsule.
In another embodiment of the present invention, the composition is a liquid solution.
In yet another embodiment of the present invention, the composition is a liquid suspension.
In a further embodiment of the present invention, the composition is an emulsion.
In yet another embodiment of the present invention, the tablet further comprises excipients selected from the group comprising one or more of diluents, disintegrants, solvents, cosolvents, isotonicity agents, chelating agents, buffers, pH-adjusting agents, solubilizers, stabilizers, dispersion agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavouring agents and diluents.
In yet another embodiment of the present invention, the capsule further comprises excipients selected from the group comprising one or more of diluents, disintegrants, solvents, cosolvents, isotonicity agents, chelating agents, buffers, pH-adjusting agents, solubilizers, stabilizers, dispersion agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavouring agents and diluents.
In another embodiment of the present invention, the liquid solution or suspension or emulsion further comprises excipients selected from the group comprising one or more of the solubilizer, suspending agent or an emulsifier.
In another embodiment of the present invention, the composition is used in the treatment and management of osteoarthritis and associated inflammation.

DETAILED DESCRIPTION OF THE INVENTION:
The term “comprising”, which is synonymous with “including”, “containing”, or “characterized by” herein defined as being inclusive or open-ended and does not exclude additional, unrecited elements or method steps, unless the context clearly requires otherwise.
The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In the context of the present disclosure, the term “about” means within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

The definition of "extract" encompasses aqueous extract, alcoholic extract, or hydroalcoholic extract unless otherwise specified.
The term “shelf life”, as used herein, refers to the amount of time the pharmaceutical composition may be stored without loss of potency and/or performance profile. In some embodiments of the present invention, shelf life refers to the amount of time the pharmaceutical composition may be stored without loss of about 4%, about 3%, about 2% or about 1% of the potency and/or performance, when stored at room temperature, i.e. about 15oC to about 25oC. The stable compositions provided herein are designed to have shelf life of at least 24 months.
As discussed in the background of the present disclosure, osteoarthritis and associated inflammation is a major concern. Conventional medications currently being used, such as NSAIDs, are associated with several adverse events.
Accordingly, the present disclosure provides a herbal composition comprising a mixture of (i) Phellodendron amurense or Berberis aristata , (ii) Tribulus terrestris, (iii) Piper nigrum, and (iv) Tinospora cordifolia.
In one embodiment, the composition comprises about 50%w/w to about 70%w/w of extract of Phellodendron amurense or Berberis aristata, about 10%w/w to about 30%w/w of extract Tribulus terrestris, about 0.5%w/w to about 5%w/w of extract of Piper nigrum, and about 5%w/w to about 25%w/w of extract of Tinospora cordifolia.
In a preferred embodiment, the composition comprises about 55%w/w to about 65%w/w of extract of Phellodendron amurense, about 15%w/w to about 25%w/w of extract of Tribulus terrestris, about 0.5%w/w to about 1.5%w/w of extract of Piper nigrum, and about 10%w/w to about 20%w/w of extract of Tinospora cordifolia.
In a highly preferred embodiment, the composition comprises about 63%w/w of extract of Phellodendron amurense, about 22%w/w of extract of Tribulus terrestris, about 1%w/w of extract of Piper nigrum, and about 14%w/w of extract of Tinospora cordifolia.

In another embodiment, the composition comprises about 50%w/w to about 70%w/w of extract of Berberis aristata, about 10%w/w to about 30%w/w of extract of Tribulus terrestris, about 0.5%w/w to about 5%w/w of extract of Piper nigrum, and about 5%w/w to about 25%w/w of extract of Tinospora cordifolia.
In one embodiment, the herbal composition is a polyherbal composition comprising hydroalcoholic extract of its components. Hydroalcoholic solvents are preferred as they are suitable for extracting the active components useful in treating inflammation, from the herbs used in the present disclosure. Especially, the active ingredients present in the polyherbal composition of the present disclosure are useful in treating inflammation, such as that of osteoarthritis, wherein the individual components are used in amounts lower than those required when each component is used alone.
In one embodiment, the composition comprises about 50%w/w to about 70%w/w of hydroalcoholic extract of Phellodendron amurense, about 10%w/w to about 30%w/w of hydroalcoholic extract of Tribulus terrestris, about 0.5%w/w to about 5%w/w of hydroalcoholic extract of Piper nigrum, and about 5%w/w to about 25%w/w of hydroalcoholic extract of Tinospora cordifolia.
In a preferred embodiment, the composition comprises about 55%w/w to about 65%w/w of hydroalcoholic extract of Phellodendron amurense, about 15%w/w to about 25%w/w of hydroalcoholic extract of Tribulus terrestris, about 0.5%w/w to about 1.5%w/w of hydroalcoholic extract of Piper nigrum, and about 10%w/w to about 20%w/w of hydroalcoholic extract of Tinospora cordifolia.
In a highly preferred embodiment, the composition comprises about 63%w/w of hydroalcoholic extract of Phellodendron amurense, about 22%w/w of hydroalcoholic extract of Tribulus terrestris, about 1%w/w of hydroalcoholic extract of Piper nigrum, and about 14%w/w of hydroalcoholic extract of Tinospora cordifolia.
In another embodiment, the composition comprises about 50%w/w to about 70%w/w of hydroalcoholic extract of Berberis aristata, about 10%w/w to about 30%w/w of hydroalcoholic extract of Tribulus terrestris, about 0.5%w/w to about 5%w/w of hydroalcoholic extract of Piper nigrum, and about 5%w/w to about 25%w/w of hydroalcoholic extract of Tinospora cordifolia.
In a preferred embodiment, the composition comprises about 55%w/w to about 65%w/w of hydroalcoholic extract of Berberis aristata, about 15%w/w to about 25%w/w of hydroalcoholic extract of Tribulus terrestris, about 0.5%w/w to about 1.5%w/w of hydroalcoholic extract of Piper nigrum, and about 10%w/w to about 20%w/w of hydroalcoholic extract of Tinospora cordifolia.
In a highly preferred embodiment, the composition comprises about 63%w/w of hydroalcoholic extract of Berberis aristata, about 22%w/w of hydroalcoholic extract of Tribulus terrestris, about 1%w/w of hydroalcoholic extract of Piper nigrum, and about 14%w/w of hydroalcoholic extract of Tinospora cordifolia.
Phellodendron amurense is a species of tree in the family Rutaceae, commonly called the Amur cork tree. It is a major source of huáng bò, one of the 50 fundamental herbs used in traditional Chinese medicine. It is native to eastern Asia: northern China, northeast China, Korea and Japan. Phellodendron amurense is used in combination with a lot of other herbs and agents in treating inflammation, but none with the specific herbs disclosed in this application, and in the quantities disclosed herein. The present invention uses the bark extract of Phellodendron amurense.
Berberis aristata, also known as Indian barberry, "chutro" or tree turmeric, is a shrub belonging to the family Berberidaceae and the genus Berberis. The genus comprises approximately 450-500 species of deciduous evergreen shrubs and is found in the temperate and sub-tropical regions of Asia, Europe, and America. Berberis aristata is native to the Himalayas in India and in Nepal. It is also naturally found in the wet zone of Sri Lanka. It is characterized by an erect spiny shrub, ranging between 2 to 3 m (6.6 to 9.8 ft) in height. It is a woody plant, with bark that appears yellow to brown from the outside and deep yellow from the inside. The bark is covered with three-branched thorns, which are modified leaves, and can be removed by hand in longitudinal strips. The leaves are arranged in tufts of five to eight and are approximately 4.9 cm (1.9 in) long and 1.8 cm (0.71 in) broad. The leaves are deep green on the dorsal surface and light green on the ventral surface. The leaves are simple with pinnate venation. The leaves are leathery in texture and are toothed, with several to many small indentations along the margin of the leaf. The present invention uses root extract of Berberis aristata.
Tribulus terrestris is an annual plant in the caltrop family (Zygophyllaceae) widely distributed around the world. It is adapted to grow in dry climate locations in which few other plants can survive. It is native to warm temperate and tropical regions in southern Eurasia and Africa. It has been unintentionally introduced to North America and Australia. An aggressive and hardy invasive species, Tribulus terrestris, commonly called gokhru, is widely known as a noxious weed because of its small woody fruit – the bur – having long sharp and strong spines. The herb has been known in traditional Chinese medicine in combination with other agents. The present invention uses fruit extract of Tribulus terrestris.
Piper nigrum, also known as black pepper is a flowering vine in the family Piperaceae, cultivated for its fruit, known as a peppercorn, which is usually dried and used as a spice and seasoning. The fruit is a drupe (stonefruit) which is about 5 mm (0.20 in) in diameter (fresh and fully mature), dark red, and contains a stone which encloses a single pepper seed. Peppercorns and the ground pepper derived from them may be described simply as pepper, or more precisely as black pepper (cooked and dried unripe fruit), green pepper (dried unripe fruit), or white pepper (ripe fruit seeds). Black pepper is native to the Malabar Coast of India. The present invention uses seed extract of Piper nigrum. The extract may be prepared through conventional extraction processes, and/or may be procured commercially.
Tinospora cordifolia, commonly known as gurjo, heart-leaved moonseed, guduchi or giloy, is a herbaceous vine of the family Menispermaceae indigenous to tropical regions of the Indian subcontinent. Most patents/applications in the art claim combination with other agents, none being similar to the polyherbal extract disclosed here. The present invention uses aqueous extract of the stem of Tinospora cordifolia.

While the individual herbs have been reported in the art for treating arthritis, and/or have been disclosed to be useful in combination with other herbs, there is nothing in the art that suggests a composition containing the specific combination of Berberis aristata or Phellodendron amurense, Tribulus terrestris, Piper nigrum, and Tinospora cordifolia for use in treating osteoarthritis and associated inflammation.
A hydroalcoholic extract of the four individual herbs, i.e. Berberis aristata or Phellodendron amurense, Tribulus terrestris, Piper nigrum, and Tinospora cordifolia, was obtained by methods conventional in the art, and which are commercially viable. The individual extracts were collected and stored under conditions such that they remain physically and chemically stable. By stable is meant that there is no microbial growth and there is no precipitation seen upon storage.
Solid/liquid extraction is used in traditional or classical procedures. It is based on the fact that when a solid component comes into touch with a solvent (liquid), the soluble parts of the solid dissolve in the solvent, and this migration of the soluble ingredients into the solvent occurs along a concentration gradient. The rate of transfer is determined by the constituent concentrations and continues until equilibrium is attained. Different types of conventional techniques are maceration (cold and hot), digestion, infusion, elution (lixiviation), decoction, reflux extraction, tincture, percolation, steam distillation, hydrodistillation, soxhlet extraction, serial exhaustive extraction, and fermentation (aqueous-alcoholic extraction).
The analysis of bioactive compounds present in the plant extracts is analysed through various techniques involving the applications of chromatographic techniques such as HPLC (High Performance Liquid Chromatography), TLC (Thin Layer Chromatography), HPTLC (High Performance Thin Layer Chromatography), OPLC (Optimum Performance Laminar Chromatography), GC (Gas Chromatography), PC (Paper Chromatography), CC (Column Chromatography) and it’s detection through Fourier Transform Infra-Red spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS).
A polyherbal extract was obtained by mixing about 55% to about 65%w/w of hydroalcoholic extract of Berberis aristata or Phellodendron amurense, about 15% to about 25%w/w of hydroalcoholic extract of Tribulus terrestris, about 0.5% to about 1.5%w/w of hydroalcoholic extract of Piper nigrum, and about 10% to about 20%w/w of hydroalcoholic extract of Tinospora cordifolia. This polyherbal extract was tested for anti-inflammatory effects, as described in the examples below. The terms “polyherbal extract” and “polyherbal mixture” may be used interchangeably herein.
The polyherbal extracts of the present disclosure are formulated into compositions for oral administration. The oral compositions of the present invention may be selected from, but are not limited to, tablets, capsules, solutions, syrups, suspension, emulsions, lozenges, solid dispersion, oral films, granules, powders filled in sachet, dry powders for reconstitution, and the like.
The compositions may contain inert excipients that are conventionally used in oral formulations, but are not limited to, one or more agents selected from diluents, disintegrants, solvents, cosolvents, isotonicity agents, chelating agents, buffers, pH-adjusting agents, solubilizers, stabilizers, dispersion agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, binders, flavouring agents, and other known additives to provide an elegant presentation of the drug, i.e., a compound described herein or pharmaceutical composition thereof, or aid in the manufacturing of the pharmaceutical product.
Examples of suitable diluents that may be used in the compositions of the present disclosure include, but are not limited to, microcrystalline cellulose, dibasic calcium phosphate, lactose, talcum, calcium phosphate, magnesium carbonate, magnesium oxide, calcium sulphate, saccharose, monosaccharides, such as glucose, maltodextrin, dextrates, dextrin and combinations thereof.
Examples of suitable disintegrants that may be used in the compositions of the present disclosure include, but are not limited to, crosslinked polymers, starches and modified starches. Preferred disintegrants are crosslinked polyvinylpyrrolidone (crospovidone), crosslinked sodium carboxymethyl cellulose (croscarmellose sodium), corn starch and sodium starch glycolate and combinations thereof.
Examples of binders that may be used in the compositions of the present disclosure include, but are not limited to, maltose syrup powder, gum arabic, sodium alginate, propylene glycol alginate ester, hydrolyzed gelatin powder, fructose, carboxylvinyl polymer, hydrous silicon dioxide, agar powder, light anhydrous silicic acid, polyvinylpyrrolidone, copolydone, Polyvinyl acetate, cellulose acetate phthalate, dioctyl sodium sulfosuccinate, starch, cornstarch, tragacanth, lactose, concentrate glycerin, sucrose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose phthalate, vinylpyrrolidone-vinyl acetate copolymer, glucose, pregelatinized starch, fumaric acid, povidone, polyvinyl alcohol, D-mannitol, and methyl cellulose and combinations thereof.
Examples of glidants that may be used in the compositions of the present disclosure include, but are not limited to, hydrous silicon dioxide, light anhydrous silicic acid, crystalline cellulose, synthetic aluminum silicate, titanium oxide, stearic acid, calcium stearate, magnesium stearate, calcium tertiary phosphate, talc and combinations thereof.
Lubricants are generally used in order to reduce sliding friction. Suitable lubricants that may be used in the compositions of the present invention include, but are not limited to, sodium stearyl fumarate, magnesium stearate, glyceryl behenate, stearic acid and adipic acid and combinations thereof.
The compositions of the present disclosure may be in the form of film coated tablets. Film-coatings can be prepared by using agents such as cellulose derivatives, poly(meth)acrylate, polyvinyl pyrrolidone, polyvinyl acetate phthalate, or natural rubbers such as carrageenan, and mixture thereof.
When the pharmaceutical composition of the present disclosure is a tablet, it may be produced by mixing pharmaceutically acceptable carriers such as an excipient, a disintegrant, and a binder with the polyherbal extracts, together or separately, and directly compression-molding the mixture, or the tablet may be produced by granulating a mixed powder by fluidized-bed granulation, stirring granulation, or the like, and then compressing the obtained granules.
When the pharmaceutical composition of the present disclosure is a capsule, it can be produced by merely filling the granules or a mixture of the polyherbal extracts and excipients, into appropriate capsule shells.
For liquid dosage forms such as solutions, suspensions and emulsions, the polyherbal extracts of the present invention can be mixed with suitable vehicle, solubilizer, suspending agent or an emulsifier to prepare the appropriate liquid formulation.
The components of the pharmaceutical compositions described herein are pharmaceutically acceptable, generally regarded as safe (GRAS), conventionally used in the pharmaceutical art, and provide a composition that is stable over its shelf life. Excipients, such as the categories defined by their functions above, are conventionally known in the pharmaceutical art. A person skilled in the art would know of the use of the right excipients in the right quantities for the desired function, to prepare a pharmaceutical carrier. The present disclosure provides the novel combination of the herbal extracts, in defined quantities, for use in treating inflammation. The use of excipients to obtain the dosage forms containing the novel polyherbal extract is within the understanding of the skilled person, i.e. while the specific polyherbal extract and its use is novel and inventive, the excipients and their use is conventional and is not the inventive aspect of the disclosure.
The composition of the present disclosure may be used for treating and/or preventing an inflammatory disorder selected from: peripheral pain, inflammatory status of different origin, pain associated with muscle spam like stiffness, stiff neck, painful lumbago, joint pain, muscle pain, osteoarthrosis, osteoarthritis, psoriatic arthritis, headache, diabetic pain, gout. Preferably, the composition is used in treating and/or preventing osteroarthritis.
The disclosure will now be illustrated with working examples, which are intended to illustrate the working of the disclosure and not intended to limit or restrict the scope of the present disclosure.

EXAMPLES:
Example 1
A polyherbal extracts A and B were obtained by mixing individual extracts of the components. About 55% to about 65%w/w of hydroalcoholic extract of Berberis aristata or Phellodendron amurense, about 15% to about 25%w/w of hydroalcoholic extract of Tribulus terrestris, about 0.5% to about 1.5%w/w of hydroalcoholic extract of Piper nigrum, and about 10% to about 20%w/w of hydroalcoholic extract of Tinospora cordifolia were mixed to obtain the polyherbal extracts A and B, each containing individual components in amounts as shown in Table 1 below.
Table 1
Ingredients Polyherbal extract A Polyherbal extract B
Phellodendron amurense - 970mg
Berberis aristata 970mg -
Tribulus terrestris 350mg 350mg
Piper nigrum 5mg 5mg
Tinospora cordifolia 220mg 220mg

The extracts were individually obtained using a hydroalcoholic solvent, by means conventional in the art. The plant parts were spread in trays and dried in an hot air oven at temperature not exceeding 50°C, followed by size reduction using a grinder mill. The powder was then sieved through 80 mesh. 2kg of the powder was then macerated with 5 litres of hydroalcoholic solvent comprising ethanol and water in the ratio of 70:30 using an extractor. Extraction time was 2 Hrs. The resultant extract was filtered through 0.2 micron centrifuge filtration unit. The filtrate was then concentrated on water bath with a temperature maintained at 100° C till it is reduced to one-fourth of the original volume. The semi-solid extract was then further dried in hot air oven below 100°C and triturated to obtain a fine powder. The extracts were then mixed in the amounts specified above. The mixtures were then stored in sealed containers.
Similarly, aqueous and alcoholic extracts of the formulation were prepared using water and any alcohol, conventionally known in the art, including ethanol.

Extract enrichment: The above hydroalcoholic extracts of all the crude drugs was concentrated on a water bath, whose temperature was maintained at 100oC. When the volume was reduced to 1/4th, the extract was cooled and transferred to a separating funnel. To this, Petroleum ether (60-80) was added. To every 100ml of concentrated extract 50 ml of petroleum ether was added. After thorough shaking, the petroleum ether layer was separated and evaporated. The remaining aqueous extract was now termed as enriched extract from whose matrix chlorophyll and other pigments were removed by petroleum ether. The presence of phytoconstituents in enriched extract was established through TLC and UV analysis.
The UV analysis was conducted using the parameters and conditions shown in Table 2 below -
Table 2
Detection Mode Conditions
Standard solution 1mg/ml methnolic solution of extract
UV system Thermoscientific evolution 300 UV-Vis spectrophotometer
Detector Thermoscientific multiwavelength detector
Software Visionchrome
Detection wavelength 200-800nm

The observation for different extracts revealed that though the yield was slightly higher with hydroalcoholic extraction, the water extract also yielded good yield. The UV analysis, when scanned between 200-800 nm, showed presence of many constituents between 200-400 nm. Giloy extract showed the removal of chlorophyll and some pigments, whereas when the petroleum ether extract was scanned, it showed presence of chlorophyll. Gokhru fruit extract did not reveal much difference between original hydroalcoholic and the enriched extract. In case of Gokhru aerial extract, the peaks of constituents were almost the same, except for chlorophyll peaks in petroleum ether. All the water extracts showed peaks matching with those of hydroalcoholic extracts. Thus, the water extraction proves to be a commercially viable option and would also have lower toxicity.
A chromatography was performed using Berberine hydrochloride as standard. Giloy and Phellodendron showed bright yellow spots indicating presence of Berberine in hydroalcoholic extracts. Phellodendron had higher quantities of berberine. The Gokhru aerial extract however did not show presence of Berberine.
The polyherbal formulation was then standardized with respect to berberine.
The extracts were individually analysed before they were mixed to obtain the polyherbal extracts. The results were as shown in Table 3(a), Table 3(b), Table 3(c) and Table 3(d) below -
Table 3(a)
Berberis Extract Result Specification
Test of Foreign organic matter Nil Not more than 2%
Loss on drying 9.56% Not more than 10%
Total Ash 2.72% NMT 5%
Acid insoluble Ash 1.64% NMT 2%
Alcohol extractive value 3.4% Not less than 2%
Aqueous extractive value 8.8% Not less than 5%
Residual solvent (By GC-HS) Ethanol 222 ppm NMT 5000 ppm

Table 3(b)
Phellodendron Extract Result Specification
Test of Foreign organic matter Nil Not more than 2%
Loss on drying 4.69% Not more than 5%
Total Ash 4.63% NMT 5%
Acid insoluble Ash 0.02% NMT 2%
Alcohol extractive value 11.8% Not less than 10%
Aqueous extractive value 13.3% Not less than 10%
Residual solvent (By GC-HS) Ethanol 1592 ppm NMT 5000 ppm

Table 3(c)
Gokhru Extract Result Specification
Test of Foreign organic matter Nil Not more than 2%
Loss on drying 4.22% Not more than 5%
Total Ash 17.4% NMT 20%
Acid insoluble Ash 4.62% NMT 5%
Alcohol extractive value 1.7% Not less than 1%
Aqueous extractive value 9.3% Not less than 5%
Residual solvent (By GC-HS) Ethanol 2111 ppm NMT 5000 ppm

Table 3(d)
Giloy Extract Result Specification
Test of Foreign organic matter Nil Not more than 2%
Loss on drying 8.72% Not more than10%
Total Ash 9.19% NMT 10%
Acid insoluble Ash 2.14% NMT 3%
Alcohol extractive value 1.4% Not less than 1%
Aqueous extractive value 7.5% Not less than 5%
Residual solvent (By GC-HS) Ethanol 83 ppm NMT 5000 ppm

Example 2
The polyherbal extracts obtained in example 1 were tested by inducing arthritis in rats using Complete Freund’s adjuvant, and evaluating the individual extracts and the polyherbal extracts for anti-arthritic and anti-inflammatory effect by physical, biochemical and histopathological studies.
The Freund’s adjuvant-induced arthritis model was used to assess the anti-arthritic activity of the polyherbal extract and the individual extracts in albino Wistar rats. Rats weighing about 150-175 gm and of 5-7 weeks age were housed as per CPCSEA guidelines in clean polypropylene cages under standard conditions of humidity (50 ±5 % RH), temperature (maximum 24 ºC and minimum 23 ºC) and light (12 h light/12 h dark cycle). The rats were fed with a standard diet (Nutrivet Lifesciences, Pune, India) and water ad libitum. All rats were handled with humane care. After acclimatization (6-7 days) in the animal quarters, they were randomly divided into 15 groups of 6 animals each, with each group comprising 3 males and 3 females.
The groups were studied as provided in Table 4 below –
Table 4
Group Details
1 Normal Control which received only saline water
2 Arthritic Control which received the Complete Freund’s adjuvant (0.1ml, subplantar) only
3 Test group 1 which received test Giloy hydroalcoholic extract (3mg/kg, po*) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
4 Test group 2 which received test Giloy water extract (3mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
5 Test group 3 which received test Gokhru hydroalcoholic extract (5mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
6 Test group 4 which received test Gokhru water extract (5mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
7 Test group 5 which received test Phellodendron hydroalcoholic extract (14mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
8 Test group 6 which received test Berberis hydroalcoholic extract (14.3mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
9 Test group 7 which received test Berberis water extract (14.3mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
10 Test group 8 which received test Piperine alcoholic extract (0.07mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
11 Test group 9 which received test Polyherbal extract A (23mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
12 Test group 10 which received test Polyherbal extract A (46mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
13 Test group 11 which received test Polyherbal extract B (23mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
14 Test group 12 which received test Polyherbal extract B (46mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
15 Reference Standard group which received diclofenac sodium (10 mg/kg, po) + Complete Freund’s adjuvant (0.1ml, subplantar) after 30 min
po* = peroral
Polyherbal extract A = mixture of extracts of Berberine aristata, Tribulus terrestris, Piper nigrum and Tinospora cordifolia
Polyherbal extract B = mixture of extracts of Phellodendron amurense, Tribulus terrestris, Piper nigrum and Tinospora cordifolia

Arthritis was induced by injecting 0.1 ml of Freund’s adjuvant into the left hind paw of all animals, except Control. Drug treatment was started from the initial day, i.e., from the day of adjuvant injection (Day-0), 30 min before adjuvant injection and was continued till the 28th day. Paw volume was measured on the 1st, 6th, 11th, 16th, 21st and 28thdays of the study with a plethysmometer. The mean changes in injected paw edema with respect to initial paw volume, were calculated on respective days and percentage inhibition of paw edema with respect to untreated group (control) was calculated. The changes in body weight were recorded daily. On the 29th day, blood was collected from the retro-orbital vein and centrifuged for 10 min to obtain the serum. The hematological and biochemical parameters assayed from the serum were total protein, albumin, globulin, fibrinogen, red blood cell (RBC), white blood cell (WBC), hemoglobin (Hb), erythrocyte sedimentation rate (ESR), TNF alpha, superoxide dismutase (SOD), catalase and lipid peroxidation.
The animals were sacrificed on Day-29 by cervical dislocation. Ankle joints were separated from the hind paw, weighed and immersed in 10% buffered formalin for 24 h followed by decalcification in 5% formic acid, processed for paraffin-embedding and sectioned at 5 ?m thickness. The sections were stained with haematoxylin and eosin and evaluated under light microscope for the presence of hyperplasia of synovium, pannus formation and destruction of joint space.
The results provided in Table 5 below are represented as mean ± SEM from n=6. The biochemical markers and physical parameters of treated groups were compared with disease control group by one-way ANOVA, followed by Dunnett’s multiple comparison test. Values <0.05 were considered as statistically significant.

Table 5 – Mean paw volumes in study animals during the study
Group Days
0 1 6 11 16 21 28
G1-Normal Control 1.0 1.1 1.3 1.1 1.2 1.3 1.4
G2-Arthritic Control 1.1 1.8a 2.0a 3.0a 3.3a 3.4a 3.6a
G3-Giloy hydroalcoholic extract 1.0 1.6* 1.8* 2.7* 2.4* 2.3* 2.3*
G4-Giloy water extract 1.1 1.5* 1.7* 2.7* 2.4* 2.3* 2.3*
G5-Gokhru hydroalcoholic extract 1.1 1.4* 1.6* 2.6* 2.2* 2.2* 2.0*
G6-Gokhru water extract 1.1 1.5* 1.7* 2.7* 2.4* 2.4* 2.3*
G7-Phellodendron hydroalcoholic extract 1.0 1.6* 1.7* 2.6* 2.2* 2.1* 2.0*
G8-Berberis hydroalcoholic extract 1.1 1.5* 1.7* 2.6* 2.3* 2.2* 2.1*
G9-Berberis water extract 1.1 1.6* 1.7* 2.8* 2.4* 2.4* 2.3*
G10-Piperine alcoholic extract 1.1 1.6* 1.8* 2.7* 2.4* 2.4* 2.3*
G11-polyherbal A extract 1.1 1.6* 1.8* 2.7* 2.4* 2.3* 2.2*
G12-polyherbal A extract 1.1 1.5* 1.6* 2.5* 2.1* 2.1* 2.1*
G13-polyherbal B extract 1.1 1.6* 1.8* 2.7* 2.4* 2.3* 2.3*
G14-polyherbal B extract 1.1 1.4* 1.6* 2.5* 2.2* 2.1* 2.0*
G15- Diclofenac 1.0 1.3* 1.5* 2.2* 1.8* 1.7* 1.6*
Note: Data is represented as Mean ±SEM, analyzed by one way ANOVA followed by the Tukey post-test.
ap<0.001 when Disease control compared with Normal control
*p<0.001 when all experimental test groups compared to Arthritic Control.

The above results indicate that the anti-inflammatory effect of the polyherbal A and B combinations is higher than the individual components of the combinations, and is also higher than the standard of care, diclofenac.
Table 6 below provides details of testing conducted on the groups -

Table 6 – Effect of treatments on biochemical and hematological parameters
Group Total Protein (g/dl) Albumin (g/dl) Globulin (g/dl) Fibrinogen (mg/dl) TNF-a (ng/L) SOD (U/mg) Catalase (U/mg) Lipid Peroxidation (nmolMDA/min/mg protein) RBC 106/mm3 WBC 103/mm3 Hb (g/dL) ESR mm/hr
G-1 6.58 3.53 2.32 42.46 105.88 46.0 21.3 4.4 5.8 7.7 14.6 3.8
G-2 4.33a 2.36a 2.87a 162.38a 154.69a 32.0a 10.7a 10.9a 4.7a 12.6a 11.5a 10.4a
G-3 4.28 2.08b 2.70 120.64* 148.28* 33.4 13.7* 8.3* 4.7 11.5b 12.6** 5.8*
G-4 4.45 2.08b 2.77 120.85* 148.62** 33.1 13.5* 7.8* 4.6 11.6b 12.7** 6.1*
G-5 5.32* 2.97* 2.61** 63.83* 119.43* 38.0* 16.6* 5.5* 5.3** 9.5* 13.5* 5.3*
G-6 4.44 2.20 2.72 117.60* 135.92* 31.8 13.6* 9.4* 4.7 10.5* 12.4b 6.3*
G-7 5.27* 3.08* 2.55* 68.92* 117.53* 34.1 13.0* 7.6* 4.7 11.2* 13.9* 6.3*
G-8 4.53 2.17 2.75 111.92* 145.32* 33.3 13.3* 6.9* 4.7 11.1* 12.6** 5.7*
G-9 4.57 2.18 2.76 110.99* 144.13* 38.0* 15.3* 6.3* 5.3** 10.9* 12.8* 5.4*
G-10 4.67b 2.22 2.77 109.42* 137.37* 31.5 15.6* 8.5* 4.7 11.4* 12.3 6.3*
G-11 4.8b 2.22 2.73 107.02* 137.45* 31.3 15.0* 8.1* 4.6 11.0* 11.8 6.4*
G-12 5.1* 3.16* 2.43* 53.13* 114.32* 38.2* 16.4* 5.8* 5.3** 8.5* 13.9* 4.8*
G-13 4.67b 2.15 2.73 109.55* 134.13* 31.6 13.6* 7.6* 4.4 11.4 11.7 6.6*
G-14 5.44* 3.10* 2.66b 66.83* 118.75* 32.0 15.1* 7.0* 4.4 9.5* 12.9* 5.2*
Note: Data is represented as Mean ±SEM, analyzed by one way ANOVA followed by the Tukey post-test.
ap<0.001 when Disease control compared with Normal control;
bp<0.05, **p<0.01, *p<0.001 when experimental test groups compared with Arthritic Control

The above results indicate that the polyherbal compositions have significantly reduced fibrinogen levels as compared to the individual components of the compositions, especially for the higher doses of polyherbal A and polyherbal B extracts. Fibrinogen levels increase with increased inflammation, and reduced levels for the polyherbal compositions is indicative of the synergy of the combination used.

Example 3
The hydroalcoholic polyherbal extracts A and B of Example 1 and aqueous polyherbal extracts C and D (which contain similar constituents as polyherbal extracts A and B, but which have been extracted using water) were subjected to HPLC analysis using 2695 Series, 2996 PDA With Empower2 software for determination of Berberine and Piperine, which are major anti-inflammatory markers. The method of analysis is described below.

Method of Analysis:
Column: Hemochrom, Intsil C8, A31, 250 x 4.6, 5µ.
Flow: 1 mL/min
Detection: 205 nm
Mobile Phase: A: Acetonitrile
B: Water containing 0.05% TFA
Injection volume:20 µl
Run time: 45 min

Standard solutions were prepared by weighing 10 mg piperine and berberine separately and dissolving each in 10ml of methanol in a volumetric flask. 1 mL of each solution was taken and diluted upto 10 mL (Stock solution 100 µg/ mL). From these stock solutions, standard solutions (10 µg/ mL) were prepared by transferring aliquot 1mL of each stock solution to 10-mL in two volumetric flask and volume was made with ethanol. For polyherbal extracts, 1 g of each of the powdered extracts A, B, C and D were macerated with 50 mL of Ethanol using an ultrasonicator extractor. Extraction time was 30 mins. The resultant extract was filtered, the filtrate concentrated & solvent recovered. 0.25 g of the concentrated extract was weighed accurately & transferred to 10-mL volumetric flask and the volume was made up to 10 ml with ethanol.

The results of the analysis are given below:

Table 7: HPLC analysis of Polyherbal extract A

Table 8: HPLC analysis of Polyherbal extract B

Table 9: HPLC analysis of Polyherbal extract C

Table 10: HPLC analysis of Polyherbal extract D

Individual extracts of Berberis aristata, Phellodendron amurense, Tribulus terrestris and Giloy were subjected to HPLC analysis using 2695 Series, 2996 PDA With Empower2 software for determination of major anti-inflammatory markers.
The HPLC analysis of the individual extracts indicated that hydroalcoholic extract of Berberis aristata contained 6.17%w/w of berberine, hydroalcoholic extract of Phellodendron amurense contained 2.58%w/w of berberine, aqueous extract of Tribulus terrestris contained 0.14%w/w of protodioscin and aqueous extract of Giloy contained 0.1048%w/w of berberine.

Example 4
The dried polyherbal extract A of Example 1 was taken in suitable quantity and the material was compacted into tablets. Each tablet contained 970mg of Berberis aristata, 350mg of Tribulus terrestris, 5mg of Piper nigrum and 220mg of Tinospora cordifolia. The tablets were analysed for contents and packed in sealed containers.

Example 5
The dried polyherbal extract B of Example 1 was taken in suitable quantity and the material was compacted into tablets. Each tablet contained 970mg of Phellodendron amurense, 350mg of Tribulus terrestris, 5mg of Piper nigrum and 220mg of Tinospora cordifolia. The tablets were analysed for contents and packed in sealed containers.
While tablets of Examples 4 and 5 were prepared by simply compacting the dried polyherbal extracts, tablets or capsules or powders for filling in sachets can be prepared by using pharmaceutically acceptable excipients, described in the specification above. Processes conventional in the art and known to persons of skill in the art may be used to obtain and package the dosage forms.

,CLAIMS:We claim:
1. A herbal composition comprising a mixture of:

(i) about 50% to about 70%w/w of Phellodendron amurense or Berberis aristata
(ii) about 10% to about 30%w/w of Tribulus terrestris,
(iii) about 0.5% to about 5%w/w of Piper nigrum, and
(iv) about 5% to about 25%w/w of Tinospora cordifolia.
2. The herbal composition as claimed in claim 1, wherein the composition comprises a hydroalcoholic extract of (i) Phellodendron amurense or Berberis aristata, (ii) Tribulus terrestris, (iii) Piper nigrum and (iv) Tinospora cordifolia.
3. The herbal composition as claimed in claim 1, wherein the composition comprises an aqueous extract of (i) Phellodendron amurense or Berberis aristata, (ii) Tribulus terrestris, (iii) Piper nigrum and (iv) Tinospora cordifolia.
4. The herbal composition as claimed in claim 1, wherein the composition comprises an alcoholic extract of (i) Phellodendron amurense or Berberis aristata, (ii) Tribulus terrestris, (iii) Piper nigrum and (iv) Tinospora cordifolia.
5. The herbal composition as claimed in claim 1, wherein the composition is in the form of a dosage selected from the group comprising tablets, capsules, solutions, syrups, suspension, emulsions, lozenges, solid dispersion, oral films, granules, powders filled in sachet, and dry powders for reconstitution.
6. The herbal composition as claimed in claim 5, wherein the composition is a tablet.
7. The herbal composition as claimed in claim 5, wherein the composition is a capsule.
8. The herbal composition as claimed in claim 5, wherein the composition is a liquid solution.
9. The herbal composition as claimed in claim 5, wherein the composition is a liquid suspension.
10. The composition as claimed in claim 5, wherein the composition is an emulsion.
11. The herbal composition as claimed in claim 6, wherein the tablet further comprises excipients selected from the group comprising one or more of diluents, disintegrants, solvents, cosolvents, isotonicity agents, chelating agents, buffers, pH-adjusting agents, solubilizers, stabilizers, dispersion agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavouring agents and diluents.
12. The herbal composition as claimed in claim 7, wherein the capsule further comprises excipients selected from the group comprising one or more of diluents, disintegrants, solvents, cosolvents, isotonicity agents, chelating agents, buffers, pH-adjusting agents, solubilizers, stabilizers, dispersion agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavouring agents and diluents.
13. The herbal composition as claimed in claims 8, 9 and 10, wherein the liquid solution or suspension or emulsion further comprises excipients selected from the group comprising one or more of the solubilizer, suspending agent or an emulsifier.
14. The herbal composition as claimed in claim 1, wherein the composition is used in the treatment and management of osteoarthritis and associated inflammation.