Polyhebal formulation to treat hyperuricemia

Abstract

The present invention provides polyherbal formulation for treating Hyperuricemia, comprising of about 20–35 %w / w of heartwood aqueous extract of Quebracho, about 32–39 %w / w of hydro-ethanolic fruit extract of Terminalia bellerica as well as Terminalia chebula along with pharmaceutically acceptable lubricant, wherein the extracts essentially consist of Tannin as the active component.

Description

[0001] POLYHEBAL FORMULATION TO TREAT HYPERURICEMIA

[0002] FIELD OF INVENTION

[0003] The present invention relates to preparation of a herbal formulation for the treating hyperuricemia. More particularly, the present invention relates to developing a polyherbal formulation consisting of the extracts of Quebracho sp, Terminalia chebula and Terminalia bellerica as active ingredient.

[0004] BACKGROUND OF THE INVENTION

[0005] Hyperuricemia is a common disorder that affects patients of all ages and genders. The most common manifestation of hyperuricemia is gout which is usually best treated with a xanthine oxidase inhibitor. Hyperuricemia is defined as an elevated serum uric acid level, usually greater than 6 mg / dL in women and 7 mg / dL in men. Recent increase in the prevalence and incidence of hyperuricemia might be explained by similar increases in adverse lifestyle habits such as purine rich diets and excess alcohol consumption, other important factors being obesity, increasing diuretic use, and increasing aging population. Asymptomatic hyperuricemia is common and found in about 5 to 8% of adult males.

[0006] When serum uric acid level is greater than 9.0 mg / dl, the probability of progression to clinical gout is six times higher. Hyperuricemia is not only a risk factor for renal disease progression, but also may induce or aggravate cardiovascular disease. Increased uric acid may reflect tissue hypoxia or increased oxygen free radical formation, which is related closely to cardiovascular pathology. Most studies have linked hyperuricemia to poor clinical outcomes due to its marked association with cardiovascular disease (CVD) and renal disease.

[0007] Xanthine oxidase (" XO") is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. This enzyme plays an important role in the catabolism of purines in some species, including humans. However, sustained elevation of uric acid in blood can cause various diseases, including gout. In patients having gout, excess uric acid result in uric acid crystals which accumulate in cartilage, ligament and surrounding tissues causing severe inflammation and pain. A xanthine oxidase inhibitor (XO inhibitor) is any substance that inhibits the enzymatic activity of XO, blocking the hepatic oxidation of xanthine to uric acid. In humans, inhibition of XO reduces the production of uric acid, and several medications that inhibit XO are indicated for treatment of hyperuricemia and related medical conditions including gout.

[0008] Many herbal formulations based on Terminalia Sp. are mentioned in prior arts for the treatment of gout, Hyperuricemia and inhibition of Xanthine oxidase. In traditional Ayurvedic medicine, Terminalia bellerica has been used as a "health-harmonizer" in combination with Terminalia chebula and Emblica officinalis. Terminalia is a tree. Three species of terminalia are used for medicine. These species are Terminalia arjuna, Terminalia bellerica and Terminalia chebula. Terminalia bellerica and Terminalia chebula have common use for many of the diseases and disorders such as high cholesterol, digestive disorders, diarrhea and constipation and gout.

[0009] Terminalia chebula

[0010] Taxonomic Classification:

[0011] Kingdom Pl antae

[0012] Phylum Tracheophyta

[0013] Class Magnoliopsida

[0014] Order Myrtales

[0015] Family Combretaceae

[0016] Genus Terminalia

[0017] Species Terminalia chebula

[0018] Terminalia chebula is considered to have constituents that can be used for treating a range of diseases and disorders. Studies have reported on treatment of vaginal infections, constipation, diarrhoea, ulcers, gastroenteritis, asthma, cough, dyspnea, dyspepsia, hemorrhoids, candidiasis, parasites, malabsorption syndrome, hepatomegaly, vesicular and renal calculi, urinary discharges, tumors, skin diseases, leprosy, rheumatism, arthritis, gout, neuropathy, paralysis, memory loss, epilepsy, depression, diabetes, cardiovascular diseases, dysentery etc.,

[0019] Terminalia bellerica

[0020] Taxonomic Classification:

[0021] Kingdom Pl antae

[0022] Phylum Streptophyta

[0023] Class Equisetopsida

[0024] Order Myrtales

[0025] Family Combretaceae

[0026] Genus Terminalia

[0027] Species Terminalia bellirica

[0028] Terminalia bellerica (Gaertn.) Roxb. is grown widely throughout India, Sri Lanka, and South East Asia. T. bellerica has been used for centuries in Ayurveda, and contains several chemical constituents in common with T. chebula (Saraswathi, et al., Inti. J. Res. Pharm. Biomed. Sci. (2012) 3(l):97-99). T. bellerica fruit is eaten as food and widely used in Indian traditional medicine to treat diseases like hypertension, cancer, dysuria and inflammation. Terminalia bellerica is used to protect the liver and to treat respiratory conditions, including respiratory tract infections, cough, and sore throat.

[0029] Generally, the chemical constituents isolated from any plant parts may vary considerably in type and / or concentration due to a number of factors, e.g., ecological variation, soil variation, and nutrient variation, as well as variations in the process of extraction.

[0030] Many of the patents have expunged on the use of Terminalia species for treatment of hyperuricemia.

[0031] EP2898891 describes a composition containing extracts of Terminalia chebula and Terminalia bellerica, or combinations thereof and methods for treatment of uricemia, hyperuricemia, and gout in a human subject are provided, wherein the composition is used for inhibition of xanthine oxidase. ES2807907T3 mentions of Terminalia bellerica extract for inhibition of xanthine oxidase and decrease of uric acid in serum.

[0032] US10953067 describes a herbal composition used as dietary supplement to prevent or treat inflammatory disorders, comprising a therapeutically effective amount of an extract of Terminalia chebula, extract of Curcuma longa and a non - acidic, water -immiscible organic solvent extract of a Boswellia serrata resin. This composition is used to treat arthritis, asthma and other inflammation related disorders.

[0033] However, there is always a hunt for herbal compositions that may provide higher efficacy in treatment and faster remedy. The compositions or formulations mentioned in the prior arts though exhibit inhibition activity of XO, there is still a need for alternate herbal drugs that effectively attenuates the Uric acid and sustains the level in the long term on par with standard drug.

[0034] The present invention enumerates a polyherbal dosage formulation comprising of a mixture of extracts of Quebracho Colorado, Terminalia bellerica and Terminalia chebula as active ingredients

[0035] Quebracho Colorado (Schinopsis balansae and Schinopsis lorentzii, commonly called quebracho is an evergreen tree that grows wild in South America, mainly in Argentina and Paraguay, in dense sub-tropical forests which also include a variety of other trees and vegetation. Quebracho derives its name from two Spanish words, quebrar and hacha, meaning 'the axe breaks’ and is named so due to its hardness. The taste is very bitter, but there is scarcely any odour. Quebracho contains six alkaloids: Aspidospermine, Aspidospermatine, Aspidosamine, Quebrachine, Hypoquebrachine and Quebrachamine, amongst which Quebrachamine is the most active. Other constituents include, two new sugars - quebrachite and laevogyrate inosite, tannin and starch. Quebracho extract is commercialized in the form of spray-dried moistened powder.

[0036] Conventionally, cold soluble or semi-soluble quebracho extracts are blended either among themselves, or with other vegetable extracts, phenolic syntans, naphthalene and phenol-naphthalene syntans and they are used in all stages of vegetable tanning or in re-tanning of chrome tanned leathers. This is due to high amount of tannin content. Very few studies have described the use of Quebracho for therapeutic purpose. Commercially available Citokain™ Lung Protector is a formulation comprising of Quebracho Extract, Osha Root Extract and is used as dietary supplement to provide relief from respiratory disorders.

[0037] The Polyherbal formulations shows presence of high content of tannins. Tannins can help reduce uric acid levels by inhibiting xanthine oxidase (XOD), an enzyme that catalyzes the formation of uric acid from xanthine. Tannins can also inhibit uric acid metabolism. Excess xanthine oxidase generates high levels of uric acid and can create crystals in joints and tissues, leading to gout. Tannins are known to exhibit good antiinflammatory activity.

[0038] Tannins may be divided into (a) hydrolysable tannoids (HTs), which are esters of a polyol or sugar, usually glucose, with one or more trihydroxybenzene carboxylic acids (i.e., gallates), and (b) derivatives of procyanidins, flavanols or flavanones, so-called condensed tannins. HTs are molecules with a polyol (polyfunctional alcohols, generally D-glucose or its derivatives and phenols, namely galloyl and ellagoyl moieties) as a central core.

[0039] Terminalia chebula is rich in tannoid principles. The chief constituent tannoids in the fruit are chebulinic acid, chebulagic acid, carilagin and a tannoid metabolite, gallic acid (J. Bruneton. Pharmacognosy, Phytochemistry, Medicinal Plants. (Paris, France: Laviosier Publishing, 1995), p. 333). Other minor hydrolysable tannoids reported in T. chebula include punicalagin, chebulanin, neochebulinic acid, 1,2,3,4,6-penta-O-galloyl-beta-D-glucose, 1,6,-di-O-galloyl-D-glucose, casuarinin, 3,4,6-tri-O-galloyl-D-glucose, and terchebulin (L. J. Juang, et al., J. Sep. Sci. (2004) 27: 718-24). Terminalia bellerica has many of the constituents common with T. chebula including the Tannoids.

[0040] In view of the above, a formulation of Quebracho sp., Terminalia chebula and Terminalia bellerica for treatment of Hyperuricemia is elaborated. The Polyherbal formulation may hence prove a boon to treat hyperuricemia with no known side effects. ADVANTAGES OF THE INVENTION

[0041] The present invention provides a formulation comprising Quebracho sp., Terminalia chebula and Terminalia bellerica for treating hyperuricemia. One of the active ingredients in all the three species is Tannin. The present formulation provides for consistent and sustained reduction of serum uric acid in a short duration of 3 - 4 weeks and provides more pronounced results compared to the known Ayurvedic extracts of Terminalia sp.

[0042] OBJECTIVE OF THE INVENTION

[0043] The main object of the invention is to provide a polyherbal dosage formulation for treating Hyperuricemia disorder.

[0044] Another object of the invention is to provide a polyherbal dosage formulation comprising extracts of Quebracho sp., Terminalia chebula and Terminalia bellerica. Another object of the invention is to optimize a dosage formulation to reduce the Uric Acid level.

[0045] SUMMARY OF THE INVENTION

[0046] The present invention provides polyherbal formulation for treating Hyperuricemia, comprising of about 20 - 35 %w / w of heartwood aqueous extract of Quebracho, about 32 - 39 %w / w of hydro-ethanolic fruit extract of Terminalia bellerica as well as Terminalia chebula along with pharmaceutically acceptable lubricant, wherein the extracts essentially consist of Tannin as the active component.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention describes a mixed herbal formulation that is used to treat hyperuricemia. The herbal formulation is a mixture of extracts of Quebracho Colorado, Terminalia bellerica and Terminalia chebula as active ingredients.

[0049] Geographical Location The hydro-ethanolic extracts of Terminalia bellerica and Terminalia chebula in powdered form is procured for research purpose from Ayush Herbs Pvt. Ltd, Kangra, Himachal Pradesh, India. The granulated form of heartwood extract of the Quebracho Colorado (Schinopsis balansae or Schinopsis lorentzii) is procured for the research purpose of the present invention from a commercial manufacturer of extracts in Argentina, South America.

[0050] Various modifications to the embodiments will be readily apparent to those skilled in the art. The present disclosure is not intended to be limited to the embodiments illustrated but is to be accorded the widest scope consistent with the invention, dosage formulation, described herein. The term “comprising”, herein defined as being inclusive or open-ended and does not exclude additional, unrecited elements known to a person skilled in the art, unless the context clearly requires otherwise.

[0051] The main embodiment of the invention provides for formulation of an oral dosage form comprising of mixture of extracts of Quebracho Colorado, Terminalia bellerica and Terminalia chebula as active components. In a more preferred embodiment, the powder form of the extracts is used.

[0052] In an embodiment, various parts of the plants Terminalia bellerica and Terminalia chebula may be used to prepare the extract. In a preferred embodiment, the pericarp of the fruit of the plants Terminalia bellerica and Terminalia chebula is used. In a more preferred embodiment, the hydro-ethanolic fruit extract characterized with >80 %w / w and not less than 25 %w / w Tannin is used in the formulation.

[0053] The extraction procedure followed in the present invention is according to Sarkar and Mandal (Ref: Hydroalcoholic extracts of Indian medicinal plants can help in amelioration from oxidative stress through antioxidant properties. J Complement Integr Med. 2012;9: Article 7). The required parts of the plants Terminalia bellerica and Terminalia chebula is shredded and loaded onto the extractor and upon addition of Hydro-Alcohol in the specific ratio, the extracts of the plant parts is obtained. The extraction is done upto 5 times repeatedly and the final concentrate is vacuum dried at 50 - 60°C.

[0054] In an embodiment, the heartwood of the Quebracho Colorado is used to prepare the extract. In a preferred embodiment, the granulated form of the heartwood extract characterized with >68%w / w of Tannins is used for the formulation. The extract is analyzed as per the International Standard ISO 14088, IULTCS / IUC 32 (Ref Quantitative analysis of tanning agents by filter method, 2ndEdition, 2020 - 03).

[0055] The extraction procedure followed according to Poaty et al, 2010 (Ref Poaty et al, 2010, Industrial Crops and Products 31: 509-515), steps including initial milling of the raw materials (ie) the Heartwood of Quebracho. The chipped wood is being subjected to suitable solvent extraction to extract the tannoids. In the present method, Water is used for extraction. The tannic liquor is then processed as per the standard methods such as concentration and subsequent subjection to Cracking, Spray-Drying and Pelletizing.

[0056] In an embodiment, a required amount of granulated extract of Quebracho Colorado, Hydro-ethanolic extract of Terminalia bellerica and Terminalia chebula in the form of powder are mixed well through any of the known methods. The method of mixing includes any of the manual mode known to a person skilled in the art such as mortar and pestle, grinding etc., Alternatively, the method of mixing may also include mechanical means such as mechanical mixers or blenders etc., without altering the nature of the extract either chemically or otherwise. The grinding and mixing are carried out until the size of the particles is in the range of 60 to 80 mesh size.

[0057] In another embodiment, the final mixture of the active ingredients is mixed with a suitable pharmaceutically acceptable lubricant or flow agent to increase its flowability and non-stickiness. The lubricant may be chosen from Magnesium stearate, Calcium stearate, Stearic acid, sodium stearyl fumarate, Talc etc. But most preferably magnesium stearate is used in the present invention. According to the present invention, the dosage formulation is in the form of tablets. However alternate dosage forms such as capsules, pellets, oral thin films, suspensions with suitable pharmaceutically acceptable excipients, conventionally known in the art and to a person skilled in the art may be prepared for the same dosage. The amount / quantity of excipients may be defined appropriately depending on the type of formulation.

[0058] The word “about” used in the specification defines the range of values inclusive of the ± 1% w / w or ± 5 mg or 70% -90% of tannins blanketing the lower limit values as well as the upper limit values of the specified range in the disclosure. The term “about” is intended to broaden the scope of value range within its therapeutic activity permissibility of the dosage formulation. The “about” thus does not limit the values only to the specified range.

[0059] In an embodiment, the dosage formulation consists in the range from about 20 %w / w to about 35 %w / w of Quebracho extract.

[0060] In a preferred embodiment, the dosage formulation comprises from about 22%w / w to about 24 %w / w of Quebracho extract.

[0061] In a more preferred embodiment, the dosage formulation essentially consists of about 23 %w / w of Quebracho extract.

[0062] In an alternate embodiment, the dosage formulation comprises from about 32%w / w to about 34 %w / w of Quebracho extract.

[0063] In a more preferred embodiment, the dosage formulation essentially consists of about 33 %w / w of Quebracho extract.

[0064] In an embodiment, the dosage formulation comprises in the range from about 32 %w / w to about 39 %w / w of Terminalia bellerica extract.

[0065] In a preferred embodiment, the dosage formulation comprises in the range from about 38 %w / w to about 39 %w / w of Terminalia bellerica extract. In a most preferred embodiment, the dosage formulation essentially consists of about 38.5 %w / wof Terminalia bellerica extract.

[0066] In an embodiment, the dosage formulation comprises in the range from about 32 %w / w to about 39 %w / w of Terminalia chebula extract.

[0067] In a preferred embodiment, the dosage formulation comprises in the range from about 38 %w / w to about 39 %w / w of Terminalia chebula extract.

[0068] In a most preferred embodiment, the dosage formulation essentially consists of about 38.5 %w / w of Terminalia chebula extract.

[0069] In an embodiment, the weight percentage of Magnesium stearate IP, Talc is in the range from about 1 %w / w to about 5 %w / w. In the present invention Magnesium stearate IP is preferably used at 1 %w / w.

[0070] In an embodiment, the dosage formulation essentially consists of about 150 mg of Quebracho extract, about 250 mg of T. bellerica extract and about 250 mg of T. chebula extract as active ingredients, henceforth referred to as Formulation XX.

[0071] In another embodiment, the dosage formulation essentially consists of about 250 mg of Quebracho extract, about 250 mg of T. bellerica and about 250 mg of T. chebula as active ingredients, henceforth referred to as Formulation YY.

[0072] In an embodiment the average weight of each tablet is in the range from 617 mg to 682 mg.

[0073] In a preferred embodiment, the dosage of the Quebracho extract in a tablet formulation is about 150 mg / tablet.

[0074] In an embodiment the average weight of each tablet is in the range from 712 mg to 787 mg / tablet.

[0075] In another preferred embodiment, the dosage of the Quebracho extract in a tablet formulation is about 250 mg / tablet.

[0076] In an embodiment, the dosage of the T. bellerica extract in a tablet formulation is about 250 mg / tablet. In an embodiment, the dosage of the T. chebula extract in a tablet formulation is about 250 mg / tablet.

[0077] In an embodiment the Percentage Friability for the tablet formulations is in the range from 4.19 % to 4.22 %.

[0078] In an embodiment the Percentage Friability for the tablet formulations weighing approximately on average 650 mg is 4.19%.

[0079] In an embodiment the Percentage Friability for the tablet formulations weighing approximately on average 750 mg is 4.22%.

[0080] In an embodiment the disintegration time for the tablet formulations is 15 mins.

[0081] In an embodiment the dissolution rate for the tablet formulations is in the range from 84 % to 100 %.

[0082] In an embodiment the dissolution rate for the tablet formulation weighing approximately on average 650 mg is in the range from 90% - 100%, preferably 95%. In an embodiment the dissolution rate for the tablet formulation weighing approximately on average 750 mg is from 81% to 85%, preferably 84%.

[0083] Evaluation Parameters:

[0084] In the present invention, the dosage formulation in the tablet form is evaluated for various parameters for quality standards compliance.

[0085] A) Hardness: The hardness of tablets is determined using Hardness Tester. It is expressed in Newtons or kg / cm2.

[0086] B) Friability: The friability test is performed using friabilator. 10 tablets are selected randomly and weighed initially and transferred into friabilator. The drum is rotated for 100 revolutions after which the tablets are dusted and reweighed.

[0087] The percentage friability is calculated by:

[0088] % Friability = (initial weight-final weight) / initial weight x 100.

[0089] C) Disintegration time: Disintegration test is carried out in the basket rack assembly consisting of 6 open ended transparent tubes and rack for holding these tubes in vertical direction. 6 tablets were placed in 1000 ml beaker containing 900 ml distilled water at 37°C. Time required to break down all the tablets completely into smaller particles or granules is noted as disintegration time.

[0090] D) Dissolution Test: Dissolution test is carried out by placing 6 tablets in dissolution apparatus (IP apparatus 2 paddle). The phosphate buffer of pH 6.8 is used as medium and temperature is set at 37°C. The apparatus operated at a set speed of 50 rpm and samples were withdrawn at predetermined intervals (5, 15, 30, 45, 60, 90, 120 mins). The amount of active drug released into the medium is quantified using UV-Vis spectrophotometry.

[0091] Pre-Clinical Study Parameters:

[0092] The therapeutic efficacy of the Formulation XX and Formulation YY is studied with reference to the standard drug Febuxostat. The therapeutic efficacy of the Formulation XX and Formulation YY are also compared with the Formulation containing only the two Terminalia sp. (B+C) as it is widely studied for hyperuricemia treatment.

[0093] The results of the present invention exhibit better efficacy with respect to various study parameters such as Serum Urea, Serum Uric Acid and Serum Creatinine as shown in Tables 7, 8, 9. The comparative study reveals that Formulation YY is more efficacious overall.

[0094] The Formulation YY is efficacious than Formulation XX in attenuating the Serum Uric Acid and Serum Urea. Amongst them, the formulation YY is more efficacious than the Formulation containing two Terminalia sp. (B+C) in attenuating the Serum Uric Acid and Serum Urea. The inhibition of Serum Uric Acid attained in the disease animals is 59.4 % by Formulation YY compared to the B+C showing a maximum of 49% (Table 1). The data shows that the attenuation is more consistent with Formulation YY and sustains low level of Uric Acid in the long term.

[0095] Table 1: % inhibition of disease value attributed to Serum Uric Acid

[0096] Serum uric acid (mg / dl)

[0097]

[0098] Treatment DAY 7 DAY 14 DAY 17 DAY 19 DAY 21 group (% (% (% (% (%

[0099] Inhibition Inhibition Inhibition Inhibition Inhibition increase increase increase increase increase over over over over over disease disease disease disease disease group) group) group) group) group) Q150+B+C

[0100] 0.25 22.7 35.1 50.7 35.6 (XX)

[0101] Q250+B+C

[0102] 13 35.7 46 59.4 51.3 (YY)

[0103] B+C 0.5 25.2 36.2 49 41.3 Q150 10 37.7 29 53 46 Q250 11 23.4 36.6 60.5 45.5 Febuxostat

[0104] 10 29 60.5 73.7 51

[0105]

[0106] (std ref)

[0107] Examples:

[0108] Example 1:

[0109] Best mode of invention

[0110] Tables 2a and 2b shows the formulations XX and YY according to the present invention.

[0111] Table 2a: Formulation XX - 650 mg tablet

[0112] Ingredients %w / w mg / tablet Quebracho extract 23 150 Terminaliabellerica extract 38.5 250 Terminaliachebula extract 38.5 250

[0113] Magnesium stearate IP 1% 6.5 mg

[0114]

[0115] Table 2b: Formulation YY - 750 mg tablet

[0116] Ingredients %w / w mg / tablet Quebracho extract 33.3 250

[0117]

[0118] Terminaliabellerica extract 33.3 250 Terminaliachebula extract 33.3 250

[0119] Magnesium stearate IP 1% 7.5 mg

[0120]

[0121] Formulation: The granulated form of the heartwood extract of the Quebracho Colorado characterized with >68%w / w of Tannins is used for the formulation. The extraction procedure followed according to Poaty et al, 2010. Hydro-ethanolic extract of Terminalia bellerica and Terminalia chebula according to Sarkar and Mandal, 2012 is prepared. Both the extracts are obtained in the form of dried powder.

[0122] Weighed amount of Quebracho extract, Terminalia bellerica and Terminalia chebula extract are mixed well using a mortar and pestle or mechanical mixers or blenders, without altering the nature of the extract either chemically or otherwise. The grinding and mixing are carried out until the size of the particles is in the range of 60 to 80 mesh size.

[0123] The final mixture was lubricated with magnesium stearate before the tablets were punched.

[0124] Tableting: The weight and hardness was adjusted. The punches used were standard flat round shape 12mm size. A single punch tablet press was used to punch the tablets. Capsule Preparation Procedure:

[0125] Dispensing:

[0126] • Dispense all raw materials as per the quantities mentioned above.

[0127] • Verify the identity and status labels of all materials before use.

[0128] Sieving:

[0129] • Pass Terminalia chebula, Terminalia bellirica and Quebracho extract through #40 mesh sieve

[0130] • Pass MCC PH 112 and Syloid through #40 mesh.

[0131] • Pass Magnesium stearate through #60 mesh and keep aside.

[0132] Dry Mixing:

[0133] • Transfer all sieved materials (except magnesium stearate) into the blender. • Mix for 20 minutes at 12 rpm to ensure uniform blending.

[0134] • Collect a sample from different locations and visually verify uniformity of mix. Slugging:

[0135] • Compress the blended powder using a slugging press with flat-faced punches.

[0136] • Adjust hardness to form firm slugs that do not break during handling.

[0137] • Record compression parameters (pressure, speed, weight, hardness).

[0138] Milling of Slugs:

[0139] • After slugging, pass slugs through a multi-mill fitted with a #16 mesh screen.

[0140] • Collect milled granules in stainless steel container.

[0141] Lubrication:

[0142] • Add Magnesium stearate to the milled granules.

[0143] • Mix in blender for 5 minutes at low speed (6-8 rpm).

[0144] • Ensure no overmixing to avoid poor compressibility.

[0145] Filling:

[0146] • Size 00 Capsule.

[0147] In-process Checks

[0148] • Uniformity of blend (visual check)

[0149] • Hardness of slugs

[0150] • Bulk density and flow properties of milled granule.

[0151] Example 2

[0152] Table 3: Characterization results of the polyherbal formulation

[0153] The characterization results showing percentage of tannin which is the active agent, is not less than 35%.

[0154] PHYSICAL TEST

[0155] s. Tests Specification Results No.

[0156] 1. Color Brown Powder Complies 2. Taste Bitter Complies 3. Odor Characteristics Complies 4. Particle Size

[0157] a Mesh Size #20 84-100% w / w Complies b Mesh Size #60 38-98% w / w Complies

[0158]

[0159] c Mesh Size #100 50-85% w / w Complies 5. Bulk Density 0.5 / 0.8 gm / ml Complies 6. Tap Density 0.8-1.0 gm / ml Complies PHYTOCHEMICAL TEST

[0160] 1. Loss on drying (at 105° C) Not more than 5.0% w / w Complies 2. Solubility in Alcohol Not less than 60% w / v Complies Complies 3. Ash Content Not more than 5.0% w / w

[0161] 4. Active Compound by UV method

[0162] a Tannin Not less than 35% w / w Complies HEAVYMETALS TEST

[0163] Impurities

[0164] 1. Lead(ppm) Not more than 2 ppm Complies 2. Arseni c(ppm) Not more than 2 ppm Complies 3. Cadmium(ppm) Not more than 0.3 ppm Complies 4. Mercury (ppm) Not more than 1 ppm Complies MICROBIOLOGICALPROFILE

[0165] 1. Total viable aerobic count Not more than 105cfu / g Complies 2. Yeast and mould Not more than 103cfu / g Complies 3. E. coli Absent Complies 4. Salmonella sp. Absent Complies 5. Staphylococcus aureus Absent Complies 6. Pseudomonas aeruginosa Complies

[0166]

[0167] Absent

[0168] Example 3

[0169] Table 4: Hardness (kg / cm2)

[0170] S. No Weight of Tablet

[0171] 650 mg (XX) 750 mg (YY)

[0172] 1. 6 6.5

[0173] 2. 6 6.5

[0174] Average 6 6.5

[0175]

[0176] The hardness of tablets is found to be 6kg / cm2and 6.5 kg / cm2for the tablet formulation XX and formulation YY respectively.

[0177] Example 4

[0178] Table 5: Friability

[0179] Formulation XX Formulation YY

[0180] Initial Weight 633.6 mg Initial Weight 778.0 mg

[0181] Final Weight 607.5mg Final Weight 745.1 mg

[0182]

[0183] According to IP, a maximum % weight loss for 10 tablets should be not more than 1.0% is acceptable. The Percentage Friability for the tablets formulation XX and formulation YY is found to be 4.19% and 4.22% respectively. None of the tablet completely broke during the test.

[0184] Example 5

[0185] Table 6a: Dissolution Rate for formulation XX

[0186] S. No. Time Tablet 1 Tablet 2 Tablet 3 Average (minutes) (%CR) (%CR) (%CR)

[0187] 1. 5

[0188] 22.670 8.730154 13.119 14.840±5.82 2. 15

[0189] 34.570 36.57293 23.154 31.432 ±5.91 3. 30

[0190] 40.826 40.54649 33.867 38.413±3.22 4. 45

[0191] 50.877 54.74905 49.321 51.649±2.28 5. 60

[0192] 87.264 78.86652 76.725 80.952±4.55 6. 90

[0193] 88.407 87.57648 84.312 86.765±1.77 7. 120

[0194]

[0195] 104.038 102.723 101.757 102.839±0.93

[0196] Table 6b: Dissolution Rate for Formulation YY S. No. Time Tablet 1 Tablet 2 Tablet 3 Average (minutes) (%CR) (%CR) (%CR)

[0197] 1. 5

[0198] 7.525 4.678 7.241 6.481 ± 1.28 2. 15

[0199] 24.985 22.053 19.717 22.252 + 2.16 3. 30

[0200] 39.445 45.950 43.588 42.994+ 2.69 4. 45

[0201] 54.735 53.115 57.160 55.003+ 1.66 5. 60

[0202] 63.356 61.706 65.779 63.614+ 1.67 6. 90

[0203] 73.270 71.296 74.168 72.911+ 1.20 7. 120

[0204]

[0205] 81.193 87.476 85.689 84.786+ 2.64

[0206] Example 6: Preclinical Study

[0207] Animals: Male and Female Sprague Dawley rats (250-270 g and age of 2-2.5 months) were procured from registered animal breeders and used for the study. The study was carried out for a duration of 3 months

[0208] Protocol for in vivo studies

[0209] Rats, 15 in numbers, are assigned for each group with a composition of 8F+7M in each group. An additional group was the satellite group which contained 6 rats (3M+3F).

[0210] Normal Control Group: Rats received normal saline

[0211] Toxic Control Group: Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 21 days

[0212] Q150: Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 7 days, followed by the Quebracho extract (150 mg / kg), orally, for 14 days and potassium oxonate (250 mg / kg), intraperitoneally for 14 more days

[0213] QI 50+B: Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 7 days, followed by a combination of Quebracho extract (150 mg / kg) and Terminalia bellerica extract (250 mg / kg), orally, for 14 days and potassium oxonate (250 mg / kg), intraperitoneally for 14 more days

[0214] Q150+B+C (XX): Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 7 days, followed by a combination of Quebracho extract (150 mg / kg), Terminalia bellerica extract (250 mg / kg), and Terminalia chebula extract (250 mg / kg), orally, for 14 days and potassium oxonate (250 mg / kg), intraperitoneally for 14 more days

[0215] B+C: Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 7 days, followed by a combination of Terminalia bellerica (250 mg / kg) and Terminalia chebula extract (250 mg / kg), orally, for 14 days and potassium oxonate (250 mg / kg), intraperitoneally for 14 more days

[0216] Q250: Group III- termed as Q: Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 7 days, followed by the Quebracho extract (250 mg / kg), orally, for 14 days and potassium oxonate (250 mg / kg), intraperitoneally for 14 more days

[0217] Q250+B: Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 7 days, followed by a combination of Quebracho extract (250 mg / kg) and Terminalia bellerica extract (250 mg / kg), orally, for 14 days and potassium oxonate (250 mg / kg), intraperitoneally for 14 more days

[0218] Q250+B+C (YY): Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 7 days, followed by a combination of Quebracho extract (250 mg / kg) + Terminalia bellerica (250 mg / kg) and Terminalia chebula extract (250 mg / kg), orally, for 14 days and potassium oxonate (250 mg / kg), intraperitoneally for 14 more days

[0219] Reference Standard: Rats received potassium oxonate (250 mg / kg), intraperitoneally every day for 7 days, followed the standard drug febuxostat (5 mg / kg), orally, for 14 days and potassium oxonate (250 mg / kg), intraperitoneally for 14 more days

[0220] Potassium oxonate is dissolved in 0.9% saline solution and administered intraperitoneally 1 h before oral administration of test samples or febuxostat every day for 14 days. Animals are fasted 2 h before test drug administration. Treatment is administered once a day by oral gavage for 14 consecutive days.

[0221] Rats are anesthetized with isoflurane 1 h after drug / extract administration on Day-0, Day-3, Day-5, Day-7, Day- 10, Day- 12 and Day- 14 in order to collect blood from retro- orbital plexus. The blood is allowed to clot for approximately 1 h at room temperature and centrifuged at 2500 × g for 10 min at 4°C. Serum is separated and used for assays of uric acid, creatinine, and urea.

[0222] Example 7

[0223] Table 7: Serum uric acid

[0224] Serum uric acid (mg / dl)

[0225] Treatment

[0226] groups

[0227] DAY 7 DAY 14 DAY 17 DAY 19 DAY 21

[0228] Normal 1.96±0.15 1.71±0.30 1.79±0.12 2.31±0.41 1.67±0.23 Disease 4.02±0.32a4.32±0.28a5.27±0.32a6.05±0.44a6.81±0.28aQ150 3.61±0.08 2.69±0.14*** 3.74±0.18** 2.84±0.28*** 3.66±0.16*** Q150+B 3.54±0.08 3.73±0.08 3.78±0.35** 3.16±0.31*** 3.54±0.32*** Q150+B+C

[0229] (XX) 4.01±0.09 3.34±0.13* 3.42±0.33*** 2.98±0.23*** 4.39±0.10*** B+C 4.04±0.05 3.23±0.24* 3.36±0.22*** 3.09±0.23*** 4.00±0.24*** Q250 3.58±0.09 3.31±0.22* 3.34±0.20*** 2.39±0.14*** 3.71±0.06*** Q250+B 3.70±0.07 3.15±0.11** 3.98±0.11* 3.40±0.35*** 3.24±0.16*** Q250+B+C

[0230] (YY) 3.50±0.06 2.78±0.23*** 2.85±0.33*** 2.46±0.14*** 3.32±0.09*** Febuxostat

[0231]

[0232] Standard 3.62±0.08 3.07±0.16** 2.08±0.28*** 1.59±0.25*** 3.34±0.24*** Note: All values are mean ± SEM; N=6 in each group; One-way ANOVA followed by Tukey -Kramer multiple comparison test is applied for statistical analysis. P values:a< 0.001 when Disease compared with Normal;*< 0.05, **< 0.01 and *** < 0.001when experimental groups compared with Disease for each day

[0233] While the Disease Control treatment elicit a significantly (P<0.001) higher serum uric acid level than Normal Control on Day 7, there was a progressive increase in uric acid levels from Day 14 to Day 21, the highest level being recorded on Day 21 as 6.81±0.28mg / dl (Table 7)

[0234] A progressive decrease in uric acid levels is exhibited in all treatment groups from Day 14 to Day 19. However, the formulation YY exhibits an immediate effect as well as sustained levels over a period of time, especially Day 19, indicating a super saturation or ceiling effect. This is evident from the Serum Uric acid level by the Group Q250 compared to B+C or Formulation YY, but not Disease group. The attenuation of Serum

[0235] Uric Acid is 29.7 % on Day 19 with Formulation YY compared to the B+C showing a maximum of 23.5% on Day 19 (Table 7A). The data shows that the attenuation is more consistent with Formulation YY and sustains low level of Uric Acid even on Day 21

[0236] (5.1% reduction) which is comparable to standard drug treatment febuxostat (7.7%) and higher compared to B+C (0.9% reduction) whereas other treatment groups show increase in Uric acid level.

[0237] Table 7A: Percentage reduction of Serum uric acid - A comparative table

[0238] Treatment Groups % Serum Uric Acid reduction

[0239] Day 14 Day 19 Day 21

[0240] (7 days of daily (12 days of daily (14 days of daily

[0241] potassium potassium potassium oxonate) oxonate) oxonate)

[0242] Disease +7.4 +50.5 +69.4

[0243] (Untreated)

[0244] B+C -20 -23.5 -0.9

[0245] Q150 -25.5 -21.3 + 1.3

[0246] Q250 -7.5 -33.2 +3.6

[0247] Q150+B+C (XX) -16.5 -25.6 +9.4

[0248] Q250+B+C (YY) -20.5 -29.7 -5.1

[0249] Febuxostat (std -15 -56 -7.7

[0250]

[0251] ref)

[0252] Example 8

[0253] Table 8: Serum urea

[0254] Treatment Serum urea (mg / dl)

[0255] groups

[0256] DAY 7 DAY 14 DAY 17 DAY 19 DAY 21 Normal 38.91+2.50 31.39+3.91 36.75+3.81 40.98+2.84 39.13+2.73 Disease 41.55+3.07 62.25±4.75b96.52±4.76a113.81+4.85a166.03+11.03aQ150 44.62+1.61 35.30+7.09** 40.08+4.70*** 40.14+5.91*** 34.45+3.79*** Q150+B 42.85+3.83 35.57+4.37** 40.35+4.85*** 34.95+3.52*** 44.33+3.70*** Q150+B+C

[0257] (XX) 46.44+5.56 22.98+3.79*** 31.43+4.06*** 35.81+5.03*** 36.78+2.98*** B+C 36.97+2.00 35.77+2.80* 31.48+4.17*** 28.22+2.59*** 35.13+4.04***

[0258]

[0259] Q250 37.96+3.22 28.15+4.82*** 29.44+3.20*** 28.35+3.08*** 31.96+3.42*** Q250+B 39.17+4.57 26.89+5.03*** 38.87+3.13*** 32.61+4.57*** 25.17+2.07*** Q250+B+C

[0260] (YY) 37.62+3.13 24.64+2.98*** 24.69+2.85*** 30.19+1.13*** 30.94+2.03***

[0261]

[0262] Febuxostat 39.34+2.95 30.47+6.90** 40.96+6.27*** 28.26+3.96*** 28.90+2.32*** Note: All values are mean ± SEM; N=6 in each group; One-way ANOVA followed by Tukey -Kramer multiple comparison test is applied for statistical analysis. P values:a< 0.001 andb< 0.01 when Disease compared with Normal; *< 0.05, **< 0.01 and *** < 0.001when experimental groups compared with Disease for each day

[0263] All groups exhibit almost similar values of urea on day 7 including Disease control, which indicate that urea did not rise in 7 days. On Day 14, a significant (P<0.01) rise in urea levels is seen in the Disease Control group when compared with the Normal group indicating the setting in of the disease. All treatment groups exhibit significant

[0264] (P< 0.05 for B+C; P< 0.01 for QI 50, QI 50+B and standard; P< 0.001 for Q150+B+C, Q250, Q250+B and Q250+B+C) attenuation of the elevated urea levels (Table 8).

[0265] Administering a uricase inhibitor (oxonate) to rats raises serum uric acid levels, which results in increased kidney weight and elevated proinflammatory cytokines in both

[0266] serum and kidney. This activates the caspase- 1 inflammasome in the kidneys, causing

[0267] a gradual decline in kidney function and subsequently raising blood urea and creatinine levels. This effect is prevented by drugs that reduce urate like Febuxostat and the test products here Q, B, C (Ref Anjana Chaudhary et al., J. of Renal Physiology (2024) Vol.326, Iss.6, Pgs F1004 - F1015).

[0268] The Formulation YY exhibits higher attenuation of the Urea level on Day 14 and Day

[0269] 17 compared to B+C and the Standard Febuxostat proving immediate activity and higher efficacy. The values stabilized, near normal, over the next few days of study.

[0270] Example 9

[0271] Table 9: Serum creatinine

[0272] Serum Creatinine (mg / dl)

[0273] Treatment groups

[0274] DAY 7 DAY 14 DAY 17 DAY 19 DAY 21 Normal 0.49+0.02 0.59+0.07 0.53+0.03 0.6+0.02 0.58+0.01

[0275]

[0276] Disease 0.64+0.08 0.82+0.19 0.75±0.04b1.69±0.07a2.55±0.49a Q150 0.50+0.07 0.51+0.04 0.44+0.05*** 0.54+0.02*** 0.52+0.03*** Q150+B 0.53+0.02 0.51+0.01 0.54+0.04** 0.65+0.03*** 0.52+0.02*** Q150+B+C (XX) 0.47+0.02 0.51+0.03 0.59+0.03 0.59+0.03*** 0.46+0.03*** B+C 0.46+0.03 0.55+0.01 0.66+0.06 0.62+0.03*** 0.48+0.03*** Q250 0.55+0.06 0.51+0.01 0.55+0.02** 0.44+0.04*** 0.49+0.01*** Q250+B 0.52+0.03 0.49+0.01* 0.54+0.01** 0.51+0.03*** 0.48+0.02*** Q250+B+C (YY) 0.56+0.03 0.48+0.02* 0.52+0.02** 0.52+0.02*** 0.39+0.04***

[0277]

[0278] Febuxostat 0.51+0.03 0.53+0.03 0.49+0.01*** 0.45+0.05*** 0.83+0.39*** Note: All values are mean ± SEM; N=6 in each group; One-way ANOVA followed by Tukey -Kramer multiple comparison test is applied for statistical analysis. P values:a< 0.001 andb< 0.01 when Disease compared with Normal;*< 0.05, **< 0.01 and *** < 0.001when experimental groups compared with Disease for each day

[0279] No significant increase in creatinine is observed in Disease Control group on Days 7 and 14. However on Day 17 onwards a significant rise in creatinine levels was observed. The treatment groups, too, brought about a statistically significant attenuation of the oxonate-elevated creatinine levels from Day 17, to elicit their strongest attenuation on Day 21. The lowest creatinine value in the study was brought about by Formulation YY on Day 21 exhibiting a strong dose-dependent activity. Similar trend is also exhibited by Formulation XX. Group B+C, on the other hand, shows very less percentage of reduction evincing the non-dependency of combination of B & C,ie., Terminalia sp (Table 9).

[0280] Example 10

[0281] In vitro xanthine oxidase (XAO) activity

[0282] The inhibitory effects of the herbal extracts and standards allopurinol and febuxostat on in vitro Xanthine oxidase activity were determined using a modified spectrophotometric method (Umamaheswari et al., (2007), J Ethnopharmacol. Feb 12; 109(3): 547-51; Murugaiyah and Chan, (2009) J Ethnopharmacol. Jul 15;124(2):233-9). Briefly, the test samples were dissolved in distilled water and 0.1 mL of the test sample at 2 concentrations(100 and 500 pg / ml) was added to 2.9 mL of 200 mM phosphate buffer solution (pH 7.5). Freshly prepared enzyme solution (0.1 ml of 0.3 units / ml in 200 mM phosphate buffer, pH 7.5) was then added and the assay mixture was incubated at 37 °C for 10 min. The enzymatic reaction was then initiated by the addition of 2.0 mL of freshly prepared xanthine substrate (1.5 mM in distilled water) and the mixture was incubated at 37 °C for 30 min. The reaction was terminated by the addition of 1.0 mL hydrochloric acid (1 N). The absorbance of the assay mixture was then measured at 292 nm. Allopurinol, a known inhibitor of XAO, was used as a positive control (2.5, 5.0, 10, 25, 50 and 100 g / ml). A blank was also prepared as above but without XAO. Each sample was assayed in triplicate.

[0283] The Xanthine Oxidase (XAO) activity is calculated from the equation:

[0284] % XAO inhibition = (1 -A / B) X 100,

[0285] Where,

[0286] A is the activity of XAO without extract

[0287] B is the activity of XAO with extract / standard

[0288] The drugs Febuxostat and Allopurinol are used as reference standards to determine the therapeutic efficacy of the dosage formulation according to the present invention. The in vitro studies exhibiting XAO inhibition activity for Formulation XX and Formulation YY is in the range from 90 % to 94% and from 103 % to 106% respectively.

[0289] The results reveal that Quebracho extract by itself pronounces a high inhibitory activity of XAO as evaluated at concentrations lOOpg / ml and 500pg / ml compared to other groups and standard. The higher values of formulation YY is appreciably pronounced confirming a synergistic activity of Quebracho sp., T.bellirica and T.chebula with tannin as main active ingredient.

[0290] Table 10: in vitro XAO activity:

[0291] Sr. | Treatment | % Inhibition of XAO |

[0292] No. § § activity §

[0293] 1. | Standard Febuxostat | 65.257±1.33 |

[0294] I (500iig / ml) I I

[0295] 2. | Standard Febuxostat(100µg / ml) | 78.514±0.72 |

[0296] | Febuxostat! I OOiig / ml) | |

[0297]

[0298] § Standard Allopurinol § 79.314±1.45 § (500ug / ml)

[0299]

[0300] § Standard Allopurinol § 7

[0301]

[0302] 6.457±1.66 § > > 605 § (lOOpg / ml) § §

[0303] 5. | Q150 (100µg / ml) | 73.943±3.20 | 6. | Q150 (500µg / ml) | 91.086±2.50 | 7. | Q150+B (100µg / ml) | 84.571±2.33 | 8. | Q150+B (500µg / ml) | 90.971±1.23 | 9. | Q150+B+C (100µg / ml) | 90.590±2.50 | 10. | Q150+B+C (500µg / ml) (XX) | 94.819±2.66 | 11. | B + C (100µg / ml) | 90.476±3.25 | 12. | B + C (500µg / ml) | 96.724±2.25 | 13. | Q250 (500 µg / ml) | 103.467±4.33 | 14. | Q250 (100µg / ml) | 99.048±3.66 | 15. | Q250+B (500 µg / ml) | 93.295±2.46 | 16. | Q250+B (100µg / ml) | 103.276±4.66 | 17. | Q250+B+ C (100µg / ml) | 95.581±0.75 | j

[0304]

Claims

ClaimsWe Claim:

1. A polyherbal formulation to treat hyperuricemia comprising,about 33 %w / w of Quebracho aqueous extract asdried powder;about 33 %w / w of hydro-ethanolic fruit extract ofTerminalia bellerica as dried powder;about 33 %w / w of hydro-ethanolic fruit extractof Terminalia chebula as dried powder;about 1 %w / w of lubricant;(OR)about 23 %w / w of Quebracho aqueous extract as dried powder; about 38 %w / w of hydro-ethanolic fruit extract of Terminalia bellerica as dried powder;about 38 %w / w of hydro-ethanolic fruit extract of Terminalia chebula as dried powder;about 1 %w / w of lubricant;Wherein, the Quebracho extract essentially consists of more than 68 % w / w of active ingredient, the extract of Terminalia bellerica and Terminalia chebula, each essentially consists of more than 80 %w / w of active ingredient.Wherein the weighed amount of dried powder extract of Quebracho, Terminalia bellerica and Terminalia chebula are ground and mixed well to obtain the size of the particles of 60 to 80 mesh size, lubricated with magnesium stearate and hard pressed to form tablets2. The polyherbal formulation as claimed in claims 1, wherein the active ingredient is tannin or tannoids.

3. The polyherbal formulation as claimed in claims 1, wherein the Quebracho sp is selected from Schinopsis balansae or Schinopsis lorentzii.

4. The polyherbal formulation as claimed in claims 1, wherein the extract is reduced to the form of dried fine powder and / or dried granules.

5. The polyherbal formulation as claimed in claims 1, wherein the formulation is a tablet.

6. The polyherbal formulation as claimed in claimsl, wherein the formulation is a capsule.

7. The polyherbal formulation as claimed in claimsl, wherein the tablet formulation is characterized with the Percentage Friability in the range from 4.19 % to 4.22 %.

8. The polyherbal formulation as claimed in claims 1, wherein the lubricant is selected from pharmaceutical grade Magnesium stearate, Calcium stearate, Stearic acid, Sodium stearyl fumarate, Talc, most preferably Magnesium stearate.

9. The Polyherbal formulation as claimed in 1, according to any of the above claims for treating Hyperuricemia, characterized in that achieving attenuation of upto 50.7-59.4 % % of Serum Uric Acid.

Images