WO2021020961A1

WO2021020961A1 - Extract of persicaria minor and use for treating or alleviating symptoms and/or conditions associated with hyperuricemia

Info

Publication number: WO2021020961A1
Application number: PCT/MY2019/050039
Authority: WO
Inventors: Tengku Shahrir Tengku Adnan; Annie George; Sasikala M. Chinnappan
Original assignee: Biotropics Malaysia Berhad
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2021-02-04

Abstract

The present invention is directed to a new kind of medicinal value or health care function of Persicaria minor extracts, particularly in the treatment or alleviation symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms. In one aspect, the present invention discloses the use of a composition comprising a therapeutically effective amount of Persicaria minor extract in the manufacture of a medicament for the alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms. The alleviation or treatment of the symptoms and/or conditions according to the present invention is characterised by inducing a change in the uric acid content of the blood. Also disclosed is the use of a pharmaceutical composition comprising the therapeutically effective amount of Persicaria minor extract.

Description

EXTRACT OF PERSICARIA MINOR AND USE FOR TREATING OR ALLEVIATING SYMPTOMS AND/OR CONDITIONS ASSOCIATED WITH

HYPERURICEMIA

FIELD OF THE INVENTION

The present invention relates generally to the medicinal use of Persicaria minor extract. More particularly, the present invention relates to the use of Persicaria minor extract in treating or alleviating symptoms and/or conditions associated with hyperuricemia.

BACKGROUND OF THE INVENTION

Uric acid is produced during the breakdown of purines, which are found in certain foods and are also formed in the body. Once produced, uric acid is carried in the blood and passes through the kidneys, where most of it is filtered out into the urine. In the pH conditions of body fluid, uric acid exists largely as urate, the ion form. The amount of urate in the body depends on the balance between the amount of purines eaten in food, the amount of urate synthesised within the body (e.g., through cell turnover), and the amount of urate that is excreted in urine or through the gastrointestinal tract. In humans, the upper end of the normal range is 360 pmol/L (6 mg/dL) for women and 400 pmol/L (6.8 mg/dL) for men.

Hyperuricemia is a metabolic disorder that is characterized by abnormally elevated levels of serum urate (sUA) or uric acid in the blood due to the imbalance between uric acid production and excretion. Elevated uric acid levels can also lead to several diseases, including a painful type of arthritis called gout. Elevated uric acid levels are also associated with health conditions such as heart disease, diabetes and kidney disease.

Gout, the most common form of inflammatory arthritis, arises from the subsequent deposition of urate crystals when concentrations become saturated. Gout has been defined as “a progressive metabolic disease characterized by symptomatic hyperuricemia and deposition of monosodium urate (MSU) crystals in joints and soft tissues due to an imbalance in uric acid uptake, synthesis or excretion”. The initial clinical sign of an acute gout attack is severe disabling pain, usually involving a single joint, which typically spontaneously resolves over a period of a few days to weeks without intervention, although treatment with anti-inflammatory drugs such as colchicine, NSAIDs (non-steroidal anti-inflammatory drugs) and corticoids will generally improve symptoms more rapidly. Upon resolution of an acute attack the patient will enter a symptom-free interval; however flares can recur with increased frequency and duration if the underlying pathology is not addressed. If sUA values remain high, MSU crystal deposits can grow and expand to other sites leading to further inflammation and associated tissue/joint injury. Ultimately a subset of individuals will transition to chronic tophaceous gout which is characterized by nodular urate crystal deposits, recurrent flares and concurrent arthritis, which takes 1 1 .6 years on average to occur from the initial flare.

It is often stated that the prevalence of hyperuricemia and gout has increased in recent years, although there are relatively few longitudinal studies in geographically diverse populations and increasing diagnosis rates may play a significant role. It has been highlighted that the distribution of gout varies significantly across the world which may reflect factors such as ethnicity, diet and socioeconomic factors. In the United States, the National Health and Nutrition Examination Survey [NHANES] 2007-2008 suggested estimating prevalence of gout hyperuricemia and gout at approximately 21 and 4%, respectively, an increase of 3.2 and 1.2% respectively when compared to the prior NHANES-III study conducted from 1988 to 1994. Likewise, a review of data collected in the Australian population suggested an increase in the prevalence of gout from 0.5 to 1 .7% from 1968 to 1995/6. However, this trend is not universal and data collected in Taiwan over a similar time period, 1993-1996 and 2005-2008, to the United States study showed a fall in the prevalence of hyperuricemia from 25.3 to 22.0% in men and from 16.7 to 9.7% in women. Some authors have argued that this is related to the epidemic of obesity and associated dietary shift toward foods rich in purines, alcohol consumption and fructose-sweetened drinks; however, this is still disputed and indeed, the impact of dietary intervention may be limited with respect to management of serum urate (sUA) or uric acid. The rapid upward trend of hyperuricemia has not only affected the quality of life of patients, but threatens the life of patients. This is a serious threat to the public health state and has brought a heavy financial burden amongst patients.

Medications most often used to treat hyperuricemia are xanthine oxidase inhibitors (e.g. allopurinol and febuxostat) and uricosuric agents (e.g. benzbromarone and probenecid). Xanthine oxidase inhibitors decrease the production of uric acid, by interfering with xanthine oxidase. Uricosuric agents, on the other hand, increase the excretion of uric acid, by reducing the reabsorption of uric acid once the kidneys have filtered it out of the blood. However, only a limited number of medications or drugs are available for the treatment of hyperuricemia, and many of the above- mentioned agents have adverse effects. Therefore, a more effective anti- hyperuricemia agent is needed.

Several other kinds of medications have potential for use in treating hyperuricemia. In people receiving hemodialysis, sevelamer can significantly reduce serum uric acid, apparently by adsorbing urate in the gut. In women, use of combined oral contraceptive pills is significantly associated with lower serum uric acid.

On the other hand, non-medication treatments for hyperuricemia include a low purine diet and a variety of dietary supplements. Treatment with lithium salts has been used as lithium improves uric acid solubility.

Persicaria minor from the family Polygonaceae, also known as Polygonum minus, is a medicinal plant commonly consumed as“ulam” for preventive health care. The plant leaves are aromatic and popularly used as an ingredient in Malaysian delicacies such as“laksa” (spicy noodle dish), “kerabu” (fried herbal rice), “tom yam” (spicy tangy soup) and“asam pedas” (spicy tamarind curry). The leaves are often sliced and sprinkled for its aroma and flavouring. In the Malaysian Traditional Medicine System, the decoction of the fresh leaves is consumed for indigestion, constipation and as a remedy for stomach disorder and pain (Vimala S., Ilham, M.A., Rashih A. A. and Rohana S. (2003), Nature’s Choice to Wellness: Antioxidant Vegetables/Ulam. Siri Alam and Rimba 7. Forest Research Institute Malaysia (FRIM), pp. 131 ). Several effects of Persicaria minor extract have been disclosed in the prior art, which include improving cognition and cosmetic uses, as well as stimulation of the immune system.

Malaysia patent application number PI 2012003882 discloses a composition comprising an extract of Persicaria minor for cognition enhancement and cosmetic uses, particularly for skin care, anti-wrinkling and protection against oxidative cell damage.

Malaysia patent application number PI 2012005248 discloses a composition comprising an extract of Persicaria minor and its use for the immunomodulation of mammals by way of stimulating under-active immune functions to enhance general or cell mediated immunity for the remedy of immunity related conditions such as auto-immune disease, pain, allergy, degenerative diseases and ailments or related conditions.

Given the wide variety of therapeutic benefits of Persicaria minor extracts, it is advantageous to provide an extract of this therapeutically valuable herbal medicine, used either alone or in combination for treating or alleviating symptoms and/or conditions associated with hyperuricemia.

SUMMARY OF THE INVENTION

The present invention provides a safe and effective treatment or alleviation of symptoms and/or conditions associated with hyperuricemia. Simultaneously, the present invention provides a new kind of medicinal value or health care function of Persicaria minor or traditionally known as Kesum.

It is therefore an object of the present invention to provide surprising and novel effects of Persicaria minor extract for treating or alleviating symptoms and/or conditions associated with hyperuricemia, characterized by reducing uric acid content in the blood.

According to an embodiment of the present invention, the effect of the Persicaria minor extract in respect of alleviating or treating symptoms and/or conditions associated with hyperuricemia is characterized by reducing uric acid content in the blood, specifically by:

• decreasing xanthine oxidase activity, which in turn inhibits uric acid biosynthesis and decreases the uric acid content,

• increasing uric acid excretion.

In particular, the present invention discloses the use of Persicaria minor extract in the manufacture of a medicament to treat or alleviate symptoms and/or conditions associated with hyperuricemia, including gout and its related symptoms, which is characterized by reducing uric acid content in the blood.

In an embodiment, the effect of reducing uric acid content in the blood is specifically characterized by:

• increasing uric acid excretion.

It is another object of the present invention to exploit the alleviation or treatment property of Persicaria minor in respect of symptoms and/or conditions associated with hyperuricemia, including gout and its related symptoms, and to develop a pharmaceutical composition using this plant extract composition as an immediate alternative and evidence-based product to treat symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms.

Accordingly, a first aspect of the present invention provides the use of a composition comprising a therapeutically effective amount of Persicaria minor extract in the manufacture of a medicament for alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, which includes gout and its related symptoms.

Preferably, the composition comprises an aqueous extract, an alcohol extract or a combination of aqueous and alcohol extract of Persicaria minor. The alcohol extract includes an alcohol having 1 to 3 carbon atoms. Preferably, the therapeutically effective amount of the extract ranges from 10 mg to 1000 mg.

More preferably, the therapeutically effective amount of the extract ranges from 50 mg to 500 mg.

The plant extract of Persicaria minor is derived from aerial parts of the plant that include stem and leaves.

The plant extract of Persicaria minor comprises quercetin-3-glucoronide.

Specifically, the plant extract of Persicaria minor comprises quercetin-3-glucoronide in an amount of about 0.2% - 1.0% w/w of the total weight of the composition.

According to the first aspect of the present invention, the medicament or composition for use in the manufacture of a medicament for alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, is characterized by inducing a change of uric acid content in the blood.

In an embodiment, the effect of the medicament or composition comprising Persicaria minor extract in respect of alleviating or treating symptoms and/or conditions associated to hyperuricemina, including gout and its related symptoms, is characterized by reducing the uric acid content in blood, specifically by:

• increasing uric acid excretion.

In a second aspect of the present invention, there is provided the use of a pharmaceutical composition comprising a therapeutically effective amount of Persicaria minor extract in the manufacture of a medicament for alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, wherein the alleviation or treatment of the symptoms and/or conditions is characterized by inducing a change of uric acid content in the blood. According to a third aspect of the present invention, there is provided a method of alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, the method comprising the step of administering to a subject in need thereof a composition having a therapeutically effective amount of an extract derived from Persicaria minor.

The method of alleviation or treatment of the present invention is characterized by inducing a change in the uric acid content of the blood.

In a preferred embodiment, the subject in need is a mammal. The mammal may be a human or an animal.

According to another preferred embodiment of the present invention, the therapeutically effective amount of the extract ranges from 10 mg to 1000 mg. More preferably, the therapeutically effective amount ranges from 50 mg to 500 mg.

According to yet another preferred embodiment, the alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, is characterized by a decrease in uric acid synthesis in the body and an increase in uric acid excretion from the body.

The symptoms and/or conditions associated to hyperuricemia include, but not limited to, gout, tophaceous gout, kidney stones and its associated symptoms.

Still another preferred embodiment of the present invention discloses that the extract is an aqueous extract, an alcohol extract or a combination thereof that is derived from Persicaria minor. The extract may contain the major bioactive compound from Persicaria minor. Specifically, the plant extract of Persicaria minor comprises quercetin-3-glucoronide.

One skilled in the art will readily appreciate that the present invention is well adapted to perform the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention. BRIEF DESCRIPTION OF THE DRAWING

Figure 1 illustrates the retention time and UV spectra of quercetin-3-glucuronide with that of the sample extract of Persicaria minor, according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The treatment or ameliorative effect against symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, according to the present invention, is provided by a new kind of medicinal value or health care function of Persicaria minor or Polygonum minus, which is traditionally known as Kesum.

Accordingly, one aspect of the present invention provides the use of a composition comprising a therapeutically effective amount of Persicaria minor extract in the manufacture of a medicament for alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms.

Preferably, the composition comprises an aqueous extract, an alcohol extract or a combination of aqueous and alcohol extract of Persicaria minor.

Preferably, the therapeutically effective amount of the extract ranges from 10 mg to 1000 mg.

According to the first aspect of the present invention, the medicament or composition for use in the manufacture of a medicament for alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, is characterized by inducing a change in the uric acid content of the blood. In an embodiment, the effect of the medicament or composition comprising Persicaria minor extract in respect of alleviating or treating symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, is characterized by reducing uric acid content in the blood, specifically by:

• increasing uric acid excretion.

According to a second aspect of the present invention, there is provided the use of a pharmaceutical composition comprising a therapeutically effective amount of Persicaria minor extract in the alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, wherein the alleviation or treatment of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms is characterized by inducing changes and reducing uric acid content in the blood.

According to a third aspect of the present invention, there is provided a method of treating or alleviating symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, the method comprising the step of administering to a subject in need thereof a composition having a therapeutically effective amount of an extract derived from Persicaria minor.

In a preferred embodiment, the subject in need is a mammal, The mammal may be a human or an animal.

According to yet another preferred embodiment, the symptoms and/or conditions associated to hyperuricemia include, but not limited to, gout, tophaceous gout, kidney stones and its associated symptoms. The symptoms include, but not limited to symptoms of gout such as severe pain in the joints, joint stiffness, difficulty in moving the affected joints, redness and swelling, misshapen joints; symptoms of tophaceous gout such as lumps under the skin, around the joints, in the curve at the top of the ear, damage of the joints, compression of the nerves; symptoms of kidney stones such as pain in the back, abdomen or groin, nausea, increased urge to urinate, pain when urinating, difficulty in urinating, blood in the urine, foul-smelling urine, and infections of the kidney and urinary tract.

The extract of Persicaria minor as described herein contains the desired active ingredients from the Persicaria minor plant, which may be further subjected to separation and characterization.

Preferably, the plant extract of Persicaria minor is derived from aerial parts of the plant that include stem and leaves.

The extract is obtained by way of extraction procedures that are known in the art, which include the basic steps of pre-washing, drying or freeze-drying of the plant materials (e.g. root), grinding the plant materials to obtain a homogeneous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with an aqueous system. Proper actions must be taken to assure that potential active ingredients/constituents are not lost, distorted or destroyed during the preparation of the extract from the Persicaria minor plant sample.

The extract thus obtained may be ready for use as a medicinal agent in its original dry extract form, the form of tinctures and fluid/alcoholic extracts, it may be further processed to be incorporated in any dosage form such as tablets or capsules, or it may be fractionated to isolate individual chemical entities. Thus, standardization of extraction procedures contributes significantly to the final quality of the herbal drug.

The preparation is preferably a pharmaceutical preparation that can be formulated into therapeutic dosage forms and tablets, capsules, oral liquids, sterile injections, aqueous or oily solutions or suspensions and the like. The preparation may be administered as nutraceuticals, comestibles or functional food by known techniques, for example oral, topical and parenteral administration (including subcutaneous injection, intravenous or intramuscular technique), in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, diluents or excipient.

The extract of the leaves and/or stem of Persicaria minor, as it is in the preparation, may be a liquid, paste or a solid powder. As used herein, the term pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars and lactose, glucose, and sucrose; starches and corn starch and potato starch; cellulose and its derivatives and sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients and cocoa butter and suppository waxes; oils and peanut oil, cotton seed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, and propylene glycol; esters, and ethyl oleate and ethyl laurate; agar; buffering agents and magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl water or alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants and sodium laryl sulphate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavouring and perfuming agents; preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

In another aspect, the present invention provides a pharmaceutical, nutraceutical, comestible or functional food preparation comprising a plant extract of Persicaria minor as an active ingredient in a therapeutically effective amount for treating or alleviating symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms.

In an embodiment, the usual dose or therapeutically effective amount of the extract varies from about 0.1 to 500 mg/kg of body weight of the subject per day. More preferably, the usual dose or therapeutically effective amount of the extract is in the range of from about 10 to 300 mg/kg of body weight of the subject administered in equal portions twice a day or thrice a day. The comestibles mentioned herein can be any common daily consumed processed food such as bread, noodles, confections, chocolates, beverages (for example instant tea preparation), and the like. One skilled in the art shall appreciate the fact that the aforesaid extract can be incorporated into the processed comestibles, capsules or tablets during the course of processing. Therefore, any modification thereon shall not depart from the scope of the present invention.

According to a preferred embodiment, the extract to be incorporated into the comestibles and medicine can be acquired from any known method not limited only to the foregoing disclosed method.

Following another embodiment, the extract is prepared in a concentrated form, preferably paste or powdery form which enables the extract to be incorporated in various formulations of the comestibles, capsules, tablets or topical products. In line with the preferred embodiment, the extract shall be the plant metabolites which are susceptible to an extraction solvent. The compounds and small peptides with the anti-hyperuricemia properties are those metabolites in the water or alcohol extracts. Therefore, the water or alcohol extract or combination of water and alcohol extract of leaves of Persicaria minor is preferably derived from the extraction solvent of water, mixture consisting water and an alcohol having 1 to 3 carbon atoms, acetone, chloroform, and any combination thereof.

In view of the prominent property of treating or preventing hyperuricemia by the extract in a subject, further embodiment of the present invention includes a method comprising the step of administering to the subject in need thereof a therapeutically effective amount of a composition comprising an extract derived from the leaves and/or stems of Persicaria minor.

The subject is a mammal and may be a human or an animal, but preferably a human. Preferably, the plant extract of Persicaria minor is derived from aerial parts of the plant that include stem and leaves.

The present invention will now be described in further detail by way of non-limiting examples.

EXAMPLE 1

Preparation of Persicaria Extract

Aerial parts of the plant Persicaria minor including stem and leaves are extracted using water and dried by oven drying with an extraction ratio of approximately 1 :10. The raw material used is dried leaves with properties of 2 to 5 cm in size, not more than 10% in loss on drying, not more than 10% ash content, not more than 2% foreign matter, not more than 10⁷ total aerobic microbial count, not more than 10⁵ total yeast and mold count. The dried leaves were then subjected to the steps of percolation using purified water and extracted at a temperature in the range of 70- 105°C preferably at 80°C to obtain the extract which is further filtered, concentrated, and dried using methods such as freeze drying, spray drying or vacuum belt drying to provide for a dry extract powder which is the packed and stored. After the drying step, the dry extract powder is package and stored at room temperature.

EXAMPLE 2

Content Analysis

The identification and quantification of actives in the extract and moisture content analysis of Persicaria minorwas studied and the results are provided hereunder.

The extract obtained was found to include actives quercetin-3-glucuronide as per the assay below: HPLC Assay: Preparation of Sample Solution for HPLC Analysis

100.0 mg of sample extract was accurately weighed in 5.0 ml. volumetric flask and DMSO was added to volume. The solution was then sonicated for 20 minutes at about 60°C, filtered with 0.20 pm RC membrane filter and was used as test solution for HPLC analysis.

Mobile Phase Preparation

Channel A - 1 .0 mL of FA was added into 1000 mL of deionised water. The solution was filtered and degassed. Channel B - 1.0 mL of FA was added into 1000 mL of acetonitrile.

Chromatographic System (Agilent 1290 Infinity)

Column PhenomenexKinetex (2.1 c 150 mm, 1.7 micron)

Temperature 60.0°C

Mobile Phase Solvent A - 0.1 % FA in Water

Solvent B - 0.1 % FA in CAN

Flow rate 0.400 mL/min (Gradient)

Table 1 : Gradient System

Wavelength UV at 360 nm, Reference at 450 nm

Run time 25 minutes (with 5.0 min post run)

Injection Volume 2.0 pL

Data Acquisition 25 minutes Quercetin-3-glucuronide was quantified by % assay in the said final extract obtained from Persicaria minor as shown in Table 2 below.

Moisture Content Analysis

Moisture content of the sample extract was determined by using a moisture analyzer, in which about 0.5 to 0.6 g of powdered sample was transferred onto an aluminium pan and heated at 105°C till a constant weight was obtained. The loss on drying (LOD) was determined by the percentage of weight loss during heating against the initial weight.

Table 2 HPLC Quantification by % Assay of quercetin-3-glucuronide and moisture content analysis of the extract

With reference to Figure 1 , a comparison of retention time and UV spectra of quercetin-3-glucuronide with that of the sample extract of Persicaria minor showed the presence of the compound in said sample. Therefore, quercetin-3-glucuronide is positively identified in the sample extract of Persicaria minor as per HPLC studies above. Persicaria minor extract was studied to reveal the anti-hyperuricemia activity, which is described below.

EXAMPLE 3

Materials and Methods

The present study was undertaken to evaluate and determine the in vivo anti- hyperuricemia effect of Persicaria minor ex tract of the present invention at a dose of 100 mg/kg, 300 mg/kg and 500 mg/kg body weight (b/w) against potassium oxonate-induced hyperuricemia Sprague Dawley rats. 48 animals were randomly divided into 6 groups consisting of Normal, Positive control, Negative control along with Test product treatment groups. On day 1 , 3 and 7, all the groups except normal control were induced with Potassium Oxonate injection through intraperitoneally followed by respective treatments for 7 days. On day 0, 1 , 3 and 7 serum uric acid levels were measured. In the end of the treatment, Xanthine oxidase levels were measured in liver homogenates.

Husbandarv and Acclimatization

The animals were housed under standard laboratory conditions for a period of one (1 ) week prior to treatment. The standard conditions include air-conditioned with adequate fresh air supply (12-15 air changes per hour), room temperature of 18.0 - 25.0°C and relative humidity 30-70%, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity was recorded once daily.

Grouping

Grouping was done by the method of body weight stratification and randomization. After acclimatization period healthy animals were weighed and randomly allotted to 6 groups (8 animals per group) as mentioned in study design. The grouping was done two days prior to the initiation of treatment.

The selected animals were assigned to control and different test groups as shown in Table 3 below:

Table 3

Preparation of Test Item

The test substance formulations were prepared daily during the treatment period. Detailed formulation methods and information was recorded in the study raw data file. 100 mg, 300 mg and 500 mg of Persicaria minor extract (i.e. Extract PM 1 ) weighed separately and added to separate mortars. Slowly small quantity of Carboxy Methyl Cellulose sodium suspension was added to each mortar with stirring continuously by a pestle. Then, quantity sufficient volume was made using the vehicle (Carboxy Methyl Cellulose sodium suspension) and stirred 5 minutes for uniform suspension. Total 10 ml of vehicle was added to each formulation. Quantity of formulation depends on animal body weight and dose volume was 10 ml/kg body weight. Dose Administration

Dose Volume: Animals were dosed at the volume of 10 ml/kg body weight.

Actual dose volumes were based on the recorded body weight on day 0.

Dose Administration for group 1 to group 8 by Oral gavage.

Dose Interval: Day of first dose was considered as study Day 1. Animals were dosed once daily for 7 days.

Dose Administration for Potassium Oxonate induction: Intraperitoneal injection of Potassium Oxonate dosed at 250 mg/kg body weight.

Inducer dose interval: Day 1 , day 3 and day 7.

Procedure

Healthy male Sprague Dawley rats were selected and body weights of the animals were 180-200 grams. After acclimatization of 1 week, the animals were divided into six groups of 8 rats in each group and average body weight was less than 20% in each group at randomization.

Group 1 was served as Control, Group 2 rats as Negative control, Group 3 rats as Positive control and Group 4 to Group 6 were served as test product treatment groups. Except group 1 , all the other group animals were induced with potassium oxonate (250 mg/kg b.w) on day 1 , 3 and 7, 1 hour before the respective treatment regimen. All the animals from respective groups received the treatment with vehicle, test and standard drugs from day 1 to day 7 orally. On day 0, 1 , 3 and 7 blood samples were collected from each animal by cutting tail tip 2 hours after the drug administration for the determination of serum uric acid (mg/dl) level. Serum was separated by centrifuging blood sample at 3000 rpm for 10 minutes and it was stored at -20°C for further biochemical analysis. EXAMPLE 4

Observation

Body Weight Measurement

Body weight was recorded before acclimatization, day 0 and day 7 of the experiment.

Feed Consumption Measurement

Feed intake was carried out daily during the study period. 200 grams feed was added daily to each group in the morning time and leftover feed was recorded on next day from the same animal group. Feed consumption was averaged, calculated and expressed as gram s/group/d ay.

Termination

On day 8, animals were sacrificed by cervical dislocation and their livers were excised immediately and washed with ice cold saline for Xanthine Oxidase estimation (spectrophotometric quantification of hepatic uric acid).

Enzyme preparation from rat liver

Rat liver was excised and homogenized in an ice cold 50 mM potassium phosphate buffer (pH 8.0). The homogenate was centrifuged at 3000 rpm for 10 min at 4°C, and then lipid layer was removed carefully. The resulting supernatant was further centrifuged at 10000 rpm for 60 min at 4°C. The supernatant obtained from the final centrifugation was used to measure Xanthine Oxidase activity. Biochemical analysis

1 . Serum Uric Acid estimation

Serum uric acid was determined by using Mispa ace AGAPPE auto analyzer using AGAPPE kit (Liqui check, Uric acid) by the uricase TOPS method. Briefly serum uric acid was assayed by adding 1000 mI of blank reagent, 25 mI of sample with 1000 mI of sample reagent and 25 mI of standard with 1000 mI of standard reagent to separate test tubes. The reagents were mixed and incubated for 5 min at 37°C. The absorbance of sample and standard was measured at 630 nm against the reagent blank.

Calculation:

Absorbance of sample

Uric acid concentration ( m a/dl ) = - x 8

Absorbance of standard

2. Assay for Xanthine Oxidase (XO) inhibition

Enzyme activity of Xanthine Oxidase was assayed by monitoring uric acid formation using the spectrophotometric method. Reaction was started by adding 100 mI_ of the supernatant to a phosphate buffer solution (pH 7.5) containing 0.12 mM xanthine and 0.192 mM EDTA. The mixture (total 5.0 ml.) was incubated for 30 min at 37°C, and the reaction was terminated by the addition of 1 M HCI (0.5 ml_). The production of uric acid was determined by measuring UV absorbance at 290 nm of uric acid. Xanthine Oxidase activity was expressed as mmol of uric acid per minute per miligram protein. Following formula was used to calculate the percentage inhibition of xanthine oxidase.

Control— Test

% Inhibition = xlOO

Control 3. Statistical analysis

The values were expressed in Mean±SEM. The significance of in vivo data was analyzed by One way anova followed by Dunnet test. P < 0.05 was considered as statistically significant.

EXAMPLE 5

Results and Discussion

Hyperuricemia is a metabolic disorder characterized by an imbalance between uric acid production and excretion and causes gout. Uric acid, the final oxidation product of purine metabolism, is eliminated mainly via urinary excretion from the kidney. Accordingly, inhibiting uric acid biosynthesis and increasing uric acid excretion may be useful therapeutic approaches for the treatment of hyperuricemia. Currently, XOD inhibitors (allopurinol (AP) and febuxostat) and uricosuric agents (benzbromarone and probenecid) are used as anti-hyperuricemia drugs in clinical practice. However, only limited numbers of drugs are available for the treatment of hyperuricemia, and many of these agents have adverse effects. Therefore, more effective antihyperuricemia agents are needed.

In the present study, the inventors have examined the effects of Persicaria minor extract on hyperuricemia and the inventors have surprisingly found that the herbal extract showed significantly decreased serum uric acid levels and Xanthine Oxidase activity (liver uric acid) in potassium oxonate induced hyperuricemic rats.

1. Serum Uric Acid Analysis

Administration of potassium oxonate resulted in significant (P<0.001 ) increase in urate level in rats, as indicated by an increase in the serum uric acid levels when compared to the control group. The positive control group treated with allopurinol at a dose of 5 mg/kg elicited significant reduction (P<0.001 ) of serum uric acid level on day 7 compared to negative control group. Treatment with the test products with different doses resulted in reduction of uric acid levels compared to the negative control group.

Treatment with Persicaria minor extract at 300 mg/kg (P<0.05) and 500 mg/kg (P<0.001 ) showed significant reduction in serum uric acid levels on day 7, when compared with the negative control group, as shown in Table 4.

Table 4: Summary of effect of Persicaria minor extract on serum uric acid in rats (mg/dl) (Mean ± SEM)

Values are expressed as mean±SEM (n=6); ^#P<0.05, ^##P<0.01 and ^###P<0.001 for Group 2 vs. Group

1 ; ^*P<0.05, ^**P<0.01 and ^***P<0.001 for Treatment groups 3 to 8 vs. Group 2. (Negative control group-2 compared against control group 1 and all the treatment groups 3-8 compared against Negative control group 2).

2. Xanthine Oxidase (Liver uric acid)

As represented in Table 5, animals treated with potassium oxonate showed significant increase in the xanthine oxidase in the liver compared to control group. Allopurinol group inhibited (56%) xanthine oxidase activity at a dose of 5 mg/kg. Treatment of rats with Persicaria minor extract at 300 mg/kg b.w indicated highly significant (P<0.001 ) xanthine oxidase inhibition when compared with the negative control group. Treatment with Persicaria minor extract 100 mg/kg b.w showed significant (P<0.05) inhibition of xanthine oxidase with 18% inhibition over the control group Table 5: Summary of effect of Persicaria minor extract on liver uric acid (Xanthine Oxidase, XO Activity) in rats (%) (Mean ± SEM)

Values are expressed as mean±SEM (n=6); ^#P<0.05, ^##P<0.01 and ^###P<0.001 for Group 2 vs. Group 1 ; ^*P<0.05, ^**P<0.01 and ^***P<0.001 for Treatment groups 3 to 8 vs. Group 2. (Negative control group-2 compared against control group 1 and all the treatment groups 3-8 compared against Negative control group 2).

CONCLUSION

In the present invention, the Persicaria minor extract showed a positive therapeutic effect in the treatment of hyperuricemia and can safely reduce the level of serum uric acid along with the reduction of xanthine oxidase activity in the liver. Further, it has been concluded that the herbal extract of Persicaria minor showed better anti- hyperuricemic activity at higher dose among the other.

Claims

1. Use of a composition comprising a therapeutically effective amount of Persicaria minor extract in the manufacture of a medicament for the treatment or alleviation of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms.

2. The use according to claim 1 , wherein the composition comprises an aqueous extract, an alcohol extract or a combination of aqueous and alcohol extract of Persicaria minor.

3. The use according to claim 2, wherein the alcohol extract includes an alcohol having 1 to 3 carbon atoms.

4. The use according to claim 1 , wherein the extract of Persicaria minor is derived from aerial parts of the plant that include stem and leaves.

5. The use according to claim 1 , wherein the extract of Persicaria minor comprises quercetin-3-glucoronide in an amount of 0.2% - 1.0% w/w of the total weight of the composition.

6. The use according to claim 1 , wherein the therapeutically effective amount of the extract ranges from 10 mg to 1000 mg.

7. The use according to claim 6, wherein the therapeutically effective amount of the extract ranges from 50 mg to 500 mg.

8. The use according to claim 1 , wherein the medicament comprising

Persicaria minor extract is characterized by inducing a change in the uric acid content of the blood.

9. The use according to claim 8, wherein the medicament comprising

Persicaria minor extract is characterized by reducing the uric acid content in the blood, specifically by: • decreasing xanthine oxidase activity, which in turn inhibits uric acid biosynthesis and decreases the uric acid content,

• increasing uric acid excretion.

10. Use of a pharmaceutical composition comprising a therapeutically effective amount of Persicaria minor extract in the manufacture of a medicament for the treatment or alleviation of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, wherein the treatment or alleviation of symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms, is characterized by inducing a change in the uric acid content..

1 1 . The use according to claim 10 wherein the medicament comprising Persicaria minor extract is characterized by reducing the uric acid content in the blood, specifically by:

• increasing uric acid excretion.

12. The use according to claim 10, wherein the therapeutically effective amount of the extract ranges from 10 mg to 1000 mg.

13. The use according to claim 12, wherein the therapeutically effective amount of the extract ranges from 50 mg to 500 mg.

14. The use according to claim 10, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

15. A pharmaceutical, nutraceutical, comestible or functional food preparation comprising a plant extract of Persicaria minor as an active ingredient in a therapeutically effective amount that is capable of treating or alleviating symptoms and/or conditions associated to hyperuricemia, including gout and its related symptoms.

16. A pharmaceutical, nutraceutical, comestible or functional food preparation according to claim 15, further comprising a pharmaceutically acceptable carrier, diluents or excipent.

17. A pharmaceutical, nutraceutical, comestible or functional food preparation according to claim 15, wherein the therapeutically effective amount of the extract comprises dosage in the range of from 0.1 to 500 mg/kg of body weight of a subject.

18. A pharmaceutical, nutraceutical, comestible or functional food preparation according to claim 17, wherein the therapeutically effective amount of the extract comprises dosage in the range of from 10 to 300 mg/kg of body weight of a subject.