WO2012081776A1 - Composition comprising an extract of phellodendri cortex for the prevention or treatment of pancreatitis. - Google Patents

Abstract

The present invention is related to a composition comprising an extract of Phellodendri cortex for the prevention or treatment of pancreatitis. Accordingly, the inventive compositions according to the present invention are useful in the prevention and treatment of pancreatitis.

Description

COMPOSITION COMPRISING AN EXTRACT OF PHELLODENDRI CORTEX FOR THE PREVENTION OR TREATMENT OF PANCREATITIS.

The present invention is related to a composition comprising an extract of Phellodendri cortex for the prevention or treatment of pancreatitis and the use thereby

Pancreatitis occurring from the inflammation in pancreas can be classified into acute pancreatitis and chronic pancreatitis. Pancreatic juice comprises various digestive enzymes such as amylase, trypsin, lipase etc and pancreatitis occurs by alcohol abuse, gallstones etc resulting in the autolysis of pancreasdue to the digestive enzymes blocked by the blocking factors. In detail, pancreatitis can be classified into two types according the severity of the diseases, i.e., mild type pancreatitis accompanying with an interstitial edema, peripancreatic fat necrosis; and severe type pancreatitis accompanying with the widely spread fat necrosis in peripancreatic and intrapancreatic lesion, hemorrhage etc (Bank PA., Am. J. Gatroenterol., 89, pp151-152, 1994).

In particular, the progress of acute pancreatitis may be divides into three steps; i.e., local inflammatory response, systemic inflammatory response resulting in one or multiple organ dysfunction syndrome and the infection of intestinal flora within pancreas. At the earlier stage, the inflow of macrophage resulting from the injury of pancreas acinar cell causes to release various cytokines such as IL-1, IL-6, TNF-alpha etc in response to them, which plays an important roles in the circulation of inflamed cell, pancreas edema and pancreatic parenchyma failure. The level of the cytokines is increased in the blood serum of the patient suffering from acute pancreatitis and significantly increased in case of the patients suffering from pancreatitis complication such as pancreas necrosis, systemic inflammatory response, multiple organ dysfunction etc.

The level of blood amylase and lipase has been reported to be an important marker to predict acute pancreatitis. The enzymes release when acute pancreatitis occurs and cleaved at pancreas acinar cells. Especially, the level of amylase abruptly increases within 3-6 hours when acute pancreatitis occurs, reaches to maximum within 1-2 days and recovered to normal level within 2-6 day after the disease. The change of blood lipase has been also regarded as an important bio-marker and the level of lipase reached to 15-20 fold in case of patientsuffering from acute pancreatitis comparing with that in case of normal person (Braunwald, Eugene et al., Harrison’s Principles of Internal Medicine, 15^th Ed., New York: McGraw-Hill, pp. 1788-1791, 2001).

It has been reported that the conventionally used agents to treat pancreatitis, for example, immunomodulating agent, analgesic and anti-inflammatory drugs such as Naproxen, Ibuprofen, Methylprednisolone etc give rise to various adverse action such as the risk in cardiovascular system such as cardiac thrombosis, myocardial infarction, stroke etc and the risk in gastrointestinal system such as the hemorrhage, ulcer or perforation of gastrointestinal system. Accordingly, three have been still needed to develop new drug to treat pancreatitis without adverse response and effective efficacy till now (Lee H.S., Hanyang Medical Reviews, 27, 2007).

Phellodendri cortex is the tree cortex of Phellodendron amurense Rupr or the same genus plant belonged to Rutaceae such as Phellodendron moll NAKAI, Phellodendron sachalinense SAKGENT, Phellodendron insulare NAKAI and the like. It has been reported to comprise various alkaloids such as berberine, palmatine, jateorrhizine, magnoflorine, phellodendrine, candicine, menisperine; bitter components such as obakunone, obakulactone, steroids etc and to show various activities for example, anti-bacterial activity (Nakamoto K. et al., J. Prosthet. Dent. 64, pp691-694, 1990); hypotensive activity, CNS depression activity (Kulkarni SK & Dhir A., Phytother. Res., 24, pp317-324, 2009), anti-cancer activity (Mitani N. et al., Cancer Lett. 165, pp35-42, 2001) etc

However, there has been not reported or disclosed about therapeutic effect for pancreatitis disease of Phellodendron cortex in any of above cited literatures, the disclosures of which are incorporated herein by reference.

Therefore, the present inventors have endeavored to find the novel agent for treating pancreatitis and study the pharmacological effect of the extract of Phellodendron cortex through various in vitro test and animal model tests, for example, inhibitoryeffect on the blood level of amylase, lipase, the morphological change of pancreatic tissue, the effect on the inflammatory cytokine in blood in C57B mouse induced by pancreatitis.

Finally, the present inventors have found that the extract of Phellodendron cortex iseffective in treating and preventing pancreatitis in a dose dependent manner.

According to one aspect, the present invention also provides a use of an extract of Phellodendron cortexfor the manufacture of medicament employed for treating or preventing pancreatitis in human or mammal.

The present invention also provides a method for treating pancreatitis in human or mammal comprising administering to said mammal an effective amount of above-mentioned extract, together with a pharmaceutically acceptable carrier thereof.

The present invention provides a pharmaceutical composition comprising an extract of Phellodendron cortex as an active ingredient for treating or preventing pancreatitis.

The present invention also provides a health functional food comprising the above-described extract for the prevention or improvement of pancreatitis as an active ingredient in an amount effective to preventing and improving pancreatitis, together with a sitologically acceptable additive.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising an extract of Phellodendron cortex as an active ingredient for treating or preventing pancreatitis, together with a pharmaceutically acceptable carrier.

It is an another object of the present invention to provide a use of an extract of Phellodendron cortexfor the manufacture of medicament employed for treating or preventing pancreatitis.in human or mammal.

It is the other object of the present invention to provide a method for treating pancreatitis in human or mammal comprising administering to said mammal an effective amount of an extract of Phellodendron cortex, together with a pharmaceutically acceptable carrier thereof.

The term "Phellodendron cotex" disclosed herein comprise the extract of Phellodendron amurense Rupr or the same genus plant belonged to Rutaceae such as Phellodendron moll NAKAI, Phellodendron sachalinense SAKGENT, Phellodendron insulare NAKAI and the like, preferably, Phellodendron amurense Rupr.

The term "extract" disclosed herein comprises the crude extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, or the mixtures thereof, preferably water or the mixture solvent with water and ethanol, more preferably, 40-60% ethanol; and purified extract using by Dianion ion column chromatograph, specifically, the purified extract prepared by the procedure comprising the steps: subjecting the crude extract to ion column chromatography using by Dianion, SP207, HP20SS or HP20 filler, preferably, Dianion filler running with solvent system increasingthe polarity started from water to mixture solvent of water and ethanol, preferably, mixture solvent of water and ethanol mixed with ratio of 1:1-3 (v/v) to fractionate into seven fractions; collecting, concentrating each fraction under vaccuo and freeze-drying each fraction to afford purposed purified extract of the invention.

The term “purified extract” disclosed herein comprise the purified extract (a) prepared by the procedure comprising the steps: subjecting the crude extract to ion column chromatography using by Dianion, SP207, HP20SS or HP20 filler, preferably, Dianion filler running with solvent system increasing the polarity started from water to mixture solvent of water and ethanol, preferably, mixture solvent of water and ethanol mixed with ratio of 1:1-3 (v/v) to fractionate into seven fractions; collecting, concentrating each fraction under vaccuo and freeze-drying each fraction to afford purposed purified extract of the invention, which include 1^st fraction (designated as “PA-P0” hereinafter), 2^nd fraction (designated as “PA-P1” hereinafter), 3^rd fraction (designated as “PA-P2” hereinafter), 4-5^th fractions (designated as “PA-P3”hereinafter), 6-7^th fractions (designated as “PA-P4” hereinafter), preferably, PA-P2 extract comprising 3-10 (w/w)% berberine chloride, 3-10 (w/w)% palmatine chloride, and 0.01-5 (w/w)% jateorrhizine, preferably, 4-7 (w/w)% berberine chloride, 4-6 (w/w)% palmatine chlorideand 0.1-2 (w/w)% jateorrhizine based on the total weight of the extract;

and purified extract (b) prepared by the procedure comprising the steps: subjecting the crude extract to fractionate with 10-100 fold volume (v/w), preferably, 20-50 fold volume (v/w), more preferably, 1-10 fold volume (v/w), most preferably, 2-5 fold volume (v/w) of saturated butanol to afford butanol soluble fraction at the 1^st step; concentrating and drying the fraction to afford butanol-soluble fraction (designated as “PA-CB”¡± hereinafter comprising 3-10 (w/w)% berberine chloride, 3-10 (w/w)% palmatine chloride, and 0.01-5 (w/w)% jateorrhizine, preferably, 4-7 (w/w)% berberine chloride, 4-6 (w/w)% palmatine chloride and 0.1-2 (w/w)% jateorrhizine based on the total weight of the extract;

In accordance with one aspect of the present invention, there provided a health functional food comprising the above-described extract for the prevention or improvement of pancreatitis as an active ingredient in an amount effective to preventing and improving pancreatitis, together with a sitologically acceptable additive.

The term "Pancreatitis" disclosed herein comprises acute pancreatitis and chronic pancreatitis, preferably, acute pancreatitis.

The Phellodendron cortex, which can be used in the present invention, but not intent to limit thereto, include the same genus plants which would be apparent to those skilled in the art and have be used for identical or similar purpose and can be substituted for the prevention and treatment of purposed diseases.

The pharmaceutical composition for treating purposed diseases could contain about 0.01 to 95 w/w%, preferably 0.5 to 50 w/w% of inventive extract of present invention based on the total weight of the composition.

An inventive extract may be prepared in accordance with the following preferred embodiment.

For the present invention, the above-described extract of Phellodendron cortex can be prepared by following procedure;

For example, dried cortex of Phellodendrin cortex is subjected to extraction with 5 to 20-fold, preferably, 10 to 15-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof, preferably, water and ethanol mixture solvent at the temperature ranging from 10℃ to 110℃ preferably, from room temperature to 50℃ for the period ranging from 0.5 hrs to 48 hours, preferably, 1 hours to 30 hours to obtain crude extract at the 1^st step (designated as “PA-C”hereinafter) the extract is subjected to ion column chromatography using by Dianion, SP207, HP20SS or HP20 filler, preferably, Dianion filler running with solvent system increasing the polarity started from water to mixture solvent of water and ethanol, preferably, mixture solvent of water and ethanol mixed with ratio of 1:1-3 (v/v) to fractionate into seven fractions at the 2^nd step; the collected fractions is concentrated under vaccuo and freeze-dried to afford purposed purified extract of the invention. i.e., 1^st fraction (designated as “PA-P0” hereinafter), 2^nd fraction (designated as “PA-P1” hereinafter), 3^rd fractions (designated as “PA-P2” hereinafter), 4-5^th fractions (designated as “PA-P3” hereinafter), 6-7^th fractions (designated as “PA-P4” hereinafter), preferably, PA-P2 extract comprising 3-10 (w/w)% berberine chloride, 3-10 (w/w)% palmatine chloride, and 0.01-5 (w/w)% jateorrhizine, preferably, 4-7 (w/w)% berberine chloride, 4-6 (w/w)% palmatine chloride and 0.1-2 (w/w)% jateorrhizine based on the total weight of the extract;

alternatively, subjecting the crude extract to fractionate with 10-100 fold volume (v/w), preferably, 20-50 fold volume (v/w), more preferably, 1-10 fold volume (v/w), most preferably, 2-5 fold volume (v/w) of saturated butanol to afford butanol soluble fraction at the 2^nd step; concentrating and drying the fraction to afford butanol-soluble fraction (designated as “PA-CB”hereinafter)comprising 3-10 (w/w)% berberine chloride, 3-10 (w/w)% palmatine chloride, and 0.01-5 (w/w)% jateorrhizine, preferably, 4-7 (w/w)% berberine chloride, 4-6 (w/w)% palmatine chloride and 0.1-2 (w/w)% jateorrhizine based on the total weight of the extract.

It is the other object of the present invention to provide a method for preparing the extract of Phellodendron cortex comprising the steps consisting of; extracting dried cortex of Phellodendrin cortexwith 5 to 20-fold, preferably, 10 to 15-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof, preferably, water and ethanol mixture solvent at the temperature ranging from 10℃ to 110℃ preferably, from room temperature to 50℃ for the period ranging from 0.5 hrs to 48 hours, preferably, 1 hours to 30 hours to obtain crude extract at the 1^st step (designated as “PA-C” hereinafter) subjecting the crude extract to ion column chromatography using by Dianion, SP207, HP20SS or HP20 filler, preferably, Dianion filler running with solvent system increasing the polarity started from water to mixture solvent of water and ethanol, preferably, mixture solvent of water and ethanol mixed with ratio of 1:1-3 (v/v) to fractionate into seven fractions at the 2^nd step; concentrating under vaccuo and freeze-drying each fraction to afford purposed purified extract of the invention. i.e., 1^st fraction (designated as “PA-P0” hereinafter), 2^nd fraction (designated as “PA-P1” hereinafter), 3^rd fraction (designated as “PA-P2” hereinafter), 4-5^th fractions (designated as “PA-P3” hereinafter), 6-7^th fractions (designated as “PA-P4” hereinafter), preferably, PA-P2 extract comprising 3-10 (w/w)% berberine chloride, 3-10 (w/w)% palmatine chloride, and 0.01-5 (w/w)% jateorrhizine, preferably, 4-7 (w/w)% berberine chloride, 4-6 (w/w)% palmatine chloride and 0.1-2 (w/w)% jateorrhizine based on the total weight of the extract; alternatively, subjecting the crude extract to fractionate with 10-100 fold volume (v/w), preferably, 20-50 fold volume (v/w), more preferably, 1-10 fold volume (v/w), most preferably, 2-5 fold volume (v/w) of saturated butanol to afford butanol soluble fraction at the 2^nd step; concentrating and drying the fraction to afford butanol-soluble fraction (designated as “PA-CB” hereinafter) comprising 3-10 (w/w)% berberine chloride, 3-10 (w/w)% palmatine chloride, and 0.01-5 (w/w)% jateorrhizine, preferably, 4-7 (w/w)% berberine chloride, 4-6 (w/w)% palmatine chloride and 0.1-2 (w/w)% jateorrhizine based on the total weight of the extract.

It is still another object of the present invention to provide a pharmaceutical composition comprising the extract obtained by above described process as an active ingredient for preventing and treating pancreatitis.

The inventive composition of the present invention significantly reduced the blood level of amylase, lipase, the inflammatory cytokines and improved the morphological change of pancreatic tissue of C57B mouse induced by pancreatitis when the inventive extract of the present invention was orally and intravenously administrated thereto. When the oral acute toxicity of the extract was tested, the extract had no apparent effect on mortality, clinical signs, body weight changes, and gross findings at necropsy.

The pharmaceutical composition for treating purposed diseases could contain about 0.01 to 99.9 w/w%, preferably 0.1 to 90 w/w% of the above crude drug composition of present invention based on the total weight of the composition.

The crude drug composition of inventive pharmaceutical composition is used in the form of pulverized form thereof, extracted form therefrom or dried extract form thereof.

The inventive composition may additionally comprise conventionalcarrier, adjuvants or diluents in accordance with a using method. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington’s Pharmaceutical Science (Mack Publishing co, Easton PA).

Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents which are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the compounds of the present invention can be formulated in the form of ointments and creams.

Pharmaceutical formulations containing inventive composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet andgranule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), suppository, or sterile injectable preparation (solution, suspension and emulsion).

The inventive composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

The desirable dose of the inventive composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.01-10g/kg, preferably, 1 to 5g/kg by weight/day of the inventivecomposition of the present invention. The dose may be administered in a single or multiple doses per day. In terms of composition, the crude drug composition should be present between 0.01 to 80% by weight, preferably 0.5 to 50% by weight based on the total weight of the composition.

The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection.

In accordance with one aspect of the present invention, there provided a health functional food comprising an extract of Phellodendron cortex for the prevention or improvement of pancreatitis as an active ingredient in an amount effective to preventing and improving the disease, together with a sitologically acceptable additive.

The crude drug composition of inventive health functional food is used in the form of pulverized form thereof, extracted form therefrom or dried extract form thereof.

Accordingly, it is the other object of the present invention to provide a functional health food comprising the above extract for the prevention or improvement of pancreatitis.

The term "a functional health food" defined herein is "the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the extract of the present invention to conventional food to prevent or improve aimed disease in human or mammal".

It is the other object of the present invention to provide a health care food comprising the above extract, together with a sitologically acceptable additive for the prevention and alleviation of aimed disease.

The term "a health care food" defined herein is "the food containing inventive extract of the present invention showing no specific intended effect but general intended effect in a smallamount of quantity as a form of additive or in a whole amount of quantity as a form of capsule, pill, tablet etc.

The term "a sitologically acceptable additive" defined herein is "any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food" for example, thickening agent, maturing agent, bleaching agent, sequesterants, humectant, anticaking agent, clarifying agents, curing agent, emulsifier, stabilizer, thickner, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetner, preservative agent, antioxidant, etc, which had been well-known in the art.

If a substance is added to a food for a specific purpose in that food, it is referred to as a direct additive and indirect food additives are those that become part of the foodin trace amounts due to its packaging, storage or other handling.

Above described health foods can be contained in food, health beverage, dietary therapy etc, and may be used as a form of powder, granule, tablet, chewing tablet, capsule, beverage etc for preventing or improving aimed disease.

The health functional food composition for preventing and improving purposed diseases couldcontain about 0.01 to 95 w/w%, preferably 0.5 to 80 w/w% of the above inventivecomposition of present invention based on the total weight of the composition.

Above described composition therein can be added to food, additive or beverage for prevention and improvement of purposed diseases. For the purpose of preventing and improving purposed diseases, wherein, the amount of above described crude drug composition in food or beverage may generally range from about 0.1 to 15 w/w %, preferably 1 to 10 w/w % of total weight of food for the health food composition and 1 to 30 g, preferably 3 to 10 g on the ratio of 100㎖ of the health beverage composition.

Providing that the health beverage composition of present invention contains above described crude drug composition as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementionedones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100㎖ of present beverage composition.

The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improvingagent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition.

Examples of addable food comprising aforementioned crude drug composition therein are various food, beverage, gum, vitamin complex, health improving food and the like.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

According to one aspect, the present invention also provides a use of an extract of Phellodendron cortexfor the manufacture of medicament employed for treating or preventing pancreatitis in human or mammal.

The present invention also provides a method for treating pancreatitis in human or mammal comprising administering to said mammal an effective amount of above-mentioned extract, together with a pharmaceutically acceptable carrier thereof.

The present invention provides a pharmaceutical composition comprising an extract of Phellodendron cortex as an active ingredient for treating or preventing pancreatitis.

The present invention also provides a health functional food comprising the above-described extract for the prevention or improvement of pancreatitis as an active ingredient in an amount effective to preventing and improving pancreatitis, together with a sitologically acceptable additive.

The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

Fig. 1, Fig. 1a, Fig. 1b, Fig. 1c, shows the microscopic observation result on mouse pancreatitis tissue prepared from Experimental example 1and 2 A: normal; B: control treated with 0.5% CMC; C: treatment with methylprednisolone (20mg/kg): D: treatment with PA-C (200mg/kg); E: treatment with PA-P2 (200mg/kg), F: treatment with PA-CB (200mg/kg), G: treatment with Berberine (30mg/kg), H: treatment with Palmatine (30mg/kg).

The following Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1. Preparation of crude extract of Phellodendron cortex

100g of the cortex of phellodendron amurense purchased from Kyung-dong market located in Seoul were dried and crushed. The dried cortex was added to 30% ethanol and left alone at room temperature for 2 hours and the solution was filtered to afford filtrate. The filtrate was concentrated under vaccuo and freeze-dried to obtain 12.8g of crude extract of Phellodendron cortex (designated as PA-C hereinafter).

Example 2. Preparation of purified extract (a) of Phellodendron cortex

30g of the PA-C extract prepared in Example 1 was further purified by using Dianion chromatography. Specifically, 30g of the PA-C extract prepared in Example 1 was loaded on HP₂O filler (Mitsubishi Chemical Corp. DianionR HP20) running with the mixture solvent system starting from water to the mixture solution with water and ethanol with increasing the polarity to afford seven fractions, The fractions were concentrated under vaccuo and freeze-dried to obtain dried purified extract (a), i.e, 8.3g of 1^st fraction (designated as “PA-P0” hereinafter), 6.9g of 2^nd fraction (designated as “PA-P1” hereinafter), 11.4 g of 3^rd fractions (designated as “PA-P2”hereinafter), 6.5 g of 4-5^th fractions (designated as “PA-P3”hereinafter), and 2.7 g of 6-7^th fractions (designated as “PA-P4” hereinafter).

Example 3. Preparation of purified extract(b) of Phellodendron cortex

30g of the PA-C extract prepared in Example 1 was further purified by using solvent fractionation. Specifically, 30g of the PA-C extract prepared in Example 1 was dissolved in 500ml of distilled water. The solution was mixed with 2L of saturated butanol to fractionateand repeated three times to collect butanol-soluble layer. The butanol soluble layer was filtered with filter paper, and concentrated under vaccuo to obtain 23g of butanol soluble purified extract (designated as “PA-CB” hereinafter).

.

Example 4. Content Analysis

The component and content of extract prepared in Examples 1-3 were determined by using HPLC analysis with the condition as follows:

<HPLC condition>

1-1. Column: YMC ODS A (5micrometer, 250 x 4.6 mm)

1-2. Mobile phase: Mixture of ACN and H₂O (6:4) containing 20 micromole octane sulfonic acid sodium salt and 30 mM KH₂PO₄

1-3. Flow rate: 1ml/min.

1-4. Injection volume: 10 microliter

1-5. Column temperature: 40℃

and puried 30g of the PA-C extract prepared in Example 1 was further purified .

At the result, it has been confirmed that PA-P2 contain 6.3 %(w/w) berberine chloride, 5.4% (w/w) palmatine, and 0.65% (w/w) jateorrhizine,and PA-CB showed almost similar component spectra to PA-P2, i.e, 6.3 %(w/w) berberine chloride, 5.4% (w/w) palmatine, and 0.60% (w/w) jateorrhizine.

Reference Example. Preparation of Experiment

1-1. Reagent and experimental animals

7-weeks aged Male C57BL/6 mice (16 to 20g) were purchased from Orient Bio Co. (Seoul, Korea) and used in the experiment and were allowed to access to feed and drinking water ad libitum. All animals were maintained in a controlled environment with temperatures at 23±2℃ and humidity at 55±5% with 12 hours of light anddark cycles for at least one week prior to use.

The mice were divided into two groups, control group and test group consisting of 6 mice respectively. Normal group (G1) did not induce acute pancreatitis without any treatment and negative control group (G2 group) was treated with only 0.5% CMC solution. Positive control group (G4 group) was treated with conventionally available anti-inflammatory drug (methylprednisolone: KunHwa Pharm. Co. Ltd. Methylon Tab.) in dose of 20mg/kg orally once prior to the pancreatitis induction. G4 group was treated with PA-C at the dose of 100-100mg/kg and G5 groups were treated with PA-P0, P1, P2 and P3 prepared in Examples at the dose of 100-400mg/kg orally. Groups G2-G5 were treated once a day prior to pancreatitis induction for 6 days.

1-2. Induction of acute pancreatitis

All the mice have been starved for 12 hours until acute pancreatitis induced. 50 microgram/kg of Cerulean(Sigma-Aldrich Co.) water solution was intraperitoneally administrated into G2-G5 mice 6 times at every 1 hour for 6 hours according to the literature (Jung WS et al., World J. Gastroenterol., 14, pp6188-6194, 2008)to induce acute pancreatitis. 6 hours after the induction, the mice were died by suffocation with CO₂ gas and pancreas tissue and lung tissue were collected. Blood was collected by cardiac puncture and centrifuged to isolate serum. The collected serum was kept on -70℃ to use in following experiment.

Experimental Example 1. Reducing effect on serum amylase and lipase

In order to investigate the inhibitory effect of the inventive extract obtained in Examples on the serum level of amylase and lipase, following experiment was performed according to the procedure disclosed in the literature (Jung WS et al., World J. Gastroenterol., 14, pp6188-6194, 2008).

The level of amylase and lipase in mouse serum isolated from pancreatitis-induced mice were determined using by ADIVA 1650 (Bayer, USA) for serumamylase determination and Cobas-mira (Roche, USA) for blood lipase determination.

At the result, the level of amylase and lipase in the group treated with PA-P2 and PA-P3 (each 200mg/kg) was significantly reduced compared with that in negative control group treated with 0.5% CMC (p<0.01) as can be seen in Table 1.

Accordingly, it has confirmed that the PA-P2 and PA-P3 reduced the level of amylase and lipase significantly.

Table 1

Group	Oral Dose	Serum amylase (U/L)	Serum lipase (U/L)
Normal		1000.4±13.5	70.3±5.1
0.5%CMC		3221.3±67.4	358.2±54.5
Methylprednisolone	20 mg/kg	2489.5±309.9***	180.8±37.1**
PA-C	200 mg/kg	3128.3±139.1	252.1±11.1*
PA-P1	200 mg/kg	3115.7±255.2	302.4±86.8
PA-P2	200 mg/kg	2779.8±258**	224.5±33.7**
PA-P3	200 mg/kg	2979.6±219.8	270.2±58.0
PA-P4	200 mg/kg	2793.6±126.7***	225.5±10.8**
P<0.05:*, p<0.01:**, p<0.001*** (N= 6 mice/group)

Experimental Example 2. Determination of inflammatory cytokines after the induction of pancreatitis

In order to investigate the reducing effect of the inventive extract obtained in Examples on the level of inflammatory cytokines such as TNF-alpha, IL-1beta and IL-6 after the induction of pancreatitis, following experiment was performed according to the procedure disclosed in the literature (Exley AR et al., Gut. 33, pp1126-1128, 1992).

The level of TNF-alpha and IL-1beta (R&D systems, USA) was determined by using ELISA method according to the protocol (R&D systems), i.e., the plate placed with primary antibody against TNF-alpha and IL-1beta was added with serum sample and standard substance and left alone at 37℃ for 2 hours. After washing the wells, enzyme treated antibodies were added to the well and left alone at 37℃ for 2 hours. The well was washed again and substrate was added thereto. The degree of colorization was determined by auto-microplate ELISA analyzer at 405nm. Standard curve was prepared by diluting recombinantTNF-alpha and IL-1beta on each plate serially.

At the result, the test groups treated with inventive extract reduced the levelof TNF-alpha and IL-1beta in a dose dependant manner (See Table 2).

Table 2

Group	Oral dose (mg/kg)	Reproduced level of cytokines (pg/ml)
		IL-1beta	TNF-alpha
Normal		1.0±0.2	3.0±0.5
0.5%CMC		6.6±2.1	44.3±3.5
Methylprednisolone	20	4.92±1.0	17.3±8.4*
PA-P2	100	3.1±1.0*	26.2±7.8
	200	2.4±0.6**	18.7±4.9**
	400	2.2±0.8**	12.9±4.9**
P<0.05:*, p<0.01:**, p<0.001*** (N= 6 mice/group)

Experimental Example 3. Reducing effect of inventive extract on the serum level of amylase and lipase after the induction of pancreatitis

In order to investigate the reducing effect of the inventive extract obtained in Examples on the serum level of amylase and lipase after the induction of pancreatitis, following experiment was performed according to the procedure disclosed in the literature(Jung WS et al., World J. Gastroenterol., 14, pp6188-6194, 2008).

The level of amylase and lipase in mouse serum isolated from pancreatitis-induced mice were determined using by ADIVA 1650 (Bayer, USA) for serum amylase determination and Cobas-mira (Roche, USA) for blood lipase determination.

The normal group was treated with none and negative control group was treated with 0.5% CMC solution. The positive control group was treated with methylprednisolone (20mg/kg) and the test groups were orally administratedwith 100mg/kg, 200mg/kg and 400mg/kg of PA-CB. Groups G2-G5 were treated once a day prior to pancreatitis induction for 6 days.

At the result, the test group treated with various concentrations of PA-CB showed potent reducing effect on the level of amylase and lipase in a dose dependant manner (p<0.001). Especially, the group treated with 400mg/kg of PA-CB showed more potent reducing effect on the level of amylase and lipase than positive control group. Moreover, In respect to the ratio of the weight of pancreas compared with that of body weight, the negative control group showed 6.87 while normal group showed 4.38 and the test group treated with inventive extract showed about 5.0 as can be seen in Table 3.

Table 3

Group	Oral dose(mg/kg)	Ratio of weight(pancreas/body )	Serum amylase(U/L)	Serum lipase(U/L)
Normal		4.38±0.16	956.7±64.0	32.2±2.7
0.5%CMC		6.87±1.04	4642.2±151.8	468.0±82.4
Methylprednisolone	20	5.05±0.43**	3113.7±225.4***	290.5±38.5***
PA-CB	100	5.30±0.34**	3233.9±47.0***	347.4±49.5***
	200	5.26±0.29*	3192.7±112.9***	321.8±63.2***
	400	5.11±0.42**	2841.0±157.1***	245.5±30.2***
P<0.05:*, p<0.01:**, p<0.001*** (N= 6 mice/group)

Experimental Example 4. Reducing effect of active ingredients on the blood level of amylase and lipase after the induction of pancreatitis

In order to investigate the reducing effect of the active ingredients of the inventive extract obtained in Examples on the blood level of amylase and lipase after the induction of pancreatitis, following experiment was performed according to the procedure disclosed in the literature (Jung WS et al., World J. Gastroenterol., 14, pp6188-6194, 2008).

The level of amylase and lipase in mouse serumisolated from pancreatitis-induced mice were determined using by ADIVA 1650 (Bayer, USA) for serum amylase determination and Cobas-mira (Roche, USA) for serum lipase determination.

The normal group was treated with none and negative control group was treated with 0.5% CMC solution. The positive control group was treated with methylprednisolone (20mg/kg) and the test groups consisting of test group G4 (PA-P2, 200mg/kg), G5 (berberine chloride cat., No. B0450, TCI Co. Ltd., 30mg/kg) and G6 (palmatine chloride, Cat. No. ASB00016049, Chromax Co., 30mg/kg) were orally administrated respectively. Groups G2-G5 were treated once a day prior to pancreatitis induction for 6 days.

At the result, the test groups treated with test group G4 (PA-P2, 200mg/kg), G5 (berberine chloride cat., No. B0450, TCI Co. Ltd., 30mg/kg) and G6 (palmatine chloride, Cat. No. ASB00016049, Chromax Co., 30mg/kg) reduced the level of amylase and lipase by 43%, and more than 45% respectively, which is almost equivalent effect to positive control group treated with methylprednisolone as can be seen in Table 4.

Table 4

Group	Oral dose(mg/kg)	Serum amylase(U/L)	Serum lipase(U/L)
Normal		787.3±68.0	31.6±3.3
0.5%CMC		3240.9±61.0	350.9±64.2
Methylprednisolone	20	2460.2±303.0**	188.6±55.5**
PA-P2	200	2638.8±236.8**	215.9±29.9*
Berberine	30	2507.2±299.9**	186.1±24.7**
palmatine	30	2417.6±127.4***	189.6±24.9**
P<0.05:*, p<0.01:**, p<0.001*** (N= 6 mice/group)

Experimental Example 5. Pathological examination of pancreas

In order to investigate the treating effect of the inventive extract obtained in Examples on thepancreatic tissue, following experiment was performed according to the procedure disclosed in the literature (Ethridge RT et al., Gastroenterol. 123, pp1311-22, 2002).

The pancreatic tissue induced acute pancreatitis prepared in Reference Example was fixed with 10% formalin solution and dipped into paraffin. The tissue was cut into 4 micrometer, stained with hematoxylin-Eosin (H&E) and read by pathologist (blind test). The degree of cell injury was divided into six scores according to degree of (1) edema, (2) inflammation (3) vacuolation (4) necrosis with the criteria of pancreas acinar cell deletion, interstitial edema, inflamed cell infiltration, peripancreatic fibrosis etc, i.e., 0 (normal), 2 (minimal), 2 (slight), 3 (moderate), 4 (marked) and 5 (severe).

All the scored result of histo-pathological speculum was expressed as mean and standard deviation and analyzed by using Kruskal-Wallis test and Mann Whitney test (nonparametric statistics) and the significance was expresses as p value.

At the result, the test group treated with the inventive extractshowed more potent treating effect on the injury of pancreas in respect to inflammation and edema than positive control group treated with methylprednisolone (p<0.01). In comparison with the total score of acute pancreatitis induction degree, the test group treated with PA-C, PA-P2 and PA-CB showed 7.04 score, 5.75 score and 5.67 score, respectively, while the negative control group treated with 0.5% CMC and positive control group treated with methylprednisolone showed about 9.33 score and 5.30 score respectively. The test group treated with berberin and palmatine as active compounds showed 6.67 score and 5.58 score

Accordingly, it has confirmed that the inventive extract showed potent treating activity of acute pancreatitis induced mouse (See Table 5)

Table 5

Group	Edema	Inflammation	Vacuolation	Necrosis	Total
Normal	0.17±0.45***	0.33±0.55***	0.33±0.55**	0.0±0.0***	0.83±0.71***
0.5%CMC	3.17± 1.41	2.50 ± 1.01	1.67 ±0.76	2.17±1.03	9.33±4.05
Methylprednisolone(20mg/kg)	1.90±0.22*	1.20±0.27*	1.40±0.41	0.80±0.44*	5.30± 0.27*
PA-C (200mg/kg)	2.29±0.21*	1.80±0.21*	1.65±0.16	1.21±0.18	7.04±0.57*
PA-P2 (200mg/kg)	2.00±0.78	1.17±0.34*	1.42±0.50	1.00±0.33	5.75 ± 1.99
PA-CB (200mg/kg)	1.83±0.87*	1.33±0.62*	1.33±0.51	1.00±0.72	5.67 ± 2.31*
Berberine 30mg/kg)	2.17±0.89	1.50±0.68	1.50±0.68	1.50±0.97	6.67 ± 2.60
Palmaine(30mg/kg)	2.00±0.66	1.25±0.58*	1.25±0.58	0.92±0.99	5.58 ± 2.50*
P<0.05:*, p<0.01:**, p<0.001*** (N= 6 mice/group)

As can be seen in Fig 1, Fig 1a, Fig 1b, Fig 1c, showing the photographs of pancreas, normal group treated with only saline solution (A) showed healthy figure and regular morphologyand interval of acinar cells whereas the negative control group showed severe edema together with irregular morphology and distant interval treated of acinar cells. All the groups treated with methylprednisolone(C), PA-P2(D), PA-CB(E), berberine(F) and palmatine(G) showed regular morphology and interval of acinar cells, of which result confirmed that the test groups treated with PA-P2 and PA-CB at the dose of 200mg/kg for 6 days showed potent improving effect on the injury of pancreatitis. In addition, berberine and palmatine,as active compounds at the dose of 30 mg/kg was showed potent improving effect. Especially, palmatine represented higher therapeutic efficacy that of berberine on the cerulein induced acute pancreatitis.

Experimental Example 6. Acute toxicity test of oral administration in rat

In order to investigate the acute toxicity of the active ingredients of the inventive extractobtained in Examples, following experiment was performed according to the procedure disclosed in the literature (Greaves P. Histopathology of preclinical toxicity studies: Interpretation and relevance in drug safety evaluation Elsevier, 2007).

The acute toxicity test was performed by administrating inventive extract to S.D rats according to Up & Down method.

2000 mg/kg of inventive extracts (PA-P2 and PA-CB, respectively) were orally administrated to each group consisting of 3 rats and the symptoms of rats were observed for 7 days. After administrating the two extracts, all the clinical changes i.e., mortality, clinical signs, body weight changes was observed and blood test such as haematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

There did not show any changes in mortality, clinical signs, body weight changes and gross findings in any group or either gender. Furthermore, there showed any toxicity in test group treated with 2000mg/kg of PA-P2 and PA-C, respectively .

Accordingly, it has been confirmed that the inventive extract prepared in the present invention was potent and safe substance showing LD₅₀ (more than 2000 mg/kg) in oral administrationof PA-P2 and PA.

Experimental Example 7. Repeated Dose 4-week oral-treatment for DRF toxicity test in rat

In order to investigate the repeated oral toxicity of the active ingredients of the inventive extractobtained in Examples, following experiment was performed according to the procedure disclosed in the literature (Jung WC et al., Lab. Anim. Res., 25, pp355-362, 2009).

The repeated oral toxicity test was performed by administrating inventive extract to S.D rats once a day for 28 days to determine systemic toxicity.

250 mg/kg, 500 mg/kg and 1000 mg/kg of inventive extracts (PA-P2 and PA-CB, respectively) were orally administrated to each group consisting of 3 rats once a day and the symptoms of rats were observed for 28days. After administrating the extract, all the clinical changes i.e., mortality, clinical signs, body weight changes was observed and blood test such as haematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

There did not show any changes in mortality, clinical signs, body weight changes and gross findings in any group or either gender. Furthermore, there showed any toxicity in test group treated with 2000mg/kg of PA-P2 and PA-C, respectively.

Accordingly, it has been confirmed that the inventive extract prepared in the present invention was potent and safe substance showing LD₅₀ (more than 2000 mg/kg) in oral administration of PA-P2 and PA.

Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

Preparation of powder

PA-C : 25mg

Corn Starch : 20mg

Lactose : 30mg

Mg stearate : optimum amount

Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of tablet

PA-P2 : 100mg

Corn Starch : 10mg

Lactose : 50mg

Magnesium stearate : optimum amount

Tablet preparation was prepared by mixing above components and entabletting.

Preparation of capsule

PA-CB : 10mg

Crystalline cellulose : 3mg

Lactose : 14.8mg

Magnesium stearate : 0.2 mg

Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of injection

PA-C : 10mg

Mannitol : 180mg

Na₂HPO₄-12H₂O : 26mg

Distilled water for injection : 1974mg

Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2㎖ ample and sterilizing by conventional injection preparation method.

Preparation of liquid

PA-P2 : 20mg

Sugar : 20g

Mannitol : 5g

Distilled water : optimum amount

Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000㎖ ample and sterilizing by conventional liquid preparation method.

Preparation of health food

PA-CB : 1000mg

Vitamin mixture : optimum amount

Vitamin A acetate : 70μg

Vitamin E : 1.0mg

Vitamin B_{1 :} 0.13mg

Vitamin B₂ : 0.15mg

Vitamin B_{6 :} 0.5mg

Vitamin B_{12 :} 0.2μg

Vitamin C : 10mg

Biotin : 10μg

Amide nicotinic acid : 1.7mg

Folic acid : 50μg

Calcium pantothenic acid : 0.5mg

Mineral mixture : optimum amount

Ferrous sulfate : 1.75mg

Zinc oxide : 0.82mg

Magnesium carbonate : 25.3mg

Monopotassium phosphate : 15mg

Dicalcium phosphate : 55mg

Potassium citrate : 90mg

Calcium carbonate : 100mg

Magnesium chloride : 24.8mg

The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

Preparation of health beverage

PA-C : 1000mg

Vitamin C : 15g

Vitamin E(powder) : 100g

Vitamin A : 0.2g

Vitamin B₁ : 0.25g

Vitamin B₂ : 0.3g

Amide nicotinic acid : 3.5g

Zinc oxide : 3.5g

Ferrous lactate : 19.75g

Distilled water : optimum amount

Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 ℃ for 1 hour, filtered and then filling all the components in 1000㎖ ample and sterilizing by conventional health beverage preparation method.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

As described in the present invention, the inventive extract significantly reduced the blood level of amylase, lipase, the inflammatory cytokines and improved the morphological change of pancreatic tissue of C57B mouse induced by pancreatitis when the inventive extract of the present invention was orally administrated thereto. When the oral acute toxicity of the extract was tested, the extract had no apparent effect on mortality, clinical signs, body weight changes, and gross findings at necropsy. The inventive compositions according to the present invention are useful in the prevention and treatment of pancreatitis.

Claims (11)

1. A pharmaceutical composition comprising an extract of Phellodendron cortex as an active ingredient for treating or preventing pancreatitis, together with a pharmaceutically acceptable carrier.
2. The pharmaceutical composition according to claim 1, wherein said Phellodendron cortex is an extract of Phellodendron amurense Rupr, Phellodendron moll NAKAI, Phellodendron sachalinense SAKGENT or Phellodendron insulare NAKAI.
3. The pharmaceutical composition according to claim 2, wherein said extract is a crude extract or purified extract.
4. The pharmaceutical composition according to claim 3, wherein said extract is a crude extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, or the mixtures thereof.
5. The pharmaceutical composition according to claim 3, wherein said purified extract is a purified extract (a) prepared by the procedure comprising the steps: subjecting the crude extract to ion column chromatography using by Dianion, SP207, HP20SS or HP20 filler, running with solvent system increasing the polarity started from water to mixture solvent of water and ethanol to fractionate into seven fractions; collecting, concentrating each fraction under vaccuo and freeze-drying each fraction to afford purposed purified extract (a), which includes 1^st fraction ( "PA-P0"), 2^nd fraction ( "PA-P1"), 3-5^th fractions ( "PA-P2"), 6-7^th fractions ( "PA-P3"), preferably, PA-P1 extract comprising 3-10 (w/w)% berberine chloride, 3-10 (w/w)% palmatine chloride, and 0.01-5 (w/w)% jateorrhizine based on the total weight of the extract or a purified extract (b) prepared by the procedure comprising the steps: subjecting the crude extract to fractionate with 10-100 fold volume (v/w) of saturated butanol to afford butanol soluble fraction at the 1^st step; concentrating and drying the fraction to afford butanol-soluble fraction ("PA-CB") comprising 3-10 (w/w)% berberine chloride, 3-10 (w/w)% palmatine chloride, and 0.01-5 (w/w)% jateorrhizine based on the total weight of the extract.
6. The pharmaceutical composition according to claim 1, wherein said pancreatitis is an acute pancreatitis or chronic pancreatitis.
7. A use of an extract of Phellodendron cortex for the manufacture of medicament employed for treating or preventing pancreatitis in human or mammal.
8. A method for treating pancreatitis in human or mammal comprising administering to said mammal an effective amount of an extract of Phellodendron cortex, together with a pharmaceutically acceptable carrier thereof.
9. A health functional food comprising an extract of Phellodendron cortex for the prevention or improvement of pancreatitis as an active ingredient in an amount effective to preventing and improving pancreatitis, together with a sitologically acceptable additive.
10. The health care food of claim 9, wherein said health care food is provided as powder, granule, tablet, capsule or beverage type.
11. A method for preparing the extract of Phellodendron cortex comprising the steps consisting of; extracting dried cortex of Phellodendrin cortex with 5 to 20-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof at the temperature ranging from 10℃ to 110℃ for the period ranging from 0.5 hrs to 48 hours to obtain crude extract at the 1^st step ("PA-C") subjecting the crude extract to ion column chromatography using by Dianion, SP207, HP20SS or HP20 filler running with solvent system increasing the polarity started from water to mixture solvent of water and ethanol to fractionate into seven fractions at the 2^ndstep; concentrating under vaccuo and freeze-drying each fraction to afford purposed purified extract selected from 1^st fraction (s "PA-P0"), 2^nd fraction ("PA-P1"), 3-5 fractions ("PA-P2"), and 6-7 fractions ("PA-P3") alternatively, subjecting the crude extract to fractionate with 10-100 fold volume (v/w) of saturated butanol to afford butanol soluble fraction at the 2^ndstep; concentrating and drying the fraction to afford butanol-soluble fraction ( "PA-CB" ).

Images