KR20100136978A - Extract from oil palm leaves comprising phenolic acids - Google Patents

Abstract

(-) A composition comprising an extract derived from palm oil leaves, characterized by comprising (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid, and (a) extracting dried or fresh palm oil leaves with a solvent (B) filtering the extract obtained in step (a), (c) filtering the extract obtained in step (b) with only a fraction comprising (-)-catechin gallate, ferulic acid, and phenolic acid. Contacting with a selectively adsorbing chromatography medium, (d) eluting the fraction from the chromatography medium using a solvent, (e) drying the eluted fraction obtained in step (d), It relates to a method for preparing the extract. There is also claimed the use of a composition for reducing or preventing oxidative stress in mammals and poultry.

Description

Extract derived from palm oil leaf containing phenolic acid {EXTRACT FROM OIL PALM LEAVES COMPRISING PHENOLIC ACIDS}

The present invention relates to palm oil leaf extracts having therapeutic activity to alleviate or prevent oxidative stress in humans and lower animals. In particular, the present invention provides enhanced (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatechuic acid having the therapeutic activity ( enriched).

Palm oil ( Elaeis ) includes two types of palm trees (Arecaceae or palm family). African palm oil (Elysian Guinea-ensis: Elaeis guineensis ) is native to West Africa, while American palm oil (Elysium oleifera: Elaeis) oleifera ) is native to Central and South America. Elysium Guinesis is also widely cultivated in Malaysia and Indonesia for oil producing fruit.

Mature palm oil is a single stem plant, growing up to 20 m high. Leaves or fronds are pinnate and can grow up to 3-5 m long. Driftwood grows about 30 leaves a year. Flowers consist of clusters; Each individual flower has three calyx and three petals.

The fruits are red, growing into large clusters where each cluster can weigh from 10 to 40 kg. The fruit comprises a fleshy outer layer of oily skin called fruit peel, with a single seed called the palm kernel. Oil is extracted from the rind and palm kernel.

Palm oil is cultivated primarily to obtain fruits that are mostly used for the production of edible oil. Palm oil extracted from palm oil fruits includes carotene, tocopherol and tocotrienols. Several studies have been conducted regarding the useful effects of extracts derived from palm oil leaves.

Abeywardena M3 et al. (Asia Pac. J. Clin. Nutr., 11: S467-S 472) disclose polyphenol-enriched extracts derived from the leaves of Elaeis guineensis . The extract can be used to promote vascular relaxation through endothelial dependent mechanisms. The presence of (-)-catechin gallate, ferulic acid, gallic acid, and protocatecuic acid has never been studied or disclosed. The use of this extract as an anti-oxidative stressor has not been studied or disclosed.

Suhaila Mohamed et al. (WHAT Medicine III Proceedings, 2007, PP 145-148) describe polyphenol-enriched palm oil leaves ( Elaeis). guineensis ) extracts disclose anti-hypercholesterol and anti-hypertensive effects. However, the presence of (-)-catechin gallate, ferulic acid, gallic acid, and protocatecuic acid has never been studied or disclosed. The use of this extract as an anti-oxidative stressor has not been studied or disclosed.

Nagendran Balasundram et al. (Asia Pac. J. Clin. Nutr. 2005; 4 (4); 319-324) disclose fractions enriched in phenol isolated from palm oil fruits. This extract exhibits biological activity, especially antioxidant effects. However, chemical compositions of extracts derived from palm oil leaves have not been studied or disclosed. In addition, the use of extracts derived from palm oil leaves as anti-oxidative stress substances has not been studied or disclosed.

Tan Y. A. et al. (The Royal Society of Chemistry 2001; 548-551) describe the presence of phenolic compounds in palm oil hulls derived from palm oil fruit and in palm oil derived from the nucleus. Such phenolic compounds include gallic acid, chlorogenic acid, protocatecuic acid, gentisic acid, coumaric acid, ferulic acid and caffeic acid, as well as catechins, hesperidine and narirutin. ), And 4-hydroxybenzoate. However, chemical compositions of extracts derived from palm oil leaves have not been studied or disclosed. In addition, the use of extracts derived from palm oil leaves as anti-oxidative stress substances has not been studied or disclosed.

Run-Cang Sun et al. (J. Agric. Food Chem., 49 (11), 5122-5129, 2001) disclose a novel method for quantitative analysis of hydroxycmnamic acid in palm oil leaf fibers. However, the presence of (-)-catechin gallate has not been studied or disclosed. The use of this extract as an anti-oxidative stressor has not been studied or disclosed.

Yew-Ai Tan et al. (European Journal of Lipid Sciences and Technology, Volume 109, Issue 4, pages 380-393, 2007) describe sterols, vitamin E, carotenoids, phosphates derived from by-products of palm oil grinding and purification obtained from palm oil fruits. The presence of phytochemicals such as lipids, squalene and phenols is disclosed. However, chemical compositions of extracts derived from palm oil leaves have not been studied or disclosed. In addition, the use of extracts derived from palm oil leaves as anti-oxidative stress substances has not been studied or disclosed.

Kinnoudo Celestin (WO2007129136) discloses an extract of palm oil ( Elaeis guineensis ) with antimalarial properties. The chemical composition of this extract has not been characterized. The presence of (-)-catechin gallate, ferulic acid, gallic acid, and protocatecuic acid in extracts derived from palm oil leaves has not been studied or disclosed. The use of these extracts as anti-oxidative stress substances has also not been studied or disclosed.

Oxidative stress

Over the years, free and bound active radicals, which are highly active and destructive molecules, have been evaluated as being of great importance for human health and disease. Most life-threatening human diseases such as atherosclerosis, cancer and aging have radical-based pathological responses as potential mechanisms of injury.

Radicals, or free radicals, are generally understood to be molecules having one or more single electrons in the outer orbital. Many molecular species with fixed radicals are other oxygen containing compounds or monooxides, commonly called active oxygen species (ROS). Such highly labile molecules tend to react rapidly with adjacent molecules, giving, taking away or sharing their outer orbital electron (s). Such reactions sometimes not only alter adjacent target molecules in an extreme and useful manner, but can also damage target molecules, or single electrons cross the target, ie, produce unwanted secondary ROS as free radicals, May react favorably or disadvantageously. Indeed, the high reactivity of ROS is due to the molecular chain reaction, which effectively multiplies the effect.

All living aerobic organisms use oxygen for energy production. However, the benefits of using oxygen indicate that it is associated with the risk that the oxidation process may damage aerobic organisms. Various free radical metabolites participate in the oxidation process and simultaneously attack various classes of biological substances. Almost all compounds are subject to oxidative damage by the oxygen compounds. For example, nucleic acids, proteins and free amino acids, lipids and hydrocarbon compounds are affected by oxygen toxicity. Humans have the most sophisticated and extensive, very important system for balancing oxidative and antioxidant processes (see H. Sies, Oxidative Stress, pages. 2-4, 1985). Naturally present antioxidant pools, however, cannot inhibit the increased production of reactive oxygen species (ROS); So-called "oxidative stress" occurs at this time.

There are two reasons for the change in balance for the oxidation process:

i. Overloading the antioxidant system due to the production of free radical metabolites, and / or

ii. Insufficient antioxidant system.

The results are summarized in the expression "oxidative stress". Today, the balance changes include various distinct diseases, especially inflammatory diseases such as hepatitis, central nervous system diseases such as epilepsy or Parkinson's disease, lung diseases such as asthma, psoriasis, side effects of anticancer drugs, chemicals such as paraquat, and radiation side effects. Rather, it is known to be caused by coronary circulatory diseases such as myocardial infarction.

While not intending to be bound by theory, oxidative stress has been implicated as a factor in a variety of diseases and conditions of injury in humans and animals, but this does not mean that oxidative stress is the cause of these diseases and many studies have been conducted. As confirmed throughout, it has been demonstrated that oxidative stress can negatively affect the progression of the disease, causing further damage to cells of living organisms.

Notable scientific evidence has shown that various dysfunctions and diseases in humans and animals are associated with oxidative stress.

Aging: Normal aging progresses faster than normal.

Heart and Cardiovascular Diseases: Atherosclerosis, Adriamycin Cardiotoxicity.

Kidney: autoimmune nephritis syndrome, heavy metal nephrotoxicity.

Solar radiation: skin wrinkles and pigmentation.

Eyes: Cataracts, degenerative retinal damage, macular degeneration.

Lungs: lung cancer (tobacco smoke), emphysema, oxidative pollutants (O ₃ , NO ₂ ), bronchopulmonary dysphasia, asbestos carcinogenicity.

Nervous system diseases: Parkinson's syndrome, Neuronal ceroid lipofuscinoses, Alzheimer's disease, muscular atrophy, multiple sclerosis.

Iron excess: Idiopathic hemochromatosis, Dietary overload, Thalassemia.

Inflammation-Immune Injury: Glomerulonephritis, Autoimmune Disease, Rheumatoid Arthritis.

Liver: Liver damage induced by alcohols, halogenated hydrocarbons, and paracetamol.

Antioxidants may have protective effects because they inhibit the spread of oxidative damage by scavenging ROS and free radicals before they damage various biological molecules, or by disrupting the radical chain reaction of lipid peroxidation. have. The human load causing the reactivity and final pathological condition of radicals in the human body is known as oxidative stress.

The efficacy of antioxidants (radical scavengers), which are believed to possess the ability to remove oxidative stress for the disease, has been studied for a long time, and anti-inflammatory agents that exhibit radical scavenging ability have been developed. Therefore, it can be seen that an antioxidant substance is useful for reducing oxidative stress.

Vitamins such as vitamin C and vitamin E, found in foods and available as supplements, help the body reduce the effects of oxidative stress. But more powerful fighters against free radicals and ROS are human self-defense systems of natural chemicals called antioxidants. This antioxidant acts to terminate the buildup of ROS or free and fixed radicals by imparting electrons to the free radicals or ROS or hindering their formation. Human antioxidant defense systems include three important natural antioxidants: superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Studies have shown that the antioxidants act synergistically because the reactions catalyzed by the antioxidants are initially initiated by SOD and are metabolically and sequentially in the form of reactions following CAT and GPX. .

However, despite the presence of SOD, CAT, and GPX, the human body's antioxidant defense system is constantly subject to oxidative stress, and the ability to produce SOD, CAT, and GPX is reduced as aging progresses, inflammation, bacteria Or is impaired by viral infection, and the progression of cancer and nervous system disorders and other pathological symptoms are caused or worsened by oxidative stress.

Recently, there has been increasing interest in natural antioxidant research for three main reasons (Dastmalchi, Dorman, Ko§ar, & Hiltunen, 2007):

(i) Many clinical and epidemiological studies have demonstrated that consumption of fruits and vegetables is associated with lowering the risk of progression of chronic diseases such as cancer, cardiovascular disease and diabetes.

(ii) consideration of the stability associated with the chronic consumption of synthetic antioxidants in food and beverages may have a potentially harmful effect; And

(iii) Public awareness that natural and dietary antioxidants are safer than synthetic analogs. As a result, interest in medicinal plants as a source of natural antioxidants has increased.

Chemical compounds from palm oil leaf extracts that result in antioxidant and anti-oxidative stress activity have never been characterized.

The present invention is to provide a novel palm oil leaf extract useful for reducing oxidative stress in mammals and poultry. The extracts fortified with (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid have anti-oxidative stress activity and have (-)-catechin gallate, ferulic acid, And synergistic effects of phenolic acids such as gallic acid and protocatecuic acid have not been reported previously.

In summary, extract fractions derived from polyphenol-enriched palm oil leaves are disclosed, which fractions can be used as anti-hypercholesterol and anti-hypertensive substances, but include (-)-catechin gallate, ferulic acid, and gallic acid and Fractions derived from palm oil leaves containing phenolic acids such as protocatecuic acid have not been studied or disclosed. The use of these extracts as anti-oxidative stress substances has not been studied or disclosed.

Accordingly, it is a primary object of the present invention to provide a composition derived from palm oil leaves having therapeutic activity in mammals and poultry.

Another object of the present invention is to provide a composition comprising (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid.

Still another object of the present invention is to provide a method for preparing an extract derived from palm oil leaves.

Another object of the present invention is to provide a pharmaceutical composition comprising the novel extract and at least one pharmaceutically acceptable carrier.

Another object of the present invention is to provide a pharmaceutical composition comprising a novel extract, stabilizer, carrier, excipient, extender, and other suitable substances.

A further object of the present invention is to provide a composition comprising a novel extract, mixed with and associated with a pharmaceutical carrier, for use in the course and prevention of oxidative stress in mammals and poultry.

The above and other objects of the present invention include (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid, wherein the composition comprising an extract derived from palm oil leaves and Using the steps, a method for preparing palm oil leaf extracts comprising (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid is achieved:

a) dried or fresh palm oil leaves by mixing with water, ethanol, methanol, acetone, ethyl acetate, chloroform, isopropyl alcohol or mixtures of these or other polar solvents and heating at 25 ° C. to 95 ° C. for 0.5 to 96 hours Extracting the herbal liquid from the;

b) filtering the herbal liquid extract obtained in step (a);

c) a column adsorption medium for selectively adsorbing the filtered herbal liquid extract obtained in step (b) to only fractions in which (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid are present; or Contacting with any other adsorption medium, an adsorption chromatography medium;

d) eluting the fraction obtained in step (c) from the adsorption chromatography medium using water, ethanol, methanol, acetone, ethyl acetate, chloroform, isopropyl alcohol or a mixture of the above solvents or other polar solvents; And

e) drying the fortified herbal liquid extract obtained in step (d) using a spray dryer, vacuum oven, conventional oven, microwave oven or freeze dryer.

Another object of the present invention is the use of the composition to reduce or prevent oxidative stress in mammals and poultry comprising administering an effective amount of the composition to a subject in need thereof.

The present invention relates to a composition comprising an extract derived from palm oil leaves, which comprises (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid.

The palm oil leaf of the present invention is Elaeis ( Elaeis) guineensis ) or Elaeis oleifera ).

The novel compositions of the present invention comprise about 0.1% to about 95% by weight of (-)-catechin gallate, about 0.1% to about 95% by weight of ferulic acid, and about 0.1% to about 95% by weight of gallic acid and protocatecu Total phenolic acids such as acids.

In a preferred embodiment of the invention, the composition comprises about 0.5% to about 10% by weight of (-)-catechin gallate, about 1% to about 5% by weight of ferulic acid, and about 5% to about 30% by weight of gallic acid And total phenolic acids such as protocatecuic acid.

In a most preferred embodiment of the invention, the composition comprises about 0.5% by weight of (-)-catechin gallate, about 1% by weight ferulic acid, and about 20% by weight of gallic acid and total phenolic acid such as protocatecuic acid do.

The present invention relates to a method for preparing the palm oil leaf extract, comprising the following steps:

a) dried or fresh palm oil leaves by mixing with water, ethanol, methanol, acetone, ethyl acetate, chloroform, isopropyl alcohol or mixtures of these or other polar solvents and heating at 25 ° C. to 95 ° C. for 0.5 to 96 hours Extracting the herbal liquid from the;

b) filtering the herbal liquid extract obtained in step (a);

c) a column adsorption medium for selectively adsorbing the filtered herbal liquid extract obtained in step (b) to only fractions in which (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid are present; or Contacting with any other adsorption medium, an adsorption chromatography medium;

d) The fraction obtained in step (c) is subjected to a temperature of about 10 ° C. to about 80 ° C., using water, ethanol, methanol, acetone, ethyl acetate, chloroform, isopropyl alcohol or a mixture of these solvents or other polar solvents. Eluting from the adsorption chromatography medium at a pressure of from about 0.1 bar to about 10 bar to obtain an extract enriched in (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid; And

e) drying the fortified herbal liquid extract obtained in step (d) using a spray dryer, vacuum oven, conventional oven, freeze dryer or any other dryer.

In a preferred embodiment of the invention, one part of palm oil leaf (dry basis) is extracted with about 5 to about 50 parts, preferably about 10 parts to about 30 parts of solvent using an extraction apparatus, wherein the solvent Is preferably mixed with palm oil leaves for about 1 hour to 96 hours, more preferably 8 hours to 24 hours. The temperature of the solvent is preferably maintained at about 25 ° C to about 95 ° C, more preferably about 50 ° C to 60 ° C.

As a further embodiment of the invention, preferred solvents for extraction are water, ethanol, isopropyl alcohol, or mixtures of said solvents, more preferably water, ethanol, or mixtures of water and ethanol, most preferably 30 A mixture of wt% water and 70 wt% ethanol.

As a preferred embodiment of the present invention, the adsorption chromatography medium comprises styrene-divinylbenzene copolymer resins, phenol-formaldehyde resins, acrylic resins, methacrylate resins, and polyamide resins. Examples of the resin include Amberlite XAD-1, Amberlite XAD-2, Amberlite XAD-4, Amberlite XAD-7, Amberlite XAD-8, Amberlite XAD-11, Amberlite XAD-12 Amberlite XAD-1180, Amberlite 7HP, Amberlite 761, and Duoolite S861 (manufactured by Rohm & Haas Co., Philadelphia, USA); Diaion HP-10, Diaion HP-20, Diaion HP-21, Diaion HP-30, Diaion HP-40, and Diaion HP-50 (manufactured by Mitsubishi Chemical Industries Ltd., Tokyo, Japan) ; And AB-8 crosslinked polystyrene and H103 crosslinked polystyrene resin (manufactured by Tianjin Nankai Hecheng Science and Technology Co., Ltd., China).

In a more preferred embodiment of the invention, the resin is AB-8 crosslinked polystyrene (manufactured by Tianjin Nankai Hecheng Science and Technology Co., Ltd., Tianjin, China), Amberlite XAD 1180, Amberlite 7HP, and Amberlite 761 ( Styrene-divinylbenzene copolymer resin manufactured by Rohm & Haas Co., Philadelphia, USA.

As the most preferred embodiment of the present invention, the resin is a styrene-divinylbenzene copolymer resin manufactured under the trade name AB-8 crosslinked polystyrene (manufactured by Tianjin Nankai Hecheng Science and Technology Co., Ltd., China).

Elution solvents for eluting the fractions enriched from the adsorption chromatography media include water, ethanol, methanol, acetone, ethyl acetate, chloroform, isopropyl alcohol, or mixtures of these solvents, or any other polar solvent known to those skilled in the art. Can be mentioned. Preferred solvents are mixtures of water and ethanol, most preferably 30% aqueous ethanol.

In a preferred embodiment of the invention, the drying method comprises the use of a spray dryer, a vacuum oven, or a conventional oven, most preferably the use of a spray dryer.

Apparatus for preparing palm oil leaf extract of the present invention; Elaeis of the present invention ( Elaeis) guineensis) and elrayi device Olay Face (Elaeis The system for preparing oleifera ) extracts includes a herb extraction tank, an adsorption chromatography column and a dryer.

According to one aspect of the invention, the hub extraction tank consists of a steam jacket and a high speed stirrer that agitates the tank contents. Steam penetrates the steam jacket to heat the tank contents.

Palm oil leaves are mixed with water, ethanol, methanol, acetone, ethyl acetate, chloroform, isopropyl alcohol, or a mixture of these solvents, or any other polar solvent known to those skilled in the art, preferably at a temperature of 25 ° C. to 95 ° C. Heat in the herb extraction tank for 1 to 96 hours in the range. When the herbal extract is obtained by the heating process, the filtrate is collected by filtration.

According to one aspect of the invention, the adsorption chromatography column is filled with an adsorption chromatography resin and preferably comprises a polystyrene resin. The filtered herbal extract is passed through an adsorption chromatography column, and the fractions enriched in (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid are adsorbed onto the resin surface. The (-)-catechin gallate is then eluted with water, methanol, ethanol, acetone, ethyl acetate, chloroform, isopropyl alcohol, or a mixture of these solvents, or any other polar solvent, Eluted fractions containing ferulic acid and phenolic acids such as gallic acid and protocatecuic acid are eluted. The resulting herbal liquid fraction is finally dried using a spray dryer, vacuum oven, conventional oven, gas range, freeze dryer or other dryers known to those skilled in the art.

(-)-Catechin in Palm Oil Leaf Extract of the Present Invention Gallate , Ferulic acid , And Gallic  Quantitative Assays to Identify Presence

How to identify the presence of (-)-catechin gallate in palm oil leaf extract using HPLC:

a) dissolving 1 mg of (-)-catechin gallate standard reference in 1 ml of 0.1% phosphoric acid to prepare a standard reference solution of (-)-catechin gallate;

b) 200 mg of the extract of the present invention is dissolved in 10 ml of ethanol to prepare a test solution;

c) The two solutions are separately injected into HPLC under the following conditions:

Appliance: Perkin Elmer Series 200 LC

Column: Sinochrom ODS-BP Column (250 mm x 4.6 mm ID, 5 μm particle size)

Mobile phase: mixture of (i) 2% (v / v) acetic acid and (ii) acetonitrile

The solvent mixture starts with 92% solvent (i) and 8% solvent (ii) and after 50 minutes is increased to a linear gradient to 31% solvent (ii).

Flow rate: 1.0 ml / min

Detection: UV 280 nm

Chromatograms obtained from standard reference material solutions and test solutions showed major peaks corresponding to (-)-catechin gallate when the residence time was about 28 minutes. The presence of the peak confirmed the presence of (-)-catechin gallate in the composition of the present invention.

How to identify the presence of ferulic acid in palm oil leaf extract using TLC identification:

a) After dissolving the extract of the invention in HPLC grade methanol, centrifuge at 4000 rpm for 15 minutes and collect the supernatant to prepare a sample.

b) The sample was prepared on a precoat silica gel 60 TLC plate (E-Merck) with a known amount of ferulic acid standard reference material, using a solvent phase consisting of a mixture of methanol / water (ratio 7: 1). Spot first.

c) The plate is air dried and checked in an ultraviolet (UV) lamp with a wavelength of 254 nm.

Principal zones corresponding to ferulic acid were observed in both the standard reference material and the sample solution. The presence of mauve highlights confirmed the presence of ferulic acid in the composition of the present invention.

How to identify the presence of gallic acid in palm oil leaf extract using HPLC:

a) dissolving 10 mg of gallic acid standard reference material in 10 ml of methanol to prepare a standard reference material solution of gallic acid;

b) 200 mg of the extract of the present invention is dissolved in 10 ml of methanol to prepare a test solution;

c) The two solutions are separately injected into HPLC under the following conditions:

Appliance: Perkin Elmer Series 200 LC

Column: Hypersil BDS C ₁₈ column (250 mm x 4.6 mm ID, 5 μm particle size)

Mobile phase: mixture of methanol / water / orthophosphoric acid (20: 79.9: 0.1)

Flow rate: 1.0 ml / min

Detection: UV 270 nm

The chromatogram obtained from the standard reference material solution and the test solution showed a major peak corresponding to gallic acid when the residence time was about 6.6 minutes. The presence of the peak confirmed the presence of gallic acid in the composition of the present invention.

HPLC  (-)-Catechin in Palm Oil Leaf Extract of the Invention by Assay Gallate  And quantitative determination of ferulic acid

Method for quantitatively determining the total amount of (-)-catechin gallate in palm oil leaf extract using HPLC:

a) Dissolve 1 mg of the (-)-catechin gallate standard reference substance in 1 ml of 0.1% phosphoric acid to prepare a standard reference substance solution of (-)-catechin gallate;

b) 200 mg of the extract of the present invention is dissolved in 10 ml of ethanol to prepare a test solution;

c) The two solutions are separately injected into HPLC under the following conditions:

Appliance: Perkin Elmer Series 200 LC

Column: Sinochrom ODS-BP Column (250 mm x 4.6 mm ID, 5 μm particle size)

Mobile phase: mixture of (i) 2% (v / v) acetic acid and (ii) acetonitrile

The solvent mixture starts with 92% solvent (i) and 8% solvent (ii) with a linear gradient for 50 minutes.

Flow rate: 1.0 ml / min

Detection: UV 280 nm

Injection volume: 20 μL

When the residence time is about 28 minutes, the peak area of (-)-catechin gallate was obtained from the HPLC data.

The amount of (-)-catechin gallate in the test solution is measured in comparison to the standard reference material solution of (-)-catechin gallate.

The following formula calculates the content (%) of (-)-catechin gallate in the test solution:

1000 Cr _t / Wr _s

Where

C = concentration of (-)-catechin gallate (mg / ml) to test solution calculated from working standard curve

W = weight of sample used to prepare test solution (mg) (mg)

r _t = peak area of (-)-catechin gallate obtained from the test solution

r _s = peak area of (-)-catechin gallate obtained from standard reference material solution

To quantitatively determine the total amount of ferulic acid in palm oil leaf extract using HPLC:

a) Dissolve the four known concentrations of ferulic acid standard reference materials of 0.15 mg / ml, 0.20 mg / ml, 0.25 mg / ml, and 0.5 mg / ml in 70% ethanol to prepare a standard reference solution of ferulic acid. To manufacture;

 b) 10 mg of palm oil leaf extract of the present invention is dissolved in 10 ml of 70% ethanol to prepare a test solution;

c) The solution is injected separately into HPLC under the following conditions to perform HPLC analysis:

Appliance: Perkin Elmer Series 200 LC

Column: Hypersil BDS C ₁₈ column (250 mm x 4.6 mm ID, 5 μm particle size)

Mobile phase: mixture of methanol and 0.1% aqueous phosphoric acid solution (30:70)

Flow rate: 1.0 ml / min

Detection: UV 325 nm

Injection volume: 20 μL

When the retention time is about 28.8 minutes, the peak area of ferulic acid was obtained from the HPLC data.

The amount of ferulic acid in the test solution is obtained by comparison with a standard reference material solution of ferulic acid.

Using the following formula, calculate the content (%) of ferulic acid in the test solution:

1000 Cr _t / Wr _s

Where

C = concentration of ferulic acid in test solution (mg / ml) calculated from working standard curve

W = weight of sample used to prepare test solution (mg) (mg)

r _t = peak area of ferulic acid obtained from the test solution

r _s = peak area of ferulic acid obtained from a standard reference material solution

UV Spectrophotometry  Of total phenolic acid in palm oil leaf extract of the present invention Quantitative Decision

A method for quantitatively determining the total amount of phenolic acid in the palm oil leaf extract of the present invention, comprising the following steps:

a) Dissolve 10.0 mg of protocatecuic acid reference material in 100 ml of HPLC grade methanol to prepare a standard reference material solution. 10 ml of this solution is diluted with HPLC grade methanol in a ratio of 1: 9;

b) test solution is prepared by dissolving 10 mg of palm oil leaf extract in 10 ml of 70% ethanol;

c) dissolving 90 mg of potassium ferricyanide in 10 ml of purified water to prepare 0.9% aqueous potassium ferricyanide solution;

d) 90 mg of ferric chloride was dissolved in 10 ml of purified water to prepare 0.9% ferric chloride aqueous solution;

e) 9 ml of 0.9% aqueous potassium ferricyanide solution is mixed with 10 ml of 0.9% ferric chloride aqueous solution to prepare a chromogenic reagent.

All solutions (standard, test and blank solutions) are left in the dark for 5 minutes. All solutions are increased in 0.1 M HCL aqueous solution, mixed well, and left in the dark for 20 minutes. Measure the absorbance of the standard and test solutions to the blank solution at 679 nm. The x-axis is the concentration of the standard solution and the y-axis is the absorbance to create a standard calibration curve. The total concentration of phenolic acid is calculated from the standard calibration curve.

Using the following formula, the content of% of total phenolic acid in the test solution is calculated:

50,000 C / W

Where

C = concentration of total phenolic acid (mg / ml) for test solution calculated from working standard curve

W = weight of sample used to prepare test solution (mg) (mg)

The present invention includes a therapeutic composition comprising an extract prepared according to the invention, wherein the composition is useful for reducing or preventing oxidative stress in humans or lower animals, teas, tablets, coated tablets, lozenges (lozenge), chewable tablets, capsules, soft capsules, granules, coated granules, powders, coated powders, solutions, syrups, emulsions and suspensions.

The present invention also encompasses pharmaceutical compositions comprising an effective amount, such as 1 mg to 800 mg, more preferably 300 mg to 500 mg of said extract, and a pharmaceutically acceptable carrier.

The present invention provides a pharmaceutical composition comprising a novel extract formulated in a form suitable for oral, buccal, rectal or transdermal administration or by inhalation or insufflation (through the mouth or nose). do.

For oral administration, the pharmaceutical composition may comprise a binder (eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); Fillers (eg, lactose, microcrystalline cellulose, starch or tricalcium phosphate); Lubricants (eg, magnesium stearate, talc or silica); Disintegrants (eg, crosslinked polyvinylpyrrolidone, croscarmellose sodium or sodium starch glycolate); Or prepared by conventional means in combination with pharmaceutically acceptable excipients such as wetting agents (e.g. sodium lauryl sulfate), e.g. teas, tablets, coated tablets, lozenges, chewable tablets , Capsules, soft capsules, granules, coated granules, powders, solutions, syrups, emulsions or suspensions.

Solutions for oral administration may be, for example, in the form of solutions, syrups, emulsions or suspensions, or may be dry products which are mixed with water or other suitable vehicle immediately before use. The solution may be a suspending agent (eg, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); Emulsifiers (eg, lecithin or acacia); Non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); And pharmaceutically acceptable additives such as preservatives (eg, methyl or propyl-p-hydroxybenzoate or sorbic acid).

Formulations for oral administration may be suitably formulated to control or delay release of the extracts of the present invention.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

The pharmaceutical compositions according to the invention can be formulated for administration via transdermal inhalation and oral inhalation. Examples of such formulations include sprays and aerosols used in inhalers or insufflators. Transdermal formulations for parenteral application can be formulated with any suitable cream, ointment or lotion base known to those skilled in the art.

The pharmaceutical compositions according to the invention may be formulated in rectal compositions such as suppositories or retention enemas, including, for example, conventional suppository bases such as cocoa butter or other fats and oils.

In a preferred embodiment of the invention, the pharmaceutical composition comprises a capsule, soft capsule or tablet, comprising from about 1% to about 95% of said extract, and from about 1% to about 95% of a pharmaceutically acceptable carrier. do.

As used herein, the term 'pharmaceutically acceptable carrier' means one or more compatible solid or liquid filled diluents, or any excipient known to those skilled in the art.

Examples of substances that act as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose derivatives such as sodium carboxymethylcellulose, cellulose acetate and microcrystalline cellulose. Can be.

Suitable doses of the compositions according to the invention administered to humans (weight of about 60 kg) are from 0.1 mg to 1 g, eg 1 mg to 500 mg of active ingredient per unit dose, based on the weight of the dry extract. Unit doses may be administered, eg, 1 to 4 times per day. The dose may vary depending on the route of administration. It will be appreciated that a dose change may be necessary depending on the age and weight of the patient as well as the severity of the condition to be treated. The dose also depends on the route of administration. The exact dosage and route will be finally determined at the discretion of the attending clinician or veterinarian.

Extracts prepared according to the present invention have been shown to exhibit antioxidant and free radical scavenging effects in in vitro test models and to exhibit anti-oxidative stress effects in in vivo test models.

According to another embodiment of the invention, the extract is used to reduce or prevent oxidative stress in mammals and poultry by administering an effective amount of the composition to a subject in need of such treatment.

Examples of compositions prepared according to the invention are described below. Toxicity tests conducted on the compositions of the present invention demonstrated that the extract was non-toxic when orally administered 30 g of palm oil leaf extract per kg of body weight in Sprague-Dawley rats.

Therefore, oral administration of 0.03 g of palm oil leaf extract per kg body weight to humans is considered adequate and stable.

1 shows the results of HPLC identification of (-)-catechin gallate in palm oil leaf extract.
Figure 2 shows the results of TLC identification of perolic acid in palm oil leaf extract.
Figure 3 shows the results of HPLC identification of gallic acid in palm oil leaf extract.
Figure 4 shows the result of HPLC quantification of (-)-catechin gallate in palm oil leaf extract.
Figure 5 shows the results of HPLC quantification of perolic acid in palm oil leaf extract.
Figure 6 shows the results of UV quantification of total phenolic acid in the palm oil leaf extract.

Example  One

Preparation of Palm Oil Leaf Extract

About 50 g of dry palm oil leaves were extracted with about 500 mL of a mixture of water / ethanol (volume ratio 3: 7) at about 60-65 ° C. for about 60 minutes. The resulting slurry was filtered through two layers of muslin cloth to give about 502 g of crude extract.

Then, column adsorption chromatography was performed on the resulting crude extract.

Amberlite XAD 16HP (Rohm & Haas) was charged to the column (5.0 cm ID x 40 cm height) to give a column volume of about 780 mL. The column was washed with about 4-5 column volumes of deionized water. The crude extract obtained in the above extraction step was pumped into the column. The column was first eluted with 1500 ml of deionized water to remove salts, sugars and other unwanted hydrophilic substances.

The column is then eluted with a mixture of 1500 ml of water / isopropanol (volume ratio 3: 7) at a constant flow rate of 25 ml / min, at a temperature of 60 ° C. and a pressure of 0.5-1 bar, to give (-)-catechin gal Purified eluates were obtained that were enriched in laterate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid.

The resulting eluent was concentrated to 50 ml on a rotary evaporator and the concentrated liquid was dried in a 60 ° C. vacuum oven for 8 hours.

2.52 gm of yellow-beige powder with the following contents were obtained:

(-)-Catechin Gallate Content-1.1% (HPLC Analysis)

Ferulic acid content-1.5% (HPLC analysis)

Total Phenolic Acid Content-10.2% (UV Spectrophotometry)

Example  2

Preparation of Palm Oil Leaf Extract

100 kg of fresh palm oil leaves were cut into small pieces about 2 cm in length and transferred to the extraction tank. 1000 L of a mixture of water / isopropyl solution (volume ratio 3: 7) was added and heated to 60 ° C. for 8 hours.

The crude extract was separated from the leaves by filtration through a cartridge filter with a pore size of 10 μm. The filtrate was concentrated at 70 ° C. under reduced pressure of 0.08-0.09 MPa to form about 200 L of concentrated flow extract (density = 1.10-1.20 g / cm ³ ).

The resulting concentrated flow extract was subjected to adsorption column chromatography using a stainless steel column 15 cm in diameter and 150 cm in length. The column was filled with polyamide resin having a particle size of 30-60 mesh.

The column is then washed with 200 L of deionized water and water / isopropyl solution (volume ratio 3: at a temperature of about 20-30 ° C. and a pressure of about 0.5 to 1 bar at a constant flow rate of about 1.5 L / min. Elution with 200 L of the mixture of 7) gave a purified eluent enriched with (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid.

The eluate produced therefrom was spray dried with a spray dryer. The inlet temperature of the spray dryer was set at about 170 ° C to about 180 ° C and the outlet temperature was set at about 100 ° C to about 105 ° C.

12.5 kg of yellow-beige powder with the following contents were obtained:

(-)-Catechin Gallate Content-1.15% (HPLC Analysis)

Ferulic acid content-1.6% (HPLC analysis)

Total Phenolic Acid Content-10.5% (UV Spectrophotometry)

Characterization of the Compositions of the Invention by Qualitative and Quantitative Analysis

Quantitative analysis was performed to confirm the presence of (-)-catechin gallate, ferulic acid and gallic acid in the compositions of the present invention using the thin film chromatography (TLC) and high performance liquid chromatography (HPLC) methods described below.

Example  3

(-)-Catechin from Palm Oil Leaf Extract Gallate HPLC  Sympathy

1 mg of (-)-catechin gallate standard reference material was dissolved in 1 ml of 0.1% phosphoric acid to form a standard reference material solution.

At the same time, 200 mg of the extract to be analyzed in 10 ml of ethanol was dissolved to prepare a test solution of the extract of the present invention obtained in Example 1.

The two solutions were separately injected into HPLC under the following conditions:

Appliance: Perkin Elmer Series 200 LC

Column: Sinochrom ODS-BP Column (250 mm x 4.6 mm ID, 5 μm particle size)

Mobile phase: mixture of (i) 2% (v / v) acetic acid and (ii) acetonitrile

The solvent mixture starts with 92% solvent (i) and 8% solvent (ii) and after 50 minutes is increased to a linear gradient to 31% solvent (ii).

Flow rate: 1.0 ml / min

Detection: UV 280 nm

Chromatograms obtained from standard reference material solutions and test solutions showed major peaks corresponding to (-)-catechin gallate when the residence time was about 28 minutes. The presence of the peak confirmed the presence of (-)-catechin gallate in the composition of the present invention (shown in FIG. 1).

Example  4

Palm Oil Leaf Extract I Ferulic acid TLC  Sympathy

The extract powder of the palm oil leaf obtained in Example 1 was dissolved in HPLC grade methanol, then centrifuged at 4000 rpm for 15 minutes and the supernatant was collected. Using a solvent phase consisting of a mixture of methanol / water (ratio 7: 1), the sample was firstly pre-mixed on a precoat silica gel 60 TLC plate (E-Merck) with a known amount of ferulic acid standard reference material. Spotted.

The plate was air dried and checked in an ultraviolet (UV) lamp with a wavelength of 254 nm. Light purple principal zones corresponding to ferulic acid were observed in both the standard reference material and the sample solution (shown in FIG. 2).

Example  5

Palm Oil Leaf Extract I Gallic HPLC  Sympathy

10 mg of gallic acid standard reference material was dissolved in 10 ml of methanol to form a standard reference material solution.

At the same time, 200 mg of the extract to be analyzed in 10 ml of methanol was dissolved to prepare a test solution of the extract of the present invention obtained in Example 1.

The two solutions were separately injected into HPLC under the following conditions:

Appliance: Perkin Elmer Series 200 LC

Column: Hypersil BDS C ₁₈ column (250 mm x 4.6 mm ID, 5 μm particle size)

Mobile phase: mixture of methanol / water / orthophosphoric acid (20: 79.9: 0.1)

Flow rate: 1.0 ml / min

Detection: UV 270 nm

The chromatogram obtained from the standard reference material solution and the test solution showed a major peak corresponding to gallic acid when the residence time was about 6.6 minutes. The presence of the peak confirmed the presence of gallic acid in the composition of the present invention (shown in FIG. 3).

To be described later HPLC  (-)-Catechin present in the composition of the present invention by the method Gallate  And Ferulic acid  Quantitative analysis was performed.

Example  6

(-)-Catechin in Palm Oil Leaf Extract Gallate HPLC  dose

To determine the amount of (-)-catechin gallate in the palm oil leaf extract obtained in Example 1, an HPLC method was used.

1 mg of (-)-catechin gallate standard reference material was dissolved in 1 ml of 0.1% phosphoric acid to form a standard reference material solution.

At the same time, 200 mg of the extract to be analyzed in 10 ml of ethanol was dissolved to prepare a test solution of the extract of the present invention obtained in Example 1.

The two solutions were separately injected into HPLC under the following conditions and subjected to HPLC analysis:

Appliance: Perkin Elmer Series 200 LC

Column: Sinochrom ODS-BP Column (250 mm x 4.6 mm ID, 5 μm particle size)

Mobile phase: mixture of (i) 2% (v / v) acetic acid and (ii) acetonitrile

The solvent mixture starts with 92% solvent (i) and 8% solvent (ii) with a linear gradient for 50 minutes.

Flow rate: 1.0 ml / min

Detection: UV 280 nm

Injection volume: 20 μL

When the retention time is about 28 minutes, the peak area of (-)-catechin gallate was obtained from the HPLC data (shown in FIG. 4).

The amount of (-)-catechin gallate in the test solution was measured in comparison to the standard reference material solution of (-)-catechin gallate.

Using the following formula, the content of (-)-catechin gallate (%) in the test solution was calculated:

1000 Cr _t / Wr _s

Where

C = concentration of (-)-catechin gallate (mg / ml) to test solution calculated from working standard curve

W = weight of sample used to prepare test solution (mg) (mg)

r _t = peak area of (-)-catechin gallate obtained from the test solution

r _s = peak area of (-)-catechin gallate obtained from standard reference material solution

(-)-Catechin Gallate Content-1.1% (HPLC Analysis)

HPLC analysis showed that the amount of (-)-catechin gallate present in the composition obtained in Example 1 was 1.1%.

Example  7

Palm Oil Leaf Extract I Ferulic acid HPLC  dose

In order to measure the amount of ferulic acid in the palm oil leaf extract obtained in Example 1, an HPLC method was used.

A solution of standard reference material of ferulic acid was prepared by dissolving four known concentrations of ferulic acid standard reference materials of 0.15 mg / ml, 0.20 mg / ml, 0.25 mg / ml, and 0.5 mg / ml in 70% ethanol. .

Simultaneously, 10 mg of the analyte extract was dissolved in 10 ml of 70% ethanol to prepare a test solution of the extract of the present invention obtained in Example 1.

The solutions were injected separately into HPLC under the following conditions and subjected to HPLC analysis:

Appliance: Perkin Elmer Series 200 LC

Column: Hypersil BDS C ₁₈ column (250 mm x 4.6 mm ID, 5 μm particle size)

Mobile phase: mixture of methanol and 0.1% aqueous phosphoric acid solution (30:70)

Flow rate: 1.0 ml / min

Detection: UV 325 nm

Injection volume: 20 μL

When the retention time is about 28.8 minutes, the peak area of ferulic acid was obtained from the HPLC data (shown in FIG. 5).

The amount of (-)-catechin gallate in the test solution was measured in comparison to the standard reference material solution of (-)-catechin gallate.

Using the following formula, the content (%) of ferulic acid in the test solution was calculated:

1000 Cr _t / Wr _s

Where

C = concentration of ferulic acid in test solution (mg / ml) calculated from working standard curve

W = weight of sample used to prepare test solution (mg) (mg)

r _t = peak area of ferulic acid obtained from the test solution

r _s = peak area of ferulic acid obtained from a standard reference material solution

Ferulic acid content-1.5% (HPLC analysis)

HPLC analysis showed that the amount of ferulic acid present in the composition obtained in Example 1 was 1.5%.

Example  8

Of Total Phenolic Acids in Palm Oil Leaf Extracts UV  dose

In order to measure the amount of total phenolic acid in the palm oil leaf extract obtained in Example 1, UV spectrophotometry was used.

A standard reference material solution was prepared by dissolving 10 mg of protocatecuic acid reference material in 100 ml of HPLC grade methanol. 10 ml of the solution was diluted with HPLC grade methanol in a ratio of 1: 9.

Different volumes of standard solutions and 1 ml of methanol (blank solution) were each separated into a 25 ml volumetric flask using a pipette. To each flask was added 5.0 ml of absolute ethanol, 2 ml of 3% sodium dodecyl sulfate solution, and 1.0 ml of chromogenic reagent [0.9% potassium ferricyanide solution: 0.9% ferric chloride solution (0.9: 1)].

The standard and blank solutions were left in the dark for 5 minutes. The solution in each flask was extended with 0.1 M HCl and mixed well. The solution was left in the dark for 20 minutes.

The absorbance of the standard solution relative to the blank solution at 679 nm was measured. The x-axis was the concentration of the standard solution and the y-axis was the absorbance to create a standard calibration curve.

The composition was dissolved in 10 ml of 70% aqueous ethanol, and 0.05 ml of the solution was transferred to a flask by pipette for analysis by UV spectrophotometry to prepare a test solution in the same manner as described above.

The absorbance of total phenolic acid in the palm oil leaf extract was measured at a wavelength of 679 nm (shown in FIG. 6).

According to the standard calibration curve, the concentration of total phenolic acid was calculated from the standard calibration curve.

Using the following formula, the total content of phenolic acid in the test solution was calculated:

50,000 C / W

Where

C = concentration of total phenolic acid (mg / ml) for test solution calculated from working standard curve

W = weight of sample used to prepare test solution (mg) (mg)

UV absorbance showed that the total amount of phenolic acid present in the composition obtained in Example 1 was 10.2%.

Example  9

In vitro  And In vivo  Determination of Antioxidant Activity of the Compositions of the Invention in a Model

The antioxidant activity of the compositions of the present invention was measured using the following in vitro and in vivo models.

Of the composition of the present invention DPPH  freedom Radical  Scavenging activity

The DPPH free radical scavenging activity of the composition of the present invention obtained in Example 1 was measured using l, l-diphenyl-2-picryl-hydrazyl (DPPH) colorimetric method detected at 517 nm. The activity was evaluated by decreasing the absorbance as a result of the DPPH color change from purple to yellow.

As a sample solution, 10 mg of the sample powder obtained in Example 1 was dissolved in 5 ml of anhydrous ethanol, and a 0.2 mmol / L DPPH solution in ethanol was prepared. 1 ml of DPPH solution was added to 4 ml of sample solution. The sample solution was then left in the dark for 30 minutes. The absorbance of the sample solution was measured at 517 nm. Low absorbance of the reaction mixture means large free radical scavenging activity. Butylated hydroxinasole (BHA) was used as a control. The higher the concentration of sample used, the stronger the free radical scavenging activity. The results showed that the compositions of the present invention showed stronger antioxidant activity than BHA.

% Of DPPH Free Radical Scavenging Activity of Palm Oil Leaf Extract vs. BHA Concentration (mg / ml)
DPPH free radical scavenging activity rate (%) BHA Palm oil leaf extract obtained in Example 1 0.125 58.15 92.52 0.25 67.79 95.78 0.5 79.76 97.18 One 86.84 96.84 2 94.12 94.34

Example lO

Reducing power of the composition of the present invention

According to the potassium ferricyanide reduction method, the reducing power of the composition of the present invention obtained in Example 1 was measured. As a sample solution, 10 mg of the sample powder obtained in Example 1 was dissolved in 10 ml of absolute ethanol. 1 ml of sample solution was diluted in 1 ml of distilled water and mixed with 2.5 ml of 0.2 mol / L phosphate buffer and 2.5 ml of 1% aqueous potassium ferricyanide solution. The mixture was incubated at 50 ° C. for 20 minutes. Thereafter, 2.5 ml of an aqueous 10% trichloroacetic acid solution was added to each sample solution. Sample solution was centrifuged at 3000 rpm for 10 minutes. 2.5 ml of supernatant was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride aqueous solution. Due to the reducing power of the composition the yellow solution (potassium ferricyanide solution) turned bright yellow. Due to the presence of the composition within the reducing agent (antioxidant), the Fe ^{+ 3} (Perry cyanide complex) is to be reduced to 2 gacheol (ferrous form). (The absorbance is measured at 700 nm) at 700 nm by the formation of a pearl Prussian blue (Perl's Prussian blue), and may be a measure Fe ^{2 +} complex. An increase in absorbance means an increase in reducing power, butylated hydroxyazole (BHA) was used as a control. The results showed that the compositions of the present invention exhibit as much reducing power as BHA.

Reducing Power of Palm Oil Leaf Extract and BHA Concentration (mg / ml)
Absorbance (measured at 700 nm) BHA Palm oil leaf extract obtained in Example 1 One 0.8897 0.2296 0.5 0.7322 0.1603 0.25 0.5777 0.1177 0.0625 0.2769 0.0015 0.125 0.4054 0.0115

Example  11

Lipid Peroxide Induced by Palm Oil Leaf Extract In vivo  restoration

In the malondialdehyde (MDA) test, the lipid peroxidation inhibitory properties of palm oil leaf extract can be measured to demonstrate the anti-oxidative stress activity of palm oil leaf extract. One of the most potent effects of oxidative stress by free radicals in living organisms is the oxidation of lipids, resulting in malondialdehyde (MDA). The level of lipid peroxide can be assessed by measuring the level of thiobarbituric acid active substance (TBARS) in the serum of an animal induced with lipid peroxidation. MDA reacts with thiobarbituric acid to form a color material measurable by calorimeter.

Carbon tetrachloride was injected intraperitoneally to induce lipid peroxidation.

group Number of rats (n = 6) Processing (1-5 days) Administration of CCL ₄ (6 days) Control 6 - - Positive control 6 Purified water 15 mg single dose CCL ₄ per kg body weight Treated group 6 1.1 g of the composition obtained in the example per 1 kg of body weight 15 mg single dose CCL ₄ per kg body weight

As shown in Table 4 below, serum MDA levels were higher in lipid peroxidation-induced rats (positive control) compared to control rats. Serum MDA levels were decreased in rats treated with the composition obtained in Example 1 (treated group), suggesting that palm oil leaf extracts have lipid peroxidation inhibitory effects and anti-oxidative stress activity.

group Number of rats (n + 6) Mean Serum MDA Level (nmol / mL) Control 6 1.913 Positive control 6 2.851 Treated group 6 1.947

Example  12

Safety of Palm Oil Leaf Extract

maximum Tolerance  Volume ( Maximum Tolerated Dose : MTD Determination of

The safety of palm oil leaf extracts has been demonstrated through animal testing. Toxicity tests conducted in Sprague Dawley rats administered with palm oil leaf extract obtained in Example 1 showed that the composition was safe even when orally administered 2 grams of Palm Oil Leaf Extract per kg body weight of Sprague Dawley rats ( No lethality).

The maximum tolerated dose was measured as follows:

The composition was weighed in groups of 3 rats of about 150-180 g 2 g, 5 g, 7 g, 9 g, 11 g, 13 g, 16 g, 19 g, 25 g, 30 g, per kg, respectively, and Orally administered at a dose of 35 g. Prior to oral administration to each animal, the composition was dissolved in purified water. Purified water was orally administered to the same number of control rats.

Animals were observed periodically every 30 minutes for the first 24 hours after administration, daily thereafter for a total of 14 days, with special attention during the first 4 hours. Observations include overall behavioral changes and general motor activity, writhing, convulsions, response to tail pinching, swelling, hairballing, pupil size, fecal output, eating behavior, and lethality. Development and the like. Weight changes were also recorded. The main observation was the occurrence of lethality.

Since no lethal or toxic effects occurred at the dose of 30 g of palm oil leaf extract per kg body weight, the maximum tolerated dose was 30 g / kg body weight.

The recommended normal daily dose for humans is about 0.03 g of palm oil leaf extract per kg body weight.

When comparing the recommended normal daily dose in humans with the maximum tolerated dose for rats,

Maximum Tolerance of Rats:

= Maximum tolerated dose for rats (g / kg body weight)

  Daily dose for humans (g / kg body weight)

= 30.0

  0.03

= 1,000 times

Conclusion: The maximum tolerated dose of palm oil leaf extract obtained in Example 1 is 30 g / kg body weight, which corresponds to 1,000 times the recommended oral dose in humans. The results show that the acute toxicity of the compositions of the invention is very low and very safe for human oral administration.

Example 13

Method of Making Soft Gelatin Capsules

The following components were mixed to form a homogeneous oily suspension.

Soft Gelatin Capsule Prescription: ingredient Volume / capsules function Palm oil leaf extract powder obtained in Example 1
(Standardized to include 1.1% (-)-catechin gallate, 1.5% ferulic acid and 10.2% total phenolic acid) 200.00mg Active ingredient Beeswax 25.00mg Suspending agent Medium chain triglycerides 275.00 mg Vehicle

The resulting mixture in oil suspension form is poured into soft gelatin capsules.

Example  14

Method of Making Tablets

Tablets with the following formula were prepared as described below.

Direct method was used to prepare the tablets. All components were mixed uniformly and the resulting mixture was compressed into round tablets (700 mg / tablet) in a tablet press.

Tablet prescription: ingredient Volume / tablet function Palm oil leaf extract powder obtained in Example 1
(Standardized to include 1.1% (-)-catechin gallate, 1.5% ferulic acid and 10.2% total phenolic acid) 350.00mg Active ingredient Starch 77.00mg diluent Microcrystalline cellulose 175.00 mg diluent Polyvinylpyrrolidone K-30 21.00mg Binder Sodium starch glycolate 70.00mg Disintegrant Magnesium stearate 7.00mg slush

Example  15

Method of making hard gelatin capsules

According to conventional methods known to those skilled in the art, the compositions of the following formulations were prepared as hard capsules.

The following ingredients were mixed to form a homogeneous powder mixture.

The resulting mixture was then injected into size # 1 hard gelatin capsules.

Hard Gelatin Capsule Prescription: ingredient Volume / capsules function Palm oil leaf extract powder obtained in Example 1
(Standardized to include 1.1% (-)-catechin gallate, 1.5% ferulic acid and 10.2% total phenolic acid) 200.00mg Active ingredient Starch 144.75mg diluent Silicon dioxide 3.50mg Anti-caking agent Magnesium stearate 1.75mg slush

Example 16

Oral suspension

A liquid oral suspension having the following composition was prepared as described below.

Thereafter, the palm oil leaf extract powder of the present invention was placed in an amber glass bottle.

Liquid Oral Suspension Prescription: ingredient Volume / tablet function Palm oil leaf extract powder obtained in Example 1
(Standardized to include 1.1% (-)-catechin gallate, 1.5% ferulic acid and 10.2% total phenolic acid) 0.50 g Active ingredient Sodium carboxymethylcellulose 2.50g Suspending agent Methyl paraben 0.09g antiseptic Profile paraben 0.10 g antiseptic Strawberry oil 0.10 g Flavor Polysorbate 80 0.10 g Wetting agent Purified Water Proper Amount 100 ml Vehicle

Claims (24)

Palm oil, characterized by containing (-)-catechin gallate, ferulic acid, and phenolic acid such as gallic acid and protocatechuic acid A composition comprising an extract derived from the leaf. The method of claim 1,
The palm oil leaf is Elaeis guineensis ) and Elaeis oleifera . The method according to claim 1 or 2,
Wherein the (-)-catechin gallate is present in an amount of 0.1% to 95% by weight. The method according to claim 1 or 2,
Wherein the ferulic acid is present in an amount of 0.1% to 95% by weight. The method according to claim 1 or 2,
The phenolic acid is a composition, characterized in that gallic acid. The method according to any one of claims 1, 2 or 5,
The content of the phenolic acid is characterized in that 0.1% to 95% by weight. (-)-Catechin gallate, ferulic acid, and phenolic acid, such as gallic acid and protocatechuic acid, comprising the following steps: Manufacturing Method of Palm Oil Leaf Extract:
a) dry or fresh by mixing with water, ethanol, methanol, acetone, ethyl acetate, chloroform, isopropyl alcohol or a mixture of these solvents or any other polar solvent and heating at 25-95 ° C. for 0.5-96 hours Extracting herbal liquid from palm oil leaves;
b) filtering the herbal liquid extract obtained in step (a);
c) a column adsorption medium for selectively adsorbing the filtered herbal liquid extract obtained in step (b) to only fractions in which (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid are present; or Contacting with any other adsorption medium, an adsorption chromatography medium;
d) eluting the fractions obtained in step (c) from the adsorption chromatography medium using water, ethanol, methanol, acetone, ethyl acetate, chloroform, isopropyl alcohol or a mixture of these solvents or any other polar solvent. step; And
e) drying the enriched herbal liquid extract obtained in step (d) using a spray dryer, vacuum oven, conventional oven, microwave oven or freeze dryer. Palm oil leaf extract comprising (-)-catechin gallate, ferulic acid, and phenolic acids such as gallic acid and protocatecuic acid, prepared by the process according to claim 7. The method of claim 7, wherein
The extract is characterized in that derived from the leaves of Elaeis guineensis ( Elaeis guineensis ). The method of claim 7, wherein
Wherein said solvent comprises at least one selected from the group consisting of water, methanol, ethanol, acetone, ethyl acetate, methyl alcohol, chloroform, and mixtures thereof. The method of claim 7, wherein
Wherein said separation medium comprises a polymer chromatography column. The method of claim 7, wherein
Wherein said separation medium comprises a synthetic polymer resin. The method of claim 7, wherein
The polymer resin is polystyrene. The method of claim 7, wherein
Wherein said drying step comprises spray drying. The method of claim 7, wherein
The eluent comprises at least one selected from the group consisting of water, methanol, ethanol, acetone, ethyl acetate, methyl alcohol, chloroform, and mixtures thereof. The method according to any one of claims 1 to 6,
The composition is characterized in the form of tea, tablets, coated tablets, lozenge, chewable tablets, capsules, soft capsules, granules, coated granules, powders, solutions, syrups, emulsions and suspensions, Therapeutic composition. A pharmaceutical composition comprising an effective amount of the composition according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. The method of claim 17,
Wherein said pharmaceutically acceptable carrier is a stabilizer, carrier, extender and other suitable material. The method according to any one of claims 1 to 6 and 16 to 18,
The extract is a pharmaceutical composition, characterized in that present in an amount of 1 mg-800 mg. The method according to any one of claims 1 to 6 and 16 to 18,
The extract is preferably a pharmaceutical composition, characterized in that it is present in an amount of 300 mg-500 mg. Mitigating oxidative stress in mammals and poultry comprising administering an effective amount of a composition according to any one of claims 1 to 6 and 16 to 20 to a subject in need of such treatment or Use of said composition for prevention. Use of a composition according to any one of claims 1 to 6 and 16 to 20, characterized in that the mammal is a human. Use of a composition according to any one of claims 1 to 6 and 16 to 20, characterized in that the mammal is a cow, buffalo, goat, sheep or any other mammal. Use of a composition according to any one of claims 1 to 6 and 16 to 20, characterized in that the poultry is a goose, chicken, duck, turkey, pigeon or any other poultry.

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