KR20100024897A - Pharmaceutical composition for appetite suppression or prevention and treatment of psychiatric disorder, containing the extract of mori folium, fraction thereof or compound isolated therefrom, having mch-1 receptor antagonistic effect, as an active ingredient - Google Patents

Abstract

PURPOSE: A composition containing Mori folium extract of a compound isolated from the same is provided to ensure antagonistic activity to Melanin Concentrating Hormone(MCH) and to control anxiety or depression. CONSTITUTION: A pharmaceutical composition for preventing and treating depression and suppressing appetite contains Mori folium fraction. A Mori folium fraction is obtained by extracting using alcohol, removing alcohol, suspending with distilled water, and fractioning dichlrodomethane, butanol or hexane. A method for preparing a compound of chemical formulas 1-6 comprises: a step 1 of adding alcohol to Mori folium to obtain Mori folium extract; a step of suspending the extract in water and fractioning with dichloromethane to obtain dichloromethane fraction a step of performing column chromatography to obtain active fraction; a step of performing column chromatography again to obtain active fraction; and a step of performing silica gel column chromatography to isolate a compound of chemical formulas 1-6.

Description

Pharmaceutical composition for suppressing appetite or preventing and treating mental disorders containing as an active ingredient upper leaf extracts having MCHC-1 receptor antagonistic activity, fractions thereof or compounds isolated therefrom {PHARMACEUTICAL COMPOSITION FOR APPETITE SUPPRESSION OR PREVENTION AND TREATMENT OF PSYCHIATRIC DISORDER, CONTAINING THE EXTRACT OF MORI FOLIUM, FRACTION THEREOF OR COMPOUND ISOLATED THEREFROM, HAVING MCH-1 RECEPTOR ANTAGONISTIC EFFECT, AS AN ACTIVE INGREDIENT}

The present invention relates to a pharmaceutical composition for inhibiting appetite or preventing and treating mental disorders, which contains an upper leaf extract having an MCH-1 receptor antagonistic activity, a fraction thereof or a compound separated therefrom as an active ingredient.

MCH is the first cyclic peptide isolated from the pituitary gland of salmon (Kawauchi et al., 1983, Nature, 305, 321-333). MCH was initially known as a factor controlling color change in tibial fish, which consists of 17 amino acids in fish and 19 amino acids in mammals, and these sequences are identical in rodents, rabbits, and humans. It is. In addition, the MCH is signaled intracellularly through the G-protein coupled receptor (GPCR). Until now, MCH-1 (SLC-1, GPR24) and MCH-2 (SLT, GPRv17) Two types of receptors have been reported. MCH-1, known in 1999, consists of 353 amino acids and is reported to have typical GPCR characteristics, with about 35% similarity to the somatostatin receptor, and the gene is known to be located at chromosome 22q13.3. [Pssios et al., 2003, Trends Endocrinol. Metab., 14, 243-248].

The role of MCH in feeding behavior in mammals has been the subject of research for many years [Qu et al., 1996, Nature, 380, 243-247; Rossi et al., 1997, Endocrinology, 138, 351-355; Shimada et al., 1998, Nature, 396, 670-674. MCH is predominantly expressed in the lateral hypothalamus and zona incerta of the central nervous system (CNS). Central administration of MCH is known to stimulate food intake and regulate energy balance. MCH is elevated and expressed in the lateral hypothalamus during fasting (Rossi et al., 1997, Endocrinology, 138, 351-355). Knockout experiments have shown that mice without MCH peptides are tingling, telling, and maintaining a high metabolic rate. MCH mRNA levels are high in both normal and obese mice. Transgenic mice overexpressing MCH are obese and insulin resistant. Genetically modified animals that do not have genes encoding MCH receptors are moderately overeaten but resistant to obesity and have elevated metabolic rates (Shimada et al., 1998, Nature, 396, 670-674). MCH is believed to exert an effect on feeding behavior by binding to MCH receptors (MCH-1 or MCH-2) to reduce intracellular calcium and reduce cyclic AMP levels. In addition, many studies have described a statistically significant reduction in food intake of rodents after short-term administration of MCH-1 receptor antagonists [Borowsky et al., 2002, Nat. Med., 8, 825-830; Souers et al., 2005, Bioorg. Med. Chem. Lett., 15, 2752-2757; Vasudevan et al., 2005, Bioorg. Med. Chem. Lett., 15, 4174-4179; Kym et al., 2005, J. Med. Chem., 48, 5888-5891; McBriar et al., 2005, J. Med. Chem., 48, 2274; Takekawa et al., 2002, Eur. J. Pharmacol., 438, 129-135; Kowalski et al., 2004, Eur. J. Pharmacol., 497, 41-47].

In addition, it has been reported that MCH may be associated with depression, anxiety and anxiety [Borowsky et al., 2002, Nat. Med., 8, 825-830]. In particular, MCH-1 receptor antagonists have been shown to inhibit depression and anxiety in rodents, which is a model of depression and anxiety in humans [Hervieu, 2003, Expert Opin. Ther. Targets, 7 (4), 495-511; Georgescu et al., 2005, J. Neurosci., 25, 2933-2940; Chaki et al., 2005, J. Pharmcol. Exp. Ther., 313, 831-839. In these papers, the rodent model includes various models of forced swimming test, vocalization and social interaction. Recent studies have also shown that MCH-1 receptors also affect cognition [Adamantidis et al., 2005, Eur. J. Neurosci., 21, 2837-2844.

According to the above series of studies, MCH-1 receptor antagonists can be used not only as an appetite suppressant but also effective in removing depression and anxiety. A number of documents, including WO 07/039462, describe the use of MCH-1 receptor antagonists for the treatment of overeating, anxiety, depression and related disorders.

Various intracerebral neuronal activities and neurotransmitters are known to be involved in the regulation of appetite in humans and animals. This neuronal activity is affected by biochemical, neurological or endocrine signals that occur in the process of nutrient digestion, absorption, metabolism and storage. Sugars and lipids as nutrients, or metabolites in fat, muscle and liver, generate biochemical signals that act temporarily or continuously on cranial nerve activity associated with appetite. During the digestion and absorption of sugars and lipids, endocrine signals (e.g., CCK, GLP1, Enterostatin, ApoAIV, etc.) or nerve signals through chemical receptors in the gastrointestinal tract or nerve signals from the enteroplexus, may be used for gastrointestinal function and It is also known to affect cranial nerve activity. It is also known that adipose tissue, a fat storage organ, produces endocrine or biochemical signals, such as leptin, adiponectin and free fatty acids, while storing and consuming fat. These signals, alone or in combination, are thought to affect the central nervous system that regulates appetite.

As a substance used as an appetite suppressant in the control of appetite, pendimethazine, phentermine, and dimethylpropion preparations are used. However, due to the indulgence of psychotropic drugs, their use is strictly restricted. Recently, noradrenaline or Drugs that regulate serotonin or drugs that regulate noradrenaline and serotonin together are used.

Drugs that regulate noradrenaline work by stimulating the central nervous system to suppress appetite. However, side effects can cause excitement, sleep disorders, nervousness, headaches, dry mouth, and blood pressure and pulse can rise, so people with high blood pressure or those with heart disease should take caution. Serotonin-modulating drugs are drugs that have been previously marketed as antidepressants and binge eating disorders, and have recently been used to suppress appetite. The involvement of neurotransmitters acts to suppress appetite, and fluorocetin and setrarin are actually being prescribed. Sibutramine (sibutramine) is marketed under the US FDA's approval in Korea under the trade name "reductil" or "slimer". The mechanism of sibutramine acts on the hypothalamus of the brain to reabsorb serotonin and noradrenaline. By suppressing uptake, prolonging their working time increases satiety and reduces appetite. However, side effects are reported to increase blood pressure and increase pulse rate 3-6 times per minute. In addition, it is known that it causes insomnia, dizziness, nausea, headache, constipation, etc., so it is necessary to take a doctor's prescription when taking medicine, and it is necessary to be careful when administering it because there is a risk of worsening memory loss, anger, and depression related to mental illness. There is a problem.

Therefore, the development of a new functional material without side effects for appetite suppression is constantly required.

Mori folium is a dried leaf of mulberry and cousin myosin. Its taste is sensitive and its properties are limited. The upper lobe is known to have effects such as nourishing blood (reducing the heat of the blood and reducing moisture), big wind nominal (removing the wind and clearing the eyes), and instructing the abdomen (when taken in powder, eliminating cold sweat). . In addition, it has been recorded that the oriental medicines such as the herbaceous tree have an effect on small thirst along with baekbaekpi and silkworm cocoons. Dohae Herbal Medicine (Medicinal Herb) Dictionary, Younglimsa, pp 545-546, 1998].

However, in relation to the efficacy of the upper lobe, there is no report on the effect of the lobe on the antagonism of the MCH-1 receptor.

Thus, the inventors of the present invention, while searching for an active substance having excellent MCH-1 receptor antagonism in natural products, the extract of the upper leaf and the compound isolated therefrom have an excellent antagonistic effect on the MCH-1 receptor, The present invention has been completed by finding out that it can be usefully used for the prevention and treatment of mental disorders such as appetite suppressants, depression, and anxiety by controlling the same emotion.

SUMMARY OF THE INVENTION An object of the present invention is to provide a pharmaceutical composition for suppressing appetite or preventing and treating mental disorders, which contains an upper leaf extract having excellent antagonistic action against MCH-1 receptor, a fraction thereof or a compound separated therefrom as an active ingredient. have.

In order to achieve the above object, the present invention provides an extract of an upper leaf, a fraction thereof, or perpurin-7 dimethyl ethyl ester, hydroxy-phephorbide A ethyl ester, phephorbide A ethyl ester, and didehydro-chlorodochlorine ethyl. Provided is a pharmaceutical composition for suppressing appetite or preventing and treating mental disorders containing a compound of methyl ester, hydroxy pheophorbide A methyl ester, or pheophorbide A methyl ester as an active ingredient.

Upper leaf extract according to the present invention, fractions thereof, or purified therefrom, purified purpurin-7 dimethyl ethyl ester, hydroxy-phephorbide A ethyl ester, phephorbide A ethyl ester, didehydro-lodochlorine ethylmethyl ester, Compositions containing a compound of hydroxy pheophorbide A methyl ester or a compound of pheophorbide A methyl ester as an active ingredient have a strong antagonist against melanin Concentration Hormone receptor subtype-1 (MCH-1). It is effective in suppressing appetite, depression and anxiety such as depression and anxiety because it exhibits a continuous effect of suppressing appetite and exhibits a control effect on emotions such as anxiety and depression. It can be usefully used.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing appetite or preventing and treating mental disorders containing the extract of the upper leaf as an active ingredient.

The upper leaf extract according to the present invention can be obtained by extracting from the upper leaf or its dried product, and the upper leaf can be used without limitation, such as those grown or commercially available.

The upper leaf extract according to the present invention can be used in the conventional extraction methods such as solvent extraction, ultrasonic extraction, filtration and reflux extraction, it can be prepared by using a solvent extraction or reflux extraction method. As a preferred embodiment, the extract of the upper leaf is added to an extract solvent of 2 to 200 times the volume of the upper leaf, preferably 10 to 30 times, to the dry leaf shredded upper leaf, and preferably 1 to 20 days at 10 to 100 ℃. After extracting for 1 hour to 10 days, filtered and concentrated under reduced pressure to prepare an upper leaf extract, but is not limited thereto.

The extract may be extracted by using water, an organic solvent or a mixture thereof as an extraction solvent. The organic solvent may be any one or a mixed solvent selected from the group consisting of C ₁ -C ₄ alcohols, n-hexane, dichloromethane and ethyl acetate, but is not limited thereto. The alcohol of C ₁ -C ₄ may be methanol, ethanol or butanol, but is not limited thereto.

The present invention also provides a pharmaceutical composition for inhibiting appetite or preventing and treating mental disorders containing the upper fraction as an active ingredient.

The upper leaf fraction according to the present invention may be obtained by fractionating the upper leaf extract alone or sequentially with one or more organic solvents and condensing under reduced pressure. As the organic solvent, dichloromethane, ethyl acetate, n-hexane or butanol may be used. It is not limited to this.

As a preferred embodiment, the upper leaf fraction according to the present invention may be obtained by suspending the upper leaf extract in distilled water and then fractionating it with n-hexane, butanol, dichloromethane or ethyl acetate, and water. Can be. According to a preferred embodiment of the present invention, the upper leaf or its dried product is cooled by 7 days using ethanol or reflux extraction twice at 85 ° C. for 2 hours, filtered and the filtrate is concentrated under reduced pressure to obtain an ethanol extract, and the ethanol extract is distilled water. Suspension in, followed by fractionation with the same amount of dichloromethane and concentrated under reduced pressure to obtain an upper leaf fraction.

In addition, the fraction may be obtained by further performing a fractionation process, such as suspending the dichloromethane fraction in 90% methanol and fractionating with hexane or methanol.

Furthermore, the present invention provides a pharmaceutical composition for suppressing appetite or preventing and treating mental disorders containing the active fraction isolated from the upper leaf fraction as an active ingredient.

The active fraction according to the present invention can be obtained by separating and purifying the upper leaf fraction by chromatography. The chromatography is preferably one or two silica gel column chromatography, but is not limited thereto. Hexane / ethyl acetate (100: 1 to 1: 1) mixed solvent can be used as a mobile phase. At this time, the solvent used is eluted by the concentration gradient elution method to sequentially raise the concentration from non-polar to polar, and the desired active fraction can be obtained by measuring the MCH-1 receptor binding inhibitory activity. At this time, the flow rate of the mobile phase is preferably 2 ~ 4 ml / min.

In addition, the present invention is a Purpurin-7 dimethyl ethyl ester (Purpurin-7 dimethyl ethylester) represented by the following formula (1), Pheophorbide A ethylester represented by the formula (2), hydroxy represented by the formula (3) -Hydroxy-pheophorbide A ethylester, Didehydro-rhodochlorin ethylmethylester represented by the formula (4), and hydroxy pheophorbide A methylester represented by the formula (5) For preventing appetite or mental disorders containing any one or a mixture thereof selected from the group consisting of Hydroxy-pheophorbide A methylester and Pheophorbide A ethylester represented by Chemical Formula 6 as an active ingredient, and Provided is a therapeutic pharmaceutical composition.

The active material of Chemical Formulas 1 to 6 may be obtained by the following preparation method:

Obtaining an upper leaf extract using any one or a mixture thereof selected from the group consisting of water, C ₁ -C ₄ alcohol, dichloromethane and ethyl acetate (step 1);

Suspending the upper leaf extract obtained in step 1 in water and fractionating with dichloromethane to obtain a dichloromethane fraction (step 2);

Dichloromethane fractions obtained in step 2 using a mixed solvent of n- hexane and ethyl acetate as a mobile phase to perform a concentration gradient column chromatography to obtain an active fraction (step 3);

Separating the active fraction from the active fraction obtained in step 3 by performing concentration gradient column chromatography again using a mixed solvent of n-hexane and ethyl acetate as a mobile phase (step 4); And

The fraction separated in step 4 is a mixed solvent of n-hexane and ethyl acetate as the in-phase, and the silica gel column chromatography is performed by varying the concentration gradient to separate the compound of formula 1-6 (step 5) .

In the above production method, step 1 is a step of obtaining an upper leaf extract as an extraction solvent, the extraction solvent is any one selected from the group consisting of water, C ₁ -C ₄ alcohol, dichloromethane and ethyl acetate It may be a mixture, preferably dichloromethane, butanol, methanol or ethanol, but is not limited thereto.

At the time of extraction, an extraction solvent of 2 to 200 times the volume of the upper leaf, preferably 10 to 30 times is added, and extracted at 10 to 100 ° C. for 1 hour to 20 days, preferably 1 hour to 10 days, filtered and concentrated under reduced pressure. By this, the upper leaf extract can be manufactured. However, it is not limited thereto.

Next, step 2 is a step of suspending the upper leaf extract obtained in step 1 in water and fractionating with dichloromethane to obtain a dichloromethane fraction.

At this time, the dichloromethane fraction may be used a method commonly used in the art, for example, the upper leaf ethanol extract obtained in step 1 is suspended in distilled water, fractionated into the same amount of dichloromethane and concentrated under reduced pressure Dichloromethane fractions can be obtained.

Next, step 3 is a step of performing a concentration gradient column chromatography using the dichloromethane fraction obtained in step 2 using a mixed solvent of n-hexane and ethyl acetate as a mobile phase to obtain an active fraction.

In the above method, the active fraction of step 3 may be obtained by performing concentration gradient silica gel column chromatography on the dichloromethane fraction obtained in step 2 using a mixed solvent of n-hexane and ethyl acetate. At this time, the concentration gradient of the mixed solvent of n-hexane and ethyl acetate is preferably from 100: 1 to 1: 1, it can be obtained by eluting at a rate of 2 ~ 4 ml / min.

Next, step 4 is a step of separating the active fraction by performing concentration gradient column chromatography on the active fraction obtained in step 3 using a mixed solvent of n-hexane and ethyl acetate.

In this case, the third fraction of the first to fourth fractions of the active fraction obtained in step 3 is a mixed solvent of n-hexane and ethyl acetate as a mobile phase, and the concentration gradient is 30: 1 to 1: 1. It is preferable to separate by chromatography, and it is preferable that the flow rate of the said mobile phase is 2-4 ml / min.

Next, in step 5, the fraction separated in step 4 is a mixed solvent of n-hexane and ethyl acetate as a mobile phase, and silica gel column chromatography is performed by varying concentration gradients to separate the compound of Chemical Formula 1-6. Step.

In step 5, the concentration gradient of the mixed solvent of n-hexane and ethyl acetate is 15: 1 to 1: 3 using silica gel column chromatography for the third to sixth fractions of the fractions separated into nine fractions in step 4. It can be obtained by separating the compound of formula 1-6 by eluting with 1, 30: 1 to 1: 1 or 5: 1 to 1: 1. At this time, it is preferable that the flow velocity of the said mobile phase is 2-4 ml / min.

Compounds of Chemical Formulas 1 to 6 isolated through the above steps were obtained by NMR analysis, respectively, for Purpurin-7 dimethyl ethyl ester, ferrophorbide A ethyl ester, hydroxy-phefobide A ethyl ester, and didehydro-chlorodochlorine ethyl. It was identified as methyl ester, hydroxy pheophorbide A methyl ester and pheophorbide A methyl ester.

Upper leaf extract according to the present invention, fractions thereof, or perpurin-7 dimethyl ethyl ester, ferrophorbide A ethyl ester, hydroxy-pheophorbide A ethyl ester, didehydro-rhodochlorine ethylmethyl ester, hydroxy separated therefrom Roxy pheophorbide A methyl ester and pheophorbide A methyl ester showed excellent MCH-1 receptor binding inhibitory activity in MCH-1 receptor binding inhibition experiment (see Tables 1 to 3).

In addition, in the experiment of appetite suppression effect using the upper leaf extract, a fraction thereof or a compound separated therefrom according to the present invention, the upper leaf extract, a fraction or a compound separated therefrom according to the present invention is sustained throughout the experimental period compared to the control group It was found to exhibit a reduction (see Fig. 2 to Fig. 1).

Through this, the leaf extract of the present invention or a compound isolated therefrom antagonizes the MCH-1 receptor, thereby continuously suppressing appetite and controlling mood such as depression or anxiety, such as appetite suppression or depression or anxiety It can be usefully used for the prevention and treatment of mental diseases.

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the above-mentioned leaf extract, fractions thereof, or compounds separated therefrom.

The pharmaceutical composition according to the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, weight agents, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used Or using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which solid preparations comprise at least one excipient such as starch, extracts from the upper leaf extract, fractions thereof, or compounds isolated therefrom. It is quiet by mixing calcium carbonate and gelatin. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized preparations. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

Dosage ranges according to the patient's worm, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and the severity of the disease. Typical daily dosages of the pharmaceutical compositions of the present invention range from 10 to 1000 mg / kg body weight, preferably 10 to 300 mg / kg body weight, based on active ingredient, and purpurin-7 dimethyl Ethyl esters, hydroxy-feophorbide A ethyl esters, pheophorbide A ethyl esters or mixtures thereof range from 1 to 300 mg / kg body weight, preferably 1 to 100 mg / kg body weight, once or several It can be administered in divided doses.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for appetite suppression or for the prevention and treatment of mental disorders such as depression or anxiety. have.

The pharmaceutical composition of the present invention may further include other medicinal herbs or extracts thereof that are pharmaceutically acceptable to enhance or supplement the desired effect, and representative examples of such herbal medicines are Baekbokryeong, Baekchul, Euiin, Red Soybean, Neckache. , A car electron, etc. are mentioned. The herbal medicine may be used in an amount of 0.01 to 50% by weight based on the total weight of the composition.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Formulation Examples.

However, the following Examples, Experimental Examples, and Formulation Examples specifically illustrate the present invention, and the content of the present invention is not limited to Examples, Experimental Examples, and Formulation Examples.

<Example 1> Preparation of the upper leaf extract 1

<1-1> Ethanol Extract of Upper Leaf

After cutting the dried upper leaf leaves 1.8 kg into an extraction container and immersed in 100% ethanol 5 times at room temperature, cooled for one week and filtered through a filter paper. The extract was concentrated under reduced pressure at 50 ° C. until the solution was completely evaporated using a concentrator to obtain 370 g of ethanol extract.

<1-2> Preparation of Water Extract of Upper Leaf

Instead of using ethanol in the manufacturing method of Example <1-1>, hot water was extracted twice at 100 ° C. for 1 hour and then filtered with filter paper. The extract was concentrated under reduced pressure using a concentrator until the solution evaporated completely to obtain 300 g of water extract of the upper leaf.

<1-3> Preparation of Methanol Extract of Upper Leaf

In the manufacturing method of Example <1-1>, the resultant was cooled by using methanol instead of ethanol, and then filtered through a filter paper. The extract was concentrated under reduced pressure using a concentrator until the solution was completely evaporated to give 270 g of methanol extract of the upper leaf.

Example 2 Preparation of Fraction of Ethanol Crude Extract of Upper Leaf 1

<2-1> Preparation of Dichloromethane Fraction

      The upper leaf ethanol extract (370 g) prepared in Example <1-1> was suspended in 2 L of distilled water, extracted with the same amount of dichloromethane, the dichloromethane layer was separated and concentrated under reduced pressure to give the upper dichloromethane. Fraction (59 g) was obtained.

<2-2> Preparation of the hexane fraction of the dichloromethane fraction

The dichloromethane fraction (59 g) prepared in Example <2-1> was suspended in 2 L of 90% methanol, extracted with the same amount of hexane, divided into hexane and methanol layers, and the hexane layer was concentrated under reduced pressure. Hexane fraction (48 g) was obtained.

<2-3> Methanol Fraction of Dichloromethane Fraction

      The hexane layer was separated from the dichloromethane fraction (59 g) prepared in Example <2-2> to obtain the remaining 90% methanol fraction (11 g).

<2-4> Preparation of ethyl acetate fraction

The dichloromethane fraction was removed in the preparation method of Example <2-1>, and the ethyl acetate layer obtained by extracting the remaining water layer with the same amount of ethyl acetate was concentrated under reduced pressure to obtain an ethyl acetate fraction (1 g) of the upper leaf.

<2-5> Preparation of Butanol Fraction

The butanol layer obtained by extracting the ethyl acetate fraction from the preparation method of Example <2-4> and extracting the remaining water layer with the same amount of butanol was concentrated under reduced pressure to obtain a butanol fraction of the upper leaf (36 g).

<2-6> Preparation of Water Fraction

In the manufacturing method of Example <2-5>, butanol fractions were separated and the remaining water layer was lyophilized to obtain a water fraction (270 g).

Example 3 Preparation of Fraction of Methanol Crude Extract of Upper Leaf 1

<3-1> Preparation of Dichloromethane Fraction

      The upper methanol extract (270 g) prepared in Example <1-3> was suspended in 2 L of distilled water, extracted with the same amount of dichloromethane, the dichloromethane layer was separated and concentrated under reduced pressure to obtain a dichloromethane fraction of the upper leaf. (64 g) was obtained.

<3-2> Preparation of the hexane fraction of the dichloromethane fraction

The dichloromethane fraction (64 g) prepared in Example <3-1> was suspended in 2 L of 90% methanol, extracted with the same amount of hexane, divided into hexane and methanol layers, and the hexane layer was concentrated under reduced pressure. Hexane fraction (51 g) was obtained.

<3-3> Methanol Fraction of Dichloromethane Fraction

      The hexane layer was separated from the dichloromethane fraction prepared in Example <3-2> to obtain the remaining 90% methanol fraction (13 g).

<3-4> Preparation of ethyl acetate fraction

The dichloromethane fraction was removed in the method of Example <3-1>, and the ethyl acetate layer obtained by extracting the remaining water layer with the same amount of ethyl acetate was concentrated under reduced pressure to obtain an ethyl acetate fraction (2 g) of the upper leaf.

<3-5> Preparation of Butanol Fraction

In the manufacturing method of Example <3-4>, the ethyl acetate fraction was removed and the remaining water layer was extracted with the same amount of butanol, and the butanol layer was concentrated under reduced pressure to obtain a butanol fraction (35 g) of the upper leaf.

<3-6> Preparation of Water Fraction

In the manufacturing method of Example <3-5>, butanol fractions were separated and the remaining water layer was lyophilized to obtain a water fraction (165 g).

Example 4 Preparation of Active Fraction from Dichloromethane Fraction of Upper Ethanol Crude Extract

Concentration gradient silica gel column chromatography with 59 g of the fraction of Example <2-1> at a flow rate of 3 ml / min using a mixed solvent of n-hexane and ethyl acetate (100: 1 to 1: 1) Photography was carried out and divided into a total of four fractions (first to fourth fractions). The third fraction was obtained as an active fraction.

Example 5 Preparation of Compounds from Upper Leaf Active Fractions

<5-1> Preparation of Compound of Formula 1 from Upper Leaf Active Fraction

The active fractions obtained in Example 4 were separated by Florisil column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1 as a mobile phase, flow rate of 3 ml / min) to obtain nine fractions (3-1). Fractions to 3-9 fractions), and then again subjected to silica gel column chromatography ( n -hexane: ethyl acetate = 15: 1 to 1: 1) for the 3-4 fractions. Purine-7 dimethyl ethyl ester was isolated.

¹ H-NMR (300 MHz, Chloroform-d) δ 9.51 (1H, s, H-10), 9.10 (1H, s, H-5), 8.45 (1H, s, H-20), 7.71 (3H, dd, J = 17.9, 11.4 Hz, H-3 ¹ ), 6.16 (1H, d, J = 17.9 Hz, H-3 ² _a ) 6.02 (1H, d, J = 11.4 Hz, H-3 ² _b ), 4.82 (1H, dd, J = 9.0, 2.0 Hz, H-17), 4.43 (1H, q, J = 7.2 Hz, H-18), 4.26 (3H, s, H-15 ³ ), 4.10 (2H, q, J = 7.1 Hz, H-17 ⁴ ), 4.00 (3H, s, H-13 ² ), 3.64 (3H, s, H-12 ¹ ), 3.49 (2H, q, J = 7.6 Hz, H- 8 ¹ ), 3.28 (3H, s, H-2 ¹ ), 3.01 (3H, s, H-7 ¹ ), 2.44 (1H, m, H-17 ¹ _a ), 2.23 (1H, m, H-7 ¹ _b ), 2.10 (2H, m, H-17 ² ), 1.91 (3H, d, J = 7.2 Hz, H-18 ¹ ), 1.20 (3H, t, J = 7.1 Hz, H-17 ⁵ ), 0.00 (2H, br.s, NH)

¹³ C-NMR (125 MHz, Chloroform-d) δ 186.6 (C-15 ¹ ), 174.5 (C-1), 173.2 (C- 17 ³ ), 168.0 (C-13 ¹ ), 166.7 (C-15 ² ), 164.3 (C-19), 156.2 (C-9), 155.8 (C-6), 149.1 (C-11), 145.7 (C-8), 138.7 (C-14), 136.7 (C-4) , 136.6 (C-16), 136.3 (C-3), 135.8 (C-7), 131.4 (C-2), 129.7 (C-12), 128.7 (C-3 ¹ ), 122.6 (C-3 ² ), 119.5 (C-13), 106.4 (C-10), 106.3 (C-15), 97.7 (C-5), 93.8 (C-20), 60.5 (C-17 ⁵ ), 53.4 (C-13 ² ), 52.8 (C-17), 52.1 (C-15 ³ ), 49.8 (C-18), 32.0 (C-17 ¹ ), 31.4 (C-17 ² ), 23.3 (C-18 ¹ ), 19.4 (C-8 ¹ ), 17.6 (C-8 ² ), 14.3 (C-17 ⁴ ), 13.1 (C-12 ¹ ), 12.0 (C-2 ¹ ), 11.0 (C-7 ¹ )

<5-2> Preparation of Compound of Formula 2 from Upper Leaf Active Fraction

The active fractions obtained in Example <4> were separated by Florisil column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1 as a mobile phase, flow rate 3 ml / min) to obtain nine fractions (third fraction). -1 to 3-9 fractions), and then subjected to silica gel column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1) for the 3-5 fraction again to obtain 22 mg of Chemical Formula 2 Pheophorbide A ethyl ester of was separated.

¹ H-NMR (300 MHz, Chloroform-d) δ 9.42 (1H, s, H-10), 9.26 (1H, s, H-5), 8.54 (1H, s, H-20), 7.91 (3H, dd, J = 17.8, 11.6Hz, H-3 ¹ ), 6.26 (1H, s, H-13 ² ), 6.15 (1H, d, J = 17.8Hz, H-3 ² _a ), 6.11 (1H, d , J = 11.6 Hz, H-3 ² _b ), 4.46 (1H, q, J = 7.2 Hz, H-18), 4.13 (1H, br.d, J = 8.1 Hz, H-17), 4.02 (2H , q, J = 6.9 Hz, H-17 ⁴ ), 3.89 (3H, s, H-13 ⁴ ), 3.66 (3H, s, H-12 ¹ ), 3.54 (2H, q, J = 7.4Hz, H -8 ¹ ), 3.36 (3H, s, H-2 ¹ ), 3.13 (3H, s, H-7 ¹ ), 2.61 (1H, m, H-17 ¹ _a ), 2.43 (1H, m, H- 17 ² _a ), 2.33 (1H, m, H-7 ¹ _b ), 2.24 (1H, m, 17 ² _b ), 1.82 (3H, d, J = 7.2 Hz, H-18 ¹ ), 1.64 (3H, t, J = 6.9 Hz, H-17 ⁵ ), 1.11 (3H, t, J = 7.4 Hz, H-8 ² ), -1.68 (2H, br.s, NH)

¹³ C-NMR (125 MHz, Chloroform-d) δ 189.9 (C-13 ¹ ), 173.2 (C-17 ³ ), 172.4 (C-19), 169.8 (C-13 ³ ), 161.4 (C-16) , 155.8 (C-6), 151.1 (C-14), 149.9 (C-9), 145.3 (C-8), 142.2 (C-1), 138.1 (C-11), 136.7 (C-4), 136.4 (C-7), 136.3 (C-3), 132.0 (C-2), 129.2 (C-3 ¹ ), 129.2 (C-12), 129.1 (C-13), 122.9 (C-3 ² ) , 105.4 (C-15), 104.6 (C-10), 97.7 (C-5), 93.3 (C-20), 64.9 (C-13 ² ), 60.7 (C-17 ⁴ ), 53.1 (C-13 ⁴ ), 51.3 (C-17), 50.3 (C-18), 31.4 (C-17 ¹ ), 30.0 (C-17 ² ), 23.3 (C-18 ¹ ), 19.6 (C-8 ¹ ), 17.6 (C-8 ² ), 14.3 (C-17 ⁵ ), 12.3 (C-12 ¹ ), 12.3 (C-2 ¹ ), 11.4 (C-7 ¹ )

<5-3> Preparation of the compound of formula 3 from the upper leaf active fraction

The active fractions obtained in Example <4> were separated by Florisil column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1 as a mobile phase, flow rate 3 ml / min) to obtain nine fractions (third fraction). -1 to 3-9 fractions), and then again subjected to silica gel column chromatography ( n -hexane: ethyl acetate = 5: 1 to 1: 1) for 3-6 fractions to obtain 130 mg of Chemical Formula 3 Hydroxy-Peophorbide A ethyl ester of was separated.

¹ H-NMR (300 MHz, Chloroform-d) δ 9.51 (1H, s, H-10), 9.34 (1H, s, H-5), 8.63 (1H, s, H-20), 7.92 (3H, dd, J = 17.8, 11.6 Hz, H-3 ¹ ), 6.23 (1H, d, J = 17.8 Hz, H-3 ² _a ) 6.13 (1H, d, J = 11.6 Hz, H-3 ² _b ), 5.54 (0.6H, s, H-13 ² ), 5.34 (0.4H, s, H-13 ² ), 4.69 (0.4H, m, H-17), 4.48 (1H, q, J = 7.0 Hz, H -18), 4.01 (2H, q, J = 7.0 Hz, H-17 ⁴ ), 3.71 (3H, s, H-13 ⁴ ), 3.68 (0.6H, m, H-17), 3.62 (3H, s , H-12 ¹ ), 3.57 (2H, q, J = 7.6 Hz, H-8 ¹ ), 3.39 (3H, s, H-2 ¹ ), 3.13 (3H, s, H-7 ¹ ), 2.89 ( 1H, m, H-17 ¹ _a ), 2.57 (1H, m, H-17 ² _a ), 2.42 (1H, m, H-7 ¹ _b ), 2.01 (1H, m, 17 ² _b ), 1.61 ( 3H, d, J = 7.0 Hz, H-18 ¹ ), 1.61 (3H, t, J = 7.6 Hz, H-8 ² ), 1.17 (3H, t, J = 7.0 Hz, H-17 ⁵ ),- 1.88 (br.s, NH)

¹³ C-NMR (125 MHz, Chloroform-d) δ 192.0 (C-13 ¹ ), 189.9 (C-17 ³ ), 172.8 (C-19), 172.4 (C-13 ³ ), 162.4 (C-16) , 155.5 (C-6), 151.0 (C-14), 150.2 (C-9), 145.1 (C-8), 142.1 (C-1), 137.8 (C-11), 136.4 (C-7), 136.3 (C-3), 136.2 (C-4), 131.9 (C-2), 129.6 (C-3 ¹ ), 129.3 (C-12), 129.3 (C-13), 122.8 (C-3 ² ) , 107.6 (C-15), 104.2 (C-10), 97.9 (C-5), 93.6 (C-20), 60.6 (C-17 ⁴ ), 60.4 (C-13 ² ), 53.8 (C-13 ⁴ ), 51.8 (C-17), 50.3 (C-18), 31.6 (C-17 ¹ ), 29.7 (C-17 ² ), 22.7 (C-18 ¹ ), 19.4 (C-8 ¹ ), 17.4 (C-8 ² ), 14.1 (C-17 ⁵ ), 12.3 (C-12 ¹ ), 12.1 (C-2 ¹ ), 11.2 (C-7 ¹ )

<5-4> Preparation of the compound of formula 4 from the upper leaf active fraction

The active fractions obtained in Example <4> were separated by Florisil column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1 as a mobile phase, flow rate 3 ml / min) to obtain nine fractions (third fraction). -1 to 3-9 fractions), and then subjected to silica gel column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1) for the third fraction to 4 mg of the general formula (4). Didehydro- rodochlorine ethylmethyl ester of was separated.

¹ H-NMR (300 MHz, Chloroform-d) δ 9.86 (1H, s, H-15), 9.72 (1H, s, H-10), 9.59 (1H, s, H-5), 8.76 (1H, s, H-20), 8.05 (3H, dd, J = 18.0, 12.0 Hz, H-3 ¹ ), 6.33 (1H, d, J = 18.0 Hz, H-3 ² _a ) 6.14 (1H, d, J = 12.0 Hz, H-3 ² _b ), 4.54 (1H, q, J = 7.2 Hz, H-18), 4.39 (3H, s, H-13 ² ), 4.14 (2H, q, J = 7.2 Hz, H-17 ⁴ ), 4.13 (1H, br.d, J = 8.1 Hz, H-17), 3.75 (2H, q, J = 7.8 Hz, H-8 ¹ ), 3.66 (3H, s, H-12 ¹ ), 3.36 (3H, s, H-2 ¹ ), 3.13 (3H, s, H-7 ¹ ), 2.79 (1H, m, H-17 ² _a ), 2.63 (1H, m, H-17 ¹ _a ), 2.52 (2H, m, H-17 ² _b ), 2.52 (2H, m, H-17 ¹ _b ), 1.93 (3H, d, J = 7.2 Hz, H-18 ¹ ), 1.73 (3H, t, J = 7.8 Hz, H-8 ² ), 1.22 (3H, t, J = 7.2 Hz, H-17 ⁵ ), 0.14 (1H, br.s, NH)

¹³ C-NMR (125 MHz, chloroform-d) δ 173.7 (C-17 ³ ), 171.5 (C-19), 167.3 (C-13 ¹ ), 166.9 (C-14), 154.4 (C-6), 149.7 (C-9), 145.0 (C-8), 140.2 (C-1), 140.1 (C-11), 137.2 (C-16), 136.3 (C-7), 135.1 (C-3), 134.7 (C-4), 130.5 (C-12), 130.3 (C-2), 129.6 (C-3 ¹ ), 122.0 (C-3 ² ), 119.1 (C-13), 102.2 (C-10), 99.0 (C-5), 96.4 (C-15), 93.1 (C-20), 60.6 (C-17 ⁴ ), 55.0 (C-17), 52.1 (C-13 ² ), 48.8 (C-18) , 32.7 (C-17 ² ), 31.5 (C-17 ¹ ), 23.7 (C-18 ¹ ), 19.8 (C-8 ¹ ), 17.9 (C-8 ² ), 14.4 (C-17 ⁵ ), 13.8 (C-12 ¹ ), 12.3 (C-2 ¹ ), 11.5 (C-7 ¹ )

<5-5> Preparation of the compound of formula 5 from the upper leaf active fraction

The active fractions obtained in Example <4> were separated by Florisil column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1 as a mobile phase, flow rate 3 ml / min) to obtain nine fractions (third fraction). -1 to 3-9 fractions), and then subjected to silica gel column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1) for the third-3 fractions to obtain 140 mg of Chemical Formula 5 Hydroxy pheophorbide A methyl ester of was separated.

¹ H-NMR (300 MHz, Chloroform-d) δ 9.47 (1H, s, H-10), 9.29 (1H, s, H-5), 8.61 (1H, s, H-20), 7.89 (1H, dd, J = 18.0 Hz, 12.0 Hz, H-3 ¹ ), 6.21 (1H, d, J = 18.0 Hz, H-3 ² ), 6.11 (1H, d, J = 12.0 Hz, H-3 ² ), 5.50 (1H, s, H-13 ^{2 /} OH), 4.48 (1H, q, J = 7.2 Hz, H-18), 4.15 (1H, dd, J = 9.0, 2.0 Hz, H-17), 3.70 ( 3H, s, H-12 ¹ ), 3.65 (3H, s, H-13 ⁴ ), 3.62 (3H, s, H-17 ⁴ ), 3.40 (2H, q, J = 7.7 Hz, H-8 ¹ ) , 3.37 (3H, s, H-2 ¹ ), 3.10 (3H, s, H-7 ¹ ), 2.96 (1H, m, H-17 ¹ a), 2.57 (1H, m, H-17 ² a) , 2.34 (1H, m, H-17 ¹ b), 2.27 (1H, m, H-17 ² b), 1.59 (3H, d, J = 7.2 Hz, H-18 ¹ ), 1.59 (2H, t, J = 7.7 Hz, H-8 ² ), -1.91 (2H, br.s, NH)

¹³ C-NMR (125 MHz, Chloroform-d) δ 192.2 (C-13 ¹ ), 174.2 (C-17 ³ ), 173.0 (C-1), 172.6 (C-13 ³ ), 162.6 (C-19) , 155.5 (C-9), 151.2 (C-11), 150.0 (C-16), 145.3 (C-8), 142.2 (C-4), 138.0 (C-14), 136.6 (C-3), 136.4 (C-6), 136.4 (C-7), 131.9 (C-2), 129.5 (C-12), 129.2 (C-3 ¹ ), 127.1 (C-13), 123.0 (C-3 ² ) , 107.9 (C-15), 104.4 (C-10), 98.1 (C-5), 93.8 (C-20), 89.2 (C-13 ² ), 53.6 (C-13 ⁴ ), 52.0 (C-17 ), 51.9 (C-17 ⁴ ), 50.5 (C-18), 31.7 (C-17 ¹ ), 31.4 (C-17 ² ), 22.9 (C-18 ¹ ), 19.6 (C-8 ¹ ), 17.6 (C-8 ² ), 12.5 (C-12 ¹ ), 12.3 (C-2 ¹ ), 11.3 (C-7 ¹ )

<5-6> Preparation of the compound of formula 6 from the upper leaf active fraction

The active fractions obtained in Example <4> were separated by Florisil column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1 as mobile phase, flow rate 3 ml / min), and nine fractions were prepared. 3-1 to 3-9 fractions), and again subjected to silica gel column chromatography ( n -hexane: ethyl acetate = 30: 1 to 1: 1) for the 3-4 fractions. Peophorbide A methyl ester of 6 was isolated.

¹ H-NMR (300 MHz, Chloroform-d) δ 9.51 (1H, s, H-10), 9.37 (1H, s, H-5), 8.56 (1H, s, H-20), 7.97 (3H, dd, J = 17.8, 11.6Hz, H-3 ¹ ), 6.28 (1H, d, J = 17.8Hz, H-3 ² _a ), 6.25 (1H, s, H-13 ² ), 6.13 (1H, d , J = 11.6 Hz, H-3 ² _b ), 4.45 (1H, q, J = 7.2 Hz, H-18), 4.14 (1H, br.d, J = 8.1 Hz, H-17), 3.87 (32H , s, H-17 ⁴ ), 3.71 (3H, s, H-13 ⁴ ), 3.59 (3H, s, H-12 ¹ ), 3.53 (2H, q, J = 7.4 Hz, H-8 ¹ ), 3.39 (3H, s, H-2 ¹ ), 3.20 (3H, s, H-7 ¹ ), 2.61 (1H, m, H-17 ¹ _a ), 2.55 (1H, m, H-17 ² _a ), 2.35 (1H, m, H-7 ¹ _b ), 2.21 (1H, m, 17 ² _b ), 1.80 (3H, d, J = 7.2 Hz, H-18 ¹ ), 1.26 (3H, t, J = 7.4 Hz, H-8 ² ), -1.68 (2H, br.s, NH)

¹³ C-NMR (125 MHz, Chloroform-d) δ 189.8 (C-13 ¹ ), 173.5 (C-17 ³ ), 172.4 (C-19), 169.8 (C-13 ³ ), 161.4 (C-16) , 155.8 (C-6), 151.1 (C-14), 149.8 (C-9), 145.4 (C-8), 142.2 (C-1), 138.1 (C-11), 136.7 (C-4), 136.4 (C-7), 136.3 (C-3), 132.0 (C-2), 129.2 (C-3 ¹ ), 129.2 (C-12), 129.1 (C-13), 123.0 (C-3 ² ) , 105.3 (C-15), 104.6 (C-10), 97.7 (C-5), 93.3 (C-20), 64.9 (C-13 ² ), 53.1 (C-13 ⁴ ), 51.9 (C-17 ⁴ ), 51.3 (C-17), 50.3 (C-18), 31.2 (C-17 ¹ ), 30.0 (C-17 ² ), 23.3 (C-18 ¹ ), 19.6 (C-8 ¹ ), 17.6 (C-8 ² ), 12.3 (C-12 ¹ ), 12.3 (C-2 ¹ ), 11.4 (C-7 ¹ )

Example 6 Preparation of Upper Leaf Extract 2

<6-1> Preparation of Ethanol Extract of Upper Leaf

After cutting the dried upper leaf, 100g was put in the extraction container and refluxed twice at 85 ° C. twice at 4 ° C. using 8 times the amount of 100% ethanol. The extract was filtered through filter paper. The extract was concentrated under reduced pressure using a concentrator until the solution evaporated completely to obtain 12.4 g of ethanol extract (WI-01).

<6-2> Preparation of 70% Ethanol Extract of Upper Leaf

28.6 g of 70% ethanol extract of the upper leaves was obtained in the same manner as in the preparation method of Example <6-1>, except that 10% of 70% ethanol was used instead of 100% ethanol (WI-07).

<6-3> Preparation of 50% Ethanol Extract of Upper Leaf

In the manufacturing method of Example <6-1>, using the same method as 50g and 50% ethanol instead of 100% ethanol to obtain 13.7g of 50% ethanol extract of the upper leaf in the same manner (WI-13).

<6-4> Preparation of Butanol Extract of Upper Leaf

2.9 g of butanol extract of the upper leaves was obtained by the same method except that 50 g of the upper leaves were used in the preparation method of Example <6-1> and butanol instead of 100% ethanol (WI-16).

<6-5> Preparation of Dichloromethane Extract of Upper Leaf-1

50 g of the dried leaf of the upper leaf was cut into an extraction container, and extracted by refluxing twice at 40 ° C. twice at 4 ° C. using 8 times dichloromethane. The extract was filtered through filter paper. The extract was concentrated under reduced pressure using a concentrator until the solution evaporated completely to obtain 1.6 g of dichloromethane extract (WI-19).

<6-6> Preparation of Dichloromethane Extract of Upper Leaf-2

After cutting the dried upper leaf leaves 50g was put in the extraction container and extracted by dipping twice at room temperature for 24 hours using 8 times the amount of dichloromethane. The extract was filtered through filter paper. The extract was concentrated under reduced pressure using a concentrator until the solution was completely evaporated to obtain 1 g of dichloromethane extract (WI-20).

Example 7 Preparation of Fraction of Ethanol Crude Extract of Upper Leaf 2

<7-1> Preparation of Dichloromethane Fraction

The upper leaf ethanol extract (12.4 g) prepared in Example <6-1> was suspended in 110 ml of distilled water, extracted with the same amount of dichloromethane, the dichloromethane layer was separated and concentrated under reduced pressure to obtain a dichloromethane fraction ( 2.9 g) was obtained (WI-02).

<7-2> Preparation of the hexane fraction of the dichloromethane fraction

The dichloromethane fraction (2.9g) prepared in Example <7-1> was suspended in 45 ml of 80% methanol, extracted with the same amount of hexane, divided into a hexane layer and a methanol layer, and the hexane layer was concentrated under reduced pressure. Hexane fraction (1.87 g) was obtained (WI-05).

<7-3> Methanol Fraction of Dichloromethane Fraction

The hexane layer was separated from the dichloromethane fraction (2.9g) prepared in Example <7-2> to obtain the remaining 80% methanol fraction (0.85g) (WI-06).

<7-4> Preparation of Butanol Fraction

The dichloromethane fraction was removed in the preparation method of Example <7-1>, and the butanol layer obtained by extracting the remaining water layer with the same amount of saturated butanol was concentrated under reduced pressure to obtain a butanol fraction (1.46 g) of the upper leaf. -03).

<7-5> Preparation of Water Fraction

In the manufacturing method of Example <7-4>, butanol fraction was removed and the remaining water layer was dried to obtain a water fraction (7.3 g) (WI-04).

Example 8 Preparation of Fractions of 70% Ethanol Crude Extracts from Upper Leaves

<8-1> Preparation of Hexane Fraction

The upper leaf 70% ethanol extract (28.6 g) prepared in Example <6-2> was suspended in 290 ml of distilled water, extracted with the same amount of hexane, the hexane layer was separated and concentrated under reduced pressure, and the hexane fraction of the upper leaf (0.59 g). ) Was obtained (WI-08).

<8-2> Preparation of Dichloromethane Fraction

The dichloromethane layer obtained by removing the hexane fraction from the method of Example <8-1> and extracting the remaining water layer with the same amount of dichloromethane was concentrated under reduced pressure to obtain a dichloromethane fraction of the upper leaf (1.7 g). (WI-09).

<8-3> Preparation of the ethyl acetate fraction

In the preparation method of Example <8-2>, the ethyl acetate layer obtained by removing the dichloromethane fraction and extracting the remaining water layer with the same amount of ethyl acetate was concentrated under reduced pressure to obtain an ethyl acetate fraction (0.98 g) of the upper leaf. -10).

<8-4> Butanol fraction

The butanol layer obtained by removing the ethyl acetate fraction from the preparation method of Example <8-3> and extracting the remaining water layer with the same amount of saturated butanol was concentrated under reduced pressure to obtain a butanol fraction (2.95 g) of the upper leaf. 11).

<8-5> Preparation of Water Fraction

The butanol fraction was separated from the preparation method of Example <8-4> and the remaining water layer was dried to obtain a water fraction (16.9 g) (WI-12).

Example 9 Preparation of Fraction of 50% Ethanol Crude Extract of Upper Leaf

<9-1> Preparation of Butanol Fraction

The upper leaf 50% ethanol extract (13.7 g) prepared in Example <6-3> was suspended in 15 ml of distilled water, extracted with the same amount of saturated butanol, the butanol layer was separated and concentrated under reduced pressure to obtain a butanol fraction of the upper leaf ( 1.85 g) was obtained (WI-14).

<9-2> Preparation of Water Fraction

In the manufacturing method of Example <9-1>, butanol fractions were separated and the remaining water layer was dried to obtain a water fraction (11.5 g) (WI-15).

Example 10 Preparation of Fraction of Butanol Crude Extract of Upper Leaf

<10-1> Preparation of Hexane Fraction

The upper leaf butanol extract (2.9 g) prepared in Example <6-4> was suspended in 50 ml of 80% methanol, extracted with the same amount of hexane, the hexane layer was separated and concentrated under reduced pressure to obtain the hexane fraction of the upper leaf (0.99). g) was obtained (WI-17).

<10-2> Preparation of 80% Methanol Fraction

The hexane fraction was separated in Example 10-1 and the remaining 80% methanol layer was concentrated under reduced pressure to obtain an 80% methanol fraction (1.67 g) of the upper leaf (WI-18).

Experimental Example 1 Measurement of MCH-1 Receptor Inhibition Effect

In order to determine the inhibitory activity of the leaf extract, fractions or active substances of the present invention MCH-1 receptor according to the present invention was carried out as follows.

The buffer solution is prepared by two types of washing solution (25 mM HEPES pH 7.4, 5 mM MgCl ₂ , 1 mM CaCl ₂ ) and experimental solution (adding 0.5% BSA to the washing solution), and MCH-1 receptor (Euroscreen, Gosselies). , Belgium) and 1 μM europium labeled melanin enriched hormone (Europium (Eu) -labeled MCH, PerkinElmer, Turku, Finland) and 1 mM melanin enriched hormone (MCH, # 070-47, Phoenix, Belmont CA, USA) Was kept at 4 ° C. 1 μM Eu-labeled MCH and 1 mM MCH were diluted to 8 nM (final reaction concentration 2 nM) and 2 μM (final reaction concentration 0.5 μM), respectively. The buffer used for all dilutions and preparations was the experimental solution, and the wash solution was used only when the plate was washed last.

MCH-1 receptor (200 assays / vial) was first homogenized by diluting in 1 ml of experimental solution. Then, after preparing the extract of the upper leaf, the upper leaf fraction or a compound separated therefrom prepared in Examples 1 to 3 and 5 to 10 as a test material, the filter paper is attached to the microplate (Multiwell 96 well filter plates PN5020, Pall Co. Ann Arbor MI, USA) using the 8-channel pipette (multi 8-channel, Eppendorf, Hamburg, Germany), so that the total volume of each well to 100 μl prepared in Examples 1 to 3 and 5 to 10 , Leaf extract, leaf fraction or compounds isolated therefrom were dispensed. In this case, non-specific binding is 25 μl of Eu-labeled MCH and 50 μl of receptor and 25 μl of MCH, and total binding is 25 μl of 10% DMSO experimental solution and 25 μl of Eu-labeled MCH and 50 μl of receptor was mixed. Finally, the experimental group mixed 25 μl of test substance with 25 μl of Eu-labeled MCH and 50 μl of receptor. After all of this was shaken gently for 15 seconds and reacted at room temperature for 90 minutes.

At the end of the reaction, the plate was partially cleaned by applying pressure to a self-made washer (microplate washer, EMBLA, Molecular Devices). The wash solution was filtered three times at 300 μl per well. This process removes the remaining Eu-labeled MCH without reacting. Then, the bottom of the water was wiped off and the dissociation solution (DELFIA Enhancement solution, PerkinElmer, Turku, Finland) was added to 150 μl per well. After shaking for 10 minutes or measured, or reacted for 2 to 4 hours as it is at room temperature to measure the time-resolved fluorescence (TRF) value. The emission wavelength was 615 nm and the excitation wavelength was 340 nm, and measured by a multi-function fluorimeter (multilabel counter, Victor2, PerkinElmer, Turku, Finland). Differential fluorescence inhibition was calculated by the following equation.

% Differential Fluorescence Inhibition = [(Total Binding Average Value-Test Material's Differential Fluorescence Value) / (Total Binding Average Value-Nonspecific Binding Average Value)] × 100

After primarily measuring the differential fluorescence inhibition at 10 μM, the IC ₅₀ value was calculated only for test substances inhibited by 50% or more. For the test substances inhibited by more than 50%, the differential fluorescence values were obtained at four concentrations of 0.01, 0.1, 1.0, and 10.0 μM, respectively, and IC ₅₀ values were calculated through linear regression analysis. The results are shown in Tables 1 to 3. .

sample IC ₅₀ (Μg / ml) Example 1-1 Ethanol Extract of Upper Leaf 2.28 ± 1.04 1-2 Water Extract of Upper Leaf > 30 2-1 Ethanol Extract of Upper Leaf → Dichloromethane Fraction 1.57 ± 0.77 2-2 Ethanol Extract of Upper Leaf → Dichloromethane Fraction → Hexane Fraction 17.12 ± 9.89 2-3 Ethanol Extract of Upper Leaf → Dichloromethane Fraction → Methanol Fraction 0.97 ± 0.46 2-4 Ethanol Extract of Upper Leaf → Ethyl Acetate Fraction 6.67 ± 1.79 2-5 Ethanol Extract of Upper Leaf → Butanol Fraction > 30 2-6 Ethanol Extract of Upper Leaf → Water Fraction > 30 1-3 Methanol Extract of Upper Leaf 3.98 ± 1.02 3-1 Methanol Extract of Upper Leaf → Dichloromethane Fraction 2.42 ± 0.89 3-4 Methanol Extract of Upper Leaf → Ethyl Acetate Fraction 6.34 ± 2.33 3-5 Methanol Extract of Upper Leaf → Butanol Fraction > 30 3-6 Methanol Extract of Upper Leaf → Water Fraction > 30

sample IC ₅₀ (Μg / ml) Example 7-1 100% ethanol extract of the upper leaf → dichloromethane fraction 4.18 ± 1.16 7-3 100% ethanol extract of the upper leaf → dichloromethane fraction → methanol fraction 1.34 ± 0.26 8-1 70% ethanol extract of the upper leaf → hexane fraction 0.89 ± 0.09 8-2 70% ethanol extract of the upper leaves → dichloromethane fraction 1.04 ± 0.36 8-3 70% ethanol extract of the upper leaves → ethyl acetate fraction 2.29 ± 0.50 9-1 50% ethanol extract of the upper leaf → butanol fraction 2.45 ± 0.86 10-1 100% butanol extract of the upper leaf → hexane fraction 3.70 ± 0.58 6-5 100% dichloromethane extract of reflux (reflux extraction) 8.44 ± 2.74 6-6 100% dichloromethane extract of cold leaf (cold extraction) 5.49 ± 1.15

sample IC ₅₀ (Μg / ml) Example 5-1 Perpurin-7 dimethyl ethyl ester 0.26 ± 0.11 5-2 Pheophorbide A ethyl ester 0.24 ± 0.08 5-3 Hydroxy-Peophorbide A Ethyl Ester 0.11 ± 0.02 5-6 Pheophorbide A Methyl Ester 4.03 ± 2.48 5-5 Hydroxy-Peophorbide A Methyl Ester 0.33 ± 0.29 5-4 Didehydro-Rodochlorine Ethyl Methyl Ester 14.94 ± 4.24

As shown in Table 1, the IC ₅₀ value for the MCH-1 receptor binding inhibitory activity of the upper leaf ethanol extract, and the dichloromethane fraction and ethyl acetate fraction thereof according to the present invention is excellent MCH by showing 0.97 ~ 6.67 ㎍ / ml It can be seen that -1 receptor binding inhibitory action. On the other hand, butanol fraction and water fraction of water extract, ethanol extract showed little MCH-1 receptor binding inhibitory activity (IC ₅₀ > 30 ㎍ / ml). The IC ₅₀ values of MCH-1 receptor binding inhibitory activity of the methanol extract of the upper leaf, and its dichloromethane fractions and ethyl acetate fractions ranged from 2.42 to 6.34 ㎍ / ml, but the butanol fraction and water fraction inhibited MCH-1 receptor binding. It showed little action (IC ₅₀ > 30 ㎍ / ml), showing a similar pattern to that of the ethanol extract of the lobe.

As shown in Table 2, the upper leaf ethanol extract (50-100% ethanol), and dichloromethane fraction, methanol fraction, hexane fraction and ethyl acetate fraction, butanol extract, and hexane fraction, dichloromethane thereof in the present invention IC ₅₀ value for MCH-1 receptor binding inhibitory activity of the extracts (reflux extraction, cold extraction) is 0.89 ~ 8.44 ㎍ / ml it can be seen that the excellent MCH-1 receptor binding inhibitory action. On the other hand, butanol fraction and water fraction of ethanol extract (70, 100% ethanol extract) showed little inhibition of MCH-1 receptor binding activity (IC ₅₀ > 30 ㎍ / ml).

As shown in Table 3, the IC ₅₀ value for the MCH-1 receptor binding inhibitory activity of the compound isolated from the leaf extract of the present invention is 0.11 ~ 14.94 ㎍ / ml, showing an excellent MCH-1 receptor binding inhibitory effect It can be seen. In particular, compounds such as Perpurin-7 dimethyl ethyl ester, pheophorbide A ethyl ester, hydroxy-phenophorbide A ethyl ester, and hydroxy- pheophorbide A methyl ester are about 10 times stronger than the extract effect. appear.

Therefore, the upper leaf extract according to the present invention or the compound isolated therefrom exhibits excellent MCH-1 receptor binding inhibitory effect, so as a pharmaceutical composition for suppressing appetite, or a pharmaceutical composition for preventing and treating mental diseases such as depression or anxiety. It can be usefully used as.

Experimental Example 2 Measurement of Appetite Suppression Effect

In order to determine the appetite suppression effect of the leaf extract, fractions or active substances thereof according to the present invention was carried out the following experiment.

The subject was a diet-induced obese mouse model. The diet-induced obese mouse model was fed a high-fat diet (60% fat in total calories: D12492. Research diet) for 9 weeks from 5 to 14 weeks of age, and then the control group (6.5% fat in total calories: Purina) A model of obesity similar to severe obesity in humans was completed, with significant weight gain (more than 25%) compared to Rodent chow 5008 (Ralston-purina). The mice used were Jacson lab, USA. C57BL / 6J mice were purchased at 4 weeks of age and were used from 5 weeks of age after one week of adaptation. The mean weight of 5 weeks old before the experiment was 18.7 ㅁ 0.88 g, and the mean weight of 14 weeks of age was 37.0 ㅁ 0.2 g (64 animals), which was significantly different from the average body weight of 29.6 ㅁ 0.5 g (9 animals). Seemed. The number of dietary-induced obese mice in each group was 10-12. The diet was maintained as a high fat diet (60% fat-containing diet of total calories: D12492. Research diet), and the extract of the upper leaf prepared in Example 1 was dissolved in 0.5% tween 80 twice a day 100, 250 , 500 mg / kg orally, and changes in feed intake were measured at 32-day 2-day intervals. The temperature of the feeding room was maintained at 25 ㅁ 2 ℃ and the day and night cycles were crossed every 12 hours, water and high-fat diet was free to eat. The control group was orally administered with only 0.5% tween 80 solution, and the control group was treated with oral sibutramin 3 mg / kg. Feed intake of the mice in each group was measured daily for 32 days.

In addition, animal experiments were performed on the methanol fraction of the dichloromethane fraction of Example <2-3> having the strongest inhibitory effect on the MCH-1 receptor among the fractions of the leaf extract. In the same manner as the animal test method described above, the methanol fraction of the dichloromethane fraction of the upper ethanol extract of Example <2-3> was dissolved in 0.5% tween 80 to 250 mg / kg twice daily Oral administration was performed, and feed intake changes were measured at 2 days intervals for 32 days. At this time, only 0.5% tween 80 solution was orally administered as a control group. Feed intake of the mice in each group was measured daily for 32 days.

The measurement results are shown in FIGS . 1 and 2 .

1 is a graph showing the change in feed intake of the mouse for administration of the extract of the upper lobe of Example 1 according to the present invention. As shown in Figure 1 , the feed intake was reduced in all the administration group administered 100, 250, 500 mg / kg of the upper leaf extract of Example 1 according to the present invention, it was observed to show a sustained appetite decrease throughout the experimental period. On the other hand, the sibutramine-administered group showed a sharp decrease in feed intake, and then increased rapidly.

Figure 2 is a graph showing the change in feed intake of the mouse to the methanol fraction of the dichloromethane fraction of the upper leaf extract of Example <2-3> according to the present invention. As shown in Fig. 2 , the feed intake was reduced in the administration group to which the methanol fraction of the dichloromethane fraction of the upper leaf extract of Example <2-3> according to the present invention was administered, and thus decreased appetite continuously throughout the experiment period. Was observed.

Therefore, the upper leaf extract and fractions thereof according to the present invention induces appetite lowering can be useful as a pharmaceutical composition for various diseases that require a decrease in appetite, such as polyphagia as an appetite suppressant.

Examples of preparations for the compositions of the present invention are illustrated below.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> powder

2 g of leaf extract of Example 1

1 g lactose

The powder was prepared by mixing the above components and filling the airtight cloth according to a conventional powder preparation method.

<1-2> tablet

100 mg of the upper leaf extract of Example 1

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

The tablets were prepared by mixing the components and then tableting according to a conventional tablet production method.

<1-3> capsules

100 mg of the upper leaf extract of Example 1

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

After mixing the following components to prepare a capsule by filling in gelatin capsules according to the conventional capsule preparation method.

<1-4> injection

100 mg of the upper leaf extract of Example 1

Suitable amount of distilled water for injection

pH adjuster

Injectables were prepared by dissolving the active substance in distilled water for injection and adjusting the pH to about 7.5 according to a conventional injection method, and then filling the 2 ml volume of the ampoule with distilled water for injection and sterilizing with the following remaining ingredients.

1 is a graph showing a change in the feeding amount of dietary-induced obese mice according to the dosage of the upper leaf ethanol extract according to the embodiment <1-1> of the present invention.

FIG. 2 is a graph showing the change in dietary induction of dietary-induced obese mice for the administration of methanol fraction of the dichloromethane fraction of the upper leaf ethanol extract according to the embodiment <2-3> of the present invention.

Claims (13)

After extracting with alcohol as a solvent, blowing off alcohol and suspending distilled water in the extract residue, the fraction obtained by fractionating with any one organic solvent selected from the group consisting of dichloromethane, butanol and hexane is effective. Containing as a component, containing any one of the following compounds 1 to 6, and exhibits antagonistic activity against melanin Concentration Hormone receptor subtype-1 (MCH-1) Pharmaceutical compositions for the prevention and treatment of depression, or the prevention and treatment of anxiety: [Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

. The pharmaceutical composition of claim 1, wherein the organic solvent is dichloromethane. The pharmaceutical composition of claim 1, wherein the alcohol is methanol or ethanol. The pharmaceutical composition of claim 1, wherein the alcohol is 50% to 100% alcohol. The dichloromethane fraction obtained by suspending the upper leaf extract in distilled water and then distilling with dichloromethane is suspended in methanol. A pharmaceutical composition comprising as an active ingredient. The dichloromethane fraction obtained by suspending the upper leaf extract in distilled water and distilling with dichloromethane is a mobile phase of a mixed solvent (100: 1 to 1: 1) of n-hexane and ethyl acetate. Pharmaceutical composition, characterized in that the active fraction obtained by performing a concentration gradient silica gel column chromatography using. The pharmaceutical composition according to claim 6, wherein the upper fraction has an antagonistic activity against Melanin Concentration Hormone receptor subtype-1 (MCH-1). A pharmaceutical composition for suppressing appetite, preventing and treating depression, or preventing and treating anxiety, containing the compound of any one of Formulas 1 to 6 or a mixture thereof as an active ingredient: [Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

. According to claim 8, wherein the compound of Formula 1 to 6 Adding alcohol to the upper leaf to obtain an upper leaf extract (step 1); Suspending the upper leaf extract obtained in step 1 in water and fractionating with dichloromethane to obtain a dichloromethane fraction (step 2); Dichloromethane fractions obtained in step 2 were subjected to concentration gradient column chromatography using a mixed solvent of n-hexane and ethyl acetate as a mobile phase to obtain an active component fraction (step 3); Separating the active fraction from the active fraction obtained in step 3 by performing concentration gradient column chromatography again using a mixed solvent of n-hexane and ethyl acetate as a mobile phase (step 4); And The fraction separated in step 4 is a mixed solvent of n-hexane and ethyl acetate as a mobile phase, but performing a silica gel column chromatography with different concentration gradients to separate the compound of formula 1-6 (step 5) A pharmaceutical composition, characterized in that it is prepared by a method comprising. The pharmaceutical composition of claim 9, wherein the alcohol of step 1 is methanol or ethanol. The pharmaceutical composition of claim 9 or 10, wherein step 3 is performed by performing silica gel column chromatography using a concentration gradient of a mixed solvent of n-hexane and ethyl acetate as a mobile phase of 100: 1 to 1: 1. Composition. The pharmaceutical composition of claim 9 or 10, wherein the concentration gradient of step 5 is 15: 1 to 1: 1, 30: 1 to 1: 1, and 5: 1 to 1: 1, respectively. The method of claim 8, wherein the compound of any one of Formulas 1 to 6 or mixtures thereof exhibits antagonistic activity against Melanin Concentration Hormone receptor subtype-1 (MCH-1). Pharmaceutical compositions.

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