KR101992808B1 - Novel compound from plasma-treated phloridzin and anti-obesity composition comprising the same as effective component - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a novel compound derived from plasma-treated florixin and an anti-obesity composition containing the same as an active ingredient, and more particularly, to a novel compound derived from a florirazine obtained by treating florazin with plasma, , Diglycosylmethylenebisfloridine and methylenebisflorethine) and an antiobesity composition containing the same as an active ingredient, wherein the floririne-derived compound inhibits the activity of pancreatic lipase as compared to floridine , The effect of inhibiting fat accumulation during differentiation of adipocytes is enhanced. In particular, the methylene bisphrheletin of the present invention, in which floretine, which is a form in which floridine is eliminated from sugar, forms a dimer by a methyl group, Therefore, it is useful as an anti-obesity therapeutic agent or an anti-obesity health functional food material. It can be used.

Description

TECHNICAL FIELD [0001] The present invention relates to novel compounds derived from plasma-treated florins and antiobesity compositions containing the same as active ingredients.

The present invention relates to a novel compound derived from plasma-treated florixin and an antiobesity composition containing the compound as an active ingredient, and more particularly to a floririzin dimer compound produced by treating florazin with plasma, To an anti-obesity composition.

Obesity is a state of excessive accumulation of adipose tissue in the body. Obesity usually causes a lot of weight. However, even if not obese, even people with a lot of muscles can lose a lot of weight. do. Excessive intake of nutrients over a long period of time in relation to energy consumption induces obesity by energy imbalance, and in addition is associated with changes in hormones, genetic, mental health problems, and socioeconomic factors. As the global obesity population is increasing, the importance of obesity has become an important issue.

In order to prevent and improve obesity and to maintain a healthy weight, a balanced diet and regular exercise are needed to maintain the lifelong lifetime. In particular, fat cells can be reduced in size once they are made, but it is impossible to remove them naturally and it is left permanently in the human body, so it is important to prevent obesity from the beginning.

Methods for reducing calories consumed, increasing activity and exercise, and taking medications such as reducing fat absorption for the prevention, improvement, or treatment of obesity have been used.

Current treatments for obesity include fenfluramine, which inhibits the serotonin nervous system, ephedrine and caffeine through the noradrenergic nervous system, sibutramine, which simultaneously acts on the serotonin and noradrenergic nervous system, and olyrie, which inhibits the lipase produced by the pancreas, There are drugs such as start. However, pfuramilamine has been prohibited because of its side effects such as primary pulmonary hypertension and heart valve lesion, and other drugs have problems such as decreased blood pressure and lactic acidosis, and are used for patients with heart failure and renal disease There is a problem that can not be done. Therefore, there is a need to prevent, ameliorate, or develop a therapeutic substance for obesity with a low side effect.

On the other hand, phloridzin is a chalcone glycoside compound in which glucose is bound to phloretin, which is contained in shells and seeds of non-edible ginseng rather than apple flesh. It is small. Therefore, if a new natural bioactive substance is developed from abandoned non-edible parts using various bioconversion methods, it is possible to save abandoned raw materials, prevent environmental problems, and become a new source of income.

On the other hand, in the ionized plasma, there exist active oxygen species including electrons, cations, anions, free radicals, and photons. In recent years, various technologies have been developed to minimize the problems of existing food disinfection technology, and non-thermal plasma processing technology has attracted attention. Nonthermal plasma technology is being developed as a disinfection and disinfection technology in environment and medical field besides the technology to inhibit microorganisms of food. However, research on the chemical and functional change of food ingredient is still insufficient.

On the other hand, Korean Patent No. 1492277 discloses a florigen analogue as an inhibitor of sodium glucose co-transporter 2. However, the novel compound derived from the plasma-treated floririn of the present invention and the anti-obesity composition containing it as an active ingredient It has not been disclosed.

Disclosure of the Invention The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a method for producing a plasma-derived dimer compound (methylenebisphloridin, diglucosylmethylenebisphloridine, methylenebisfloretin) Which comprises the novel compound as an active ingredient, and that the composition inhibits the activity of pancreatic lipase and inhibits the fat accumulation, thereby completing the present invention.

In order to solve the above-mentioned problems, the present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

또는 수소(H)이다.Wherein R < ¹ > and R < ² &

Or hydrogen (H).

In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity containing the above-mentioned compound as an active ingredient.

The present invention also provides a health functional food composition for preventing or ameliorating obesity containing the above-mentioned compound as an active ingredient.

The present invention relates to a novel compound derived from plasma-treated florixin and an antiobesity composition containing the same as an effective ingredient. The present invention relates to a novel compound derived from plasma-treated floririne and a method of treating the florisin with plasma to prepare a dimeric compound derived from three kinds of florirines (methylene bis- (Methylenebisfloridine, methylenebisflorin, methylenebisfloridine, methylenebisflorin and methylenebisflorethine). These compounds inhibit the activity of pancreatic lipase and inhibit fat accumulation during the differentiation of adipocytes, The methylene bis floretine of the present invention in which floretine, a sugar-removed form of floridin, forms a duplex by a methyl group, is superior to floretin in its effect of inhibiting fat accumulation, and thus is useful as a pharmaceutical composition for preventing or treating obesity, Or as a health functional food composition for prevention or improvement.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the results of the HMBC performance of a novel compound of the present invention, methylenebisphloridzin (A), deglucosylmethylenebisphloridzin (B), and methylenebisphloretin (C) As a result, the relationship between hydrogen and carbon is shown.
Fig. 2 shows the result of confirming the fat accumulation inhibitory effect by the novel compound of the present invention. MDI (a mixture of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin) was used as a differentiation inducing substance to induce the differentiation of 3T3-L1 as a lipogenic precursor cell. Control was a negative control without compound treatment, 1 was treated with floridine, 2 was treated with methylenebisphloridzin compound, 3 was treated with deglucosylmethylenebisphloridzin compound 4 is a group treated with a methylenebisphloretin compound, and 5 is a group treated with a floretine. * Indicates a significant decrease in adipocyte differentiation compared to control group treated with MDI alone, which means that p <0.05.

The present invention relates to a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

또는 수소(H)이다.Wherein R &lt; ¹ &gt; and R &lt; ² &

Or hydrogen (H).

인 메틸렌비스플로리진(methylenebisphloridzin) 화합물일 수 있으나, 이에 제한하지 않는다. In one embodiment of the present invention, R &lt; ¹ &gt; and R &lt; ² &

But are not limited to, methylenebisphloridzin compounds.

이고, R²는 수소(H)인 디글루코실메틸렌비스플로리진(deglucoslymethylenebisphloridzin) 화합물일 수 있으나, 이에 제한되지 않는다.In one embodiment of the invention, R &lt; ^{1 &gt;} is

And R ² is hydrogen (H), but it is not limited thereto.

In one embodiment of the present invention, R &lt; ¹ &gt; and R &lt; ² &gt; may be methylenebisphloretin compounds which are hydrogen (H), but are not limited thereto.

In one embodiment of the present invention, the compound may be prepared by treating the plasma with a floridine, wherein the plasma may be a dielectric barrier discharge, a corona discharge, a microwave discharge or an arc discharge plasma, In the manner of dielectric barrier discharge, for 30 to 80 minutes at a frequency of 200 to 300 W power and 10 to 20 kHz frequency, preferably at a 250 W power and 15 kHz frequency in a manner of dielectric barrier discharge But is not limited to, treating for 40 to 60 minutes, and most preferably for 60 minutes at Floritz in 250 W power and 15 KHz frequency conditions in the manner of dielectric barrier discharge.

The present invention also relates to a pharmaceutical composition for preventing or treating obesity, which comprises the above compound as an active ingredient.

The plasma-treated novel compound derived from Florisin inhibits the activity of pancreatic lipase and inhibits fat accumulation upon differentiation of adipocytes, but is not limited thereto.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the active ingredient. Such a carrier is usually used at the time of formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, Calcium carbonate, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals Oils, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components.

The appropriate dosage of the pharmaceutical composition according to the present invention may vary depending on such factors as the formulation method, the administration method, the patient's age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, .

The pharmaceutical composition of the present invention may be administered orally or parenterally. In the case of parenteral administration, the composition may be administered topically to the skin, intravenously, subcutaneously, intramuscularly, intraperitoneally, or transdermally.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the inhibition and treatment of obesity.

The concentration of the active ingredient contained in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.

The pharmaceutical composition of the present invention may be manufactured in a unit dosage form by formulating it using a pharmaceutically acceptable carrier or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs Into a capacity container. The formulations may be formulated into any one of the formulations selected from injectables, creams, patches, sprays, ointments, alerts, lotions, liniments, pastes and cataplasms, and may additionally contain dispersing or stabilizing agents have.

The present invention also relates to a health functional food composition for preventing or ameliorating obesity containing the above-mentioned compound as an active ingredient.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient can be suitably used depending on its use purpose (prevention or improvement). Generally, the health functional food composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight based on the raw material, when the food or beverage is produced. However, in the case of long-term consumption intended for health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount of more than the above range.

There is no particular limitation on the kind of the health functional food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when it is made of a drink, it may contain, in addition to the active ingredient, a natural carbohydrate or a flavoring agent as an additional ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, , Xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the health functional food composition, it is also possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. Although the ratio of the above-mentioned ingredients is not critical, it is generally selected in the range of 0.01 to 0.1 part by weight based on 100 parts by weight of the health functional food composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  One. Plasma  Treated Florian  Preparation and Separation of Derived New Compounds

A dielectric barrier discharge (DBD) (250 W, 15 kHz, ambient air) plasma device (240 × 200 × 100 mm) was used for the plasma treatment. 1.0 g of floridine was dissolved in 7.0 L of methanol (MeOH), treated with DBD plasma apparatus for 20, 40 and 60 minutes, and the formation of the new substance was monitored by reversed phase high performance liquid chromatography (HPLC).

 1% formic acid (A) and acetonitrile (B) were used as mobile phase solvents for HPLC analysis. Material analysis was carried out for 5 to 10 minutes with 85% (v / v) of A solvent and 5% (v / v) With a B solvent composition of 15% (v / v), a solvent composition of 70% (v / v) A solvent and 30% (v / v) B for 10-15 minutes, The solvent was analyzed with a solvent composition of 100% (v / v) A solvent and 50% (v / v) B solvent for 20-30 minutes, 1.0 ml was maintained and the compound was detected at a wavelength of 280 nm. The column for HPLC analysis was a YMC-Pack ODS A-302 column (4.6 mm id x 150 mm; YMC Co., Ltd., Tokyo, Japan).

When the plasma was treated for 60 minutes, it was confirmed that various new materials were produced. After the plasma was treated for 60 minutes, the produced flurbine reactant was dried and dissolved in methanol (MeOH), and then YMC GEL ODS AQ 120- 50S column (2.5㎝ id × 40㎝) compound a (100.8㎎, t _R 16.6 min) through a step-by-step gradient, compound B (76.1㎎, t _R 19.4 min) and compound C (2.3㎎, t _R 22.6 min) on and the compound D (38.5㎎, t _R 16.9 min) was isolated. Compound D was a previously reported floretine (J Nat Prod (1980), 43, 739-751).

Example  2. Plasma  Treated Florian  Structural analysis of the resulting new compounds

The structures of the compounds A, B, and C isolated in Example 1 were analyzed.

New compound A: amorphous amorphous powder. [a] ²⁰ _D -18.1 [deg.] ( c 0.1, MeOH); UV? _Max MeOH nm (log?): 203 (4.10), 226 (4.01), 288 (3.83); _{^{1 H NMR (CD 3 OD,}} 600MHz): δ7.04 (4H, d, J = 8.4Hz, H-2, -2a, -6, -6a), 6.67 (4H, d, J = 8.4Hz, H -3, -5, -5a), 6.26 (2H, s, H-3 ', -3a'), 5.03 (2H, d, J = 7.8Hz, H- 3.88 (2H, dd, J = 2.4, 12.0Hz, H-6 ", -6a"), 3.79 (2H, s, H-CH 2), 3.68 (2H, dd, J = 6.0, 12.0Hz, H- (2H, m, H), 3.70 (2H, m, H-2 ", -2a"), 3.50 (4H, t, J = 7.8Hz, , 3.38 (2H, m, H-5 ", -5a"), 2.87 (4H, t, J = 7.8 Hz, H-7, 7a); _{^{13 C NMR (CD 3 OD,}} 150MHz): δ207.2 (C-9, -9a), 164.0 (C-4 ', -4a'), 163.6 (C-6 ', -6a'), 160.4 (C -2 ', -2a'), 156.4 (C-4, -4a), 133.8 (C-1, -1a), 130.4 , -3a, -5, -5a), 109.1 (C-5 ', -5a'), 106.7 (C-1 ', -1a'), 102.1 -3 ', -3a'), 78.4 (C-3 '', -3a ''), 78.3 (C-5 ', -5a''), 74.7 ", -4a"), 62.4 ( C-6 ", -6a"), 47.0 (C-8, -8a), 30.8 (C-7, -7a), 16.2 (C-CH 2); FABMS m / z 833 [MH] ^- , HRFABMS m / z 833.2657 [MH] ^- (calcd for C ₄₃ H ₄₇ O ₂₀ , 833.2661).

In conclusion, the novel compound A was identified as methylenebisphloridzin of the molecular formula C ₄₃ H ₄₇ O ₂₀ .

New compound B: white amorphous powder. _{^{[α] 20 D -11.2 ° (}} c 0.1, MeOH); UV? _Max MeOH nm (log?): 205 (4.08), 225 (4.06), 289 (3.81); _{^{1 H NMR (CD 3 OD,}} 600MHz): δ7.05 (2H, d, J = 7.8Hz, H-2, -6), 7.01 (2H, d, J = 8.4Hz, H-2a, -6a) , 6.68 (2H, d, J = 8.4 Hz, H-3, -5), 6.67 (2H, d, J = 8.4 Hz, H- , 5.92 (1H, s, H -3a '), 5.04 (1H, d, J = 7.8Hz, H-1 "), 3.87 (1H, dd, J = 2.4, 12.0Hz, H-6"), 3.71 (2H, s, H-CH 2), 3.68 (1H, dd, J = 6.0, 12.0Hz, H-6 "), 3.49 (1H, m, H-2"), 3.45 (1H, m, H- M, H-4 "), 3.27 (2H, t, 3H), 3.44 (2H, t, J = 7.8Hz, H- J = 7.8Hz, H-8a) , 2.86 (2H, t, J = 7.8Hz, H-7), 2.82 (2H, t, J = 7.8Hz, H-7a); 13 C NMR (CD 3 OD, 163.3 (C-6 '), 163.3 (C-2a'), 163.2 (C-4 '), 163.6 , 162.7 (C-4a '), 160.6 (C-2'), 156.4 (C-4), 156.3 , -6.3), 130.3 (C-2a, -6a), 116.1 (C-3, -5), 116.0 , 106.6 (C-5 '), 106.5 (C-5a'), 105.6 (C-3a '), 102.0 (C-3), 74.7 (C-2), 71.1 (C-4), 62.4 ), 30.7 (C-7a) , 16.2 (C-CH 2); FABMS m / z 721 [MH] - , HRFABMS m / z 721.2141 [MH] ^- (calcd for C ₃₇ H ₃₇ O ₁₅ , 721.2132).

In conclusion, the novel compound B was identified as deglucosylmethylenebisphloridzin of the molecular formula C ₃₇ H ₃₇ O ₁₅ .

New compound C: White amorphous powder. UV? _Max MeOH nm (log?): 205 (4.12), 227 (4.03), 289 (3.78); _{^{1 H NMR (CD 3 OD,}} 600MHz): δ7.04 (4H, d, J = 9.0Hz, H-2, H-2a, -6, -6a), 6.68 (4H, d, J = 8.4Hz, (2H, s, H-CH ₂ ), 3.34 (4H, t, 3H, J = 7.8 Hz, H-8, H-8a), 2.85 (4H, t, J = 7.8 Hz, H-7, H-7a); _{^{13 C NMR (CD 3 OD,}} 150MHz): δ206.9 (C-9, -9a), 163.4 (C-6 ', -6a'), 163.3 (C-2 ', -2a'), 162.7 (C -4 ', -4a'), 156.5 (C-4, -4a), 133.9 (C-1, -1a), 130.3 , -3a, -5, -5a), 107.1 (C-5 ', -5a'), 105.3 -8, -8a), 31.6 (C -7, -7a), 14.4 (C-CH 2); FABMS m / z 559 [MH] ^- , HRFABMS m / z 559.1599 [MH] ^- (calcd for C ₃₁ H ₂₇ O ₁₀ , 559.1604).

Taking the above results into consideration, the novel compound C was identified as methylenebisphloretin of the molecular formula C ₃₁ H ₂₇ O ₁₀ .

As a result of HMBC of the new compounds, methylenebisphloridzin (A), deglucosylmethylenebisphloridzin (B) and methylenebisphloretin (C), hydrogen and carbon The interaction relationship is as shown in Fig.

Example  3. Plasma  Treated Florian  Of pancreatic lipase inhibitory activity of the novel compounds

The pancreatic lipase inhibitory activity was measured by modifying the method of Kim et al. That is, porcine pancreatic lipase was dissolved in an enzyme buffer solution (10 mM MOPS, 1 mM EDTA, pH 6.8) at a concentration of 0.5 g / 200 ml while being kept at 4 캜, and the lysate was centrifuged at 4000 rpm , And the supernatant was mixed with a Tris buffer solution (100 mM Tris-HCl, 5 mM CaCl ₂ , pH 7.0) and an enzyme buffer. The novel compounds isolated in Example 1 were dissolved in DMSO to a final concentration of 3% (w / v) and then stirred with the porcine pancreatic lipase enzyme for 15 min at 37 &lt; 0 &gt; C. Subsequently, the substrate solution p-NPB (p-nitrophenyl butyrate) dissolved in DMF to 10 mM was added and reacted at 37 ° C for 35 minutes. The reaction was shaken and then absorbance was measured at 405 nm using an ELISA reader. Orlistat was used as a positive control. The pancreatic lipase inhibitory activity was expressed as the absorbance reduction rate of the sample solution addition group and the no addition group.

As a result, as shown in Table 1, pancreatic lipase inhibitory activity of each of the novel compounds florisine, methylene bis-flurbine, diglycosyl methylene bis-fluorine and methylene bis- Was increased compared with the conventional floridine, and diglucosylmethylenebisphloridase and methylenebisflorethine exhibited enhanced pancreatic lipase inhibitory activity compared to floretin, and the IC ₅₀ value of methylenebisflorethine Was 5.2 ± 0.5 μM and showed the best pancreatic lipase inhibitory activity.

compound IC ₅₀ value Plasma treated Floridin (60 min) 29.8 + - 2.5 [mu] g / ml Florian > 300 μM Novel compound A: methylene bisphosphorin 257.1 ± 5.2 μM New compound B: diglucosyl methylene bisphosphorin 14.7 ± 0.8 μM Novel compound C: Methylenebisflorethine 5.2 ± 0.5 μM Florettin 50.4 ± 1.8 μM Orr start 0.5 ± 0.2 μM

Example  4. Plasma  Treated Florian  Measurement of inhibitory effect of new compounds on fat accumulation

In order to confirm the anti-obesity effect of plasma-treated novel floririn compounds, 3T3-L1 lipid precursor cells were differentiated and the degree of fat accumulation in adipocytes was measured through Oil-red-O staining.

3T3-L1 cells were dispensed into 24-well plates at 5 × 10 ⁴ cells per well and then maintained for another 2 days after the well was 100% filled with cells. Cells were induced for 2 days with adipocyte differentiation in 10% FBS DMEM medium containing MDI (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, 1 μM insulin) and after 48 h incubation, 2 μM insulin Containing 10% FBS DMEM for 2 days. Subsequently, the cells were replaced with 10% FBS DMEM medium for 2 days every 4 days. During the induction of adipocyte differentiation, the new compounds of the present invention isolated in Example 1 were treated at 25 μM, 50 μM and 100 μM concentrations, and in order to confirm the degree of accumulation of fat on the 8th day when the differentiation was completed, -red-O staining was performed.

As a result, as shown in FIG. 2, the effect of inhibiting fat accumulation by the novel compounds of the present invention (methylenebisfloridine, diglucosylmethylenebisfloridine, methylenebisfloretin) was increased as compared with florixin, Methylene bisphosphretin of the present invention in which a sugar-removed form of floretine was formed by a methyl group showed a further enhanced fat accumulation inhibitory effect than that of floretin.

Claims (8)

1. A compound represented by the following formula (1): &lt; EMI ID =
[Chemical Formula 1]

In this formula,
R ¹ and R ² are each

Lt; / RTI &
R ¹ is

And R ² is hydrogen (H). delete delete delete 2. The compound of claim 1, wherein the compound is prepared by treating plasma with a plasma in the range of 200-300 W for 30-80 minutes. A compound represented by the formula (1) or (2): Or a pharmaceutically acceptable salt thereof, as an active ingredient, for the prophylaxis or treatment of obesity.
(2)

The pharmaceutical composition for preventing or treating obesity according to claim 6, wherein the composition inhibits the activity of pancreatic lipase or inhibits fat accumulation of adipocytes. A compound represented by the formula (1) or (2): Or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or ameliorating obesity.
(2)

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