NOVEL COMPOUND ISOLATED FROM SPINACH, AND COMPOSITION COMPRISING SAME FOR PREVENTING OR TREATING INFLAMMATORY DISEASES

Abstract

The present invention relates to a novel compound isolated from spinach, and a composition comprising same for preventing or treating inflammatory diseases, and, more particularly, to a novel medicagenic acid glycoside compound isolated from spinach, a preparation method therefor, and a composition comprising same for preventing or treating inflammatory diseases.

Claims

1. A compound represented by any one of the following formulae 1 to 6: ##STR00017## ##STR00018## pharmaceutically acceptable salts thereof, isomers thereof, hydrates thereof or solvates thereof.

2. The compound of claim 1, wherein the compound is isolated from spinach.

3. A method for preparing the compound of claim 1, comprising: (a) extracting spinach with a solvent selected from the group consisting of water, organic solvents, subcritical fluids, supercritical fluids and mixtures thereof; (b) fractionating the spinach extract by chromatography to obtain fractions; and (c) isolating the compound from the fractions.

4. The method of claim 3, wherein, in step (a), the organic solvent is selected from the group consisting of alcohol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether having 1 to 6 carbon atoms.

5. The method of claim 3, wherein, in step (b), the chromatography is sequentially developed according to a concentration gradient by using water and a non-polar solvent as mobile phases.

6. The method of claim 5, wherein, in step (b), fractions fractionated from 20 to 100% (v/v) of a non-polar solvent aqueous solution are obtained.

7. A pharmaceutical composition for preventing or treating inflammatory diseases, comprising as an active ingredient one or more selected from the group consisting of the compounds according to claim 1 ##STR00019## ##STR00020## pharmaceutically acceptable salts thereof, isomers thereof, hydrates thereof, and solvates thereof.

8. The pharmaceutical composition of claim 7, wherein the inflammatory disease is selected from the group consisting of inflammatory respiratory disease, dermatitis, atopic dermatitis, allergy, psoriasis, bronchitis, ulcerative colitis, retinitis, uveitis, conjunctivitis, arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, nephritis, nephritis, autoimmune pancreatitis, chronic pelvic inflammatory disease, endometritis, otitis media, cystitis, and chronic prostatitis.

9. The pharmaceutical composition of claim 8, wherein the inflammatory respiratory disease is selected from the group consisting of asthma, pneumonia, acute lung injury, acute respiratory failure syndrome, chronic obstructive pulmonary disease, allergic rhinitis, bronchitis, pharyngitis, laryngitis, pharyngitis, and tonsillitis.

10.-16. (canceled).

17. A method of treating inflammatory diseases, comprising administering to a subject in need thereof an effective amount of a composition comprising as an active ingredient one or more selected from the group consisting of the compounds represented by any one of the following formulae 1 to 6: ##STR00021## ##STR00022## pharmaceutically acceptable salts thereof, isomers thereof, hydrates thereof, and solvates thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0114] FIG. 1 is a diagram showing the results of UPLC analysis of a spinach extract.

[0115] FIG. 2 shows the preparation process of an effective fraction (Fr.2) comprising five (5) novel substances and celosin I compound from a spinach extract.

[0116] FIGS. 3A and 3B are diagrams showing the results of CAD chromatogram analysis of a spinach extract, fraction 1 (Fr.1) and fraction 2 (Fr.2).

[0117] FIGS. 4A and 4B show the results of UPLC-QTOF-MS analysis of five (5) novel substances (i.e., SOA1, SOA2, SOA4, SOA5 and SOA6) and celosin I compound (i.e., SOA 3) isolated from a spinach extract.

[0118] FIGS. 5A and 5B show the results of evaluating the cytotoxicity (5A) and the ability to inhibit the secretion of TNF-alpha (5B) by a spinach extract (SO_total), its butanol fraction (SO_BuOH), fraction 1 (SO_Fr.1) and fraction 2 (SO_Fr.2) in macrophages stimulated with LPS.

[0119] FIG. 6 is a table showing the results of evaluating the cytotoxicity and the ability to inhibit the secretion of TNF-alpha and NO by five (5) novel substances isolated from a spinach extract (i.e., SOA1, SOA2, SOA4, SOA5, SOA6), celosin I compound (SOA 3) and medicagenic acid (MA) in macrophages stimulated with LPS.

[0120] FIGS. 7A to 7C show the results of evaluating the ability to inhibit the secretion of NO and PGE2 (7A, 7B) and the ability to inhibit the expression of COX-2 and iNOS protein (7C) by Fraction 2 of a spinach extract (SO_Fr.2) in LPS-stimulated macrophages, according to treatment with different concentrations.

[0121] FIGS. 8A to 8C show the results of evaluating the ability to inhibit the secretion of inflammatory cytokines IL-4 (9A), IL-5 (9B) and IL-13 (9C) by fraction 2 (SO_Fr.2) of a spinach extract in EL4 cells stimulated with PMA and lonomycin.

[0122] FIGS. 9A and 9B show the results of evaluating the cytotoxicity (9A) and the ability to inhibit the secretion of mucus (9B) by a spinach extract (SO_total), its butanol fraction (SO_BuOH), fraction 1 (SO_Fr.1) and fraction 2 (SO_Fr.2) in PMA-stimulated respiratory epithelial cells.

[0123] FIGS. 10A and 10B show the results of evaluating the cytotoxicity 10A) and the ability to inhibit the secretion of mucus (10B) by five (5) novel substances isolated from a spinach extract (i.e., SOA1, SOA2, SOA4, SOA5, SOA6) and celosin I compound (SOA 3) in respiratory epithelial cells stimulated with PMA.

[0124] FIGS. 11A to 11D show the results of evaluating the ROS (11A), elastase (11B), TNF-alpha (11C) and IL-6 (11D) secretion inhibitory ability of fraction 2 of a spinach extract in bronchoalveolar lavage fluid after administered at the indicated concentrations (S5: 5 mg/kg, S10: 10 mg/kg) in mouse models of chronic obstructive pulmonary disease (COPD).

[0125] FIGS. 12A to 12D show the results of evaluating the IL-4 (12A), IL-5 (12B), IL-13 (12C) and IgE (12D) inhibitory ability of fraction 2 of a spinach extract in bronchoalveolar lavage fluid after administered at the indicated concentrations (S5: 5 mg/kg, S10: 10 mg/kg) in asthma animal models.

[0126] FIG. 13 shows the results of measuring the number of inflammatory cells in bronchoalveolar lavage fluid by fraction 2 of a spinach extract after administered at the indicated concentrations (S5: 5 mg/kg, S10: 10 mg/kg) in pneumonia mouse models.

[0127] FIGS. 14A to 14C show the results of evaluating the ability to inhibit the expression of ROS (14A), TNF-alpha (14B) and IL-6 (14C) in bronchoalveolar lavage fluid by fraction 2 of a spinach extract after administered at the indicated concentrations (S5: 5 mg/kg, S10: 10 mg/kg) in pneumonia mouse models.

MODE FOR INVENTION

[0128] Hereinafter, the present invention will be described in detail by the following embodiments. However, the following embodiments are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1

Preparation of a Spinach Extract and Analysis of its Component Profile

[0129] 100 kg of spinach was extracted at 50? C. for 90 minutes by using 18 times 30% alcohol, and then filtered/concentrated.

[0130] The components of the prepared spinach extract were analyzed by UPLC.

[0131] Specifically, for UPLC analysis, the spinach extract was filtered once through a 0.25 mm membrane filter for UPLC. A UPLC instrument (Waters UPLC-Q-TOF) was equipped with a column (Waters BEH C18 column, 2.1?100 mm, 1.7 ?m), and each filtered fraction was loaded in an amount of 0.3 ?l.

[0132] At this time, the solvent used for UPLC analysis was acetonitrile+0.1% formic acid/water+0.1% formic acid 10:90->100:0 (v/v), and the elution rate was 0.4 ml/min.

[0133] The separation degree of materials separated from UPLC was confirmed in chromatographic format by using Mass spectrometry (MS) and Charged Aerosol Detector (CAD) as detectors.

[0134] The UPLC-QTOF-MS analysis conditions and results are shown in FIG. 1 and Table 1.

TABLE-US-00001 TABLE 1 Time(min) Flow(ml/min) % A % B 0.00 0.400 90 10 1.00 0.400 90 10 7.00 0.400 85 15 13.00 0.400 70 30 16.00 0.400 60 40 23.00 0.400 20 80 25.00 0.400 0 100 28.00 0.400 0 100 28.30 0.400 90 10 30.00 0.400 90 10

[0135] As can be seen in FIG. 1, five (5) novel substances (i.e., SOA1, SOA2, SOA4, SOA5, SOA6) and celosin I compound (SOA 3) were identified from the spinach extract through UPLC analysis.

Example 2

Fractionation of a Spinach Alcohol Extract

[0136] Effective fractions and novel compounds were isolated from the extract obtained in Example 1 by the following method.

[0137] Specifically, an MPLC instrument (YMC LAB-300) was equipped with a column (YMC-DAD-50-700S (50?700 mm, 10 ?m), and a spinach extract (20 g) was loaded. At this time, the solvent was methanol/water 10:90->100:0 (v/v), and the elution rate was 100 ml/min, and detection at the wavelengths of UV 210, 254, 280 nm was conducted to obtain small fractions (i.e., SO Fr.1 and Fr.2) (FIG. 2).

[0138] Specifically, an MPLC instrument (YMC Lc-Forte/R) was equipped with a column (Waters X-bridge, 19?250 mm, 5 ?m), and effective fraction SO Fr.2 (16.5 g) was loaded. At this time, the solvent was acetonitrile+0.1% formic acid/water+0.1% formic acid 15:85->100:0 (v/v), and the elution rate was 11 ml/min. Detection was conducted at UV wavelengths of 210, 254, and 280 nm, and compounds SO peak 1 (SOA1, 238.6 mg), 2 (SOA2, 170.2 mg), 3 (SOA3, 39.4 mg) and 4 (SOA4, 24.2 mg) with new structures represented by formulae 1, 2, 3, and 4 having the following physical properties were obtained (FIGS. 3A, 3B, 4A and 4B).

Example 3

Structural Analysis of Five (5) Novel Substances

[0139] The molecular weight and molecular formula of the oleanan-type triterpene saponin compound obtained in Example 2 were determined by using a high-resolution Qtof-MS mass spectrometer (Vion IMS-Qtof-MS (Waters, USA)). In addition, the molecular structure was determined by using spectroscopic data .sup.1H-NMR, .sup.13C-NMR and 2D NMR (COZY, HSQC, HMBC, ROESY) through nuclear magnetic resonance (NMR) analysis (Bruker Avance-800 MHz, Bruker Avance-900 MHz Bruker, Germany).

[0140] As a result of comparing the instrumental analysis results with those of published literature, the oleanan type triterpene saponin represented by the following formulae 1 to 6 was identified. The specific analysis results are as follows.

[0141] In Compound 1, the molecular ion M+H.sup.+ of m/z 1133.5383 was observed in the HR-Qtof-MS spectrum, and, through this, the molecular formula of C.sub.54H.sub.84O.sub.25 was determined. Through MS cleavage pattern analysis, a cleavage ion at m/z 971 due to loss of glucose (162 Da), and molecular ions of m/z 825 and 679 due to loss of the pentose sugars rhamnose and fucose (146 Da) were observed. In addition, the molecular ion of sapogenin m/z 503 due to the loss of glucuronic acid (176 Da) was detected, which is the same as the molecular ion value of medicagenic acid. In the .sup.1H NMR spectrum, eight methyl protons (i.e., ?.sub.H 1.89, 1.64, 1.45, 1.40, 1.19, 1.03, 0.79 and 0.77), one olefinic proton (i.e., ?.sub.H 5.33), four anomeric protons (i.e., ?.sub.H 6.20, 5.91, 5.13 and 5.06), and a methine proton with two adjacent oxygens (i.e., ?.sub.H 4.74 and 4.63) were observed. In the .sup.13C NMR spectrum, three carbonyl carbons (i.e., ?.sub.C 180. 9, 176. 6 and 172. 7), a pair of olefinic carbons (i.e., ?.sub.C 143.8 and 122.4), four enomeric carbons (i.e., ?.sub.C 106.2, 105.2, 101.1 and 94.5), and methylene carbon substituted with one oxygen (i.e., ?.sub.C 62.4), and 18 methine carbons (i.e., ?.sub.C 85.7, 84.5, 78.1, 77.9, 77.2, 76.9, 75.9, 75.8, 74.4, 74.1, 72.8, 72.6, 72.1, 71.9, 71.3, 71.2, 70.0 and 68.2) were confirmed. As a result of confirmation through 2D NMR, a signal that the methyl proton peak ?.sub.H 1.89 (3H, s, H3-24) was adjacent to the carbonyl peak (?.sub.C 180. 9) was observed. In addition, through this NMR spectrum pattern and existing prior literature (J. Agric. Food Chem., 2005, 53, 2164-2170, J. Asian Nat. Prod. Res., 2014, 16, 240-247, Phytochemistry 2020, 169, 112162), it was confirmed that Compound 1 is an oleananic saponin compound with medicagenic acid (2?,3?-dihydroxyolean-12-ene-23,28-dioic acid) as an aglycone. In addition, 2D NMR (COZY, HSQC and HMBC) confirmed that among the four enomeric protons, the peak at ?.sub.H 5.13 (1H, d, J=8.1 Hz, , H-1, GlcA) was connected to the peak at ?.sub.C 85.7 (C-3), and the peak at ?.sub.H 5.91 (1H, d, J)=8.0 Hz, H-1, Fuc) was connected to the carbonyl group at ?.sub.C 176.6 (C-28). It was also confirmed that the peak at ?.sub.H 6.20 (1H, d, J=1.8 Hz, H-1, Rham) was connected to carbon at position 2 of fucose (?.sub.C 74.1, C-2), and the peak at ?.sub.H 5.06 (1H, d, J=7.2 Hz, H-1, Glu) was bound to the carbon at position 4 of rhamnose (?.sub.C 84.5 C-4). Combining previous literature and the above-described results, Compound 1 was identified as 3-O-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-D-glucopyranosyl-(1.fwdarw.4)-?-L-rhamnopyranosyl-(1.fwdarw.2)-?-D-fucopyranoside, and was named spinasaponin C.

[0142] In Compound 2, the molecular ion M+H.sup.+ of m/z 1265.5810 was observed in the HR-Qtof-MS spectrum, and, through this, the molecular formula of C.sub.59H.sub.92O.sub.29 was determined. Through MS cleavage pattern analysis, a molecular ion of m/z 1133 due to loss of xylose (132 Da), a pentose sugar, was detected. In addition, a cleavage ion at m/z 971 due to loss of glucose (162 Da) and molecular ions at m/z 825 and 679 due to loss of pentose sugars rhamnose and fucose (146 Da) were observed. In addition, m/z 503, the molecular ion of sapogenin due to the loss of glucuronic acid (176 Da) was detected, and the molecular ion value was identical to that of medicagenic acid. As a result of comparative analysis of the 1D and 2D NMR spectra and the prior literature, Compound 2 showed a spectrum similar to Compound 1, and it was confirmed that xylose sugar, a pentose sugar, was additionally connected to Compound 1. 2D NMR data confirmed that the enomeric proton of the xylose sugar (?.sub.H 5.30, 1H, d, J=7.2 Hz, H-1, Xyl) was connected to C-3 of glucuronic acid (?.sub.C 86.1, C-3, GlcA). Therefore, combining the prior literature and the above-described results, Compound 2 was identified as 3-O-?-D-xylopyranosyl-(1.fwdarw.3)-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23, 28-dioic acid-28-O-?-D-glucopyranosyl-(1.fwdarw.4)-?-L-rhamnopyranosyl-(1.fwdarw.2)-?-D-fucopyranoside, and was named spinasaponin D.

[0143] In Compound 3, the molecular ion M+H.sup.+ of m/z 1103.5301 was observed in the HR-Qtof-MS spectrum, and, through this, the molecular formula of C.sub.53H.sub.82O.sub.24 was determined. Through MS cleavage pattern analysis, a molecular ion of m/z 971 due to loss of xylose (132 Da), a pentose sugar, was detected. Molecular ions of m/z 825 and 679 were observed due to the loss (146 Da) of the pentose sugars rhamnose and fucose. In addition, m/z 503, the molecular ion of sapogenin due to the loss of glucuronic acid (176 Da) was detected, and the molecular ion value was identical to that of medicagenic acid. As a result of comparative analysis of 1D and 2D NMR spectra and the prior literature, Compound 3 showed a spectrum similar to Compound 1. In addition, it was confirmed that the position of the glucose sugar in Compound 1 was replaced with xylose sugar, a pentose sugar, and connected to Compound 1. 2D NMR data confirmed that the enomeric proton of xylose sugar (?.sub.H 5.03, 1H, d, J=7.2 Hz, H-1, Xyl) was connected to C-4 of rhamnose (?.sub.C 85.8, C-4, Rham). When combining the above-described results, it was confirmed that Compound 3 was the compound 3-O-?-D-glucuronopyranosyl-2?,3?,-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-d-xylopyranosyl-(1.fwdarw.4)-?-1-rhamnopyranosyl-(1.fwdarw.2)-?-D-fucopyranoside (Celosin I) reported in the prior literature (J. Asian Nat. Prod. Res., 2014, 16, 240-247).

[0144] In Compound 4, the molecular ion M+H.sup.+ of m/z 1235.5725 was observed in the HR-Qtof-MS spectrum, and, through this, the molecular formula of C58H90028 was determined. Through MS cleavage pattern analysis, a molecular ion of m/z 1103 due to loss of xylose (132 Da), a pentose sugar, was detected. Molecular ions of m/z 957, 825 and 679 due to the loss of rhamnose (146 Da), xylose, and fucose (146 Da) were observed. In addition, m/z 503, the molecular ion of sapogenin, due to the loss of glucuronic acid (176 Da) was detected, the molecular ion value was identical to that of medicagenic acid. As a result of comparative analysis of the 1D and 2D NMR spectra and the prior literature, Compound 4 showed a spectrum similar to Compound 2, and it was confirmed that the glucose sugar of Compound 2 was substituted with xylose sugar, a pentose sugar, and was connected to Compound 2. 2D NMR data confirmed that an additional enomeric proton of the xylose sugar (?.sub.H 4.94, 1H, d, J=7.2 Hz, H-1, Xyl-II) was connected to C-4 of rhamnose (?.sub.C 84.6, C-3, Rham). Therefore, by combining prior literature and the above-described results, Compound 4 was identified as 3-O-?-D-xylopyranosyl-(1.fwdarw.3)-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-D-xylopyranosyl-(1.fwdarw.4)-?-L-rhamnopyranosyl-(1.fwdarw.2)-?-D-fucopyranoside, and was named spinasaponin E.

[0145] In Compound 5, the molecular ion M+H.sup.+ of m/z 1175.5487 was observed in the HR-Qtof-MS spectrum, and, through this, the molecular formula of C.sub.56H.sub.86O.sub.26 was determined. Through MS cleavage pattern analysis, a cleavage ion of m/z 1013 due to loss of glucose (162 Da) and a molecular ion of m/z 867 due to loss of rhamnose, a pentose sugar (146 Da), were observed. In addition, the molecular ion of m/z 679 due to loss of acetylated fucose (188 Da) and the molecular ion of sapogenin, m/z 503, due to loss of glucuronic acid (176 Da) were detected, and the molecular ion value was identical to that of medicagen acid. As a result of comparative analysis of the 1D and 2D NMR spectra and the prior literature, Compound 5 showed a spectrum similar to Compound 1, and it was confirmed that Compound 5 had an acetyl group added to the skeleton of Compound 1 (?.sub.H 4.94 3H, s, OAcCH3; ?.sub.C 171.3, OAc, 20.6, OAcCH3). 2D NMR data confirmed that the acetyl group was substituted at position 4 of the fucose sugar (?.sub.C 74.5, C-4, Fuc), and the enomeric proton of the acetylated fucose sugar (?.sub.H 5.95, 1H, d, J=8.1 Hz, H-1, Fuc) was connected to the carbonyl group of methikagenic (?.sub.C 176. 6, C-28). Therefore, by combining the prior literature and the above-described results, Compound 5 was identified as 3-O-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-D-glucopyranosyl-(1.fwdarw.4)-?-L-rhamnopyranosyl-(1.fwdarw.2)-4-O-acetyl-?-D-fucopyranoside, and was named spinasaponin F.

[0146] In Compound 6, the molecular ion M+H.sup.+ of m/z 1307.5915 was observed in the HR-Qtof-MS spectrum, and, through this, the molecular formula of C61H94O30 was determined. Through MS cleavage pattern analysis, a molecular ion of m/z 1175 due to loss of xylose (132 Da), a pentose sugar, was detected, and a cleaved ion of m/z 1013 due to loss of glucose (162 Da) and a molecular ion of m/z 867 due to loss of rhamnose, a pentose sugar (146 Da), were observed, and a molecular ion of m/z 679 due to loss of acetylated fucose (188 Da) was detected. In addition, m/z 503, the molecular ion of sapogenin, due to the loss of glucuronic acid (176 Da) was detected, and the molecular ion value was identical to that of medicagenic acid. As a result of comparative analysis of the 1D and 2D NMR spectra and the prior literature, Compound 6 showed a spectrum similar to Compound 5, and it was confirmed that xylose sugar, a pentose sugar, was additionally connected to Compound 5. 2D NMR data confirmed that the enomeric proton of xylose sugar (?.sub.H 5.15, 1H, d, J=7.2 Hz, H-1, , GlcA) was connected to C-3 of glucuronic acid (?.sub.C 85.6, C-3, GlcA). Therefore, by combining the prior literature and the above-described results, Compound 6 was identified as 3-O-?-D-xylopyranosyl-(1.fwdarw.3)-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23, 28-dioic acid-28-O-?-D-glucopyranosyl-(1.fwdarw.4)-?-L-rhamnopyranosyl-(1.fwdarw.2)-4-O-acetyl-?-D-fucopyranoside, and was named spinasaponin G.

[0147] Compound 1 (SOA1) 3-O-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-d-glucopyranosyl-(1.fwdarw.4)-?-1-rhannopyranosyl-(1.fwdarw.2)-?-D-fucopyranoside (Spinasaponin C)

##STR00011## [0148] 1) Physical properties: Amorphous white crystals [0149] 2) Molecular weight: 1133.24 [0150] 3) Molecular formula: C.sub.54H.sub.84O.sub.25 [0151] 4) .sup.1H-NMR (Pyridine-d.sub.5, 900 MHZ) ?.sub.H 6.20 (1H, d, J=1.8 Hz, H-1, Rham), 5.91 (1H, d, J=8.1 Hz, H-1, Fuc), 5.33 (1H, s, H-12), 5.13 (1H, d, J=8.1 Hz, H-1, GlcA), 5.06 (1H, d, J=7.2 Hz, H-1, Glu), 4.74 (1H, d, J=2.7 Hz, H-2), 4.63 (1H, d, J=2.7 Hz, H-3), 3.02 (1H, dd, J=13.5, 3.6 Hz, H-18), 2.21 (1H, d, J=12.6 Hz, H-1a), 1.89 (3H, s, H.sub.3-24), 1.64 (3H, d, J=6.3 Hz, H.sub.3-6, Rham), 1.45 (3H, s, H.sub.3-25), 1.40 (3H, d, J=6.3 Hz, H.sub.3-6, Fuc), 1.19 (3H, s, H.sub.3-27), 1.03 (3H, s, H.sub.3-26), 0.79 (3H, s, H.sub.3-30), 0.77 (3H, s, H.sub.3-29); .sup.13C-NMR (Pyridine-d.sub.5, 225 MHZ) ?.sub.C 44.1 (C-1), 70.0 (C-2), 85.7 (C-3), 52.6 (C-4), 52.3 (C-5), 20.9 (C-6), 32.7 (C-7), 40.1 (C-8), 48.4 (C-9), 36.5 (C-10), 23.7 (C-11), 122.4 (C-12), 143.8 (C-13), 42.0 (C-14), 28.0 (C-15), 23.1 (C-16), 46.8 (C-17), 41.8 (C-18), 46.0 (C-19), 30.4 (C-20), 33.6 (C-21), 32.0 (C-22), 180.9 (C-23), 13.8 (C-24), 16.6 (C-25), 17.2 (C-26), 25.8 (C-27), 176.6 (C-28), 32.8 (C-29), 23.5 (C-30), 105.2 (C-1, GlcA), 74.4 (C-2, GlcA), 77.2 (C-3, GlcA), 72.8 (C-4, GlcA), 76.9 (C-5, GlcA), 172.7 (C-6, GlcA), 94.5 (C-1, Fuc), 74.1 (C-2, Fuc), 75.8 (C-3, Fuc), 72.6 (C-4, Fuc), 72.1 (C-5, Fuc), 16.5 (C-6, Fuc), 101.1 (C-1, Rham), 71.2 (C-2, Rham), 71.9 (C-3, Rham), 84.5 (C-4, Rham), 68.2 (C-5, Rham), 18.3 (C-6, Rham), 106.2 (C-1, Glu), 75.9 (C-2, Glu), 78.1 (C-3, Glu), 71.3 (C-4, Glu), 77.9 (C-5, Glu), 62.4 (C-6, Glu).

[0152] Compound 2 (SOA2) 3-O-?-D-xylopyranosyl-(1.fwdarw.3)-?-d-glucuronopyranosyl-2?,3?,-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-d-glucopyranosyl-(1.fwdarw.4)-?-1-rhamnopyranosyl-(1.fwdarw.2)-?-D-fucopyranoside (Spinasaponin D)

##STR00012## [0153] 1) Physical properties: Amorphous white crystals [0154] 2) Molecular weight: 1265.36 [0155] 3) Molecular formula: C.sub.59H.sub.92O.sub.29 [0156] 4) .sup.1H-NMR (Pyridine-d.sub.5, 800 MHZ) ?.sub.H 6.36 (1H, s, H-1, Rham), 6.04 (1H, d, J=8.0 Hz, H-1, Fuc), 5.46 (1H, br s, H-12), 5.30 (1H, d, J=7.2 Hz, H-1, Xyl), 5.28 (1H, d, J=8.0 Hz, H-1, GlcA), 5.17 (1H, d, J=8.0 Hz, H-1, Glu), 4.84 (1H, br s, H-2), 4.77 (1H, br s, H-3), 3.12 (1H, d, J=13.6, 3.2 Hz, H-18), 2.26 (1H, d, J=12.0 Hz, H-1a), 2.01 (3H, s, H.sub.3-24), 1.70 (3H, d, J=5.6 Hz, H.sub.3-6, Rham), 1.58 (3H, s, H.sub.3-25), 1.48 (3H, d, J=6.4 Hz, H.sub.3-6, Fuc), 1.26 (3H, s, H.sub.3-27), 1.14 (3H, s, H.sub.3-26), 0.90 (3H, s, H.sub.3-30), 0.85 (3H, s, H.sub.3-29); .sup.13C-NMR (Pyridine-d.sub.5, 200 MHZ) ?.sub.C 44.9 (C-1), 71.0 (C-2), 86.8 (C-3), 53.3 (C-4), 53.1 (C-5), 21.6 (C-6), 33.4 (C-7), 40.8 (C-8), 49.0 (C-9), 37.3 (C-10), 24.3 (C-11), 123.1 (C-12), 144.3 (C-13), 42.7 (C-14), 28.6 (C-15), 23.8 (C-16), 47.3 (C-17), 42.5 (C-18), 46.6 (C-19), 31.1 (C-20), 34.3 (C-21), 32.7 (C-22), 180.9 (C-23), 14.5 (C-24), 17.3 (C-25), 17.8 (C-26), 26.5 (C-27), 177.0 (C-28), 33.5 (C-29), 24.2 (C-30), 106.3 (C-1, GlcA), 74.4 (C-2, GlcA), 86.1 (C-3, GlcA), 71.8 (C-4, GlcA), 77.0 (C-5, GlcA), 170.6 (C-6, GlcA), 106.5 (C-1, Xyl), 75.8 (C-2, Xyl), 78.4 (C-3, Xyl), 71.8 (C-4, Xyl), 67. 7 (C-5, Xyl), 95.1 (C-1, Fuc), 74.7 (C-2, Fuc), 76.8 (C-3, Fuc), 73.5 (C-4, Fuc), 72.7 (C-5, Fuc), 17.2 (C-6, Fuc), 101.8 (C-1, Rham), 72.1 (C-2, Rham), 72.8 (C-3, Rham), 85.8 (C-4, Rham), 68.9 (C-5, Rham), 19.0 (C-6, Rham), 107.4 (C-1, Glu), 76.8 (C-2, Glu), 79.2 (C-3, Glu), 72.2 (C-4, Glu), 78.8 (C-5, Glu), 63.3 (C-6, Glu).

[0157] Compound 3 (SOA3) 3-O-?-D-glucuronopyranosyl-2?,3?,-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-d-xylopyranosyl-(1.fwdarw.4)-?-1-rhamnopyranosyl-(1.fwdarw.2)-?-D-fucopyranoside (Celosin I)

##STR00013## [0158] 1) Physical properties: Amorphous white crystals [0159] 2) Molecular weight: 1103.22 [0160] 3) Molecular formula: C.sub.53H.sub.82O.sub.24 [0161] 4) .sup.1H-NMR (Pyridine-d.sub.5, 900 MHZ) ?.sub.H 6.41 (1H, br s, H-1, Rham), 6.04 (1H, d, J=8.1 Hz, H-1, Fuc), 5.41 (1H, s, H-12), 5.22 (1H, br s, H-1, GlcA), 5.03 (1H, d, J=7.2 Hz, H-1, Xyl), 4.85 (1H, m, H-2), 4.75 (1H, m, H-3), 3.12 (1H, d, J=12.6 Hz, H-18), 2.31 (1H, br s, H-1a), 2.00 (3H, s, H.sub.3-24), 1.70 (3H, d, J=6.3 Hz, H.sub.3-6, Rham), 1.57 (3H, s, H.sub.3-25), 1.48 (3H, d, J=5.4 Hz, H.sub.3-6, Fuc), 1.26 (3H, s, H.sub.3-27), 1.14 (3H, s, H.sub.3-26), 0.89 (3H, s, H.sub.3-30), 0.84 (3H, s, H.sub.3-29); .sup.13C-NMR (Pyridine-d.sub.5, 225 MHZ) ?.sub.C 44.9 (C-1), 70.7 (C-2), 86.7 (C-3), 53.3 (C-4), 52.9 (C-5), 21.6 (C-6), 33.4 (C-7), 40.8 (C-8), 49.0 (C-9), 37.2 (C-10), 24.3 (C-11), 123.1 (C-12), 144.3 (C-13), 42.7 (C-14), 28.6 (C-15), 23.7 (C-16), 47.4 (C-17), 42.4 (C-18), 46.6 (C-19), 31.1 (C-20), 34.3 (C-21), 32.7 (C-22), 181.0 (C-23), 14.6 (C-24), 17.3 (C-25), 17.8 (C-26), 26.4 (C-27), 177.0 (C-28), 33.5 (C-29), 24.1 (C-30), 106.0 (C-1, GlcA), 75.3 (C-2, GlcA), 78.2 (C-3, GlcA), 73.7 (C-4, GlcA), 77.6 (C-5, GlcA), 173.0 (C-6, GlcA), 95.1 (C-1, Fuc), 74.4 (C-2, Fuc), 77.0 (C-3, Fuc), 73.5 (C-4, Fuc), 72.7 (C-5, Fuc), 17.3 (C-6, Fuc), 101.7 (C-1, Rham), 71.3 (C-2, Rham), 72.9 (C-3, Rham), 85.8 (C-4, Rham), 68.6 (C-5, Rham), 18.9 (C-6, Rham), 108.0 (C-1, Xyl), 76.6 (C-2, Xyl), 79.2 (C-3, Xyl), 72.2 (C-4, Xyl), 67.9 (C-5, Xyl).

[0162] Compound 4 (SOA4) 3-O-?-D-xylopyranosyl-(1.fwdarw.3)-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-D-xylopyranosyl-(1.fwdarw.4)-?-L-rhamnopyranosyl-(1.fwdarw.2)-?-D-fucopyranoside (Spinasaponin E)

##STR00014## [0163] 1) Physical properties: Amorphous white crystals [0164] 2) Molecular weight: 1235.33 [0165] 3) Molecular formula: C.sub.58H.sub.90O.sub.28 [0166] 4) .sup.1H-NMR (Pyridine-d.sub.5, 900 MHZ) ?.sub.H 6.25 (1H, s, H-1, Rham), 5.91 (1H, d, J=8.1 Hz, H-1, Fuc), 5.34 (1H, br s, H-12), 5.16 (1H, d, J=8.1 Hz, H-1, GlcA), 5.15 (1H, d, J=8.1 Hz, H-1, Xyl-I), 4.94 (1H, d, J=7.2 Hz, H-1, Xyl-II), 4.72 (1H, br s, H-2), 4.65 (1H, d, J=3.6 Hz, H-3), 3.02 (1H, dd, J=13.5, 3.6 Hz, H-18), 2.18 (1H, d, J=12.6 Hz, H-1a), 1.88 (3H, s, H.sub.3-24), 1.60 (3H, d, J=6.3 Hz, H.sub.3-6, Rham), 1.44 (3H, s, H.sub.3-25), 1.40 (3H, d, J=6.3 Hz, H.sub.3-6, Fuc), 1.19 (3H, s, H.sub.3-27), 1.03 (3H, s, H.sub.3-26), 0.78 (3H, s, H.sub.3-30), 0.76 (3H, s, H.sub.3-29); .sup.13C-NMR (Pyridine-d.sub.5, 200 MHZ) ?.sub.C 44.1 (C-1), 70.1 (C-2), 85.7 (C-3), 52.6 (C-4), 52.4 (C-5), 20.9 (C-6), 32.7 (C-7), 40.1 (C-8), 48.4 (C-9), 36.5 (C-10), 23.7 (C-11), 122.4 (C-12), 143.8 (C-13), 42.1 (C-14), 28.0 (C-15), 23.0 (C-16), 46.8 (C-17), 41.8 (C-18), 46.0 (C-19), 30.4 (C-20), 33.6 (C-21), 32.0 (C-22), 180.7 (C-23), 13.8 (C-24), 16.6 (C-25), 17.1 (C-26), 25.8 (C-27), 176.6 (C-28), 32.8 (C-29), 23.5 (C-30), 104.9 (C-1, GlcA), 73.6 (C-2, GlcA), 85.5 (C-3, GlcA), 71.0 (C-4, GlcA), 76.4 (C-5, GlcA), 173.5 (C-6, GlcA), 105.5 (C-1, Xyl-I), 74.8 (C-2, Xyl-I), 77.3 (C-3, Xyl-I), 70.4 (C-4, Xyl-I), 66.7 (C-5, Xyl-I), 94.5 (C-1, Fuc), 73.8 (C-2, Fuc), 76.0 (C-3, Fuc), 72.6 (C-4, Fuc), 72.1 (C-5, Fuc), 16.6 (C-6, Fuc), 101.0 (C-1, Rham), 71.2 (C-2, Rham), 71.9 (C-3, Rham), 84.6 (C-4, Rham), 68.0 (C-5, Rham), 18.2 (C-6, Rham), 107.0 (C-1, Xyl-II), 75.7 (C-2, Xyl-II), 78.0 (C-3, Xyl-II), 70.4 (C-4, Xyl-II), 67.0 (C-5, Xyl-II).

[0167] Compound 5 (SOA5) 3-O-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-D-glucopyranosyl-(1.fwdarw.4)-?-L-rhamnopyranosyl-(1.fwdarw.2)-4-O-acetyl-?-D-fucopyranoside (Spinasaponin F)

##STR00015## [0168] 1) Physical properties: Amorphous white crystals [0169] 2) Molecular weight: 1175.28 [0170] 3) Molecular formula: C.sub.56H.sub.86O.sub.26 [0171] 4) .sup.1H-NMR (Pyridine-d.sub.5, 900 MHZ) ?.sub.H 6.13 (1H, s, H-1, Rham), 5.95 (1H, d, J=8.1 Hz, H-1, Fuc), 5.35 (1H, s, H-12), 5.14 (1H, d, J=7.2 Hz, H-1, GlcA), 5.07 (1H, d, J=8.1 Hz, H-1, Glu), 4.74 (1H, br s, H-2), 4.64 (1H, br s, H-3), 3.02 (1H, dd, J=13.5, 3.6 Hz, H-18), 2.22 (1H, d, J=12.6 Hz, H-1a), 1.95 (3H, s, OAcCH3), 1.90 (3H, s, H.sub.3-24), 1.72 (3H, d, J=6.3 Hz, H.sub.3-6, Rham), 1.46 (3H, s, H.sub.3-25), 1.20 (3H, s, H.sub.3-27), 1.16 (3H, d, J=6.3 Hz, H3-6, Fuc), 1.02 (3H, s, H3-26), 0.78 (3H, s, H3-30), 0.77 (3H, s, H.sub.3-29); .sup.13C-NMR (Pyridine-d.sub.5, 225 MHz) ?.sub.C 44.1 (C-1), 70.0 (C-2), 85.7 (C-3), 52.6 (C-4), 52.3 (C-5), 20.9 (C-6), 32.8 (C-7), 40.1 (C-8), 48.4 (C-9), 36.5 (C-10), 23.7 (C-11), 122.4 (C-12), 143.8 (C-13), 42.1 (C-14), 27.9 (C-15), 23.1 (C-16), 46.8 (C-17), 41.7 (C-18), 46.0 (C-19), 30.4 (C-20), 33.6 (C-21), 32.1 (C-22), 180.9 (C-23), 13.8 (C-24), 16.7 (C-25), 17.2 (C-26), 25.8 (C-27), 176.6 (C-28), 32.8 (C-29), 23.5 (C-30), 105.2 (C-1, GlcA), 74.4 (C-2, GlcA), 77.1 (C-3, GlcA), 72.8 (C-4, GlcA), 77.1 (C-5, GlcA), 172.0 (C-6, GlcA), 94.3 (C-1, Fuc), 74.4 (C-2, Fuc), 73.3 (C-3, Fuc), 74.5 (C-4, Fuc), 70.2 (C-5, Fuc), 16.2 (C-6, Fuc), 171.3 (OAc), 20.6 (OAcCH.sub.3), 101.5 (C-1, Rham), 71.1 (C-2, Rham), 71.8 (C-3, Rham), 84.4 (C-4, Rham), 68.4 (C-5, Rham), 18.5 (C-6, Rham), 106.2 (C-1, Glu), 75.8 (C-2, Glu), 78.1 (C-3, Glu), 71.3 (C-4, Glu), 78.1 (C-5, Glu), 62.4 (C-6, Glu).

[0172] Compound 6 (SOA6) 3-O-?-D-xylopyranosyl-(1.fwdarw.3)-?-D-glucuronopyranosyl-2?,3?-dihydroxyolean-12-ene-23,28-dioic acid-28-O-?-D-glucopyranosyl-(1.fwdarw.4)-?-L-rhamnopyranosyl-(1.fwdarw.2)-4-O-acetyl-?-D-fucopyranoside (Spinasaponin G)

##STR00016## [0173] 1) Physical properties: Amorphous white crystals [0174] 2) Molecular weight: 1307.39 [0175] 3) Molecular formula: C.sub.61H.sub.94O.sub.30 [0176] 4) .sup.1H-NMR (Pyridine-d.sub.5, 900 MHZ) ?.sub.H 6.13 (1H, s, H-1, Rham), 5.94 (1H, d, J=8.1 Hz, H-1, Fuc), 5.34 (1H, s, H-12), 5.15 (1H, d, J=7.2 Hz, H-1, Xyl), 5.16 (1H, d, J=7.2 Hz, H-1, GlcA), 5.06 (1H, d, J=8.1 Hz, H-1, Glu), 4.72 (1H, br s, H-2), 4.65 (1H, br s, H-3), 3.02 (1H, dd, J=13.5, 2.7 Hz, H-18), 2.19 (1H, d, J=12.6 Hz, H-1a), 1.94 (3H, s, OAcCH.sub.3), 1.89 (3H, s, H.sub.3-24), 1.72 (3H, d, J=6.3 Hz, H.sub.3-6, Rham), 1.46 (3H, s, H.sub.3-25), 1.20 (3H, s, H.sub.3-27), 1.16 (3H, d, J=6.3 Hz, H.sub.3-6, Fuc), 1.02 (3H, s, H.sub.3-26), 0.81 (3H, s, H.sub.3-30), 0.77 (3H, s, H.sub.3-29); .sup.13C-NMR (Pyridine-d.sub.5, 225 MHZ) ?.sub.C 44.1 (C-1), 70.1 (C-2), 85.7 (C-3), 52.6 (C-4), 52.4 (C-5), 20.9 (C-6), 32.7 (C-7), 40.1 (C-8), 48.4 (C-9), 36.5 (C-10), 23.7 (C-11), 122.4 (C-12), 143.8 (C-13), 42.1 (C-14), 27.9 (C-15), 23.2 (C-16), 46.8 (C-17), 41.7 (C-18), 46.0 (C-19), 30.4 (C-20), 33.6 (C-21), 32.0 (C-22), 180.7 (C-23), 13.8 (C-24), 16.6 (C-25), 17.2 (C-26), 25.8 (C-27), 176.6 (C-28), 32.8 (C-29), 23.5 (C-30), 105.0 (C-1, GlcA), 73.6 (C-2, GlcA), 85.6 (C-3, GlcA), 73.3 (C-4, GlcA), 75.7 (C-5, GlcA), 173.9 (C-6, GlcA), 105.5 (C-1, Xyl), 74.8 (C-2, Xyl), 77.3 (C-3, Xyl), 70.4 (C-4, Xyl), 66.7 (C-5, Xyl), 94.3 (C-1, Fuc), 74.4 (C-2, Fuc), 73.6 (C-3, Fuc), 74.5 (C-4, Fuc), 70.2 (C-5, Fuc), 16.2 (C-6, Fuc), 171.2 (OAc), 20.6 (OAcCH3), 101.5 (C-1, Rham), 71.1 (C-2, Rham), 71.8 (C-, Rham), 84.4 (C-4, Rham), 68.4 (C-5, Rham), 18.4 (C-6, Rham), 106.2 (C-1, Glu), 75.8 (C-2, Glu), 78.0 (C-3, Glu), 71.2 (C-4, Glu), 77.9 (C-5, Glu), 62.3 (C-6, Glu).

Example 4

In Vitro Evaluation of Inflammatory Index Improvement Efficacy of a Spinach Extract, Fractions, Five (5) Novel Substances, and Celosin I Compound

[0177] In order to evaluate the effect of a spinach extract, fractions (butanol fraction, Fr.1 and Fr.2), five (5) novel substances (SOA1, SOA2, SOA4, SOA5, SOA6) and celosin I compound (SOA 3) prepared/obtained through Examples 1 to 3 for improving inflammatory indicators, macrophages (RAW264.7) were treated with LPS and treated with each of the test substances at the concentrations indicated in the figures. Then, the cell culture fluid was collected, and the amount of inflammatory cytokines was measured by ELISA according to a previously reported method.

[0178] As shown in FIGS. 5A and 5B, it was confirmed that LPS increased the secretion of TNF-alpha in macrophages, but the secretion of TNF-alpha decreased in the experimental group treated with the spinach extract or fractions at a non-cytotoxic concentration (FIG. 5B).

[0179] In particular, it was confirmed that Fr.2 fraction comprising the novel glycosides of formulae 1 to 6 more effectively inhibited the secretion of TNF-alpha induced by LPS, as compared to the spinach extract, butanol fraction and Fr.1.

[0180] As shown in FIG. 6, it was confirmed that the novel glycoside compounds of Formulae 1 to 6 more effectively inhibited the secretion of TNF-alpha and nitric oxide (NO) in macrophages by LPS, as compared to medicagenic acid (MA).

[0181] Meanwhile, as shown in FIGS. 7A to 7C, it was confirmed that Fr.2 comprising the novel glycoside compounds of Formulae 1 to 6 inhibited the secretion of NO and PEG2 in macrophages treated with LPS in a concentration-dependent manner (FIGS. 7A and 7B), and also inhibited the expression of COX-2 and iNOS proteins in a concentration-dependent manner (FIG. 7C).

[0182] In addition, as a result of stimulating EL4 cells with PMA and lonomycin and treating them with Fr.2 fraction of the spinach extract, as shown in FIGS. 8A to 8C, it was confirmed that the secretion of inflammatory cytokines (IL-4, IL-5 and IL-13) was inhibited in a concentration-dependent manner.

Example 5

Evaluation of Effect on MUC5AC Secretion in Respiratory Epithelial Cells by PMA Stimulation

[0183] In order to confirm whether the spinach extract, fractions (butanol fraction, Fr.1 and Fr.2) and five (5) novel substances (SOA1, SOA2, SOA4, SOA5, SOA6) and celosin I compound (SOA 3) prepared/obtained through Examples 1 to 3 may control the excessive secretion of mucus in inflammatory respiratory diseases, respiratory epithelial cells NCI-H292 were stimulated with PMA, and treated with each of the test substances at the concentrations indicated in the figures. Next, the amount of MUC5AC secreted from the cells was measured.

[0184] As a result, as shown in FIGS. 9A and 9B, the effect of Fr.2 fraction on inhibition of mucus secretion from respiratory epithelial cells was significantly higher, as compared to the spinach extract, butanol fraction, and Fr.1 fraction (FIG. 9B).

[0185] Meanwhile, the saponin compounds of Formulae 1 to 6 comprising novel glycoside compounds also very effectively inhibited mucus secretion from respiratory epithelial cells caused by PMA stimulation (FIGS. 10A and 10B).

Example 6

Evaluation of Efficacy in Inflammatory Respiratory Disease Animal Models

[0186] To confirm the anti-COPD effect of effective fractions of spinach in COPD mouse models, COPD mouse models in which COPD was induced by cigarette smoke and lipopolysaccharide were prepared. In order to confirm the effect of the effective fractions of spinach for inhibiting ROS, elastase, TNF-?, IL-6 in bronchoalveolar lavage fluid in COPD mice, the produced amount of each of them was analyzed by using ELISA assay technique.

[0187] As a result, as shown in FIGS. 11A to 11D, it was confirmed that the levels of ROS, elastase, TNF-?, and IL-6 in the bronchoalveolar lavage fluid of experimental group S10 administered orally with the prepared effective fraction of spinach (Fr.2), was significantly decreased, as compared to the COPD-induced group, and, through this, it was confirmed that the effective fraction of spinach (Fr.2) were effective for inhibiting inflammatory cytokines, ROS and elastase in COPD mice.

[0188] In order to confirm the anti-asthmatic effect of the effective fraction of spinach (Fr.2) in asthma mouse models, asthma mouse models in which bronchial asthma was induced by using ovalbumin were prepared.

[0189] As a result, as shown in FIGS. 12A to 12D, the levels of IL-4, IL-5, IL-13 in the bronchoalveolar lavage fluid of experimental groups S5 and S10 administered orally with the effective fraction of spinach (Fr.2) were significantly decreased, as compared to the asthma-induced group. In addition, the serum IgE levels of experimental groups S5 and S10 administered orally with the effective fraction of spinach (Fr.2) were significantly decreased, as compared to the asthma-induced group. Through this, it was confirmed that the effective fraction of spinach (Fr.2) were effective for inhibiting Th2 cytokines and IgE in asthmatic mice.

[0190] In order to confirm the anti-pneumonia effect of the effective fraction of spinach (Fr.2) in pneumonia mouse models, pneumonia mouse models in which bronchopneumonia was induced by using lipopolysaccharide were prepared. The increased influx of neutrophils and macrophages is an important feature of the mechanism of pneumonia development and, therefore, in order to confirm the inhibitory effect of spinach effective fraction (Fr.2) on inflammatory cells in bronchoalveolar lavage fluid in mice with pneumonia, each of the cells was isolated and the number of cells were measured by using Diff quik? staining (FIG. 13).

[0191] As a result, it was confirmed that the number of neutrophils and macrophages was significantly increased in the bronchoalveolar lavage fluid of mice with pneumonia induced by lipopolysaccharide, and the experimental groups S5 and S10 administered orally with the effective fraction of spinach were effective for decreasing the number of inflammatory cells.

[0192] In order to confirm the effect of the effective fraction of spinach (Fr.2) for inhibiting ROS, TNF-? and IL-6 in bronchoalveolar lavage fluid in mice with pneumonia, the produced amount of each of them was quantitatively analyzed by using ELISA assay technique.

[0193] As a result, as shown in FIGS. 14A to 14C, it was

[0194] confirmed that the levels of a, b, and c in the bronchoalveolar lavage fluid of experimental groups S5 and S10 administered orally with the effective fraction of spinach (Fr.2) were significantly decreased, as compared to the pneumonia-induced group, and, through this, the effective fraction of spinach was effective for inhibiting ROS and inflammatory cytokines in mice with pneumonia.

INDUSTRIAL APPLICABILITY

[0195] The novel glycoside compound and the spinach extract fraction comprising same provided by the present invention are effective for effectively inhibiting the secretion of excessive inflammatory cytokines from immune cells and suppressing excessive secretion of mucus in inflammatory respiratory diseases such that they may be used in preventing or treating various inflammatory diseases such as inflammatory respiratory diseases. Therefore, the present invention has a high industrial applicability.