KR102484245B1 - Antidiabetic Composition comprising flovonoid derivatives isolated from outer skins of Allium cepa - Google Patents

Abstract

The present invention relates to an antidiabetic composition containing flavonoid derivatives isolated from onion skins, specifically, 9 novel compounds of Compounds 5 to 12 and 14 (Sefaflava A to I) and previously known compounds 1 to 4 , 13, relates to an anti-diabetic composition comprising six types of 15, through which it is expected to contribute to the treatment and research of oxidative stress and diabetes.

Description

Antidiabetic composition comprising flavonoid derivatives isolated from outer skins of Allium cepa

The present invention relates to an antidiabetic composition containing flavonoid derivatives isolated from onion skins, and more specifically, to an antidiabetic composition comprising 9 new compounds of Cefaflava A to I and compounds 1 to 15 including 6 previously known compounds. It relates to an anti-diabetic composition.

Diabetes is one of the four major non-communicable diseases, along with cancer, cardiovascular and chronic lung diseases, and caused 1.6 million deaths in 2015 alone. Diabetes is also a serious chronic disorder that is the leading cause of blindness, kidney failure, heart attack and stroke, and is responsible for an economic burden of up to $1.3 trillion worldwide in 2015 alone.

Type 2 diabetes, which accounts for more than 90% of all diabetes cases, usually occurs in adults and is closely related to overweight, obesity and physical inactivity. In type 2 diabetes, the body cannot use insulin properly, resulting in abnormally high blood sugar levels. Normally, when glucose levels increase, pancreatic β-cells release insulin to induce insulin secretion. Insulin binds to and activates plasma membrane receptors, triggering autophosphorylation of tyrosine residues in the receptors, triggering downstream signal transduction.

Protein tyrosine phosphatase 1B (PTP1B) disrupts the insulin signaling pathway by tyrosine dephosphorylation of insulin receptors and their substrates. Therefore, PTP1B is considered a negative regulator of leptin and insulin signaling pathways directly related to obesity and diabetes. In an in vitro experiment, overexpression of PTP1B in rat adipocytes dramatically inhibited the movement of insulin-stimulated glucose transporter type 4 (GLUT4). In addition, in a mouse model, knockout of the PTP1B gene improved insulin sensitivity and promoted weight loss in the group fed a high-fat diet. Therefore, compounds that inhibit PTP1B could be promising therapeutic agents for obesity and related metabolic diseases.

Another treatment for postprandial hyperglycemia is to reduce the rate of carbohydrate absorption from the gastrointestinal tract by inhibiting α-glucosidase. α-glucosidase inhibitors competitively bind to the carbohydrate binding site of α-glucosidase enzyme located in the brush border of the small intestine, thereby delaying carbohydrate decomposition and alleviating postprandial hyperglycemia. However, commercially available drugs targeting this enzyme cause some gastrointestinal side effects.

On the other hand, onion (Allium cepa) is a perennial plant belonging to the Liliaceae family, and has been used as a food, spice, and medicine with a unique taste and aroma, along with a long cultivation history. Onion is a perennial plant with a spherical or circular inflorescence, and the scales are aponeurotic and purple-brown, but the inner ones are thick, overlapping layers, and have a strong spicy taste. Its origin is considered to be Western Asia, and from there it reached the Mediterranean coast, such as Egypt and Italy, through the Middle East, and crossed over to the United States around the 15th century via Europe. Due to the typical pungent odor associated with sulfur-containing compounds and a rich source of several phytonutrients, onions are an essential kitchen vegetable in many cuisines and have been used as folk remedies for a variety of heart ailments, headaches, insect bites and tumors. It is said that ancient Olympians ate onion juice to improve their stamina, and Chinese people who eat a lot of greasy food also enjoy eating onions, and it is known that the incidence of heart disease is low.

Onions are known to have various biological activities such as anti-cancer, antioxidant, anti-platelet, anti-diabetic, and anti-inflammatory. Among various secondary metabolites having such biological activity, sapogenin, saponin, and flavonoids have been reported as major components. In particular, onions contain a very high amount of quercetin, which shows antioxidant activity, compared to other vegetables or fruits. Quercetin is a plant-derived flavonoid phenolic compound. It is not only a strong antioxidant, but also has anti-atherosclerosis, antibacterial, cholesterol-lowering, anti-cancer, antiviral and anti-allergic activities, and has little toxicity. It is known not to appear. Therefore, if you consume foods high in quercetin, it is effective in reducing blood pressure, diabetes, weight loss, etc., strengthening immune function, inhibiting DNA damage, and inhibiting lipid peroxidation of red blood cell membranes.

It is known that onion skins contain about 10 to 100 times higher flavonoid compounds, including quercetin, than edible parts of onions. Onion skin is a discarded part in the food industry, but these recent research results suggest the possibility that onion skin can be used as a repository of high value-added functional ingredients.

While studying the biological activity of compounds isolated from onion skins, the present inventors confirmed the antioxidant and antidiabetic activities of 15 compounds isolated from onion skins and completed the present invention. Korean Patent Registration No. 1453052 discloses a composition for preventing, improving or treating diabetes and vascular diseases containing hot water extract of onion peel as an active ingredient, but the compound of the present invention is not described.

Korean Patent Registration No. 1453052 and Korean Patent Publication No. 10-2007-0097868 disclose a method for preparing a quercetin extract from onion, a use of the quercetin extract, and a composition for preventing and treating diabetes containing an onion peel extract, but also Compounds of the present invention are not described.

Korean Patent Registration No. 1453052, Composition for Preventing, Improving or Treating Diabetes and Vascular Disease Containing Onion Peel Hot Water Extract as an Active Ingredient, Registered on October 14, 2014. Korean Patent Registration No. 1300667, Manufacturing method of quercetin extract from onion and use of the quercetin extract, registered on August 21, 2013. Korean Patent Publication No. 10-2007-0097868, composition for preventing and treating diabetes containing onion peel extract, published on October 5, 2007.

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Islam, SMS; Purnat, TD; Phuong, NTA; Mwingira, U.; Schacht, K.; Frschl, G. Glob. Health 2014, 10, 81-81. World Health Organization, others, 2016. Global Report on Diabetes. Nazir, MA; AlGhamdi, L.; AlKadi, M.; AlBeajan, N.; AlRashoudi, L.; AlHussan, M. Open Access Maced J. Med. Sci. 2018, 6, 1545-1553. Ha, MT; Seong, SH; Nguyen, TD; Cho, W K.; Ah, KJ; Ma, JY; Woo, M.H.; Choi, J.S.; Min, B. S. Phytochemistry 2018, 155, 114-125. Moller, DE Nature 2001, 414, 821-827. Goldstein, BJ; Ahmad, F.; Ding, W.; Li, PM; Zhang, WR Mol. Cell Biochem. 1998, 182, 91-99. Zabolotny, J.M.; Bence-Hanulec, K.K.; Stricker-Krongrad, A.; Haj, F.; Wang, Y.; Minokoshi, Y.; Kim, Y. B.; Elmquist, JK; Tartaglia, LA; Kahn, B.B.; Neel, B. G. Dev. Cell 2002, 2, 489-495. Hui, C.; Stanley, JW; Chung, H.L.; Susan, L.K.; Kurt, E. A; Paul, B.; Michael JQJ Biol. Chem. 1997, 272, 8026-8031. Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins, S.; Loy, AL; Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan, CC; Ramachandran, C.; Gresser, MJ; Tremblay, M.L.; Kennedy, BP Science 1999, 283, 1544-1548. Liu, M.; Zhang, W.; Wei, J.; Lin, X. Mar. Drugs 2011, 9, 1554-1565. Nguyen, TTA; Ha, MT; Park, SE; Choi, J.S.; Min, B.S.; Kim, JAJ Nat. Prod. 2020, 83, 323-332. Thuong, PT; Hung, TM; Ngoc, TM; Ha, DT; Min, B.S.; Kwack, SJ; Kang, T.S.; Choi, J.S.; Bae, K. Phytother. Res. 2010, 24, 101-106. Kim, DH; Paudel, P.; Yu, T.; Ngo, TM; Kim, J. A.; Jung, H. A.; Yokozawa, T.; Choi, JS Chem. Biol. Interact. 2017, 278, 65-73. Li, T.; Zhang, XD; Song, YW; Liu, JW Chin. J. Clin. Pharmacol. Therapeut. 2005, 10, 1128-1134. Cornish-Bowden, A. Biochem. J. 1974, 137, 143-144. Dixon, M. Biochem. J. 1953, 55, 170-171. Lineweaver, H.; Burk, DJ Am. Chem. Soc. 1934, 56, 658-666. Yoo, NH; Jang, DS; Yoo, JL; Lee, Y. M.; Kim, YS; Cho, J.-H.; Kim, JSJ Nat. Prod. 2008, 71, 713-715. Mustafa, KA; Perry, N.; Weavers, R. Phytochemistry 2004, 64, 1285-1293. Gaffield, W. Chem. Pharm. Bull. 1996, 44, 1102-1103. Ly, TN; Hazama, C.; Shimoyamada, M.; Ando, H.; Kato, K.; Yamauchi, RJ Agric. Food Chem. 2005, 53, 8183-8189. Li, Q.; Gao, W.; Cao, J.; Bi, X.; Chen, G.; Zhang, X.; Xia, X.; Zhao, Y. Fitoterapia 2014, 94, 148-154. Price, KR; Rhodes, MJ Sci. Food Agric. 1997, 74, 331-339. Nakano, K.; Takatani, M.; Tomimatsu, T.; Nohara, T. Phytochemistry 1983, 22, 2831-2833. Park, JC; Young, HS; Yu, Y. B.; Lee, JH Planta Med. 1995, 61, 377-378. Furusawa, M.; Tanaka, T.; Nakaya, K.I.; Iinuma, M.; Tuchiya, H. Heterocycles 2002, 57, 2175-2177. Barford, D.; Das, A.K.; Egloff, M. P. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 133-164

An object of the present invention is to provide an antidiabetic composition comprising a flavonoid derivative isolated from onion skin.

The present invention relates to quercetin (compound 1), kaempferol (compound 2), isorhamnetin (compound 3), quercetin 4'-O-β-D- Glucopyranoside (quercetin 4'-O-β-D-glucopyranoside, Compound 4), Cepaflava A (Compound 5), Cepaflava B (Cepaflava B, Compound 6), Cepaflava C ( Cepaflava C, Compound 7), Cepaflava D, Compound 8, Cepaflava E, Compound 9, Cepaflava F, Compound 10, Cepaflava G , compound 11), Cepaflava H (compound 12), 1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-one -2-yl) -5a- [4- (β-D-glucopyranosyloxy) -3-hydroxyphenyl] -5,6,11-hexahydro-5,6,11-trioxanaphthacene-12 -one (1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-on-2-yl) -5a- [4-( β -D-glucopyranosyloxy) -3-hydroxyphenyl] -5,6,11-hexahydro-5,6,11-trioxanaphthacene-12-one, compound 13), Cepaflava G (compound 14) and 1,3,11a-trihydride Roxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-one-2-yl)-5a-[1,3,11a-trihydroxy-5a-(3,4 -Dihydroxyphenyl) -5,6,11-hexahydro-5,6,11-trioxanaphthacen-12-one-9-yl] -5,6,11-hexahydro-5,6,11 -trioxanaphthacen-12-one (1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-on-2-yl)-5a-[1,3 ,11a-trihydroxy-5a-( 3,4-dihydroxyphenyl)-5,6,11-hexahydro-5,6,11-trioxanaphthacene-12-on-9-yl]-5,6,11-hexahydro-5,6,11-trioxanaphthacene-12- One, compound 15) to provide an antidiabetic composition comprising, as an active ingredient, an onion skin extract containing at least one compound selected from the group consisting of.

[Formula 1]

The onion skin extract may be obtained by extracting the onion skin with water, C1 to C4 alcohol or a mixture solvent thereof, and is preferably a C1 to C4 alcohol or a mixture solvent thereof. Water, C1 to C4 alcohol, or a mixture thereof used during extraction can be used in an amount of 1 to 50 times the volume of the onion skin used, and the above process can be repeated 1 to 5 times.

The extraction time of the extract is not particularly limited, but it is preferable to extract within 10 minutes to 1 day, and the extraction method is all commonly used extraction methods, such as pressure extraction, immersion (cold needle, warm needle), ultrasonic extraction, A reflux extraction method or the like may be used, and as an extraction device, a conventional extraction device, an ultrasonic crusher or a fractionator may be used.

The extract may further comprise filtering to remove floating solid particles. Particles can be filtered out using cotton, nylon, etc., or ultrafiltration, cryofiltration, centrifugation, etc. can be used, and known methods used for separation and extraction of plants can be used alone or in a suitable combination.

In addition, the extract is formed using the extract itself and the extract, such as an extract obtained from the extraction process, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a purified product or a purified product of the extract, or a mixture thereof. Includes extracts of all possible formulations.

⁺ 소거 활성일 수 있으며, DPPH 또는 ABTS

⁺ 소거 실험을 통하여 항산화 활성을 측정할 수 있다.The compound may be synthesized according to a conventional method in the art and may be prepared as a pharmaceutically acceptable salt, but is preferably obtained by chromatographic fractionation from an onion skin extract. The chromatography is silica gel column chromatography, flash column chromatography, Sephadex LH-20 column chromatography, RP-18 column chromatography (RP -18 column chromatography), thin layer chromatography (TLC), medium pressure liquid chromatography and high performance liquid chromatography (HPLC). The anti-diabetic composition has antioxidant activity. Antioxidant activity of the antidiabetic composition of the present invention is DPPH or ABTS

⁺ can be scavenging active, DPPH or ABTS

⁺ Antioxidant activity can be measured through a clearing experiment.

The anti-diabetic composition also has an inhibitory activity against protein tyrosine phosphatase 1B or alpha-glucosidase.

The present invention relates to quercetin (compound 1), kaempferol (compound 2), isorhamnetin (compound 3), quercetin 4'-O-β-D-glu quercetin 4'-O-β-D-glucopyranoside (Compound 4), Cepaflava A (Compound 5), Cepaflava B (Cepaflava B, Compound 6), Cepaflava C (Cepaflava C, compound 7), Cepaflava D (compound 8), Cepaflava E (Cepaflava E, compound 9), Cepaflava F (compound 10), Cepaflava G (Cepaflava G, Compound 11), Cepaflava H (Compound 12), 1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-one- 2-yl)-5a-[4-(β-D-glucopyranosyloxy)-3-hydroxyphenyl]-5,6,11-hexahydro-5,6,11-trioxanaphthacene-12- On (1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-on-2-yl)-5a-[4-(β-D-glucopyranosyloxy)-3 -hydroxyphenyl] -5,6,11-hexahydro-5,6,11-trioxanaphthacene-12-one, compound 13), Cepaflava G (compound 14) and 1,3,11a-trihydroxy- 9-(3,5,7-trihydroxy-4H-1-benzopyran-4-one-2-yl)-5a-[1,3,11a-trihydroxy-5a-(3,4-di Hydroxyphenyl) -5,6,11-hexahydro-5,6,11-trioxanaphthacen-12-one-9-yl] -5,6,11-hexahydro-5,6,11-tri Oxanaphthacen-12-one (1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4H-1-benzopyran-4-on-2-yl)-5a-[1,3,11a -trihydroxy-5a-(3,4-d ihydroxyphenyl)-5,6,11-hexahydro-5,6,11-trioxanaphthacene-12-on-9-yl]-5,6,11-hexahydro-5,6,11-trioxanaphthacene-12-one, compound 15 ) It provides an antidiabetic composition comprising at least one compound selected from the group consisting of as an active ingredient.

⁺ 소거 능력을 갖는 항산화 활성 및 단백질 티로신 포스파타제 1B (protein tyrosine phosphatase 1B) 또는 알파-글루코시다제에 대하여 억제활성을 갖는다.In addition, the present invention is Cepaflava A (Cepaflava A, compound 5), Cepaflava B (Cepaflava B, compound 6), Cepaflava C (Cepaflava C, compound 7) having the structure of [Formula 2], Cepaflava D (Compound 8), Cepaflava E (Compound 9), Cepaflava F (Compound 10), Cepaflava G (Cepaflava G, Compound 11), Cepaflava H (Cepaflava H, compound 12) and Cepaflava G (Cepaflava G, compound 14). The compound having the structure of [Chemical Formula 2] is a new material isolated from onion skin, DPPH or ABTS.

⁺ Antioxidant activity with scavenging ability and inhibitory activity against protein tyrosine phosphatase 1B or alpha-glucosidase.

[Formula 2]

The antidiabetic composition provides an antidiabetic pharmaceutical composition comprising a pharmaceutically acceptable excipient.

Preferably 0.001 to 50% by weight, more preferably 0.001 to 40% by weight, most preferably 0.001 to 30% by weight based on the total weight of the antidiabetic pharmaceutical composition.

The pharmaceutical composition of the above composition is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. and can be used. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, sweeteners, and acidulants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient for the antidiabetic composition of the present invention, for example, starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, and acidulants are used. can be included Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base for the suppository, witepsol, macrogol, tween-61, cacao butter, laurin paper, glycerogeratin, and the like may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages range from 0.01 mg/kg/day to approximately 500 mg/kg/day. A preferred dose is 0.1 mg/kg/day to 200 mg/kg/day, and a more preferred dose is 1 mg/kg/day to 200 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, by oral, rectal or intravenous, intramuscular or subcutaneous injection and dermal application. Since the antidiabetic composition of the present invention has little toxicity and side effects, it can be safely used even when taken for a long period of time for preventive purposes.

The present invention relates to an antidiabetic composition containing flavonoid derivatives isolated from onion skins, specifically, 9 new compounds of Cefaflava A to I and compounds 1 to 15 including 6 previously known compounds. It relates to an antidiabetic composition, and is expected to contribute to research and treatment of oxidative stress and diabetes.

1 is a structure of a flavonoid derivative isolated and confirmed from the onion skin of the present invention.
Figure 2 shows the main HMBC and COZY correlations of compounds 5, 9, 10, 11 and 14 of the present invention.
Figure 3 depicts the predicted biosynthetic pathway of compound 7 from two precursors, quercetin and protocatechuic acid.
Figure 4 shows the ECD spectra of compounds 5-12 and 14 in methanol.
Figure 5 shows the process of determining the location of the carboxylic acids of compounds 7-9 and 14. (A) Structures of compounds 7, 8 and two regioisomers 7a, 7b (B) Experimental ¹ H NMR spectra of compounds 7, 8 (left) and 7a at δ _{H -2''-H-5''} values, Calculated ¹ H NMR spectrum of 7b (right) (C) the δ _{H -2''-H-5''} values and structures of compounds 9 and 14
6 shows the structure determination process of compounds 7, 8, and 14.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Materials and methods>

Structure determination and chromatography

High-resolution electrospray ionisation mass spectrometry (HRESIMS), Thin-Layer Chromatography (TLC), and High-performance liquid chromatography (HPLC) are used for separation and structure determination of compounds. , circular dichroism spectroscopy, etc. were used. For optical rotation, a JASCO P-2000 digital polarimeter was used. Electron circular dichroism (ECD) and UV spectra were recorded using a J-1500 circular dichroism spectrophotometer. IR spectra were recorded using a JASCO FT/IR-4100 spectrometer. NMR spectra were acquired using Varian Unity Inova 400 MHz and Bruker Ascend ^™ 500 MHz spectrometers. High resolution electrospray ionization mass spectrometry (HRESIMS) was performed using an AB SCIEX TripleTOF ^™ 5600 spectrometer. High-resolution electron impact mass spectrometry (HREIMS) was performed using a JEOL JMS-AX 300L spectrometer (Jeol, Akishima, Tokyo, Japan). Column chromatography (CC) was performed using silica gel 60 (Merck, 230-400 mesh) and (RP)-C ₁₈ (Merck, 75 mesh). Thin layer chromatography (TLC) was performed using coated silica gel F ₂₅₄ and RP-18 F _254s plates Merck. The prepared high-performance liquid chromatography (HPLC) was performed using a Waters 2487 with a UV detector (UV/VIS-156) and a YMC Pak ODS-A column (20 × 250 mm, 5 μm particle size; YMC Co., Ltd., Japan). This was done using a controller system.

plant material

The hull of the onion used in the present invention was obtained from A. cepa purchased from the Jungang Market in Daejeon, Republic of Korea in May 2017. The specimen (CUD-0814-1) was kept in the Herbarium of the College of Pharmacy, Daegu Catholic University.

Extraction and Separation

The skins of air-dried onions (10 kg) were extracted with methanol (10 L x 3) under reflux and the combined extracts were concentrated in vacuo. The resulting extract (0.8 kg) was suspended in distilled water (1 L) and fractionated successively between n-hexane, CH ₂ Cl ₂ , EtOAc and n-BuOH. After concentration in vacuo, the EtOAc-soluble fraction (405 g) was subjected to column chromatography (CC) on silica gel (CH ₂ Cl ₂ : MeOH, 10: 1, v/v) to give 30 fractions (E1-E30). . Of these, fraction E23 (1 g) was chromatographed on a silica gel column (n-hexane: EtOAc, 20: 1, v/v) to obtain compound 2 (50 mg). In addition, fraction E30 (80 g) was fractionated with CC (n-hexane: EtOAc, 3: 1, v/v) to obtain 15 small fractions (E30.1-E30.15), and small fraction E30.5 ( 2.5 g) was applied with RP-18 silica gel CC (MeOH: H2O, 1:2, v/v) to obtain compound 1 (150 mg). Small fraction E30.4 (510 mg) was chromatographed on a silica gel column (n-hexane : acetone, 2 : 1, v/v) to give compound 5 (10 mg) and compound 6 (15 mg). Subfraction E30.8 (3 g) was fractionated on silica gel CC (CH ₂ Cl ₂ :MeOH, 20:1, v/v) to give 20 sub-fractions (E30.8.1-E30.8.20). Subfraction E30.8.8 (136 mg) was chromatographed using a RP-18 silica gel column (MeOH : H ₂ O, 1 : 1, v/v) to give compound 3 (10 mg). Small fraction E30.11 (6 g) was applied to silica gel CC (n-hexane : acetone, 1 : 1, v/v) to give compound 4 (50 mg) and compound 13 (80 mg). A wide range of preparative RP-HPLC [Waters 2487 controller system; YMC Pak ODS-A column (see Section 2.1); UV detection at 210 nm] and sub-fraction E30.11.18 (450 mg) was further purified using MeOH:HO (60:40, v/v) at a flow rate of 5 mL/min as the mobile phase; Compound 10 (3 mg), compound 11 (4 mg) and compound 12 (6 mg) were obtained. Subfraction E30.12 (2.5 g) was fractionated on silica gel CC (CH ₂ Cl ₂ : MeOH, 15 : 1, v/v) to obtain 20 sub-fractions (E30.12.1-E30.12.20), and a wide range Preparative RP-HPLC [Waters 2487 Controller System; YMC Pak ODS-A column (see Section 2.1); UV detection at 210 nm] from sub-fraction E30.12.6 (450 mg) using MeOH:HO (55:45, v/v) as mobile phase at a flow rate of 5 mL/min, compound 7 (3 mg), compound 8 (6mg), compound 9 (4mg), compound 14 (10mg) and compound 15 (20mg) were isolated.

acid hydrolysis

For hydrolysis, 1 mg of each compound was weighed, then 3 mL of 2N CF ₃ COOH was added and then boiled at 95° C. for 3 hours. The solution was partitioned with methylene chloride (3 x 3 mL). The aqueous layer was dried in vacuo and analyzed by TLC (MeOH:CHCl ₃ :H ₂ O, 5:8:1) on silica gel and compared to standards. The monosaccharide fraction was obtained using preparative TLC with the same solvent system. The optical rotation of the sugar was measured and identified as D-glucose (positive [ α ] _D ).

Structure determination and calculation method

Systematic structural searches for compounds 5, 6, 11 and 12 were performed with Spartan 14 software (Wavefunction, Inc., Irvine, CA, USA) under molecular dynamic force field (MMFF). Suitable conformers were selected for optimization by density functional theory (DFT) calculations at the B3LYP/6-31+G(d,p) level using the Gaussian 09 package (Gaussian, Inc., Wallingford, CT, USA), Time-dependent DFT ECD calculations of the optimized conformers were performed at the CAM-B3LYP/TZVP level using a conductor polarizability continuum (CPCM) solvent model in MeOH. Calculated ECD spectra of different conformers were simulated with half-bandwidths of 0.2–0.3 eV and ECD curves were generated with SpecDis 1.64 software. ECD spectra of compounds 5, 6, 11 and 12 were weighted with Boltzmann distribution after UV correction.

dinger LLC, New York, NY, USA) 모듈을 사용하여 발견하였다. 검색은 모든 가능한 컨포머를 탐색하기 위하여 5 kJ/mol 에너지 윈도우 한계와 10,000 개의 최대 단계로 가스 상에서 실시하였다. Polak-Ribi

re 접합 구배 방법은 10,000 회 반복 및 RMS (root mean square) 구배에서 0.001 kJ (mol Å) -1 수렴 임계 값의 컨포머를 최소화하기 위하여 사용되었다. CP3 (화합물 7 및 8) 및 DP4 (화합물 14) 분석의 경우, Gaussian 16 패키지를 사용하여 분극성 연속체 모델 (PCM; MeOH)의 mPW1PW91 / 6-311G^* 수준에서 기하 최적화없이 컨포머의 화학적 이동을 계산하였다. 계산된 NMR 특성은 각각의 볼츠만 모집단을 기준으로 평균화되었으며 CP3 및 DP4 분석을 위해 http://www-jmg.ch.cam.ac.uk/tools/nmr 에 있는 애플릿으로 분석되었다. 화합물 9의 특정 회전 계산의 경우, 모든 컨포머들은 B3LYP/6-31+G(d, p) 레벨의 기체상에서 기하 최적화를 수행했으며, 1% 이상의 모집단을 가진 컨포머는 B3LYP/6-31+G(d,p)에서 특정 회전 값 계산을 수행하였다. 계산된 특정 회전 값은 각 컨포머의 계산된 깁스 자유 에너지에 기초하여 볼츠만 평균화하였다. ECD 계산을 위해, 모든 컨포머는 BP86/SVP 레벨의 기체 상에서 기하 최적화되었으며, PCM(MeOH)에서 BP86/ aug-cc-pVDZ 레벨에서 여기 에너지, 발진기 강도 및 회전 강도 계산을 수행하였다. ECD 스펙트럼은 각 컨포머의 계산된 깁스 자유 에너지에 기초하여 볼츠만 평균화하였고, σ/γ 값이 0.6 eV인 SpecDis 소프트웨어 (버전 1.71)를 사용하여 시각화되었다.Conformers of compounds 7–10 and 14 were modeled in MacroModel (Version 2015-2; Schr.

dinger LLC, New York, NY, USA) module. The search was performed in the gas phase with a 5 kJ/mol energy window limit and a maximum step of 10,000 to search all possible conformers. Polak-Ribi

The re junction gradient method was used to minimize conformers with a convergence threshold of 0.001 kJ (mol Å) -1 at 10,000 iterations and a root mean square (RMS) gradient. For CP3 (compounds 7 and 8) and DP4 (compound 14) analyses, chemical shifts of the conformers without geometric optimization were performed at the mPW1PW91/6-311G ^* level of the polarizable continuum model (PCM; MeOH) using the Gaussian 16 package. Calculated. The calculated NMR properties were averaged for each Boltzmann population and analyzed with the applet available at http://www-jmg.ch.cam.ac.uk/tools/nmr for CP3 and DP4 analysis. For the calculation of the specific rotation of compound 9, all conformers performed geometry optimization on the B3LYP/6-31+G(d,p) level gas phase, and conformers with more than 1% population were B3LYP/6-31+ In G(d,p), specific rotation value calculations were performed. The calculated specific rotation values were Boltzmann averaged based on the calculated Gibbs free energy of each conformer. For ECD calculations, all conformers were geometrically optimized in the gas phase of the BP86/SVP level, and excitation energy, oscillator intensity and rotational intensity calculations were performed at the BP86/aug-cc-pVDZ level in PCM (MeOH). ECD spectra were Boltzmann averaged based on the calculated Gibbs free energy of each conformer and visualized using SpecDis software (version 1.71) with a σ/γ value of 0.6 eV.

DPPH Radical scavenging assay

DPPH radical scavenging assay was performed in the same way as Thuong et al. (2006). 5 μl of the methanol extract of the sample and 195 μl of 150 μM DPPH were mixed and dispensed into a 96-well plate, and then left at room temperature for 30 minutes. Then, the absorbance of the reaction mixture at 520 nm was measured with a microplate reader spectrophotometer (VERSAmax, Molecular Devices, Sunnyvale, CA, USA). The free radical scavenging activity is expressed as:

DPPH scavenging activity (%) = 100 x [(Ac-As)/(Ac-Ab)]

At this time, Ac is the absorbance of the control group, As is the absorbance of the sample, and Ab is the absorbance of the blank (methanol). Each sample was repeated in triplicate, and the IC ₅₀ value was defined as the 50% scavenged concentration of generated radicals.

⁺ ) 라디칼 소거 분석 Peroxyl ( ABTS )

⁺ ) radical scavenging assay

⁺ 라디칼 양이온은 ABTS를 칼륨퍼설페이트와 후속 반응으로 반응시켜 생성되었다. ABTS를 10 mL H₂O에 7 mM 농도로 용해시킨 후, 60 mm K₂S₄O₈ (최종 농도 2.45 mM) 400 μL를 첨가하고, 그 혼합물을 실온의 암실에서 16시간 보관하였다. 라디칼은 이 형태에서 2일 이상 동안 안정적이었다. ABTS

⁺ 라디칼 용액을 734 nm에서 0.700±0.020의 흡광도가 되도록 물로 희석하였다. 소거 분석을 위하여 희석 ABTS

⁺ 라디칼 용액 990 μL에 테스트 시료 또는 트롤록스 기준품(최종농도 0-20μM) 10 μL를 혼합한 다음 정확히 6분 후에 734 nm에서 흡광도를 측정하고 흡광도 감소율을 계산하였다.ABTS

The ⁺ radical cation was produced by reacting ABTS with potassium persulfate in a subsequent reaction. After dissolving ABTS in 10 mL H ₂ O at a concentration of 7 mM, 400 μL of 60 mm K ₂ S ₄ O ₈ (final concentration 2.45 mM) was added, and the mixture was stored in the dark at room temperature for 16 hours. The radical was stable for more than 2 days in this form. ABTS

⁺ The radical solution was diluted with water to obtain an absorbance of 0.700 ± 0.020 at 734 nm. Dilute ABTS for clearance assay

⁺ 990 μL of the radical solution was mixed with 10 μL of the test sample or Trolox reference product (final concentration 0-20 μM), and then, exactly 6 minutes later, the absorbance was measured at 734 nm and the absorbance reduction rate was calculated.

PTP1B inhibition assay

Analysis of protein-tyrosine phosphatase 1B (PTP1B) inhibition of the isolated compound (1-15) was performed using p-nitrophenyl phosphate (p-NPP) as a substrate in the same manner as Kim et al. (2017). PTP1B enzyme diluted in reaction buffer, 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA and 1 mM DTT were added to each well of a 96-well plate, and 2 mM pNPP dissolved in reaction buffer was added. 50 μL was added. The mixture was left in the dark at 37° C. for 20 minutes, and the reaction was terminated by adding 10 μL of 10 M NaOH. The amount of p - nitrophenolate produced by enzymatic dephosphorylation of pNPP was measured by absorbance at 405 nm. Ursolic acid was used as a positive control, and the inhibition rate (%) was calculated by the formula {(AC - AS) / AC} × 100, and IC ₅₀ was calculated.

α- glucosidase inhibition assay

α-glucosidase inhibition was assayed using p-nitrophenyl α-D-glucopyranoside (p-NPG) as a substrate. α-glucosidase activity was assayed by measuring the emission of p-NPG at 405 nm using a microplate spectrophotometer. Acarbose was used as a positive control.

PTP1B Enzyme kinetics analysis using

In order to determine the PTP1B inhibitory mechanism of the most active compounds 13-15, Lineweaver-Burk and Dixon plots were used in the same way as Kim et al. (2017).

PTP1B Molecular Docking Simulation in Inhibition

To clarify the interaction and binding modes of PTP1B and compounds 13 and 15, molecular docking simulations were implemented using AutoDock 4.2 software. Docking protocol was performed with AutoDock 4.2 program.

< Example 1. Identification of flavonoids isolated from onion skin >

The skin of the onion was extracted with MeOH to obtain a crude extract which was partitioned successively between n-hexane, CH ₂ Cl ₂ , EtOAc and n-BuOH. The EtOAc fraction (405 g) was separated on silica gel and semi-preparative HPLC to give 9 new flavonoids (Cefaflava AI, compounds 5-12, 14) and 6 known compounds (1-4, 13, 15) (Fig. 1). 1H (400 MHz) and 13C (100 MHz) NMR data of the nine new flavonoids are shown in Tables 1-3.

position compound 5 ^a compound 6 ^a compound 14 ^b δ _H mult.,
( J in Hz) δC , _type δ _H mult.,
( J in Hz) δC , _type δ _H mult.,
( J in Hz) δC , _type 2 108.8, C. 109.4, C. 101.8, C 3 79.9, C. 80.0,C 91.9, C 4 196.4, C=O 199.0, C=O 189.7, C=O 4a 100.7, C 101.9, C. 101.3, C. 5 165.5, C 164.4, C. 165.3, C. 6 5.94, d (2.0) 97.6, CH 5.95, s 97.8, CH 5.93 ^{, c d (2.7)} 97.4, CH 7 168.4, C. 168.3, C. 169.7, C 8 5.98, d (2.0) 96.7, CH 6.00, s 97.1, CH ^{6.00; c d (2.7)} 97.4, CH 8a 159.9, C. 158.8, C. 161.1, C. One' 126.5, C. 126.3, C. 126.8, C 2' 7.01, d (1.9) 117.3, CH 7.11, s 117.1, CH 7.21, s 116.8, CH 3' 145.9, C 145.8, C 145.8, C 4' 147.5, C. 147.5, C. 148.0,C 5' 6.80, d (8.2) 115.6, CH 6.80, d (7.8) 115.6, CH 6.69, d (8.4) 115.4, CH 6' 6.87, dd (8.2, 1.9) 121.4, CH 7.02, d (7.8) 121.6, CH 7.03 d (8.4) 121.2, CH One'' 3.05, d (16.0)
2.25, d (16.0) 41.3 CH ₂ 2.56, d (13.5)
2.42, d (13.5) 49.8 CH ₂ 2'' 214.2, C=O 209.3, C=O 101.5, C 3'' 2.23, s 31.9 CH ₃ 1.98, s 32.3 CH ₃ 91.7, C 4'' 189.3, C=O 4''a 101.3, C. 5'' 165.5, C 6'' ^{5.93; c d (2.7)} 98.4, CH 7'' 169.9, C 8'' 6.00, ^{c d (2.7)} 97.9, CH 8''a 160.8, C One''' 130.0,C 2''' 7.44, d (2.0) 119.0, CH 3''' 141.7, C. 4''' 143.7, C. 5''' 6.93, d (8.5) 118.0, CH 6''' 7.34, dd(8.5, 2.0) 123.8, CH One'''' 126.2, C. 2'''' 7.63, s 120.0, CH 3'''' 142.1, C. 4'''' 146.1, C. 5'''' 7.18, d (8.5) 118.3, CH 6'''' 7.71, d (8.5) 125.5, CH One''''' 167.7, C=O 2-OCH ₃ 3.12, s 51.4, OCH ₃ 2.98, s 51.4, OCH ₃ COO C H ₃ 3.88, s 52.6, OCH ₃

^{a 1} H (400 MHz) and ¹³ C (100 MHz).

^{b 1} H (500 MHz) and ¹³ C (125 MHz).

^c Overlapped with other signals.

position compound 7 compound 8 compound 9 δ _H mult., ( J in Hz) δC , _type δ _H mult., ( J in Hz) δC , _type δ _H mult., ( J in Hz) δC , _type 2 101.8, C 101.9, C. 101.9, C. 3 92.1, C. 91.8, C 91.8, C 4 189.6, C=O 189.7, C=O 189.7, C = O 4a 101.3, C. 101.3, C. 101.3, C. 5 165.4, C. 165.3, C. 165.4, C. 6 5.94, d (2.1) 98.0, CH 5.96, s 98.0, CH 5.89, s 98.0, CH 7 169.7, C 169.6, C 169.7, C 8 5.93, d (2.1) 97.3, CH 5.96, s 97.3, CH 5.89, s 97.3, CH 8a 161.1, C. 161.0,C 161.0,C One' 126.7, C. 126.6, C. 126.5, C 2' 7.23, d (2.3) 116.8, CH 7.24, d (2.2) 116.7, CH 7.17, d (2.0) 116.7, CH 3' 145.8, C 145.7, C. 145.8, C 4' 148.1, C. 148.0,C 148.1, C. 5' 6.69, d (8.5) 115.5, CH 6.71, d (8.5) 115.4, CH 6.64, d (8.4) 115.4, CH 6' 7.05, dd(8.5, 2.3) 121.3, CH 7.07, dd(8.5, 2.2) 121.2, CH 7.00, dd(8.4, 2.0) 121.2, CH One'' 126.4, C. 126.5, C. 126.0,C 2'' 7.682, d (2.0) 119.9, CH 7.61, d (1.8) 120.1, CH 7.55, s 119.9, CH 3'' 141.7, C. 142.0,C 142.2, C. 4'' 146.6, C. 146.1, C. 146.3, C. 5'' 7.02 d (9.0) 118.4, CH 7.12, d (8.5) 118.1, CH 7.08 d (8.5) 118.3, CH 6'' 7.676, dd(9.0, 2.0) 125.8, CH 7.70, d d (8.5, 1.8) 125.6, CH 7.65, d (8.5) 125.4, CH One''' 169.1, C=O 169.1, C=O 167.8, C=O 2''' 3.84, s 52.6, OCH ₃

position compound 10 compound 11 compound 12 δ _H mult.,
( J in Hz) δC , _type δ _H mult.,
( J in Hz) δC , _type δ _H mult.,
( J in Hz) δC , _type 2 119.4, C. 93.7, C 93.6, C 3 81.8, C 103.2, C. 104.3, C. 4 192.6, C=O 186.9, C=O 186.9, C=O 4a 100.2, C. 100.2, C. 100.2, C. 5 165.5, C 166.4, C. 166.4, C. 6 5.90, s 97.9, CH 5.99, d (2.1) 97.7, CH 5.93, s 97.7, CH 7 169.3, C. 170.6, C. 170.4, C. 8 5.85, s 96.1, CH 6.02, d (2.1) 96.3, CH 5.97, s 96.2, CH 8a 162.6, C. 164.3, C. 164.3, C. One' 125.7, C. 127.1, C. 127.1, C. 2' 6.95, s 115.3, CH 6.97, d (2.1) 116.9 6.94, d (1.8) 117.0 3' 145.9, C 146.2 146.2 4' 147.6, C. 147.0 147.0 5' 6.73, d (8.4) 115.7, CH 6.82, d (8.5) 115.9, CH 6.76, d (8.4) 116.0, CH 6' 6.86, d (8.4) 120.0, CH 6.86, dd(8.5, 2.1) 120.6, CH 6.81, d d (8.4, 1.8) 120.6, CH 2'' 148.4, C. 146.9, C. 146.9, C. 3'' 138.5, C. 138.2, C. 138.2, C. 4'' 177.8, C = O 177.8, C = O 177.8, C = O 4''a 106.7, C. 106.3, C. 106.3, C. 5'' 166.0,C 165.7, C 165.7, C 6'' 6.54, s 95.8, CH 6.44, s 95.5, CH 6.39, s 95.5, CH 7'' 167.3, C. 167.6, C. 167.6, C. 8'' 107.3, C. 108.8, C. 108.8, C. 8''a 153.3, C. 153.1, C. 153.1, C. One''' 127.2, C. 126.8, C 126.8, C 2''' 7.95, s 117.34, CH 7.80, d (2.2) 117.3, CH 7.78, s 117.3, CH 3''' 147.9, C. 147.4, C. 147.2, C. 4''' 148.1, C. 148.2, C. 148.4, C. 5''' 7.29, d (8.2) 117.29, CH 7.02 d (8.8) 117.2, CH 6.99, d (8.8) 117.3, CH 6''' 7.85, d (8.2) 122.1, CH 6.33, d d (8.8, 2.2) 120.8, CH 6.13, d (8.8) 120.8, CH One'''' 4.92, d (8.6) 103.5, CH 4.90 ^a 103.1, CH 4.80, d (6.5) 103.6, CH 2'''' 3.50 ^a 74.8, CH 3.52 ^a 74.8, CH 3.48 ^a 74.7, CH 3'''' 3.45 ^a 78.4, CH 3.54 ^a 78.4, CH 3.56 ^a 78.2, CH 4'''' 3.45 ^a 71.2, CH 3.44 m 71.4, CH 3.38 m 71.5, CH 5'''' 3.50 ^a 77.5, CH 3.55 ^a 77.6, CH 3.46 ^a 77.6, CH 6'''' 3.91, d (12.3)
3.73, d d (12.3, 6.1) 62.3 CH ₂ 4.06, dd(12.2, 2.1)
3.78, d d (12.2, 5.8) 62.6 CH ₂ 4.00, d (13.2)
3.73, d d (12.1, 5.9) 62.7 CH ₂

^a Overlapped with other signals

cefaflava A ( Cepaflava A, compound 5)

-20.7 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 291 (2.28), 252 (1.75), 198 (2.57) nm; ECD (MeOH) λ_max (Δε) 200 (2.89), 234 (-0.01), 261 (0.20), 299 (-0.18); IR (KBr) v_max 3324, 2941, 2831, 1637, 1448, 1024; ¹H (400MHz, 메탄올-d₄) 및 ¹³C NMR (100MHz, 메탄올-d ₄) 데이터, 표 1 참조; HREIMS m/z 390.0948 ([M]⁺, calcd for C₁₉H₁₈O₉, 390.0951).Reddish-brown, amorphous powder;

-20.7 ( c 0.1, MeOH); UV (MeOH) λ _max (log ε) 291 (2.28), 252 (1.75), 198 (2.57) nm; ECD (MeOH) λ _max (Δε) 200 (2.89), 234 (-0.01), 261 (0.20), 299 (-0.18); IR (KBr) v _max 3324, 2941, 2831, 1637, 1448, 1024; ¹ H (400 MHz, methanol-d ₄ ) and ¹³ C NMR (100 MHz, methanol- d ₄ ) data, see Table 1; HREIMS m/z 390.0948 ([M] ⁺ , calcd for C ₁₉ H ₁₈ O ₉ , 390.0951).

Compound 5 was isolated as a reddish-brown amorphous powder with molecular formula C ₁₉ H ₁₈ O ₉ and was deduced from HREIMS showing a molecular ion peak at m/z 390.0948 [M] ⁺ . The UV spectrum showed absorption maxima at 198, 252 and 291 nm, and the IR spectrum showed hydroxyl (3324 cm ^-1 ), carbonyl (1637 cm ^-1 ) and double bond functional groups (1448 cm ^-1 ). ¹ H and ¹³ C NMR spectra of compound 5 were obtained using the ABX coupling system [δ _H 7.01 (1H, d, J = 1.9 Hz, H-2'), 6.87 (1H, dd, J = 8.2, 1.9 Hz, H-2'). 6′) and 6.80 (1H, d, J = 8.2 Hz, H-5′)], two metacouple doublets [ _δH 5.98 (1H, d, J = 2.0 Hz, H-8) and 5.94 (1H , d, J = 2.0 Hz, H-6)] and a carbonyl signal at δ _C 196.4 (C-4), which can generally be a flavanone backbone. And, the methyl signal (δ _H 2.23, s, H3-3'') describing the CH ₂ -CO-CH ₃ fragment, the methylene group [δ _H 3.05 (1H, d, J = 16.0 Hz, H-1'' a), 2.25 (1H, d, J = 16.0 Hz, H-1″b)] and a carbonyl carbon (δ _C 214.2, C-2″).

In addition, a singlet δ _H 3.12 (3H, s, 2-OCH ₃ ) of a methoxy group was detected in the ¹ H NMR spectrum of compound 5. The ¹³ C NMR spectrum of compound 5 shows two carbonyls [δ _C 214.2 (C-2″) and 196.4 (C-4)], a ketal (δ _C 108.8, C-2), oxygenation (δ _C 79.9, C -3), methoxy (δ _C 51.4, 2-OCH ₃ ), methyl (δ _C 31.9 , C-3″), methylene (δ _C 41.3, C-1″) and 12 quaternary carbons showed 19 signals that As a result of comparing the NMR data of Compound 5 with Erigeroflavanone, the methoxy group (2''-OCH ₃ ) of Erigeroflavanone was replaced with the methyl group (2''-CH3) in Compound 5, except that and their structures were very similar. This reasoning supports the downfield chemical shift of C-2'' (δ _C 214.2) in compound 5 compared to C-2'' (δ _C 171.4) of erythroflavanone and C-2'' (δ _C 214.2). and methyl protons H ₃ -3″ (δ _H 2.23) for C-1″ (δ _C 41.3), H-1′′a (δ _H 3.05) for C-2″ (δ _C 214.2). and H-1''b (δ _H 2.25) can be confirmed by HMBC correlation (FIG. 2). In addition, the positions of the methoxy group at C-2 and the acetonyl group at C-3 are H-1''a (δ _H 3.05) and H-1''b (δ _H 2.25), C-4 (δ _C 196.4) , the correlations of C-2 (δ _C 108.8) and C-3 (δ _C 79.9), as well as H-2' (δ _H 7.01), H-6' (δ _H 6.87) and 2-OCH ₃ ( _δH 3.12) respectively (Fig. 2).

Based on the 1D and 2D NMR data of compound 5, the planar structure of 5 was inferred. These C-2 and C-3 oxygenated flavonoids are actually uncommon structures (FIG. 3).

cefaflava B ( Cepaflava B, compound 6)

-28.8 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 290 (2.31), 252 (1.69), 200 (2.66) nm; ECD (MeOH) λ_max (Δε) 200 (1.40), 236 (-0.35), 260 (-0.17), 293 (-0.61); IR (KBr) v_max 3318, 2942, 2832, 1636, 1448, 1116, 1024; ¹H (400MHz, 메탄올-d₄) 및 ¹³C NMR (100MHz, 메탄올 -d₄) 데이터, 표 1 참조; HREIMS m/z 390.0954 ([M]⁺, calcd for C₁₉H₁₈O₉, 390.0951).Pale yellow, amorphous powder;

-28.8 ( c 0.1, MeOH); UV (MeOH) λ _max (log ε) 290 (2.31), 252 (1.69), 200 (2.66) nm; ECD (MeOH) λ _max (Δε) 200 (1.40), 236 (-0.35), 260 (-0.17), 293 (-0.61); IR (KBr) v _max 3318, 2942, 2832, 1636, 1448, 1116, 1024; ¹ H (400 MHz, methanol-d ₄ ) and ¹³ C NMR (100 MHz, methanol-d ₄ ) data, see Table 1; HREIMS m/z 390.0954 ([M] ⁺ , calcd for C ₁₉ H ₁₈ O ₉ , 390.0951).

Cefaflava B (Compound 6) was obtained as a pale yellow amorphous powder having the same molecular formula C ₁₉ H ₁₈ O ₉ as Compound 5 based on the molecular ion peak of m/z 390.0954 in the HREIMS spectrum. ¹ H and ¹³ C NMR data of compound 6 showed H ₂ -1′′ [δ _H 2.42 and 2.56 (6) and 2.25 and 3.05 (5)], C-4 [δ _C 199.0 (6), 196.4 (5) ], compound 5 except for the difference in chemical shifts of C-1''[δ _C 49.8 (6), 41.3 (5)] and C-2'' [δ _C 209.3 (6), 214.2 (5 )]. were very similar to those of (Table 1). This suggests that compounds 5 and 6 may be stereoisomers. 2-OCH ₃ in compounds 5 and 6 and 3-OH groups their ¹³ C NMR chemical shifts into stereoisomers, trans-2,5-dihydroxy-6/8-methyl- with hydroxyl groups at both C-2 and C-3. It was determined by comparing 7-methoxyflavanone ( trans -DMM) and cis -DMM DMM)dhk. The ¹³ C NMR chemical shift values of trans -DMM ( _δC 103.7/103.4, 75.4/75.2 and 197.5/197.3) were smaller than those of cis -DMM ( _δC 105.4/104.8, 77.4 and 197.8/197.6). The same pattern was observed for compounds 5 and 6 [ _δC 108.8, 79.9 and 196.4 (5) and δC 109.4, 80.0 and 199.0 (6)], indicating that compounds 5 and 6 have the relative structures of the trans- and cis -forms, respectively. means to have

As shown in FIG. 4, the ECD spectra of compounds 5 and 6 were similar to isoastilbin (2 R -form), which showed a negative Cotton effect at 300 nm or less (FIG. 4). Therefore, the absolute composition of compounds 5 and 6 was determined as (2R, 3R) and (2R, 3S), and they were named cepaflava A and cepaflava B, respectively (FIG. 1).

cefaflava C ( Cepaflava C, compound 7)

-23.8 (c 0.07, MeOH); UV (MeOH) λ_max (log ε) 292 (2.46), 268 (2.20), 209 (2.69) nm; ECD (MeOH) λ_max (Δε) 299 (0.03), 263 (0.29), 243 (0.25), 203 (3.04); IR (KBr) v_max 3324, 2945, 2831, 1733, 1450, 1116, 1036; ¹H (500MHz, 메탄올-d₄) 및 ¹³C NMR (125MHz, 메탄올-d₄) 데이터, 표 2 참조; HRESIMS m/z 477.0436 ([M + Na]⁺, calcd for C₂₂H₁₄O₁₁Na, 477.0434).Green, amorphous powder;

-23.8 ( c 0.07, MeOH); UV (MeOH) λ _max (log ε) 292 (2.46), 268 (2.20), 209 (2.69) nm; ECD (MeOH) λ _max (Δε) 299 (0.03), 263 (0.29), 243 (0.25), 203 (3.04); IR (KBr) v _max 3324, 2945, 2831, 1733, 1450, 1116, 1036; ¹ H (500 MHz, methanol-d ₄ ) and ¹³ C NMR (125 MHz, methanol-d ₄ ) data, see Table 2; HRESIMS m/z 477.0436 ([M + Na] ⁺ , calcd for C ₂₂ H ₁₄ O ₁₁ Na, 477.0434).

cefaflava D ( Cepaflava D, compound 8)

-9.4 (c 0.08, MeOH); UV (MeOH) λ_max (log ε) 292 (2.32), 268 (2.05), 209 (2.54) nm; ECD (MeOH) λ_max (Δε) 299 (-0.51), 261 (-0.14), 248 (-0.21), 203 (3.94); IR (KBr) v_max 3324, 2945, 2831, 1733, 1450, 1116, 1036; ¹H (500MHz, 메탄올-d₄) 및 ¹³C NMR (125MHz, 메탄올-d₄) 데이터, 표 2 참조; HRESIMS m/z 477.0436 ([M + Na]⁺, calcd for C₂₂H₁₄O₁₁Na, 477.0434).light brown, amorphous powder;

-9.4 ( c 0.08, MeOH); UV (MeOH) λ _max (log ε) 292 (2.32), 268 (2.05), 209 (2.54) nm; ECD (MeOH) λ _max (Δε) 299 (-0.51), 261 (-0.14), 248 (-0.21), 203 (3.94); IR (KBr) v _max 3324, 2945, 2831, 1733, 1450, 1116, 1036; ¹ H (500 MHz, methanol-d ₄ ) and ¹³ C NMR (125 MHz, methanol-d ₄ ) data, see Table 2; HRESIMS m/z 477.0436 ([M + Na] ⁺ , calcd for C ₂₂ H ₁₄ O ₁₁ Na, 477.0434).

Sepaflavas C (Compound 7) and D (Compound 8) were isolated as green and light brown amorphous powders, respectively. The molecular formulas of compounds 7 and 8 were determined to be the same C ₂₂ H ₁₄ O ₁₁ based on sodium addition molecular ion peaks in the HRESIMS spectra. ¹ H NMR data of compound 7 showed a pattern similar to that of compound 8. That is, [ δ _H 7.676 (1H, dd, J = 9.0, 2.0 Hz, H-6″), 7.682 (1H, d, J = 2.0, H-2″), and 7.02 (1H, d, J = 9.0 Hz, _H -5″)] and [ δH of the B-ring 7.23 (1H, d, J = 2.3 Hz, H-2′), 7.05 (1H, dd, J = 8.5, 2.3 Hz, H-6'), and 6.69 (1H, d, J = 8.5 Hz, _H -5')] and the [ δH of the D-ring of compound 8 7.70 (1H, dd, J = 8.5 , 1.8 Hz, H-6″), 7.61 (1H, d, J = 1.8, H-2″), and 7.12 (1H, d, J = 8.5 Hz, H-5″)], B- Ring's [ δH 7.24 ( _1H , d, J = 2.2 Hz, H-2'), 7.07 (1H, dd, J = 8.5, 2.2 Hz, H-6'), and 6.71 (1H, d, J = 8.5 Hz, H-5′)] and [ δ _H of compound 7, two meta-coupled proton signals. 5.93 (1H, d, J = 2.1 Hz, H-8), 5.94 (1H, d, J = 2.1 Hz, _H -6)] and [ δH of compound 8 5.96 (2H, s, H-6 and H-8)] showed a similar pattern.

Analysis of the data of ¹³ C NMR revealed a total of 22 carbon resonances, including: of compounds 7 and 8 Ester carbonyl signal at C-1''' ( δ _C 169.1), ketone carbonyl signal at C-4, i.e. δ _C 189.6 for compound 7, 189.7 for compound 8, two quaternary carbon signature signals, i.e. C In the case of -3, δ _C 92.1 for compound 7, 91.8 for compound 8, δ _C 101.8 for compound 7, 101.9 for compound 8, and 18 aromatic carbons for C-2 showed carbon resonance. The signatures of these two quaternary carbon characters at C-2 and C-3 were similar to the 2,3-deoxygenated flavanone structures of compounds 5 and 6. However, C-3 is similar to compounds 5 ( δ _C 79.9) and 6 ( δ _C 80.0) was not shielded. These NMR data of compounds 7 and 8 are almost identical to the two previously reported regioisomers, suggesting that they are condensed compounds derived from quercetin and protocatechuic acid. H-2″ for δ _C 169.1 ( δ _H 7.682 for Compound 7, 7.61 for Compound 8) and H-6″ ( δ _H 7.676 for Compound 7, 7.70 for Compound 8), C-3″ (Compound 8) 7 is _C 141.7, Compound 8 is 142.0) and C-4'' ( δ _C 146.6 for Compound 7, 146.1 for Compound 8) with H-5'' (Compound 7 is 7.12, Compound 8 is 7.12) along with HMBC correlations. , ABX coupling system, a carboxyl group, and it was confirmed that a protocatechuic acid moiety exists in compounds 7 and 8. The oxidative coupling of the conjugated olefin bond (C-2/C-3) of the C-ring of quercetin and the ortho -dihydroxy group (C-3''/C-4) of protocatechuic acid is more It gives rise to two positional isomers with C2-C4″/C3-C3″ or C2-C3″/C3-C4″ dioxane linkages in the preferred cis -configuration (FIG. 3). Thus, a total of four cis-isomers (7a, 7b, ent-7a and ent-7b) are possible for compounds 7 and 8 (Fig. 5A). Considering that the HMBC correlation cannot distinguish between 7a and 7b (or ent-7a and ent-7b), we calculated the ¹ H and ¹³ C NMR chemical shifts and compared them with the experimental values, and found that the main difference was H-2 '' and H-5''. As shown in Fig. 5B, the experimental δΔ _{H -2''/H-5''} was 0.67 for compound 7 and 0.50 for compound 8, and the calculated values were 0.61 for 7a and 0.54 for 7b. The most probable structures for compounds 7 and 8 are those of 7a and 7b (or ent-7a and ent-7b), respectively. The proposed structures of compounds 7 and 8 were analyzed by comprehensive statistical analysis using the CP3 probability method for full calculations and experimental ¹ H and ¹³ C NMR chemical shifts with 98% probability. The absolute configurations of compounds 7 and 8 were confirmed by observing similar ECD Cotton effects and negative specific rotation values identical to those of compound 9 (methyl ester of 8) having a (2S, 3S) structurally similar configuration (FIG. 4). Therefore, as shown in Figure 1, the structures of compounds 7 and 8 were identified and named as Sepaflava C and D, respectively.

cefaflava E ( Cepaflava E, compound 9)

-11.8 (c 0.09, MeOH); UV (MeOH) λ_max (log ε) 290 (2.58), 252 (2.31), 200 (2.80) nm; ECD (MeOH) λ_max (Δε) 295 (-0.95), 206 (4.62); IR (KBr) v_max 3324, 2945, 2831, 1733, 1450, 1116, 1036; ¹H (500MHz, 메탄올-d₄) 및 ¹³C NMR (125MHz, 메탄올-d₄) 데이터, 표 2 참조; HRESIMS m/z 491.0588 ([M + Na]⁺, calcd for C₂₃H₁₆O₁₁Na, 491.0590) 및 m/z 469.0763 ([M + H]⁺, calcd for C₂₃H₁₇O₁₁, 469.0771).gray-green, amorphous powder;

-11.8 ( c 0.09, MeOH); UV (MeOH) λ _max (log ε) 290 (2.58), 252 (2.31), 200 (2.80) nm; ECD (MeOH) λ _max (Δε) 295 (−0.95), 206 (4.62); IR (KBr) v _max 3324, 2945, 2831, 1733, 1450, 1116, 1036; ¹ H (500 MHz, methanol-d ₄ ) and ¹³ C NMR (125 MHz, methanol-d ₄ ) data, see Table 2; HRESIMS m/z 491.0588 ([M + Na] ⁺ , calcd for C ₂₃ H ₁₆ O ₁₁ Na, 491.0590) and m/z 469.0763 ([M + H] ⁺ , calcd for C ₂₃ H ₁₇ O ₁₁ , 469.0771).

Sepaflava E (Compound 9) was isolated as a gray-green amorphous powder. Molecular formula of compound 9 is established as C ₂₃ H ₁₆ O ₁₁ based on sodium addition molecular ion peak at m/z 491.0588 [M+Na] ⁺ (calcd for C ₂₃ H ₁₆ O ₁₁ Na, 491.0590) by HRESIMS did The ¹ H and ¹³ C NMR data of compound 9 showed the resonance of the methoxy group [δ _H 3.84 (3H, s), δ _C 52.6] and the presence of a masked carboxyl signal at C-1''' (δC 169.1 in compound 8). and 167.8 in 9), but similar to compound 8 data. This difference indicates that compound 9 is a methyl ester derivative of compound 8.

The HMBC correlation between the carbon of C-1''' (δ _C 167.8) and the methoxy proton δ _H 3.84 confirmed the location of the methoxy group in the carboxyl group of protocatechuic acid. The relatively small Δδ _{H -2''/H-5''} value of compound 9 (0.47 ppm) indicates that the position of the carboxylic acid is the same as that of compound 8 (Fig. 5C).

The experimental specific rotation values of compound 9 were compared with the calculated specific rotation values of the two possible isomers (2R, 3R) and (2S, 3S). The calculated optical rotations for (2S, 3S)-9 and (2R, 3R)-9 were −276.8 and +276.8 at the B3LYP/6-311+G (d, p) level, respectively, whereas the recorded values for compound 9 was -11.8, which was closer to the calculated value of the (2S, 3S) form. Therefore, the absolute configuration (cepaflava E) of compound 9 was determined to be (2S, 3S) (Fig. 1).

cefaflava F ( Cepaflava F, compound 10)

-26.3 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 299 (2.51), 260 (2.46), 203 (2.90) nm; ECD (MeOH) λ_max (Δε) 319 (7.37), 288 (-2.90), 254 (0.04), 234 (-2.44); IR (KBr) v_max 3316, 2946, 2831, 1450, 1112, 1024; ¹H (400MHz, 메탄올-d₄) 및 ¹³C NMR (100MHz, 메탄올-d₄) 데이터, 표 3 참조; HRESIMS m/z 787.1119 ([M + Na]⁺, calcd for C₃₆H₂₈O₁₉Na, 787.1122) 및 m/z 765.1329 ([M + H]⁺, calcd for C₃₆H₂₉O₁₉, 765.1303).Bronze, amorphous powder;

-26.3 ( c 0.10, MeOH); UV (MeOH) λ _max (log ε) 299 (2.51), 260 (2.46), 203 (2.90) nm; ECD (MeOH) λ _max (Δε) 319 (7.37), 288 (-2.90), 254 (0.04), 234 (-2.44); IR (KBr) v _max 3316, 2946, 2831, 1450, 1112, 1024; ¹ H (400 MHz, methanol-d ₄ ) and ¹³ C NMR (100 MHz, methanol-d ₄ ) data, see Table 3; HRESIMS m/z 787.1119 ([M + Na] ⁺ , calcd for C ₃₆ H ₂₈ O ₁₉ Na, 787.1122) and m/z 765.1329 ([M + H] ⁺ , calcd for C ₃₆ H ₂₉ O ₁₉ , 765.1303).

Cefaflava F (Compound 10) was obtained as a bronze-yellow amorphous powder whose HRESIMS was m/z 787.1119 [M+Na] ⁺ (calcd for C ₃₆ H ₂₈ O ₁₉ Na, 787.1122) as sodium addition molecular ion A peak was obtained to obtain molecular formula C ₃₆ H ₂₈ O ₁₉ .

The ¹ H NMR spectrum of compound 10 shows two trisubstituted benzene ring sets [δ _H 6.95 (1H, s, H-2'), 6.86 (1H, d, J = 8.4 Hz, H-6 for the B-ring). '), 6.73 (1H, d, J = 8.4 Hz, H-5') and δ _H 7.95 (s, 1H, H-2''), 7.85 (1H, d, J = 8.4 Hz, H-5') and B'-ring 8.2 Hz, H-6'''), 7.29 (1H, d, J = 8.2 Hz, H-5''')]; Two meta-coupled proton signals at δ _H 5.90 (1H, s, H-6) and 5.85 (1H, s, H-8) for the A-ring, δ _H 6.54 (1H, s, H-6'') for aromatic proton signals and δH 4.92 for anomeric protons (1H, d, J = 8.6 Hz, H-1'''').

Acid hydrolysis of compound 10 with CF ₃ COOH yielded D-glucose, which was confirmed by comparing TLC shifts and specific rotations to those of real samples. The β-structure of the D-glucose moiety was deduced from the large coupling constant ( J = 8.6 Hz) of the anomeric proton (H-1''''). In the ¹³ C NMR spectrum of compound 10, 30 resonances were correlated with the aglycon motif, including three characteristic signals at δC 119.4 ( _C -2), 81.8 (C-3) and 192.6 (C-4). Comparison of these spectroscopic data with the biflavone A of the known compound Lawsonia suggested that compound 10 may be a biflavonoid. The structural difference between compound 10 and Lawsonia biflavone A is the presence of a D-glucose moiety and a 3-substituted benzene ring (B-ring), whereas Lawsonia biflavone A has a 2-substituted benzene ring instead. The HMBC correlation of C-4''' (δ _C 148.1) with the anomeric proton H-1'''' (δ _H 4.92) confirms the link between the sugar group and the aglycone (Fig. 2). As shown in Figure 3, similar to the beneficial syn -addition between quercetin and protocatechuic acid in the proposed biosynthetic pathway of compounds 7-9, the association of the two quercetin molecules occurs via syn -addition to form the cis-form of compound 10. can form

The calculated ECD spectrum of compound 10 having a (2S, 3S)-structure was in good agreement with the experimental results, confirming the absolute configuration of 10 (FIG. 4). Thus, the structure of compound 10 was characterized as shown in Figure 1 as a new biflavonoid glucoside (Cefaflava F).

cefaflava G ( Cepaflava G, compound 11)

+60.3 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 303 (2.39), 255 (2.40), 201 (2.85) nm; ECD (MeOH) λ_max (Δε) 304 (7.02), 252 (-1.40), 239 (-2.32), 218 (2.10); IR (KBr) v_max 3316, 2946, 2831, 1450, 1112, 1024; ¹H (400MHz, 메탄올-d₄) 및 ¹³C NMR (100MHz, 메탄올-d₄) 데이터, 표 3 참조; HRESIMS m/z 787.1119 ([M + Na]⁺, calcd for C₃₆H₂₈O₁₉Na, 787.1122) 및 m/z 765.1329 ([M + H]⁺, calcd for C₃₆H₂₉O₁₉, 765.1303).Bronze, amorphous powder;

+60.3 ( c 0.1, MeOH); UV (MeOH) λ _max (log ε) 303 (2.39), 255 (2.40), 201 (2.85) nm; ECD (MeOH) λ _max (Δε) 304 (7.02), 252 (-1.40), 239 (-2.32), 218 (2.10); IR (KBr) v _max 3316, 2946, 2831, 1450, 1112, 1024; ¹ H (400 MHz, methanol-d ₄ ) and ¹³ C NMR (100 MHz, methanol-d ₄ ) data, see Table 3; HRESIMS m/z 787.1119 ([M + Na] ⁺ , calcd for C ₃₆ H ₂₈ O ₁₉ Na, 787.1122) and m/z 765.1329 ([M + H] ⁺ , calcd for C ₃₆ H ₂₉ O ₁₉ , 765.1303).

cefaflava H ( Cepaflava H, compound 12)

-30.0 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 303 (2.38), 255 (2.40), 201 (2.83) nm; ECD (MeOH) λ_max (Δε) 304 (-4.79), 276 (1.79), 251 (0.69), 239 (1.28), 223 (-2.14), 199 (3.05); IR (KBr) v_max 3316, 2946, 2831, 1450, 1112, 1024; 1H (400MHz, 메탄올-d₄) 및 ¹³C NMR (100MHz, 메탄올-d₄) 데이터, 표 3 참조; HRESIMS m/z 787.1119 ([M + Na]⁺, C₃₆H₂₈O₁₉Na에 대한 계산치, 787.1122) 및 m/z 765.1329 ([M + H]⁺, calcd for C₃₆H₂₉O₁₉, 765.1303).Bronze, amorphous powder;

-30.0 ( c 0.1, MeOH); UV (MeOH) λ _max (log ε) 303 (2.38), 255 (2.40), 201 (2.83) nm; ECD (MeOH) λ _max (Δε) 304 (-4.79), 276 (1.79), 251 (0.69), 239 (1.28), 223 (-2.14), 199 (3.05); IR (KBr) v _max 3316, 2946, 2831, 1450, 1112, 1024; 1H (400 MHz, methanol-d ₄ ) and ¹³ C NMR (100 MHz, methanol-d ₄ ) data, see Table 3; HRESIMS m/z 787.1119 ([M + Na] ⁺ , calcd for C ₃₆ H ₂₈ O ₁₉ Na, 787.1122) and m/z 765.1329 ([M + H] ⁺ , calcd for C ₃₆ H ₂₉ O ₁₉ , 765.1303) .

Both Cefaflava G (compound 11) and H (compound 12) were obtained as bronze-yellow amorphous powders. These two compounds have the same molecular formula C ₃₆ H ₂₈ O ₁₉ based on HRESIMS data (m/z 787.1105 for compound 11, 787.1115 for compound 12 [M+Na] ⁺ , calculated molecular formula C ₃₆ H ₂₈ O ₁₉ Na 787.1122). shared. The ¹ H and ¹³ C NMR data of compounds 11 and 12 are very similar to each other, but the ECD spectra are mirror images (FIG. 4), suggesting that they may be a pair of stereoisomers. Examination of ¹ H NMR spectra of compounds 11 and 12 revealed that two sets of ABX coupling systems [ δ _H 6.97 (1H, d, J = 2.1 Hz, H-2′), 6.86 (1H for the B-ring of compound 11) , dd, J = 8.5, 2.1 Hz, H-6'), 6.82 (1H, d, J = 8.5 Hz, _H -5'), δH 7.80 ( 1H , d, J = 2.2 for the B'-ring) Hz, H-2'''), 6.33 (1H, dd, J = 8.8, 2.2 Hz, H-6'''), 7.02 (1H, d, J = 8.8 Hz, H-5''') and δH 6.94 ( _1H , d, J = 1.8 Hz, H-2'), 6.81 (1H, dd, J = 8.4, 1.8 Hz, H-6'), 6.76 (1H, d, J = 8.4 Hz, H-5') and δH 7.78 (1H, s, _H -2''), 6.13 ( 1H , d, J = 8.8 Hz, H-6') for the B'-ring ''), 6.99 (1H, d, J = 8.8 Hz, H-5''')]; Two meta coupled proton signals [ δ _H for the A-ring of compound 11 5.99 (1H, d, J = 2.1 Hz, H-6) and 6.02 (1H, d, J = 2.1 Hz, H-8), δ _H for the A-ring of compound 12 5.93 (1H, s, H-6) and 5.97 (1H, s, H-8)]. ¹³ C NMR spectra of compounds 11 and 12 show one carbonyl carbon ( δ _C 186.9 in compounds 11 and 12 ) , one oxygenated carbon (C-3, δ _C 103.2 for compound 11, 104.3 for compound 12) and one It exhibited highly unshielded ketal carbons (C-2, δ _C 93.7 for compound 11 and 93.6 for compound 12). By comparing these NMR data with the literature, compounds 11 and 12 are 2-[4-(β-D-glucopyranosyloxy)-3-hydroxyphenyl]-3,5,7-trihydroxy-9-[ 2-(3,4-dihydroxyphenyl)-2,3-dihydro-2,3-epoxy-5,7-dihydroxy- 4H -1-benzopyran-4-one-8-yl] It was identified as two stereoisomers of -4H-1-benzopyran-4-one.

In previous studies, individual stereoisomers were not purified and their absolute composition was not determined. For assignment of the absolute configuration of the 2,3-epoxide present in compounds 11 and 12, the experimental ECD spectra of compounds 11 and 12 were compared with the simulated ECD spectra. Based on the results shown in Figure 4, the absolute configurations of compounds 11 and 12 were inferred to be (2S, 3S) and (2R, 3R) forms, respectively. Thus, the structures of compounds 11 and 12 were established and named cepaflava G and cepaflava H, respectively (Fig. 1).

cefaflava I ( Cepaflava I, compound 14)

- 23.5 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 299 (2.53), 264 (2.26), 202 (2.87) nm; ECD (MeOH) λ_max (Δε) 303 (0.26), 269 (0.49), 241 (0.43), 203 (3.15); IR (KBr) v_max 3324, 2945, 2832, 1684, 1449, 1037; ¹H (500MHz, 메탄올-d₄) 및 ¹³C NMR (125MHz, 메탄올-d₄) 데이터, 표 1 참조; HRESIMS m/z 791.0848 ([M + Na]⁺, calcd for C₃₈H₂₄O₁₈Na, 791.0860) 및 m/z 769.1047 ([M + H]⁺, calcd for C₃₈H₂₅O₁₈에 대한 계산치, 769.1041).Bronze, amorphous powder;

- 23.5 ( c 0.1, MeOH); UV (MeOH) λ _max (log ε) 299 (2.53), 264 (2.26), 202 (2.87) nm; ECD (MeOH) λ _max (Δε) 303 (0.26), 269 (0.49), 241 (0.43), 203 (3.15); IR (KBr) v _max 3324, 2945, 2832, 1684, 1449, 1037; ¹ H (500 MHz, methanol-d ₄ ) and ¹³ C NMR (125 MHz, methanol-d ₄ ) data, see Table 1; HRESIMS m/z 791.0848 ([M + Na] ⁺ , calcd for C ₃₈ H ₂₄ O ₁₈ Na, 791.0860) and m/z 769.1047 ([M + H] ⁺ , calcd for C ₃₈ H ₂₅ O ₁₈ , 769.1041).

Cefaflava I (Compound 14) was isolated as a bronze yellow amorphous powder. Molecular formula determined as C ₃₈ H ₂₄ O ₁₈ suggesting 27 levels of unsaturation by sodium addition molecular ion peak in HRESIMS at m/z 791.0848 [M+Na] ⁺ (calcd for C ₃₈ H ₂₄ O ₁₈ Na, 791.0860) It became. ¹ H NMR spectrum of compound 14 shows δ _H 5.93 (2H, d, J = 2.7 Hz, H-6, H-6″) and δ _H Two sets of meta-bonded aromatic hydrogen resonances at 6.00 (2H, d , J = 2.7 Hz, H-8, _H -8″), one methoxy group at δH 3.88 (3H, s), D-ring At [ δ _H 7.71 (1H, d, J = 8.5 Hz, H-6'''), 7.63 (1H, s, H-2''''), 7.18 (1H, d, J = 8.5 Hz, H-5'''')], in the B-ring [ δ _H 7.21 (1H, s, H-2'), 7.03 (1H, d, J = 8.4 Hz, H-6'), and 6.69 (1H, d, J = 8.4 Hz, H-5')], B' In the -ring [ δ _H 7.44 (1H, d, J = 2.0 Hz, H-2'''), 7.34 (1H, dd, J = 8.5, 2.0 Hz, H-6'''), and 6.93 (1H, d, J = 8.5 Hz , H-5″″)] indicated the presence of three trisubstituted benzene rings. The ¹³ C NMR spectrum of compound 14 shows two carbonyl groups (δ _C 189.7 and 189.3), one ester carbonyl group (δ _C 167.7), one methoxy group (δ _C 52.6), and four characteristic quaternary carbon signals (δ _C 167.7). 101.8, 101.5, 91.9, 91.7), representing 12 oxygenated aromatic carbons and 18 sp2 methine carbons. The NMR data were similar to 9 except that an additional quercetin moiety was present in compound 14 (Tables 1, 2 and Figure 5C). The chemical shifts of C-2/C-2'' or C-3/C-3'' in compound 14 are similar, but C-3' and C-4' are C-3''' and C-4' A downward shift (~4 ppm) rather than a shift of '' suggested that additional quercetin units could be attached to C-3' and C-4'.

The chemical shifts of C-2″ (δ _C 101.5) and C-3″ (δ _C 91.7) were almost identical to those of C-2 (δ _C 101.8) and C-3 (δ _C 91.9), respectively. , this similarity suggests that additional quercetin units are linked via the same dioxane bonds as in C-2 and C-3. As described above for compounds 7-9, all dioxane linkages in compound 14 were proposed to be in cis-form, and based on these data, a total of eight isomers are possible (14a-14d and ent-14a-14d). To determine the planar structure and relative configuration of compound 14, chemical shifts of 14a–14d (enantiomer chemical shifts of each isomer are identical) were calculated and DP4 analysis with experimental values was performed. Statistical results showed that compound 14 showed structural equivalence to 14b with 100% probability. The absolute composition of compound 14 was inferred by comparing the experimental ECD data with the calculated ECD of 14b and its enantiomer. As shown in Figure 4, the experimental ECD of compound 14 was in good agreement with that of 14b, and its absolute configuration was inferred to be 2R, 3R, 2''S, 3''S, and from these data, compound 14 (Sefaflava I) was determined as shown in FIG. 1 .

Based on a comprehensive analysis of the spectroscopic data and comparison with the literature, the chemical structures of six known compounds were quercetin (compound 1), kaempferol (compound 2), isorhamnetin (compound 3), Quercetin 4'-O- β -D-glucopyranoside (Compound 4), 1,3,11a-trihydroxy-9-(3,5,7- Trihydroxy-4H-1-benzopyran-4-one-2-yl)-5a-[4-(β-D-glucopyranosyloxy)-3-hydroxyphenyl]-5,6,11-hexa Hydro-5,6,11-trioxanaphthacen-12-one (1,3,11a-trihydroxy-9-(3,5,7-trihydroxy-4 H -1-benzopyran-4-on-2-yl )-5a-[4-( β -D-glucopyranosyloxy)-3-hydroxyphenyl]-5,6,11-hexahydro-5,6,11-trioxanaphthacene-12-one, compound 13) and 1,3,11a- Trihydroxy-9-(3,5,7-trihydroxy- 4H -1-benzopyran-4-one-2-yl)-5a-[1,3,11a-trihydroxy-5a-( 3,4-dihydroxyphenyl) -5,6,11-hexahydro-5,6,11-trioxanaphthacen-12-one-9-yl] -5,6,11-hexahydro-5, 6,11-trioxanaphthacen-12-one ( 1,311a -trihydroxy-9-(3,5,7-trihydroxy-4h-1-benzopyran-4-on-2-yl)-5a-[1, 3,11a-trihydroxy-5a-(3,4-dihydroxyphenyl)-5,6,11-hexahydro-5,6,11-trioxanaphthacene-12-on-9-yl]-5,6,11-hexahydro-5 ,6,11-trioxanaphthacene-12-one, compound 15).

< Example 2. Antioxidant activity>

⁺ 에 대해 화합물 5-15의 소거 용량을 조사하였다. DPPH 및 ABTS

⁺ 소거에서의 억제 결과를 확인하기 위해 아스코르브산 및 트롤록스를 기준 화합물로 사용하였다.As shown in Table 4 below, two free radicals DPPH and ABTS in a concentration dependent manner

The clearing capacity of compounds 5-15 was investigated for ⁺ . DPPH and ABTS

⁺ Ascorbic acid and Trolox were used as reference compounds to confirm inhibition results in clearance.

⁺ 억제를 나타냈다. 특히 화합물 10-12는 뛰어난 라디칼 소거 능력을 보였다. 화합물 11의 경우, DPPH 및 ABTS

⁺의 IC₅₀은 각각 4.25 및 7.12 μM로 가장 뛰어난 항산화능을 나타내었으며, 화합물 10은 DPPH 및 ABTS

⁺의 IC₅₀ 가 각각 6.27 7.84 μM을, 화합물 12는 각각 8.88 및 8.14 μM의 IC₅₀ 을 나타내었다.As a result, compounds 5-15 all had excellent DPPH and ABTS with IC ₅₀ ranges of 4.25-47.69 and 7.12-23.45 μM.

⁺ showed inhibition. In particular, compounds 10-12 showed excellent radical scavenging ability. For compound 11, DPPH and ABTS

The IC ₅₀ of ⁺ was 4.25 and 7.12 μM, respectively, showing the most excellent antioxidant activity, and compound 10 was DPPH and ABTS.

The IC ₅₀ of ⁺ was 6.27 and 7.84 μM, respectively, and the IC ₅₀ of compound 12 was 8.88 and 8.14 μM, respectively.

compounds IC ₅₀ (μM) ^a enzyme kinetic analysis with PTP1B DPPH scavenging activity

⁺ scavenging activityABTS

⁺ scavenging activity PTP1B α -glucosidase K _i (μM) ^h inhibition type ^g One - - 25.34 ± 0.91 20.14 ± 0.92 - - 2 - - 62.42 ± 1.73 61.44 ± 1.16 - - 3 - - 56.36 ± 0.81 17.75 ± 0.02 - - 4 - - >100 >100 - - 5 12.50 ± 2.18 7.93 ± 0.83 >100 74.44 ± 2.60 - - 6 39.87 ± 0.88 9.84 ± 0.79 >100 >100 - - 7 28.05 ± 1.29 9.13 ± 0.67 >100 13.17 ± 0.47 - - 8 13.51 ±3.09 9.87 ± 0.28 22.55 ± 0.71 15.81 ± 1.23 - - 9 14.89 ± 0.11 10.09 ± 0.37 22.33 ± 0.81 12.55 ± 0.06 - - 10 6.27 ± 0.43 7.84 ± 0.41 17.01 ± 0.01 9.71 ± 0.23 - - 11 4.25 ± 3.40 7.12 ± 1.47 24.07 ± 1.74 11.10 ± 0.03 - - 12 8.88 ± 0.12 8.14 ± 0.28 14.29 ± 0.03 10.92 ± 0.16 - - 13 14.53 ± 2.28 8.82 ± 0.35 6.82 ± 0.08 6.80 ± 0.07 3.72 mixed 14 47.69 ± 1.18 23.45 ± 4.67 1.68 ± 0.02 2.30 ± 0.01 1.25 competitive 15 46.65 ± 1.37 9.86 ± 0.29 1.13 ± 0.01 0.89 ± 0.01 2.18 competitive ascorbic acid ^b 3.50 ± 0.05 - - - trolox ^c - 5.14 ± 0.18 - - ursolic acid ^d - - 12.09 ± 0.01 - sodium orthovanadate ^e - - 23.2 ± 1.15 - acarbose ^f - - - 257.41 ± 0.57

^a These data are expressed as the mean ± SEM of triplicate experiments.

⁺ scavenging assays, respectively. ^{b , c} Positive controls for DPPH and ABTS

⁺ scavenging assays, respectively.

^{d , e} Positive controls for PTP1B inhibitory assay.

^f Positive controls for α -glucosidase inhibitory assay.

^g Determined by Lineweaver-Burk plots.

^h Determined by Dixon plots.

< Example 3. PTP1B and α-glucosidase inhibitory activity>

Inhibition of α-glucosidase and PTP1B enzymes is an effective strategy to treat metabolic diseases such as diabetes and obesity. Table 4 shows the results of analyzing the PTP1B and α-glucosidase inhibitory activities of Compounds 1 to 15.

As shown in Table 4, the ability of compounds 1 to 3 and 8 to 15 to inhibit PTP1B was dose-dependent, with an IC ₅₀ ranging from 1.13 to 62.42 μM. In particular, compounds 15 (IC ₅₀ = 1.13 μM), 14 (IC ₅₀ = 1.68 μM) and 13 (IC ₅₀ = 6.82 μM) showed a strong PTP1B inhibitory ability, more potent than the two positive controls [ursolic acid (IC ₅₀ = 6.82 μM)]. = 12.09 μM) and sodium orthovanadate (IC ₅₀ = 23.20 μM)]. Compounds 10 and 12 also showed IC ₅₀ values of 17.01 and 14.29 μM, respectively, and compounds 8, 9, 11 and 1-3 also showed constant PTP1B inhibitory activity.

As also shown in Table 4 above, compounds 1 to 3, 5 and 7 to 15 exhibited α-glucosidase inhibitory activity in the IC ₅₀ range of 0.89-74.44 μM, and this α-glucosidase inhibitory activity was positive. It was significantly higher than the activity of acarbose used as a control (IC ₅₀ = 257.41 μM). In particular, Compounds 14 and 15 exhibited strong PTP1B inhibitory activity and excellent α-glucosidase inhibitory activity, respectively, 100-fold and 250-fold higher activity than that of acarbose. Compound 13 also exhibited strong α-glucosidase inhibitory activity while having high PTP1B inhibitory activity (IC ₅₀ = 6.80 μM).

Compounds 3 and 7 to 12 also showed high α-glucosidase inhibitory activity with an IC ₅₀ value of 20 μM or less, and compounds 1 and 2 also showed significantly higher α-glucosidase activity than that of acarbose used as a positive control. showed inhibitory activity.

< Example 4. PTP1B Enzyme Kinetics of Inhibition>

The enzyme inhibition properties of compounds 13-15 were determined using Lineweaver-Burk and Dixon plots. As a result of the experiment, compounds 14 and 15 were identified as competitive inhibitors as the regression lines for each compound intersected on the y-axis. Also, from the Dixon plot, inhibition constant (Ki) values for compounds 14 and 15 were determined to be 1.25 and 2.18 μM, respectively (Table 4). In addition, increasing concentrations of compound 13 generated a series of lines sharing a common y-axis intercept but with different gradients, confirming that compound 13 was a mixed inhibitor with a Ki value of 3.72 μM (Table 4).

< Example 5. PTP1B Molecular Docking Simulation of Inhibition>

Compounds 13 and 15 were docked to the binding site of the crystallographic structure of PTP1B using AutoDock 4.2 software to confirm the interaction and binding mode of these active compounds. To validate and optimize the docking procedure, we docked two unique co-ligands, namely Compound A (allosteric inhibitor) and Compound C (catalytic inhibitor), to the allosteric and catalytic sites of PTP1B, respectively. Previous studies have shown that binding interference of the α7 helix to the α3-α6 helix of PTP1B can lead to allosteric inhibition of enzyme activity. As a result of this docking experiment, the PTP1B-13 inhibitor complex at the allosteric site showed a binding energy of -7.92 kcal/mol, and was associated with four hydrogen bond interactions and Ala189 of α3 helix and Glu276, Gly277 and Lys279 residues of α6 helix. have an interaction In addition, compound 13 and compound A shared the same allocetric residues, that is, Glu276 of the α6 helix through a hydrogen bond interaction, and Met282 through a Van der Waals interaction. Catalytic inhibition of compound 13 on PTP1B (−6.41 kcal/mol) revealed six hydrogen bonds with residues Gln266, Thr263, Gly183, Arg221, Arg24 and Asp48. Similar to compound C, compound 13 is also involved in two hydrogen bond interactions with key catalytic residues Arg221 and Asp48. In addition, compound 13 showed hydrophobic interactions with Phe182, Ala27, Gly259, Val49, Ile219, Ala217, Tyr46, Gly220, Asp265 and Val184, enhancing the protein-ligand interaction between PTP1B and compound 13. Compound 15 was bound to the catalytic domain of PTP1B with a binding energy of -8.01 kcal/mol, indicating a high affinity for the PTP1B moiety. The predicted binding mode showed that compound 15 forms 5 hydrogen bond interactions with residues Arg221, Asp48, Gln 262, Arg24 and Gly183 through the hydroxy group. Among them, Arg221 is a representative residue of PTP1B essential for catalytic inhibition. In addition, compound 15 and compound C share the same residues Gln266, Trp179 and Val49 through hydrophobic interaction and Met258 residue through π-sulfur interaction. From these docking results, it was confirmed that compounds 13 and 15 are mixed and competitive inhibitors of PTP1B, respectively.

Claims (9)

delete delete delete delete An antidiabetic composition characterized in that it contains Cepaflava F (Compound 10) having the chemical structure of [Formula 1] as an active ingredient.
[Formula 1]

Cepaflava F (Cepaflava F, Compound 10) having the chemical structure of [Formula 1] below.
[Formula 1]

According to claim 5,
The Cepaflava F (Cepaflava F, compound 10) is an antidiabetic composition, characterized in that separated from the onion skin extract extracted with water, C1 to C4 alcohol or a mixed solvent thereof. According to claim 5,
The antidiabetic composition, characterized in that the Cepaflava F (Compound 10) has an antioxidant activity. According to claim 5,
The Cepaflava F (Compound 10) is an antidiabetic composition, characterized in that it has an inhibitory activity against protein tyrosine phosphatase 1B or alpha-glucosidase.

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