Aldose-Reductase- and Protein-Glycation-Inhibitory Principles from theWhole Plant of Duchesnea chrysantha

by Jong Min Kima), Dae Sik Janga), Yun Mi Leea), Jeong Lim Yooa), Young Sook Kima),Joo-Hwan Kimb), and Jin Sook Kim*a)

a) Department of Herbal Pharmaceutical Development, Korea Institute of Oriental Medicine, Daejeon,305-812, Korea (phone: þ82-42-868-9465; e-mail: jskim@kiom.re.kr)

b) Division of Life Science, Daejeon University, Daejeon 300-716, Korea

Ellagic acid (1), 3,3’-di-O-methylellagic acid (2), 3,3’,4-tri-O-methylellagic acid (3), isovitexin (4),kaempferol 3-O-b-d-glucuronide methyl ester (5), quercetin 3-O-a-l-arabinopyranosyl-(1!6)-b-d-galactopyranoside (6), ursolic acid, pomolic acid, tormentic acid, euscaphic acid, euscaphic acid 28-O-b-d-glucopyranoside, and maslinic acid were isolated from the AcOEt- and BuOH-soluble MeOH extractofDuchesnea chrysantha (whole plant). The isolates were subjected to in vitro bioassays to evaluate theirinhibitory activity on rat-lens aldose reductase (RLAR) and formation of advanced glycation endproducts (AGEs). The ellagic acids and flavonoids, compounds 1–6, exhibited moderate inhibitoryeffects on RLAR. However, compounds 1 and 4–6 showed excellent inhibitory activities towards theformation of AGEs. This is the first report that 4 and 6 exhibit inhibitory activity towards AR and AGEsformation.

Introduction. – Hyperglycemia plays an important role in the pathogenesis of long-term complications, and diabetic patients with poor blood-glucose control areparticularly at risk [1]. Persistent hyperglycemia induces abnormal changes such asthe formation of advanced glycation end products (AGEs) [2], the increase of sorbitolthrough the polyol pathway [3], or the overactivation of protein-kinase-C isoforms dueto the synthesis of DAG [4]. Enhanced formation and accumulation of AGEs havebeen implicated as a major pathogenesis process, leading to diabetic complications,normal aging, atherosclerosis, and Alzheimer [5] [6]. Aldose reductase (AR), the keyenzyme in the polyol pathway, has also been demonstrated to play important roles inthe pathogenesis of diabetic complications and cataract formation [7]. Thus, the designand discovery of inhibitors of the formation of AGEs or AR can offer a promisingtherapeutic approach for the prevention of diabetic or other pathogenic complications[8] [9].

In our ongoing project directed toward the discovery of preventive agents fordiabetic complications from herbal medicines [10] [11], Duchesnea chrysantha (wholeplant) was chosen for more detailed investigation, since the AcOEt- and BuOH-solublefractions of the 80% EtOH extract showed significant in vitro inhibitory activities onboth the formation of AR and AGEs. Duchesnea chrysantha (Zoll. etMoritz.) Miq.(Rosaceae) is a perennial plant, and has traditionally been used to treat congenitalfever, toothache, inflammation, and cancers in Korea and China [12]. It has shown

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E 2008 Verlag Helvetica Chimica Acta AG, ZGrich

various biological and pharmacological activities, including cytotoxicity towards humancancer cell lines and antioxidation [13] [14].

Previous phytochemical investigations on the whole plant ofD. chrysantha resultedin the isolation of several ellagic acids, triterpenoids, and phenolic compounds [13] [15].In the present study, repeated chromatography of the AcOEt- and BuOH-solublefractions of this plant led to the isolation of three ellagic acids, compounds 1–3, threeflavonoids, compounds 4–6, and six triterpenoids. The known isolates obtained in thepresent study were evaluated for their potential to inhibit the formation of AGEs andfor their activity towards rat-lens aldose reductase (RLAR). The biological evaluationof the isolates is described herein.

Results and Discussion. – Twelve compounds were isolated from the AcOEt- andBuOH-soluble fractions of the whole plant of D. chrysantha, and were identified asellagic acid (1) [13], 3,3’-di-O-methylellagic acid (2) [16], 3,3’,4-tri-O-methylellagicacid (3) [17], isovitexin (4) [18], kaempferol 3-O-b-d-glucuronide methyl ester (5)[19], quercetin 3-O-a-l-arabinopyranosyl-(1!6)-b-d-galactopyranoside (6) [20],ursolic acid [15], pomolic acid [21], tormentic acid [15], euscaphic acid [15], euscaphicacid 28-O-b-d-glucopyranoside [15], and maslinic acid [22] by physical and spectro-scopic methods, as well as by comparison of their data with literature values. To the bestof our knowledge, compounds 2–6, pomolic acid, and maslinic acid were isolated forthe first time from this plant.

In this study, we evaluated the isolates obtained from D. chrysantha for theirpotential to inhibit RLAR activity and AGEs formation (Table). The six isolatedtriterpenoids were inactive in these assays. The ellagic acids 1–3 and the flavonoids 4–6, respectively, exhibited moderate inhibitory effects on RLAR, with IC50 valuesranging from 4.6 to 24.3 mm. However, compounds 1 and 4–6 showed significantinhibitory activities against the formation of AGE, with IC50 values ranging from 26.0 to108.8 mm, better than the positive control, aminoguanidine, which showed an IC50 valueof 961 mm. To the best our knowledge, isovitexin (4) and quercetin 3-O-a-l-arabinopyranosyl-(1!6)-b-d-galactopyranoside (6) have not been reported beforeto exhibit inhibitory activity towards AR and AGEs formation. Therefore, these twocompounds seem especially worth of additional biological tests to more fully evaluatetheir potential as therapeutic agents for diabetic complications and related diseases.

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Ellagic acids such as 3,3’,4-tri-O-methylellagic acid-4’-sulfate (IC50¼0.080 mm) arewell-known natural AR inhibitors [23], and their inhibitory effects on sorbitolaccumulation, both in vitro and in vivo, have also been reported [24]. Moreover, anumber of flavonoids, including desmanthin-1 (IC50¼0.082 mm towards AR), querci-trin (IC50¼0.15 mm towards AR), baicalein (IC50¼93 mm towards AGEs), and luteolin(IC50¼99 mm towards AGEs), have been isolated from various natural sources, andtheir structure–activity relationships (SARs) have been reported [23] [25] [26].Recently, a new 2-arylbenzofuran, puerariafuran, and coumestrol were isolated fromthe roots of Pueraria lobata and identified as potent glycation inhibitors in ourlaboratories [11].

In summary, the whole plant of D. chrysantha was found to exhibit significantinhibitory activity towards AR and AGEs, and the ellagic acids 1–3 and the flavonoids4–6 were identified to be major active principles of this plant.

We thank the Korea Basic Science Institute (KBSI) for recording NMR and mass spectra. Thisresearch was supported by a grant (L06010) from the Korea Institute of Oriental Medicine.

Experimental Part

General. TLC: Kieselgel 60 F254 (0.25 mm; Merck). Column chromatography (CC): silica gel 60A(70–230 or 230–400 mesh;Merck) or Sephadex LH-20 (Amersham Pharmacia Biotech). Melting points:IA-9100melting-point apparatus (Barnstead International, USA). Optical rotations: Jasco P-2000 digitalpolarimeter. NMR Spectra: Bruker DRX-300 FT-NMR apparatus. LR-ESI-MS: Autospec apparatus(Micromass, UK).

Plant Material. Whole plants of Duchesnea chrysantha (Zoll. et Moritz.) Miq. (Rosaceae) werecollected at Mount Sikjang, Dong-gu, Daejeon City, Korea (36819’45.4’’ N, 127827’48.4’’ E), in May 2005,and identified by one of us (J.-H. K.). A voucher specimen (20050507-001) has been deposited at theHerbarium of the Department of Herbal Pharmaceutical Development, Korea Institute of OrientalMedicine, Korea.

Extraction and Isolation. The dried and cut plant material (4.6 kg) was extracted with MeOH (3�20 l) by maceration for 2 d at r.t. The solvent was evaporated in vacuo at 408 to afford a MeOH extract

Table. Inhibitory Effects of 1–6 on Rat-Lens Aldose Reductase (RLAR) and the Formation of AdvancedGlycation End Products (AGEs) in vitro. Inhibitory effects are expressed as mean �S.D. of triplicateexperiments. IC50 values were calculated from dose-dependent inhibition curves. For details, see Exper.

Part.

Compound IC50 [mm]

RLAR AGEs formation

1 6.9�0.6 26.0�0.032 6.7�3.6 n.d.a)3 24.3�1.9 n.d.4 10.1�6.5 85.2�0.705 4.6�0.6 108.8�1.56 19.1�3.0 58.6�0.49AGb) – 961�34EPb) 0.067�0.009 –

a) Not determined due to only minute amounts of material. b) Aminoguanidine (AG) and epalrestat(EP) were used as positive controls.

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(440 g), which was suspended in H2O (1.5 l) and sequentially extracted with hexane (3�1.5 l), AcOEt(3�1.5 l), and BuOH (3�1.5 l). TheAcOEt-soluble fraction (120 g) was subjected to CC (SiO2, 70–230mesh; hexane/AcOEt 1 :1!0 :1, then neat MeOH) to yield twelve pooled fractions: Fr. E1–Fr. E12.Fr. E4 (eluted with hexane/AcOEt 1 :1; 10.0 g) was purified by CC (SiO2; 230–400 mesh; CHCl3/MeOH30 :1!1 :1) to yield ursolic acid (120 mg) and pomolic acid (44 mg). Next, Fr. E5 (eluted with hexane/AcOEt 2 :3; 9.7 g) was further fractionated by CC (SiO2, 230–400 mesh, hexane/AcOEt 1 :4) to affordeuscaphic acid (40 mg),maslinic acid (43 mg), and tormentic acid (50 mg). The ellagic acids 2 (2 mg) and3 (4 mg) were obtained from two combined fractions, Fr. E6þFr. E7 (5.2 g), eluted with hexane/AcOEt1 :3, by repeated CC. Fr. E9 (7.7 g), eluted with neat AcOEt, was subjected to CC (Sephadex LH-20 ;MeOH) to afford euscaphic acid 28-O-b-d-glucopyranoside (16 mg). Compound 1 (17 mg) was obtainedfrom Fr. E10 (eluted with AcOEt; 8.5 g) by repeated CC. The BuOH-soluble fraction (112 g) of the plantextract was purified by CC (Diaion HP-20; MeOH/H2O 1 :1!1 :0) to yield eight pooled fractions:Fr. B1–Fr. B8. Fr. B2 (12 g), eluted with MeOH/H2O 1 :1, was subjected to CC (SiO2, 230–400 mesh;CHCl3/MeOH/H2O 8 :2 :0.2) to afford 4 (100 mg) and 6 (150 mg). Compound 5 (25 mg) was purifiedfrom Fr. B3 (3.0 g), eluted with MeOH/H2O 3 :2, by repeated CC.

Measurement of RLAR Activity. Rat lenses were removed from the eyes of eight-weeks-oldSprague–Dawley rats (Dae-Han Bio Link, Co., Umsung, Korea), weighing 100–150 g, and homogenizedin twelve volumes of 135 mm Na/K phosphate buffer (pH 7.0) containing 0.5 mm phenylmethanesulfonylfluoride and 10 mm 2-mercaptoethanol. The homogenate was centrifuged at 100,000 g for 30 min, and thesupernatant fluid was used as crude rat-lens aldose reductase (RLAR). RLAR Activity was assayedaccording to the methods described in [25] and [27], with slight modification. The incubation mixture(1.0 ml in total) contained 135 mm Na/K phosphate buffer (pH 7.0), 100 mm lithium sulfate, 0.03 mm

NADPH, 1 mm dl-glyceraldehyde as a substrate, and enzyme fraction (50 ml), with or without samplesoln. (25 ml). The reaction was initiated by addition of NADPH at 378, and stopped by addition of 0.5maq. HCl (0.3 ml). Then, 6m aq. NaOH soln. containing 10 mm 1H-imidazole (1 ml) was added, and thesoln. was heated at 608 for 10 min to convert NADP to a fluorescent product. Fluorescence was measuredwith a spectrofluorometric detector (Shimadzu RF-5301PC, Japan) at excitation and emissionwavelengths of 360 and 460 nm, resp. Both AGEs and RLAR assays were performed in triplicate. Theconcentration of each test sample giving rise to 50% inhibition of activity (IC50) was estimated from theleast-squares regression line of the logarithmic concentration plotted against the remaining activity.

Determination of AGEs Formation. According to an established method [28], the reaction mixture,containing 700 ml of bovine serum albumin (BSA, 10 mg/ml; Sigma) in 50 mm phosphate buffer (pH 7.4)with 0.02% sodium azide, was added to 0.2m fructose and glucose (100 ml). In screw-cap tubes (1.5 ml),the reaction mixture was mixed with 200 ml of serially diluted compounds, or aminoguanidine (Sigma) aspos. control. The tubes were incubated at 378 for 14 d, and the fluorescent reaction products (200 ml) weretransferred to 96-well plates and assayed on a spectrofluorometric detector (BIO-TEK ; Synergy HT,USA) at excitation and emission wavelengths of 350 and 450 nm, resp.

REFERENCES

[1] N. Ahmed, Diabetes Res. Clin. Pract. 2005, 67, 3.[2] Z. Makita, S. Radoff, E. J. Rayfield, Z. H. Yang, E. Skolnik, V. Delaney, E. A. Friedman, A. Cerami,

H. N. Vlassara, N. Eng. J. Med. 1993, 325, 836.[3] R. Shinohara, T. Mano, A. Nagasaka, Y. Sawai, K. Uchimura, R. Hayashi, N. Hayakawa, M. Nagata,

M. Makino, H. Kakizawa, Y. Itoh, A. Nakai, M. Itoh, Biochim. Biophys. Acta 1998, 1425, 577.[4] R. G. Larkins, M. E. Dunlop, Diabetologia 1992, 35, 499.[5] S. Rahbar, K. K. Yerneni, S. Scott, N. Gonzales, I. Lalezari,Mol. Cell Biol. Res. Commun. 2000, 3,

360.[6] M. Kalousova, T. Zima, V. Tesar, S. Stipek, S. Sulkova, Kidney Blood Press. Res. 2004, 27, 18.[7] A. Beyer-Mears, E. Cruz, Diabetes 1985, 34, 15.[8] J. M. Forbes, M. E. Cooper, M. D. Oldfield, M. C. Thomas, J. Am. Soc. Nephrol. 2003, S254.[9] C. Yabe-Nishimura, Pharmacol. Rev. 1998, 50, 21.

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 355

[10] G. Y. Lee, D. S. Jang, Y. M. Lee, J. M. Kim, J. S. Kim, Arch. Pharm. Res. 2006, 29, 587.[11] D. S. Jang, J. M. Kim, Y. M. Lee, Y. S. Kim, J.-H. Kim, J. S. Kim, Chem. Pharm. Bull. 2006, 54, 1315.[12] G. Stuart, MChinese Materia MedicaN, Vegetable Kingdom, Shanghi, 1991, p. 178.[13] I. R. Lee, M. Y. Yang, Arch. Pharm. Res. 1994, 17, 476.[14] I. G. Kim, T. J. Jung, K. C. Oh, H. W. Kim, Biotechnol. Lett. 2002, 24, 1299.[15] L. Ye, J. S. Yang, Yao Xue Xue Bao 1996, 31, 844.[16] G. Pakulski, J. Budzianowski, Phytochemistry 1996, 41, 775.[17] D. D. Khac, S. T. Van, A. M. Campos, J. Y. Lallemand, M. Fetizon, Phytochemistry 1990, 29, 251.[18] U. L. B. Jayasinghe, B. A. I. S. Balasooriya, A. G. D. Bandara, Y. Fujimoto,Nat. Prod. Res. 2004, 18,

499.[19] H. A. Jung, J. E. Kim, H. Y. Chung, J. S. Choi, Arch. Pharm. Res. 2003, 26, 279.[20] M. Takemura, R. Nishida, N. Mori, Y. Kuwahara, Phytochemistry 2003, 61, 135.[21] P. J. Hougton, L. M. Lian, Phytochemistry 1986, 25, 1939.[22] X. Wen, H. Sun, J. Liu, G. Wu, L. Zhang, X. Wu, P. Ni, Bioorg. Med. Chem. Lett. 2005, 15, 4944.[23] K. Kawanishi, H. Ueda, M. Moriyasu, Curr. Med. Chem. 2003, 10, 1353.[24] H. Ueda, K. Kawanishi, M. Moriyasu, Biol. Pharm. Bull. 2004, 27, 1584.[25] H. Matsuda, T. Morikawa, I. Toguchida, M. Yoshikawa, Chem. Pharm. Bull. 2002, 50, 788.[26] H. Matsuda, T. Wang, H. Managi, M. Yoshikawa, Bioorg. Med. Chem. 2003, 11, 5317.[27] H.-Y. Kim, J. H. Oh, Biosci. Biotechnol. Biochem. 1999, 63, 184.[28] J. A. Vinson, T. B. Howard III, J. Nutr. Biochem. 1996, 7, 659.

Received July 25, 2007

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