Journal of Ethnopharmacology 158 (2014) 437–441
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Inhibitory effects of flavonoids from stem bark of Derris indica on the formation of advanced glycation end products Pornpat Anusiri a, Siwattra Choodej b, Pranom Chumriang c, Sirichai Adisakwattana d, Khanitha Pudhom b,n a
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand c Mangrove Extension, Learning and Development Center 5, Satun 91000, Thailand d Department of Nutrition and Dietetics, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 May 2014 Received in revised form 7 October 2014 Accepted 21 October 2014 Available online 4 November 2014
Ethnopharmacological relevance: Derris indica (Lamk.) Bennet has been used in traditional medicine in many countries for the treatment of bronchitis, whooping cough, rheumatic joints and dipsia in diabetes. In addition, several studies have revealed that this plant displayed various pharmacological activities including anti-diabetic. The present study was designed to isolate the active compounds from its stem bark and evaluate their inhibitory activity on the formation of advanced glycation end products. Material and methods: The EtOAc extract of the stem bark of Derris indica was isolated by column chromatographic techniques. The structures of isolated compounds were established on the basis of extensive spectroscopic methods. All compounds were assayed for their inhibitory effects on advanced glycation end products formation using BSA–methylglyoxal assay. Results: Chromatographic fractionation of the EtOAc extract of Derris indica stem bark led to the isolation of two new pyranoflavonoids, derrisins A and B (1˗2), along with 11 known flavonoids (3–13). The inhibitory activities of the compounds on the formation of advanced glycation end products were evaluated. Derrisin B (2) was the most active compound with IC50 value of 18.0 mM, and displayed stronger inhibitory activity compared with positive control aminoguanidine. Conclusions: This study provided the possibility that a pyranoflavonoid (2) found in Derris indica might have therapeutic potential as an inhibitor against the formation of advanced glycation end products. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Derris indica Flavonoid Pyranoflavonoid Advanced glycation end products Antiglycation
1. Introduction Advanced glycation end products (AGEs) are unstable and irreversible substances generated in the glycation process, a nonenzymatic reaction that initiates from a complex cascade of several reactions between reducing sugars and free amino groups. AGEs can react with other free amino groups leading to protein modification such as alternative protein half-life, immune system, and enzyme function. Thus they contribute to the pathophysiology of age-related diseases, including diabetes, atherosclerosis, end-stage renal disease, and neurodegenerative disease (Brownlee, 1995). More particularly, the presence and accumulation of AGEs play an important role in the development and complications of diabetes (Ahmed, 2005; Goldin et al., 2006). Due to the emerging evidence about the adverse effects of AGEs on the patients with diabetes, the use of anti-AGEs therapy
n
Corresponding author. Tel.: þ 66 22 187 641; fax: þ66 22 541 309. E-mail address: [email protected] (K. Pudhom).
http://dx.doi.org/10.1016/j.jep.2014.10.053 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
has thus attracted considerable attention from the researchers in recent years. Derris indica (Fabaceae), also known as Pongamia pinnata (L.) Pierre, Pongamia pinnata (L.) Merr., Pongamia glabra Vent. and Millettia pinnata (L.) Panigrahi, is widely distributed throughout the mangrove areas in the southern part of Thailand. This plant is traditionally used for bronchitis, rheumatic joints and to stop dipsia in diabetes (Yadav et al., 2004). The stem⧸bark of this plant has been used for treatment of diabetes, malaria, bleeding piles, beriberi, anthelmintic, elexteric hemorrhoid, ophthalmopathy, vaginopathy, skin diseases, gentitalia, sinus, stomach pain, intestinal disorder and wound treatment (Al Muqarrabun et al., 2013). Various extracts from the leaves, pods, roots, and stems of this plant displayed significant anti-diabetic activity (Rao et al., 2009; Badole and Bodhankar, 2009; Semalty et al., 2012; Sikarwar and Patil, 2012), as well as the ethanolic extract of the flowers considerably reduced the blood glucose concentration in alloxan-induced diabetic rat (Punitha and Manoharan, 2006). However, the inhibitory activity against the AGEs formation of this plant has not been reported yet. In the present
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P. Anusiri et al. / Journal of Ethnopharmacology 158 (2014) 437–441
study, we report the isolation and structure elucidation of two new pyranoflavonoids, derrisins A and B (1–2), together with 11 known flavonoids, from the stem bark of Derris indica, and their inhibitory effects on the formation of AGEs.
2. Materials and methods 2.1. General Optical rotations were measured on a Perkin-Elmer 341 polarimeter using methanol as a solvent. Melting points were measured using a Fisher-Johns melting point apparatus. UV spectra were recorded on a Shimadzu UV-160 UV–visible spectrometer. NMR spectra were acquired on a Varian Mercury-400 Plus NMR spectrometer with TMS as internal standard. HR-ESI-MS was carried out on a micrOTOF-Q II ESI mass spectrometer. Single-crystal X-ray diffraction analysis was performed on an Oxford Gemini S Ultra diffractometer. Silica gel 60 (Merck, 230–400 mesh) and Sephadex LH20 (GE healthcare life science) were used for column chromatography. 2.2. Plant material Stem bark of Derris indica was collected from mangrove area in Satun province, Thailand, in November 2012. The plant sample was identified by one of the authors (P. Chumriang). Avoucher specimen (CUCHEM-2012-04) has been deposited at Department of Chemistry, Faculty of Science, Chulalongkorn University. 2.3. Extraction and isolation 2.3.1. Preparation of the EtOAc extract The air-dried stem bark of Derris indica (2 kg) was roughly ground and macerated in MeOH ( 3, each for two days), and filtered. The MeOH extract was concentrated, suspended in H2O, and then partitioned with EtOAc to obtain the EtOAc crude extract (27 g). The extract was subjected to quick column chromatography over silica gel using a gradient of increasing EtOAc in hexane from 10% to 100%, and a gradient of increasing MeOH in CH2Cl2 from 5% to 20% to give 11 fractions (F1–F11) in consideration of a similar TLC pattern. Fraction F3 was separated by silica gel column chromatography using mixtures of EtOAc and hexane (from 3:17 to 1:1), and further recrystallization with CHCl3 afforded compound 4 (50.6 mg, 0.19% w/w). Fraction F6 was subjected to silica gel column chromatography with a gradient system of acetone– hexane (from 3:17 to 6:4) to furnish compound 7 (14.7 mg). Fraction F7 was reapplied to a silica gel column with EtOAc– hexane (from 1:9 to 1:1) to afford compound 13 (3.7 mg, 0.017% w/w) and 12 subfractions. Subfraction F7.2 was further subjected to a Sephadex LH-20 column using MeOH as eluent to yield compound 8 (7.1 mg, 0.026% w/w), while subfraction F7.5 was separated by silica gel column chromatography eluted with EtOAc–hexane (from 2:8 to 1:1) to yield compounds 1 (4.0 mg, 0.015% w/w) and 5 (248.6 mg, 0.92% w/w). Subfraction F7.6 was also purified by silica gel column chromatography using EtOAc-hexane (from 2:8 to 1:1) to give compound 11 (3.9 mg, 0.014% w/w). Subfraction F7.7 was chromatographed on silica gel eluted with a gradient system of acetone–hexane from 3:17 to 4:6 to yield compound 3 (21.1 mg, 0.078% w/w), and subfraction F7.10 afforded compound 9 (9.6 mg, 0.036% w/w) after purification with silica gel column chromatography using the same solvent system as that of subfraction F7.7. Isolation of fraction F8 by silica gel column chromatography eluted with acetone–hexane (from 1:9 to 1:1) yielded compound 12 (47.4 mg, 0.18% w/w) and 18 subfractions. Subfractions 8.14 and 8.17 were subsequently repeated by column chromatography using the same solvent system to give compounds 2 (25.5 mg, 0.094% w/w) and 6 (171.8 mg, 0.64% w/w),
respectively. Fraction F10 was chromatographed on silica gel eluted with acetone–CH2Cl2 (from 3:17 to 4:6), followed by MeOH–CH2Cl2 (from 1:19 to 1:9) to obtain compound 10 (7.4 mg, 0.027% w/w). 2.3.2. Derrisin A (1) Colorless crystals; mp. 195–198 1C; [α]25 D 13.0 (c 0.1, MeOH); UV (MeOH) λmax 254, 316 nm; 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; HR-ESI-MS m⧸z 441.1598 [MþH] þ , calcd. for C24H25O8, 441.1544. X-ray data of 1: C24H24O8, Mr¼440.43, triclinic, a¼ 9.2968(7) Å, b¼12.0179(12) Å, c¼ 20.457 (2) Å, space group P1, Z¼4 and V¼2220.5(4) Å3, m(Mo Kα)¼ 0.099 mm 1, and F(000)¼928. Crystal dimensions: 0.34 0.08 0.04 mm3. Independent reflections: 13,081 (Rint ¼0.025). The final R1 values were 0.056, wR2 ¼0.136 (I42σ (I)). CCDC number: 938525. 2.3.3. Derrisin B (2) þ6.5 (c 0.1, MeOH); UV White solid; mp. 160–162 1C; [α]25 D (MeOH) λmax 258, 313 nm; 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; HR-ESI-MS m⧸z 367.1217 [M–H] , calcd. for C21H19O6, 367.1176. 2.4. Antiglycation assay in bovine serum albumin (BSA)–methylglyoxal (MGO) model The BSA–MGO assay was used for investigation of an inhibitor on the middle stage of the glycation of protein, and was slightly modified according to Peng et al. (2007). BSA solution (10 mg/mL in 0.1 M PBS, 125 mL, pH 7.4) was mixed with MGO (1 mM, 115 mL). Then, the indicated concentrations (0.1, 0.05, 0.025, 0.0125 and 0.00625 mM) of test compounds were added in BSA–MGO reaction, and incubated at 37 1C for 7 days. After incubation, the fluorescence intensity was measured at the excitation wavelength of 370 nm and an emission wavelength of 420 nm by using the EnSpire Multilabel Plate Reader. The % inhibition of AGEs was calculated based on the following equation and IC50 values were Table 1 1 H (400 MHz) and No.
13
C (100 MHz) NMR data (CDCl3) of compounds 1 and 2.
1
2
δH (mult, J in Hz)
δC
5.10 d (12.4) 4.58 dd (2.0, 12.4)
83.9 72.8 192.1 129.2 112.7 161.9 107.3 160.6 111.9 136.1 127.3 129.3 128.5 129.3 127.3 77.5 70.7 61.0 25.6 21.9 20.6 169.8 20.4 169.5
2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 2″ 3″ 4″ 2″-Me 2″-Me 3″-OAc
5.17 d (4.8) 6.26 d (4.8) 1.41 s 1.45 s 2.05 s
4″-OAc
1.94 s
3-OH 3-OMe 3″-OH 4″-OH
3.70 d (1.6)
7.85 d (8.8) 6.63 d (8.8)
7.46m 7.39 m 7.40 m 7.39 m 7.46 m
δH (mult, J in Hz)
7.93 d (8.8) 6.83 d (8.8)
8.10 m 7.48 m 7.46 m 7.48 m 8.10 m 3.90 dd (5.2, 6.4) 5.20 dd (5.2, 5.2) 1.44 s 1.55 s
3.80 s 3.28 d (5.6) 3.54 d (5.6)
δC 154.8 141.3 174.5 126.9 116.2 155.6 110.0 157.5 118.1 130.8 128.3 130.7 128.7 130.7 128.3 79.2 71.5 62.0 25.0 22.0
60.1
P. Anusiri et al. / Journal of Ethnopharmacology 158 (2014) 437–441
obtained from line graph between the % inhibition and the concentration of compound. Aminoguanidine (AG) was used as positive control. %inhibition ¼ ðF 0 –F t Þ=F 0 100 Ft and F0 respectively represent the fluorescence intensity of the sample and the control of mixtures. 2.5. Statistical analysis All data were expressed as mean 7S.E.M. of five determinations. The data analysis was performed by one-way analysis of variance (ANOVA), followed by Tukey's HSD test. The p value o0.05 was considered to be significant.
3. Results and discussion 3.1. Isolation and structure elucidation Repeated open column chromatography on silica gel and Sephadex LH-20 of the EtOAc crude extract led to the isolation of two new pyranoflavonoids (1–2) and 11 known compounds, including pongachromene (3), pongaflavone (4), karanjin (5), pongaglabol methyl ether (6), lacneolatin B (7), pongaglabrone (8), pongapin (9), 5-methoxy-3″,4″-methylenedioxy(8,7,4″,5″)-flavone (10), 3,7-dimethoxy-2-(4-methoxy phenyl)-flavone (11), fisetin tetramethyl ether (12), and desmethoxy kanugin (13) (Fig. 1). The structures of the known compounds were determined by comparison of their NMR spectroscopic data with those in the literature (Talapatra et al., 1980, 1982; Gupta et al., 1982; Garcez
439
et al., 1988; Tanaka et al., 1992; Das et al., 1994; Yoshioka et al., 2004; Koysomboon et al., 2006; Lee and Park, 2012 ). Compound 1 was isolated as colorless needle-like crystals and its molecular formula C24H24O8 was deduced from HR-ESI-MS at m⧸z 441.1598 [Mþ H] þ (calcd 441.1544), implying 13 degrees of unsaturation. The 1H NMR data, taken in conjunction with the UV absorption maxima at 254 and 316 nm, were indicative of a flavanone skeleton. The 1H NMR spectrum of 1 (Table 1) displayed characteristic signals for two tertiary methyls (δH 1.41 s, 1.45 s), two acetyl methyls (δH 1.94 s, 2.05 s), and one unsubstituted phenyl ring (δH 7.39 m, 2H; 7.40 m, 1H; 7.46 m, 2H). In addition, signals for ortho-coupled aromatic protons at δH 6.63 and 7.85 (each d, J¼8.8 Hz) were observed, attributable for an additional aromatic ring. Analysis of 13C NMR and HSQC data further revealed the presence of two tertiary methyls, two acetyl methyls, four oxygenated methines, one oxygenated quaternary carbon, 12 aromatic carbons (two oxygenated), and three carbonyls (two esters and one ketone). On the basis of the above NMR data, compound 1 had a tetracyclic skeleton due to nine units of the 13 unsaturations coming from three carbonyl groups and six carbon– carbon double bonds of two aromatic rings. The existence of a 2,2dimethyl-3,4-diacetyl-3,4-dihydro-2H-pyran moiety was corroborated by strong COSY correlations between H-3″ and H-4″, and HMBC correlations from both tertiary methyls (2″-Me 2) to oxygenated C-2″ and C-3″ (Fig. 2). The location of two acetyl groups at C-3″ and C-4″ was confirmed by HMBC correlations from H-3″ and H-4″ to their carbonyl carbons. Indeed, the NMR data of 1 were very similar to those of 3-methoxy-(3″,4″-dihydro-3″,4″-diacetoxy)-2″,2″-dimethylpyrano-(7,8:5″,6″)-flavone (Koysomboon et al., 2006), except for the replacement of the Δ2,3 double bond by the –CH(2)–CH(OH)(3)– unit in 1. Finally, a single-crystal X-ray diffraction study confirmed the gross structure of 1 and allowed the determination of its relative
Fig. 1. Structures of compounds 1–13 isolated from the stem bark of Derris indica.
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configuration as depicted (Fig. 3). Compound 1 has thus been named derrisin A. Compound 2, a white solid, showed a [M–H]– ion at m⧸z 367.1217 (calcd 367.1176), consistent with a molecular formula of C21H20O6. It provided a typical flavone UV spectrum (λmax 239, 258, and 312 nm). The NMR data suggested that the structure of 2 was closely related to that of pongaflavone (4) (Koysomboon et al., 2006), except for the appearance of two additional oxygenated methines [δH 3.90 dd (J¼5.2, 6.4 Hz), 5.20 dd (J¼5.2, 5.2 Hz); δC 71.5, 62.0] as well as the loss of a carbon–carbon double bond in 4. Moreover, two exchangeable protons, observed at 3.28 (d, J¼8.0 Hz) and 3.54 (d, J¼8.0 Hz), were assigned to OH-3″ and OH-4″, respectively, by the COSY correlations with their vicinal protons (Fig. 4a). An HMBC correlation between
Fig. 2. 1H–1H COSY and selected HMBC correlations of 1.
Fig. 3. ORTEP diagram of compound 1.
the methoxyl protons and the double bond carbon C-3 confirmed its location at the C-3 position. The relative configuration at C-3″ and C-4″ was deduced from the NOESY interactions to be the same as that of 1, due to the correlations of H-3″⧸H-4″ and OH-3″ and OH-4″ (Fig. 4b). Therefore compound 2 was identified as a new pyranoflavone and was named derrisin B.
3.2. Inhibitory effects on AGEs formation of compounds isolated from stem bark of Derris indica All isolated compounds, except for compound 1, were further evaluated for their inhibitory effects on the AGEs formation by using the bovine serum albumin (BSA)–methylglyoxal (MGO) antiglycation model (Peng et al., 2007). Since it is noted that not only AGEs are derived from glucose, but reactive carbonyl species such as MGO are crucial intermediates formed during glycation of proteins by glucose (Thornelly et al., 1999; Nohara et al., 2002), thus MGO should be included as a target. Aminoguanidine (AG) was used as a positive control. At the screening dose of 0.1 mM, compound 2 displayed the most potent activity with 84.5% inhibition, whereas compounds 5, 7 and 9 gave much weaker activity with the inhibition of 14.4%, 8.8% and 18.5%, respectively. The remaining compounds did not show any detectable activity at this dose. This also suggested that the loss of the C-3″, C-4″ diol moiety caused a significant loss of activity, revealing from no activity observed in compound 4. Additionally, it was subsequently found that the inhibitory effect of compound 2 was in a dose-dependent manner (Fig. 5) with an IC50 value of 18.070.4 μM. Since the current result revealed that derrisin B (2) showed remarkable antiglycation activity, much greater than
Fig. 5. % AGEs inhibition of compound 2 in the BSA–MGO model. Each value represents the mean7SEM (n¼ 5), (A) 27.72%72.24 at concentration of 6.25 mM, (B) 61.89%71.82 at concentration of 25 mM, (C) 96.53%72.22 at concentration of 50 mM, and (D) 100.65%74.22 at concentration of 100 mM.
Fig. 4. (a) 1H–1H COSY and selected HMBC correlations of compound 2. (b) Diagnostic NOESY correlations of compound 2.
P. Anusiri et al. / Journal of Ethnopharmacology 158 (2014) 437–441
the antiglycative standard AG (IC50 ¼477.1710.0 μM), it provided the possibility that this compound might have therapeutic potential as an antiglycative agent for the treatment of diabetes. 4. Conclusion This work demonstrates that the medicinal mangrove plant Derris indica is a prolific source of flavonoids. This is the first report of the inhibitory effects on the AGEs formation of flavonoids isolated from Derris indica. In particular, derrisin B (2), a new pyranoflavonoid, displaying potent activity when compared to aminoguanidine, might be useful for the development of novel AGEs inhibitor for the treatment of diabetes. Acknowledgments This work was supported by The 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund), and the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (RES560530208-AS). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.10.053. References Ahmed, N., 2005. Advanced glycation endproducts—role in pathology of diabetic complications. Diabetes Research and Clinical Practice 67, 3–21. Goldin, Alison, Beckman, Joshua A., Schmidt, Ann Marie, Creager, Mark A., 2006. Advanced glycation end product: sparking the development of diabetic vascular injury. Circulation 114, 597–605. Al Muqarrabun, L.M.R., Ahmat, N., Ruzaina, S.A.S., Ismail, N.H., Sahidin, I., 2013. Medicinal uses, phytochemistry and pharmacology of Pongamia pinnata (L.) Pierre: a review. Journal of Ethnopharmacology 150, 115–120. Badole, S.L., Bodhankar, S.L., 2009. Investigation of antihyperglycaemic activity of aqueous and petroleum ether extract of stem bark of Pongamia pinnata on serum glucose level in diabetic mice. Journal of Ethnopharmacology 123, 115–120.
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Brownlee, M.M., 1995. Advanced protein glycation in diabetes and aging. Annual Review of Medicine 46, 223–234. Das, B., Chakravarty, A.K., Masuda, K., Suzuki, H., Ageta, H., 1994. A diterpenoid from roots of Gelonium multiflorum. Phytochemistry 37, 1363–1366. Garcez, F.R., Scramin, S., Nascimento, M.C.D., Mors, W.B., 1988. Prenylated flavonoids as evolutionary indicators in the genus Dahlstedtia. Phytochemistry 27, 1079–1083. Gupta, S.C., Singh, F.P., Cook, I.B., Ternai, B., 1982. 13C NMR studies of some complex natural oxygen heterocycles. V—13C NMR spectra of furanoflavones. Organic Magnetic Resonance 20, 221–223. Koysomboon, S., Altena, I., Kato, S., Chantrapromma, K., 2006. Antimicrobacterial flavonoids from Derris indica. Phytochemistry 67, 1034–1040. Lee, J.I., Park, S.B., 2012. An effective synthesis of 3-methoxyflavones via 1-(2hydroxyphenyl)-2-methoxy-3-phenyl-1,3-propanediones. The Bulletin of the Korean Chemical Society 33, 1379–1382. Nohara, Y., Ususi, T., Kinoshita, T., Watanabe, M., 2002. Generation of superoxide anions during the reaction of guanidine compounds with methylglyoxal. Chemical and Pharmaceutical Bulletin 50, 179–184. Peng, X., Zheng, Z., Cheng, K.-W., Shan, F., Ren, G.-X., Chen, F., Wang, M., 2007. Inhibitory effect of mung bean extract and its constituents vitexin and isovitexin on the formation of advanced glycation end products. Food Chemistry 106, 475–481. Punitha, R., Manoharan, S., 2006. Antihyperglycemic and antilipidperoxidative effects of Pongamia pinnata (Linn.) Pierre flowers in alloxan induced diabetic rats. Journal of Ethnopharmacology 105, 39–46. Rao, R.R., Tiwari, A.K., Reddy, P.P., Babu, K.S., Ali, A.Z., Madhusdana, K., Rao, J.M., 2009. New furanoflavanoids, intestinal alpha-glucosidase inhibitory and freeradical (DPPH) scavenging, activity from antihyperglycemic root extract of Derris indica (Lam.). Bioorganic & Medicinal Chemistry 17, 5170–5175. Semalty, A., Semalty, M., Kumar, P., Mir, S.R., Ali, M., Amin, S., 2012. Isolation and hypoglycemic activity of a novel pongamiaflavonylflavonol from Pongamia pinnata pods. International Journal of Pharmacology 8, 265–270. Sikarwar, M.S., Patil, M.B., 2012. Antidiabetic activity of Pongamia pinnata leaf extracts in alloxan-induced diabetic rats. International Journal of Ayurveda Research 1, 199–204. Talapatra, S.K., Mallik, A.K., Talapatra, B., 1980. Pongaglabol, a new hydroxyfuranoflavone, and aurantiamide acetate, a dipeptide from the flowers of Pongamia glabra. Phytochemistry 19, 1199–1202. Talapatra, S.K., Mallik, A.K., Talapatra, B., 1982. Isopongaglabol and 6-methoxyisopongaglabol, two new hydroxyfuranoflavones from Pongamia glabra. Phytochemistry 21, 761–766. Tanaka, T., Iinuma, M., Yuki, K., Fujii, Y., Mizuno, M., 1992. Flavonoids in root bark of Pongamia pinnata. Phytochemistry 31, 993–998. Thornelly, P.J., Langborg, A., Minhas, H.S., 1999. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochemical Journal 344, 109–116. Yadav, P.P., Ahmad, G., Maurya, R., 2004. Furanoflavonoids from Pongamia pinnata fruits. Phytochemistry 65, 439–443. Yoshioka, T., Inokuchi, T., Fujioka, S., Kimura, Y., 2004. Phenolic compounds and flavonoids as plant growth regulators from fruit and leaf of Vitex rotundifolia. Zeitschrift für Naturforschung C 59, 509–514.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Inhibitory effects of flavonoids from stem bark of Derris indica on the formation of advanced glycation end products Pornpat Anusiri a, Siwattra Choodej b, Pranom Chumriang c, Sirichai Adisakwattana d, Khanitha Pudhom b,n a
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand c Mangrove Extension, Learning and Development Center 5, Satun 91000, Thailand d Department of Nutrition and Dietetics, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 May 2014 Received in revised form 7 October 2014 Accepted 21 October 2014 Available online 4 November 2014
Ethnopharmacological relevance: Derris indica (Lamk.) Bennet has been used in traditional medicine in many countries for the treatment of bronchitis, whooping cough, rheumatic joints and dipsia in diabetes. In addition, several studies have revealed that this plant displayed various pharmacological activities including anti-diabetic. The present study was designed to isolate the active compounds from its stem bark and evaluate their inhibitory activity on the formation of advanced glycation end products. Material and methods: The EtOAc extract of the stem bark of Derris indica was isolated by column chromatographic techniques. The structures of isolated compounds were established on the basis of extensive spectroscopic methods. All compounds were assayed for their inhibitory effects on advanced glycation end products formation using BSA–methylglyoxal assay. Results: Chromatographic fractionation of the EtOAc extract of Derris indica stem bark led to the isolation of two new pyranoflavonoids, derrisins A and B (1˗2), along with 11 known flavonoids (3–13). The inhibitory activities of the compounds on the formation of advanced glycation end products were evaluated. Derrisin B (2) was the most active compound with IC50 value of 18.0 mM, and displayed stronger inhibitory activity compared with positive control aminoguanidine. Conclusions: This study provided the possibility that a pyranoflavonoid (2) found in Derris indica might have therapeutic potential as an inhibitor against the formation of advanced glycation end products. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Derris indica Flavonoid Pyranoflavonoid Advanced glycation end products Antiglycation
1. Introduction Advanced glycation end products (AGEs) are unstable and irreversible substances generated in the glycation process, a nonenzymatic reaction that initiates from a complex cascade of several reactions between reducing sugars and free amino groups. AGEs can react with other free amino groups leading to protein modification such as alternative protein half-life, immune system, and enzyme function. Thus they contribute to the pathophysiology of age-related diseases, including diabetes, atherosclerosis, end-stage renal disease, and neurodegenerative disease (Brownlee, 1995). More particularly, the presence and accumulation of AGEs play an important role in the development and complications of diabetes (Ahmed, 2005; Goldin et al., 2006). Due to the emerging evidence about the adverse effects of AGEs on the patients with diabetes, the use of anti-AGEs therapy
n
Corresponding author. Tel.: þ 66 22 187 641; fax: þ66 22 541 309. E-mail address: [email protected] (K. Pudhom).
http://dx.doi.org/10.1016/j.jep.2014.10.053 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
has thus attracted considerable attention from the researchers in recent years. Derris indica (Fabaceae), also known as Pongamia pinnata (L.) Pierre, Pongamia pinnata (L.) Merr., Pongamia glabra Vent. and Millettia pinnata (L.) Panigrahi, is widely distributed throughout the mangrove areas in the southern part of Thailand. This plant is traditionally used for bronchitis, rheumatic joints and to stop dipsia in diabetes (Yadav et al., 2004). The stem⧸bark of this plant has been used for treatment of diabetes, malaria, bleeding piles, beriberi, anthelmintic, elexteric hemorrhoid, ophthalmopathy, vaginopathy, skin diseases, gentitalia, sinus, stomach pain, intestinal disorder and wound treatment (Al Muqarrabun et al., 2013). Various extracts from the leaves, pods, roots, and stems of this plant displayed significant anti-diabetic activity (Rao et al., 2009; Badole and Bodhankar, 2009; Semalty et al., 2012; Sikarwar and Patil, 2012), as well as the ethanolic extract of the flowers considerably reduced the blood glucose concentration in alloxan-induced diabetic rat (Punitha and Manoharan, 2006). However, the inhibitory activity against the AGEs formation of this plant has not been reported yet. In the present
438
P. Anusiri et al. / Journal of Ethnopharmacology 158 (2014) 437–441
study, we report the isolation and structure elucidation of two new pyranoflavonoids, derrisins A and B (1–2), together with 11 known flavonoids, from the stem bark of Derris indica, and their inhibitory effects on the formation of AGEs.
2. Materials and methods 2.1. General Optical rotations were measured on a Perkin-Elmer 341 polarimeter using methanol as a solvent. Melting points were measured using a Fisher-Johns melting point apparatus. UV spectra were recorded on a Shimadzu UV-160 UV–visible spectrometer. NMR spectra were acquired on a Varian Mercury-400 Plus NMR spectrometer with TMS as internal standard. HR-ESI-MS was carried out on a micrOTOF-Q II ESI mass spectrometer. Single-crystal X-ray diffraction analysis was performed on an Oxford Gemini S Ultra diffractometer. Silica gel 60 (Merck, 230–400 mesh) and Sephadex LH20 (GE healthcare life science) were used for column chromatography. 2.2. Plant material Stem bark of Derris indica was collected from mangrove area in Satun province, Thailand, in November 2012. The plant sample was identified by one of the authors (P. Chumriang). Avoucher specimen (CUCHEM-2012-04) has been deposited at Department of Chemistry, Faculty of Science, Chulalongkorn University. 2.3. Extraction and isolation 2.3.1. Preparation of the EtOAc extract The air-dried stem bark of Derris indica (2 kg) was roughly ground and macerated in MeOH ( 3, each for two days), and filtered. The MeOH extract was concentrated, suspended in H2O, and then partitioned with EtOAc to obtain the EtOAc crude extract (27 g). The extract was subjected to quick column chromatography over silica gel using a gradient of increasing EtOAc in hexane from 10% to 100%, and a gradient of increasing MeOH in CH2Cl2 from 5% to 20% to give 11 fractions (F1–F11) in consideration of a similar TLC pattern. Fraction F3 was separated by silica gel column chromatography using mixtures of EtOAc and hexane (from 3:17 to 1:1), and further recrystallization with CHCl3 afforded compound 4 (50.6 mg, 0.19% w/w). Fraction F6 was subjected to silica gel column chromatography with a gradient system of acetone– hexane (from 3:17 to 6:4) to furnish compound 7 (14.7 mg). Fraction F7 was reapplied to a silica gel column with EtOAc– hexane (from 1:9 to 1:1) to afford compound 13 (3.7 mg, 0.017% w/w) and 12 subfractions. Subfraction F7.2 was further subjected to a Sephadex LH-20 column using MeOH as eluent to yield compound 8 (7.1 mg, 0.026% w/w), while subfraction F7.5 was separated by silica gel column chromatography eluted with EtOAc–hexane (from 2:8 to 1:1) to yield compounds 1 (4.0 mg, 0.015% w/w) and 5 (248.6 mg, 0.92% w/w). Subfraction F7.6 was also purified by silica gel column chromatography using EtOAc-hexane (from 2:8 to 1:1) to give compound 11 (3.9 mg, 0.014% w/w). Subfraction F7.7 was chromatographed on silica gel eluted with a gradient system of acetone–hexane from 3:17 to 4:6 to yield compound 3 (21.1 mg, 0.078% w/w), and subfraction F7.10 afforded compound 9 (9.6 mg, 0.036% w/w) after purification with silica gel column chromatography using the same solvent system as that of subfraction F7.7. Isolation of fraction F8 by silica gel column chromatography eluted with acetone–hexane (from 1:9 to 1:1) yielded compound 12 (47.4 mg, 0.18% w/w) and 18 subfractions. Subfractions 8.14 and 8.17 were subsequently repeated by column chromatography using the same solvent system to give compounds 2 (25.5 mg, 0.094% w/w) and 6 (171.8 mg, 0.64% w/w),
respectively. Fraction F10 was chromatographed on silica gel eluted with acetone–CH2Cl2 (from 3:17 to 4:6), followed by MeOH–CH2Cl2 (from 1:19 to 1:9) to obtain compound 10 (7.4 mg, 0.027% w/w). 2.3.2. Derrisin A (1) Colorless crystals; mp. 195–198 1C; [α]25 D 13.0 (c 0.1, MeOH); UV (MeOH) λmax 254, 316 nm; 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; HR-ESI-MS m⧸z 441.1598 [MþH] þ , calcd. for C24H25O8, 441.1544. X-ray data of 1: C24H24O8, Mr¼440.43, triclinic, a¼ 9.2968(7) Å, b¼12.0179(12) Å, c¼ 20.457 (2) Å, space group P1, Z¼4 and V¼2220.5(4) Å3, m(Mo Kα)¼ 0.099 mm 1, and F(000)¼928. Crystal dimensions: 0.34 0.08 0.04 mm3. Independent reflections: 13,081 (Rint ¼0.025). The final R1 values were 0.056, wR2 ¼0.136 (I42σ (I)). CCDC number: 938525. 2.3.3. Derrisin B (2) þ6.5 (c 0.1, MeOH); UV White solid; mp. 160–162 1C; [α]25 D (MeOH) λmax 258, 313 nm; 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; HR-ESI-MS m⧸z 367.1217 [M–H] , calcd. for C21H19O6, 367.1176. 2.4. Antiglycation assay in bovine serum albumin (BSA)–methylglyoxal (MGO) model The BSA–MGO assay was used for investigation of an inhibitor on the middle stage of the glycation of protein, and was slightly modified according to Peng et al. (2007). BSA solution (10 mg/mL in 0.1 M PBS, 125 mL, pH 7.4) was mixed with MGO (1 mM, 115 mL). Then, the indicated concentrations (0.1, 0.05, 0.025, 0.0125 and 0.00625 mM) of test compounds were added in BSA–MGO reaction, and incubated at 37 1C for 7 days. After incubation, the fluorescence intensity was measured at the excitation wavelength of 370 nm and an emission wavelength of 420 nm by using the EnSpire Multilabel Plate Reader. The % inhibition of AGEs was calculated based on the following equation and IC50 values were Table 1 1 H (400 MHz) and No.
13
C (100 MHz) NMR data (CDCl3) of compounds 1 and 2.
1
2
δH (mult, J in Hz)
δC
5.10 d (12.4) 4.58 dd (2.0, 12.4)
83.9 72.8 192.1 129.2 112.7 161.9 107.3 160.6 111.9 136.1 127.3 129.3 128.5 129.3 127.3 77.5 70.7 61.0 25.6 21.9 20.6 169.8 20.4 169.5
2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 2″ 3″ 4″ 2″-Me 2″-Me 3″-OAc
5.17 d (4.8) 6.26 d (4.8) 1.41 s 1.45 s 2.05 s
4″-OAc
1.94 s
3-OH 3-OMe 3″-OH 4″-OH
3.70 d (1.6)
7.85 d (8.8) 6.63 d (8.8)
7.46m 7.39 m 7.40 m 7.39 m 7.46 m
δH (mult, J in Hz)
7.93 d (8.8) 6.83 d (8.8)
8.10 m 7.48 m 7.46 m 7.48 m 8.10 m 3.90 dd (5.2, 6.4) 5.20 dd (5.2, 5.2) 1.44 s 1.55 s
3.80 s 3.28 d (5.6) 3.54 d (5.6)
δC 154.8 141.3 174.5 126.9 116.2 155.6 110.0 157.5 118.1 130.8 128.3 130.7 128.7 130.7 128.3 79.2 71.5 62.0 25.0 22.0
60.1
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obtained from line graph between the % inhibition and the concentration of compound. Aminoguanidine (AG) was used as positive control. %inhibition ¼ ðF 0 –F t Þ=F 0 100 Ft and F0 respectively represent the fluorescence intensity of the sample and the control of mixtures. 2.5. Statistical analysis All data were expressed as mean 7S.E.M. of five determinations. The data analysis was performed by one-way analysis of variance (ANOVA), followed by Tukey's HSD test. The p value o0.05 was considered to be significant.
3. Results and discussion 3.1. Isolation and structure elucidation Repeated open column chromatography on silica gel and Sephadex LH-20 of the EtOAc crude extract led to the isolation of two new pyranoflavonoids (1–2) and 11 known compounds, including pongachromene (3), pongaflavone (4), karanjin (5), pongaglabol methyl ether (6), lacneolatin B (7), pongaglabrone (8), pongapin (9), 5-methoxy-3″,4″-methylenedioxy(8,7,4″,5″)-flavone (10), 3,7-dimethoxy-2-(4-methoxy phenyl)-flavone (11), fisetin tetramethyl ether (12), and desmethoxy kanugin (13) (Fig. 1). The structures of the known compounds were determined by comparison of their NMR spectroscopic data with those in the literature (Talapatra et al., 1980, 1982; Gupta et al., 1982; Garcez
439
et al., 1988; Tanaka et al., 1992; Das et al., 1994; Yoshioka et al., 2004; Koysomboon et al., 2006; Lee and Park, 2012 ). Compound 1 was isolated as colorless needle-like crystals and its molecular formula C24H24O8 was deduced from HR-ESI-MS at m⧸z 441.1598 [Mþ H] þ (calcd 441.1544), implying 13 degrees of unsaturation. The 1H NMR data, taken in conjunction with the UV absorption maxima at 254 and 316 nm, were indicative of a flavanone skeleton. The 1H NMR spectrum of 1 (Table 1) displayed characteristic signals for two tertiary methyls (δH 1.41 s, 1.45 s), two acetyl methyls (δH 1.94 s, 2.05 s), and one unsubstituted phenyl ring (δH 7.39 m, 2H; 7.40 m, 1H; 7.46 m, 2H). In addition, signals for ortho-coupled aromatic protons at δH 6.63 and 7.85 (each d, J¼8.8 Hz) were observed, attributable for an additional aromatic ring. Analysis of 13C NMR and HSQC data further revealed the presence of two tertiary methyls, two acetyl methyls, four oxygenated methines, one oxygenated quaternary carbon, 12 aromatic carbons (two oxygenated), and three carbonyls (two esters and one ketone). On the basis of the above NMR data, compound 1 had a tetracyclic skeleton due to nine units of the 13 unsaturations coming from three carbonyl groups and six carbon– carbon double bonds of two aromatic rings. The existence of a 2,2dimethyl-3,4-diacetyl-3,4-dihydro-2H-pyran moiety was corroborated by strong COSY correlations between H-3″ and H-4″, and HMBC correlations from both tertiary methyls (2″-Me 2) to oxygenated C-2″ and C-3″ (Fig. 2). The location of two acetyl groups at C-3″ and C-4″ was confirmed by HMBC correlations from H-3″ and H-4″ to their carbonyl carbons. Indeed, the NMR data of 1 were very similar to those of 3-methoxy-(3″,4″-dihydro-3″,4″-diacetoxy)-2″,2″-dimethylpyrano-(7,8:5″,6″)-flavone (Koysomboon et al., 2006), except for the replacement of the Δ2,3 double bond by the –CH(2)–CH(OH)(3)– unit in 1. Finally, a single-crystal X-ray diffraction study confirmed the gross structure of 1 and allowed the determination of its relative
Fig. 1. Structures of compounds 1–13 isolated from the stem bark of Derris indica.
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configuration as depicted (Fig. 3). Compound 1 has thus been named derrisin A. Compound 2, a white solid, showed a [M–H]– ion at m⧸z 367.1217 (calcd 367.1176), consistent with a molecular formula of C21H20O6. It provided a typical flavone UV spectrum (λmax 239, 258, and 312 nm). The NMR data suggested that the structure of 2 was closely related to that of pongaflavone (4) (Koysomboon et al., 2006), except for the appearance of two additional oxygenated methines [δH 3.90 dd (J¼5.2, 6.4 Hz), 5.20 dd (J¼5.2, 5.2 Hz); δC 71.5, 62.0] as well as the loss of a carbon–carbon double bond in 4. Moreover, two exchangeable protons, observed at 3.28 (d, J¼8.0 Hz) and 3.54 (d, J¼8.0 Hz), were assigned to OH-3″ and OH-4″, respectively, by the COSY correlations with their vicinal protons (Fig. 4a). An HMBC correlation between
Fig. 2. 1H–1H COSY and selected HMBC correlations of 1.
Fig. 3. ORTEP diagram of compound 1.
the methoxyl protons and the double bond carbon C-3 confirmed its location at the C-3 position. The relative configuration at C-3″ and C-4″ was deduced from the NOESY interactions to be the same as that of 1, due to the correlations of H-3″⧸H-4″ and OH-3″ and OH-4″ (Fig. 4b). Therefore compound 2 was identified as a new pyranoflavone and was named derrisin B.
3.2. Inhibitory effects on AGEs formation of compounds isolated from stem bark of Derris indica All isolated compounds, except for compound 1, were further evaluated for their inhibitory effects on the AGEs formation by using the bovine serum albumin (BSA)–methylglyoxal (MGO) antiglycation model (Peng et al., 2007). Since it is noted that not only AGEs are derived from glucose, but reactive carbonyl species such as MGO are crucial intermediates formed during glycation of proteins by glucose (Thornelly et al., 1999; Nohara et al., 2002), thus MGO should be included as a target. Aminoguanidine (AG) was used as a positive control. At the screening dose of 0.1 mM, compound 2 displayed the most potent activity with 84.5% inhibition, whereas compounds 5, 7 and 9 gave much weaker activity with the inhibition of 14.4%, 8.8% and 18.5%, respectively. The remaining compounds did not show any detectable activity at this dose. This also suggested that the loss of the C-3″, C-4″ diol moiety caused a significant loss of activity, revealing from no activity observed in compound 4. Additionally, it was subsequently found that the inhibitory effect of compound 2 was in a dose-dependent manner (Fig. 5) with an IC50 value of 18.070.4 μM. Since the current result revealed that derrisin B (2) showed remarkable antiglycation activity, much greater than
Fig. 5. % AGEs inhibition of compound 2 in the BSA–MGO model. Each value represents the mean7SEM (n¼ 5), (A) 27.72%72.24 at concentration of 6.25 mM, (B) 61.89%71.82 at concentration of 25 mM, (C) 96.53%72.22 at concentration of 50 mM, and (D) 100.65%74.22 at concentration of 100 mM.
Fig. 4. (a) 1H–1H COSY and selected HMBC correlations of compound 2. (b) Diagnostic NOESY correlations of compound 2.
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the antiglycative standard AG (IC50 ¼477.1710.0 μM), it provided the possibility that this compound might have therapeutic potential as an antiglycative agent for the treatment of diabetes. 4. Conclusion This work demonstrates that the medicinal mangrove plant Derris indica is a prolific source of flavonoids. This is the first report of the inhibitory effects on the AGEs formation of flavonoids isolated from Derris indica. In particular, derrisin B (2), a new pyranoflavonoid, displaying potent activity when compared to aminoguanidine, might be useful for the development of novel AGEs inhibitor for the treatment of diabetes. Acknowledgments This work was supported by The 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund), and the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (RES560530208-AS). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.10.053. References Ahmed, N., 2005. Advanced glycation endproducts—role in pathology of diabetic complications. Diabetes Research and Clinical Practice 67, 3–21. Goldin, Alison, Beckman, Joshua A., Schmidt, Ann Marie, Creager, Mark A., 2006. Advanced glycation end product: sparking the development of diabetic vascular injury. Circulation 114, 597–605. Al Muqarrabun, L.M.R., Ahmat, N., Ruzaina, S.A.S., Ismail, N.H., Sahidin, I., 2013. Medicinal uses, phytochemistry and pharmacology of Pongamia pinnata (L.) Pierre: a review. Journal of Ethnopharmacology 150, 115–120. Badole, S.L., Bodhankar, S.L., 2009. Investigation of antihyperglycaemic activity of aqueous and petroleum ether extract of stem bark of Pongamia pinnata on serum glucose level in diabetic mice. Journal of Ethnopharmacology 123, 115–120.
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