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Xanthones inhibitors of α-glucosidase and glycation from Garcinia nobilis
Phytochemistry Letters 5 (2012) 236–239
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Xanthones inhibitors of a-glucosidase and glycation from Garcinia nobilis Hugues Fouotsa a,b, Alain Meli Lannang c,d,*, Celine Djama Mbazoa a, Saima Rasheed b, Bishnu P. Marasini b, Zulfiqar Ali b, Krishna Prasad Devkota e, Augustin Ephrem Kengfack a, Farzana Shaheen b, Muhammad Iqbal Choudhary b, Norbert Sewald d a
Department of Organic Chemistry, Faculty of Science, University of Yaounde´ I, P.O. Box 812, Yaounde´, Cameroon H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan Department of Organic Chemistry, Higher Teachers’ Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon d Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany e Institute of Forestry, Pokhara Campus, Tribhuvan University, P.O. Box. 43, Pokhara, Kaski, Nepal b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 September 2011 Received in revised form 18 December 2011 Accepted 2 January 2012 Available online 12 January 2012
One new xanthone, caroxanthone (1) together with six known xanthones, 4-prenyl-2-(3,7-dimethyl-2,6octadienyl)-1,3,5,8-tetrahydroxyxanthone (2), smeathxanthone A (3), gartanin (4), euxanthone (5), 8hydroxycudraxanthone G (6) and morusignin I (7) were isolated from the stem bark of Garcinia nobilis. The structures were determined by 1D- and 2D-NMR techniques. All these compounds were tested for anti-glycation, a-glucosidase and a-chymotrypsin activities. Some of them exhibited strong to moderate a-glucosidase activities, while none of them inhibited a-chymotrypsin. Compounds 6 and 7 were found to be modest a-glucosidase inhibitors with IC50 values of 76 mM and 84 mM, respectively. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Garcinia nobilis Clusiaceae Xanthone a-Glucosidase a-Chymotrypsin Anti-glycation
1. Introduction Plants of the genus Garcinia (family Clusiaceae), widely distributed in tropical Africa, Asia, New Caledonia, and Polynesia, have yielded an abundance of biologically active and structurally intriguing natural products (Ampofo and Waterman, 1986). Garcinia species are known to contain a wide variety of oxygenated and prenylated xanthones, as well as polyisoprenylated benzophenones such as the guttiferones (Nilar Nguyen et al., 2005). Xanthones show a wide range of biological and pharmacological properties such as antioxidant, antiinflammatory, antimicrobial, anticholinesterase and cytotoxic activities (Chin et al., 1998; Louh et al., 2008). Guttiferones have been reported as anti-HIV, trypanocidal and cytotoxic agents (Gustafson et al., 1992; Merza et al., 2006; Komguem et al., 2005). As part of our ongoing research program on the identification of bioactive constituents from plants in the genus Garcinia, we have investigated the methanol extract of the stem of Garcinia nobilis. In this paper, we describe the isolation
* Corresponding author at: Higher Teachers’ Training College, Department of Chemistry, University of Maroua, P.O. Box 55, Maroua, Cameroon. Tel.: +237 77 53 48 30; fax: +237 22 22 91 16. E-mail addresses: [email protected], [email protected] (A.M. Lannang).
and characterization of one new xanthone (1) together with six known xanthones (2–7) and their in vitro a-glucosidase, antiglycation and a-chymotrypsin inhibitory properties. 2. Results and discussion The methanol aqueous extract of the stem bark of G. nobilis was partitioned with n-hexane and ethyl acetate and subjected to silica gel column chromatography affording a new xanthone derivative named caroxanthone (1), along with six known xanthones: 3dimethyl-2-geranyl-4-prenylbellidifolin (2) (Ricaldez et al., 2000), smeathxanthone A (3) (Komguem et al., 2005), gartanin (4) (Parveen and Khan, 1988), euxanthone (5) (Han et al., 2009), 8hydroxycudraxanthone G (6) (Marcy et al., 2008) and morusignin I (7) (Chihiro et al., 1998). Compound 1 was isolated as a yellow oil. Its molecular formula C18H16O5 was deduced from the HREIMS. A positive test with alcoholic ferric chloride revealed it to be phenolic in nature. The UV spectrum showed absorption bands at lmax 352, 264, 241 and 201 nm indicating a xanthone derivative (Meli et al., 2005). The IR spectrum showed absorptions at 3730, 3370, 2922, 1739 and 1622 cm1 suggesting a xanthone skeleton with a chelated carbonyl group (Meli et al., 2005). The 1H NMR spectrum of compound 1 showed signals at d 5.30 (1H, t, J = 7.5 Hz, H-20 ), 3.37
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2012.01.002
H. Fouotsa et al. / Phytochemistry Letters 5 (2012) 236–239
(2H, d, J = 7.5 Hz, H2-10 ), 1.72 (3H, s, H3-40 ) and 1.75 (3H, s, H3-50 ) suggesting the presence of a 3-methyl-2-butenyl moiety, which was connected to C-4 (dC 124.07) on the basis of HMBC correlations. The protons at H2-10 showed HMBC correlations with C-4a (dC 153.8), C-3 (dC 137.6) and C-4 (dC 124.0). The analysis of an aromatic AB type proton at d 7.49 (1H, d, J = 8.5 Hz, H-3) and 6.88 (1H, d, J = 8.5 Hz, H-2) on one hand, then d 7.28 (1H, d, J = 8.3 Hz, H-6) and 6.69 (1H, d, J = 8.3 Hz, H-7) on the other hand, suggested the presence of two different ortho-protons in the skeleton. H-3 connected to C-3 showed HMBC correlations with C1 (dC 158.8), C-10 (dC 27.0) and C-4a, H-2 connected to C-2 showed long range correlation with C-1, C-9a (dC 107.3) and C-4 (dC 124.0). Similarly, H-6 correlated with C-10a (dC 142.9), C-5 (dC 135.7) and C-8 (dC 154.0) and H-7 with C-8a (dC 107.6), C-8, C-5. The signal resonating at dC 186.2 (C-9), dH 12.14 (1H, s, 1-OH) and 11.16 (1H, s, 8-OH) indicated the presence of a chelated carbonyl group and a chelated hydroxyl in compound 1. In the HMBC spectrum, dH 12.14 (1-OH) correlated with the signals at dC 107.33 (C-9a), 158.8 (C-1) and 106.1 (C-2). That at dH 11.16 (8-OH) showed a cross peak with a signal at dC 154.0 (C-8), 109.8 (C-7) and 107.6 (C-8a). From these spectral data, compound 1 was confirmed as a tri-oxygenated xanthone which we named caroxanthone (1,5,8-trihydroxy-4prenylxanthone) (Fig. 1). All the compounds were evaluated for in vitro anti-glycation activity as well as for a-glucosidase and a-chymotrypsin inhibition. Compounds were first screened at 1 mM concentration, at which % inhibition was measured, to identify the active compounds. Compounds for which the percent inhibition was greater than 50% were selected for IC50 determination. Results of anti-glycation activity are shown in Table 1. Only compounds 3 and 4 were found to be moderately active inhibitors of the process of protein glycation, compared with the standard inhibitor rutin (IC50 294.5 1.5 mM). These results revealed that in the xanthone skeleton, the 3-methyl-2butenyl moiety at the 2- and 4-positions is apparently essential for these compounds to inhibit the process of glycation. Apart from antiglycation all xanthones were also investigated for a-glucosidase inhibition. As shown in Table 2, most compounds possessed a significant degree of a-glucosidase inhibition; however the activity
Table 1 The inhibitory effects of compounds 1–7 on AGE formation. Compounds
% Inhibition @ 1 mM
IC50 (mM) SEM
1 2 3 4 5 6 7 a Rutin
48 40 64 67 47 24 29 86
Inactive Inactive 690.0 4.1 662.9 3.4 Inactive Inactive Inactive 294.5 1.5
IC50 values expressed as SEM (Standard Error of Mean), where n = 3. a Standard inhibitor.
237
was significantly affected by the changes in structure motifs. Inactive xanthones, i.e. compounds 1 and 7 and the most active one differed only by the relative position of the 3-methyl-2-butenyl moiety. Among all compound screened, compounds 4 and 6 were found to be potent and have little difference in their efficiency. 6 found to be most active (IC50 76 4.1 mM) followed by compound 4 (IC50 84 0.2 mM). IC50 values clearly indicate that along with the 3-methyl-2-butenyl moiety at position-2 and 4, C3 O-methylation would makes the compound an effective inhibitor. On the other hand the demethylated analogue i.e. compound 4 was found to be less effective. This implicates hydrogen bond donor ability or polarity as an important factor in defining structure activity relationship. Chain lengthening at position-2 also causes gradual decrease in activity, as in the case of compound 2 (IC50 227 0.5 mM). Along with chain lengthening, absence of a 3-methyl2-butenyl group at position-4 is mainly responsible for diminishing the potency of inhibitors, as in the case of compound 3 (IC50 394 2.4 mM). Therefore, we can conclude that xanthones having a 3-methyl-2butenyl substituent at position-2 and 4 along with a OCH3/OH moiety at position-3 mainly involved in activity while other positions showed significant tolerance. None of them were found to inhibit achymotrypsin at 500 mM. 3. Experimental 3.1. General IR spectra were recorded with a Shimadzu FTIR-8900 spectrophotometer. UV absorption spectra were determined in chloroform solution on a Thermo Scientific UV-visible spectrophotometer. Optical rotation was measured on a JASCO P.2000 polarimeter. 1H and 13C NMR spectra were recorded in deuteriochloroform on Bruker Avance 500 and 600 MHz NMRs. EIMS were obtained on a Jeol JMS.600H and HREIMS were performed on a Thermo Finnigan Mat 95 XP. TLC was performed using silica gel GF254 (Merck). Column chromatography (CC) was done with silica gel (Merck) type 100 (70-230 Mesh, ASTM) eluted either with a gradient system of n-hexane–CH2Cl2–MeOH, or Sephadex LH-20 eluted with MeOH. All the solvents were distilled commercial grade. The isolated compounds were crystallized from the same solvent and their purity was checked by TLC. Precoated plates of silica gel 60 GF254 were used for this purpose; the spots were detected with an UV lamp at 254 and 366 nm and by spraying with 50% H2SO4 or ceric sulfate followed by heating. 3.2. Plant material The stem bark of G. nobilis Engl. was collected from Okola, Central Province, Cameroon in April 2010, and identified by Mr. Victor Nana of the Cameroon National Herbarium (Yaounde´) where a voucher specimen (50779/HNC/Cam/Mt Zamangoue´) was deposited. 3.3. Extraction and isolation
Table 2 a-Glucosidase inhibition of compounds 1–7. Compounds
Concentration (mM)
% Inhibition
IC50 (mM) SEM
1 2 3 4 5 6 7 1-Deoxynojirimycina
250 250 200 125 250 250 150 1000
Inactive 74 68 84 91 81 Inactive 71
– 227 0.5 394 2.4 84 0.2 145 1.3 76 4.1 – 441 0.01
IC50 values expressed as SEM (Standard Error of Mean), where n = 3. a Standard inhibitor.
The air-dried and ground stems of G. nobilis (2 kg) was extracted three times with MeOH at room temperature. The resulting extract was concentrated under reduce pressure to obtain a crude extract (100 g). The obtain extract was dissolve in the mixture MeOH–H2O 10%. The aqueous extract was partitioned with n-hexane (15 g) and ethyl acetate (30 g). The n-hexane extract (15 g) was then subjected to silica gel flash chromatography (8 cm 14 cm), eluting with n-hexane-EtOAc of increasing polarity (10:0 1 L; 8:2 1 L, 6:4 750 mL, 4:6 500 mL, 0:10 500 mL), which were collected into 150 mL fractions and subsequently combined based on their TLC into 7 fractions A–G. Fraction B (3.0 g) was subjected to column chromatography on silica gel (25–40 mm,
H. Fouotsa et al. / Phytochemistry Letters 5 (2012) 236–239
238
compounds. After 9 days of incubation, each sample was examined for the development of specific fluorescence (excitation, 330 nm; emission, 440 nm), against sample blank on a microtitre plate spectrophotometer (Spectra Max, Molecular Devices). Rutin (Harris et al., 2011) was used as a positive control (IC50 = 294.5 1.5 mM). The percent inhibition of AGE formation in the test sample versus control was calculated for each inhibitor compound by using the following formula:
4.0 cm 60 cm) eluting with n-hexane/CH2Cl2 and CH2Cl2/MeOH by increasing polarity, collected 150 mL of each fraction and subsequently combined on the basis of similar TLC into 4 fractions B1–B4. 8-hydroxycudraxanthone G (6, 7 mg), morusignin I (7, 11 mg) were obtained from fraction B2 by using preparative thin layer chromatography (PTLC) following by further purification on silica gel chromatography (25–40 mm; 3.0 cm 15 cm) eluting with n-hexane/CH2Cl2 solvent system by increasing the polarity. Fraction B3 (2.5 g) was then further purified using column chromatography and silica gel (25–40 mm; 4.0 cm 60 cm) eluting with n-hexane/CH2Cl2 to afford euxanthone (5, 9 mg). Fraction C (700 mg) was subjected to column chromatography on silica gel (25–40 mm; 3 cm 15 cm) eluting with n-hexane/CH2Cl2 (150 mL) and subsequently combined on the basis of TLC to yield 10 fractions C1–C10. C2 (70 mg) was further purified by silica gel in the same column chromatography using n-hexane/CH2Cl2/MeOH by increasing polarity then with silica gel Normal Phase HPLC eluting with 25% ethyl acetate-hexane to afford 4-prenyl-2-(3,7dimethyl-2,6-octadienyl)-1,3,5,8-tetrahydroxyxanthone (2, 7 mg). Fraction C3 and C4 (200 mg) were combined due to similar TLC and then subjected to silica gel column chromatography (25–40 mm; 3.0 cm 15 cm) using n-hexane/CH2Cl2/MeOH by increasing polarity and then silica gel Normal Phase HPLC with n-hexane/ ethyl acetate (7.5–3.5) to afford the new xanthone (1, 5 mg) and gartanin (4, 10 mg). The high chlorophyll containing fraction D (3.0 g) was not investigated. Fractions E–G (4.0 g) were combined on the basis of TLC and subjected to Sephadex LH-20 chromatography eluting with methanol. The resultant fraction was then subjected to PTLC and further purification was done using silica gel column chromatography (25–40 mm; 4.0 cm 60 cm) to afford smeathxanthone A (3, 15 mg).
The inhibitory activity of a-chymotrypsin was performed in 50 mM Tris–HCl buffer pH 7.6 with 10 mM CaCl2 as reported by Cannell et al. (1988) with slight modification. The enzyme; achymotrypsin (12 Units/mL prepared in buffer mentioned above) with the test compound (0.5 mM) prepared in DMSO (final concentration 7%) was incubated at 30 8C for 25 min. The reaction was started by the addition of the substrate N-succinyl-Lphenylalanine-p-nitroanilide (SPpNA; 0.4 mM prepared in the buffer as above). The change in absorbance by released pnitroanilide was continuously monitored at 410 nm. Positive controls without test compound and negative controls without enzyme or with standard inhibitors were run in parallel. The percent inhibition was calculated as: % inhibition = 100 (OD of test sample/OD of the control) 100. Chymostatin was used as a positive control (IC50 = 5.7 0.13 mM).
3.4. In vitro a-glucosidase inhibition assay
3.7. Statistical analysis
a-Glucosidase activity was assayed in 0.1 M sodium phosphate buffer (pH 6.8) with p-nitrophenyl-a-D-glucopyranoside as a substrate. The concentration of a-glucosidase was 0.2 U/mL in each experiment. The enzyme (20 mL) along with 100 mL of
Results are presented as means SEM from three experiments as indicated in each figure legend. IC50 values were determined by using EZ-FIT, Enzyme kinetics software by Perrella Scientific, Inc. USA. All reagents, chemicals, and solvents used were of analytical grade. Bovine serum albumin (BSA) was purchased from Merck Marker Pvt. Ltd. Rutin, methyl glyoxal (MGO) (40% aqueous solution), a-glucosidase (from Saccharomyces cerevisiae), p-nitrophenyl-a-D-glucopyranoside, 1-deoxynojirimycin, sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), dimethyl sulfoxide (DMSO), a-chymotrypsin (from bovine pancreas), N-succinyl-L-phenylalanine-p-nitroanilide (SppNA) all were purchased from Sigma Aldrich.
phosphate buffered saline was incubated with various concentrations of tested compounds at 37 8C. The pre-incubation time was 15 min, then 20 mL of substrate (0.7 mM) was added and reaction was carried out at 37 8C for 30 min. Enzymatic activity was quantified by measuring the absorbance of p-nitrophenol at 400 nm on a microtitre plate spectrophotometer (Spectra Max, Molecular Devices, USA). One unit of a-glucosidase was defined as the amount of enzyme liberating 1.0 mmol of p-nitrophenol per minute under the conditions specified (Choudhary et al., 2010). 1Deoxynojirimycin was used as the positive control (IC50 = 441 0.01 mM). The percent inhibition of p-nitrophenol formation in the test sample versus control was calculated for each compound by using the following formula:
% inhibition ¼ 100
OD of test sample 100 OD of the control
% inhibition ¼
1 fluorescence of test sample 100 fluorescence of the control
3.6. In vitro a-chymotrypsin inhibition assay
3.8. Caroxanthone (1) Yellow powder, UV(MeOH): lmax 352, 264, 241 and 201 nm IR (KBr): ymax 3730, 3370, 2922, 1739 and 1622 cm1. EI-MS: m/ z = 312 [M]+ (98), 297 (98), 269 (96), 257 (100), 244 (14); HR-EIMS: m/z = 312.0978 (calc 312.0998) for C18H16O5. 1H (CDCl3, 500 MHz) and 13C (CDCl3, 125 MHz). See Table 3.
OH
1
9
3.5. In vitro antiglycation assay The BSA-MGO assay was performed by using the method described by Rahbar and Figarola (2003) with slight modifications. Triplicate samples of BSA at 10 mg/mL, 14 mM MGO, in 0.1 M phosphate buffer (pH 7.4) containing sodium azide (30 mM) were incubated under aseptic conditions, with each well containing 50 mL BSA, 50 mL MGO, and 20 mL test sample, at 37 8C for 9 days in the presence or absence of various concentrations of the
OH
O
8
2
8a
5
10a
OH
O 1
4a
4 1'
Fig. 1. Structure compound 1.
H. Fouotsa et al. / Phytochemistry Letters 5 (2012) 236–239 Table 3 1 H-(600 MHz), HMBC and
13
C NMR (125 MHz) data for 1 in CDCl3.
Position
13
1
1 2 3 4 4a 10a 5 6 7 8 8a 9 9a 10 20 30 40 50 1-OH 5-OH 8-OH
158.8 106.1 137.6 124.0 153.8 142.9 135.7 123.7 109.8 154.0 107.6 186.2 107.3 27.0 121.3 133.8 17.8 27.8 – – –
– 6.88 7.49 – – – – 7.28 6.69 – – – – 3.37 5.30 – 1.72 1.75 12.14 5.21 11.16
C
H [m, J (Hz)]
HMBC correlations
(d, 8.5) (d, 8.5)
158.8; 107.3; 124.0 158.8; 153.8; 27.0
(d, 8.3) (d, 8.3)
135.7; 142.9; 154.0 135.7; 154.0; 107.6
(d, 7.5) (t)
121.3; 137.6; 133.8; 124.0; 153.8 17.8; 25.8; 124.0
(s) (s) (s) (s) (s)
121.3; 121.3; 158.8; 135.7; 154.0;
133.8; 133.8; 107.3; 142.9 107.6;
27.8 17.8 106.1 109.8
Acknowledgments FOUOTSA Hugues is thankful to the TWAS for financial support for ICCBS-TWAS fellowship at the H.E.J. R.I., ICCBS, University of Karachi, Pakistan. Dr. Alain Meli Lannang would like to thank TWAS Research Grand N: 09-092 RG/CHE/AF/AC_I; UNESCO: 3240230323 of 30 April 2010, and the Alexander von Humboldt Stiftung to provided postdoctoral fellowship. We would like to acknowledge Dr. John A. Beutler, National Cancer Institute, Frederick, MD 21702, USA for English editing of the manuscript. This paper is also dedicated to the memory of Professor David Lontsi who passed away on December 22, 2008. References Ampofo, A.S., Waterman, G.P., 1986. Xanthones from three Garcinia species. Phytochemistry 25, 2351–2355.
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Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Xanthones inhibitors of a-glucosidase and glycation from Garcinia nobilis Hugues Fouotsa a,b, Alain Meli Lannang c,d,*, Celine Djama Mbazoa a, Saima Rasheed b, Bishnu P. Marasini b, Zulfiqar Ali b, Krishna Prasad Devkota e, Augustin Ephrem Kengfack a, Farzana Shaheen b, Muhammad Iqbal Choudhary b, Norbert Sewald d a
Department of Organic Chemistry, Faculty of Science, University of Yaounde´ I, P.O. Box 812, Yaounde´, Cameroon H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan Department of Organic Chemistry, Higher Teachers’ Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon d Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany e Institute of Forestry, Pokhara Campus, Tribhuvan University, P.O. Box. 43, Pokhara, Kaski, Nepal b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 September 2011 Received in revised form 18 December 2011 Accepted 2 January 2012 Available online 12 January 2012
One new xanthone, caroxanthone (1) together with six known xanthones, 4-prenyl-2-(3,7-dimethyl-2,6octadienyl)-1,3,5,8-tetrahydroxyxanthone (2), smeathxanthone A (3), gartanin (4), euxanthone (5), 8hydroxycudraxanthone G (6) and morusignin I (7) were isolated from the stem bark of Garcinia nobilis. The structures were determined by 1D- and 2D-NMR techniques. All these compounds were tested for anti-glycation, a-glucosidase and a-chymotrypsin activities. Some of them exhibited strong to moderate a-glucosidase activities, while none of them inhibited a-chymotrypsin. Compounds 6 and 7 were found to be modest a-glucosidase inhibitors with IC50 values of 76 mM and 84 mM, respectively. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Garcinia nobilis Clusiaceae Xanthone a-Glucosidase a-Chymotrypsin Anti-glycation
1. Introduction Plants of the genus Garcinia (family Clusiaceae), widely distributed in tropical Africa, Asia, New Caledonia, and Polynesia, have yielded an abundance of biologically active and structurally intriguing natural products (Ampofo and Waterman, 1986). Garcinia species are known to contain a wide variety of oxygenated and prenylated xanthones, as well as polyisoprenylated benzophenones such as the guttiferones (Nilar Nguyen et al., 2005). Xanthones show a wide range of biological and pharmacological properties such as antioxidant, antiinflammatory, antimicrobial, anticholinesterase and cytotoxic activities (Chin et al., 1998; Louh et al., 2008). Guttiferones have been reported as anti-HIV, trypanocidal and cytotoxic agents (Gustafson et al., 1992; Merza et al., 2006; Komguem et al., 2005). As part of our ongoing research program on the identification of bioactive constituents from plants in the genus Garcinia, we have investigated the methanol extract of the stem of Garcinia nobilis. In this paper, we describe the isolation
* Corresponding author at: Higher Teachers’ Training College, Department of Chemistry, University of Maroua, P.O. Box 55, Maroua, Cameroon. Tel.: +237 77 53 48 30; fax: +237 22 22 91 16. E-mail addresses: [email protected], [email protected] (A.M. Lannang).
and characterization of one new xanthone (1) together with six known xanthones (2–7) and their in vitro a-glucosidase, antiglycation and a-chymotrypsin inhibitory properties. 2. Results and discussion The methanol aqueous extract of the stem bark of G. nobilis was partitioned with n-hexane and ethyl acetate and subjected to silica gel column chromatography affording a new xanthone derivative named caroxanthone (1), along with six known xanthones: 3dimethyl-2-geranyl-4-prenylbellidifolin (2) (Ricaldez et al., 2000), smeathxanthone A (3) (Komguem et al., 2005), gartanin (4) (Parveen and Khan, 1988), euxanthone (5) (Han et al., 2009), 8hydroxycudraxanthone G (6) (Marcy et al., 2008) and morusignin I (7) (Chihiro et al., 1998). Compound 1 was isolated as a yellow oil. Its molecular formula C18H16O5 was deduced from the HREIMS. A positive test with alcoholic ferric chloride revealed it to be phenolic in nature. The UV spectrum showed absorption bands at lmax 352, 264, 241 and 201 nm indicating a xanthone derivative (Meli et al., 2005). The IR spectrum showed absorptions at 3730, 3370, 2922, 1739 and 1622 cm1 suggesting a xanthone skeleton with a chelated carbonyl group (Meli et al., 2005). The 1H NMR spectrum of compound 1 showed signals at d 5.30 (1H, t, J = 7.5 Hz, H-20 ), 3.37
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2012.01.002
H. Fouotsa et al. / Phytochemistry Letters 5 (2012) 236–239
(2H, d, J = 7.5 Hz, H2-10 ), 1.72 (3H, s, H3-40 ) and 1.75 (3H, s, H3-50 ) suggesting the presence of a 3-methyl-2-butenyl moiety, which was connected to C-4 (dC 124.07) on the basis of HMBC correlations. The protons at H2-10 showed HMBC correlations with C-4a (dC 153.8), C-3 (dC 137.6) and C-4 (dC 124.0). The analysis of an aromatic AB type proton at d 7.49 (1H, d, J = 8.5 Hz, H-3) and 6.88 (1H, d, J = 8.5 Hz, H-2) on one hand, then d 7.28 (1H, d, J = 8.3 Hz, H-6) and 6.69 (1H, d, J = 8.3 Hz, H-7) on the other hand, suggested the presence of two different ortho-protons in the skeleton. H-3 connected to C-3 showed HMBC correlations with C1 (dC 158.8), C-10 (dC 27.0) and C-4a, H-2 connected to C-2 showed long range correlation with C-1, C-9a (dC 107.3) and C-4 (dC 124.0). Similarly, H-6 correlated with C-10a (dC 142.9), C-5 (dC 135.7) and C-8 (dC 154.0) and H-7 with C-8a (dC 107.6), C-8, C-5. The signal resonating at dC 186.2 (C-9), dH 12.14 (1H, s, 1-OH) and 11.16 (1H, s, 8-OH) indicated the presence of a chelated carbonyl group and a chelated hydroxyl in compound 1. In the HMBC spectrum, dH 12.14 (1-OH) correlated with the signals at dC 107.33 (C-9a), 158.8 (C-1) and 106.1 (C-2). That at dH 11.16 (8-OH) showed a cross peak with a signal at dC 154.0 (C-8), 109.8 (C-7) and 107.6 (C-8a). From these spectral data, compound 1 was confirmed as a tri-oxygenated xanthone which we named caroxanthone (1,5,8-trihydroxy-4prenylxanthone) (Fig. 1). All the compounds were evaluated for in vitro anti-glycation activity as well as for a-glucosidase and a-chymotrypsin inhibition. Compounds were first screened at 1 mM concentration, at which % inhibition was measured, to identify the active compounds. Compounds for which the percent inhibition was greater than 50% were selected for IC50 determination. Results of anti-glycation activity are shown in Table 1. Only compounds 3 and 4 were found to be moderately active inhibitors of the process of protein glycation, compared with the standard inhibitor rutin (IC50 294.5 1.5 mM). These results revealed that in the xanthone skeleton, the 3-methyl-2butenyl moiety at the 2- and 4-positions is apparently essential for these compounds to inhibit the process of glycation. Apart from antiglycation all xanthones were also investigated for a-glucosidase inhibition. As shown in Table 2, most compounds possessed a significant degree of a-glucosidase inhibition; however the activity
Table 1 The inhibitory effects of compounds 1–7 on AGE formation. Compounds
% Inhibition @ 1 mM
IC50 (mM) SEM
1 2 3 4 5 6 7 a Rutin
48 40 64 67 47 24 29 86
Inactive Inactive 690.0 4.1 662.9 3.4 Inactive Inactive Inactive 294.5 1.5
IC50 values expressed as SEM (Standard Error of Mean), where n = 3. a Standard inhibitor.
237
was significantly affected by the changes in structure motifs. Inactive xanthones, i.e. compounds 1 and 7 and the most active one differed only by the relative position of the 3-methyl-2-butenyl moiety. Among all compound screened, compounds 4 and 6 were found to be potent and have little difference in their efficiency. 6 found to be most active (IC50 76 4.1 mM) followed by compound 4 (IC50 84 0.2 mM). IC50 values clearly indicate that along with the 3-methyl-2-butenyl moiety at position-2 and 4, C3 O-methylation would makes the compound an effective inhibitor. On the other hand the demethylated analogue i.e. compound 4 was found to be less effective. This implicates hydrogen bond donor ability or polarity as an important factor in defining structure activity relationship. Chain lengthening at position-2 also causes gradual decrease in activity, as in the case of compound 2 (IC50 227 0.5 mM). Along with chain lengthening, absence of a 3-methyl2-butenyl group at position-4 is mainly responsible for diminishing the potency of inhibitors, as in the case of compound 3 (IC50 394 2.4 mM). Therefore, we can conclude that xanthones having a 3-methyl-2butenyl substituent at position-2 and 4 along with a OCH3/OH moiety at position-3 mainly involved in activity while other positions showed significant tolerance. None of them were found to inhibit achymotrypsin at 500 mM. 3. Experimental 3.1. General IR spectra were recorded with a Shimadzu FTIR-8900 spectrophotometer. UV absorption spectra were determined in chloroform solution on a Thermo Scientific UV-visible spectrophotometer. Optical rotation was measured on a JASCO P.2000 polarimeter. 1H and 13C NMR spectra were recorded in deuteriochloroform on Bruker Avance 500 and 600 MHz NMRs. EIMS were obtained on a Jeol JMS.600H and HREIMS were performed on a Thermo Finnigan Mat 95 XP. TLC was performed using silica gel GF254 (Merck). Column chromatography (CC) was done with silica gel (Merck) type 100 (70-230 Mesh, ASTM) eluted either with a gradient system of n-hexane–CH2Cl2–MeOH, or Sephadex LH-20 eluted with MeOH. All the solvents were distilled commercial grade. The isolated compounds were crystallized from the same solvent and their purity was checked by TLC. Precoated plates of silica gel 60 GF254 were used for this purpose; the spots were detected with an UV lamp at 254 and 366 nm and by spraying with 50% H2SO4 or ceric sulfate followed by heating. 3.2. Plant material The stem bark of G. nobilis Engl. was collected from Okola, Central Province, Cameroon in April 2010, and identified by Mr. Victor Nana of the Cameroon National Herbarium (Yaounde´) where a voucher specimen (50779/HNC/Cam/Mt Zamangoue´) was deposited. 3.3. Extraction and isolation
Table 2 a-Glucosidase inhibition of compounds 1–7. Compounds
Concentration (mM)
% Inhibition
IC50 (mM) SEM
1 2 3 4 5 6 7 1-Deoxynojirimycina
250 250 200 125 250 250 150 1000
Inactive 74 68 84 91 81 Inactive 71
– 227 0.5 394 2.4 84 0.2 145 1.3 76 4.1 – 441 0.01
IC50 values expressed as SEM (Standard Error of Mean), where n = 3. a Standard inhibitor.
The air-dried and ground stems of G. nobilis (2 kg) was extracted three times with MeOH at room temperature. The resulting extract was concentrated under reduce pressure to obtain a crude extract (100 g). The obtain extract was dissolve in the mixture MeOH–H2O 10%. The aqueous extract was partitioned with n-hexane (15 g) and ethyl acetate (30 g). The n-hexane extract (15 g) was then subjected to silica gel flash chromatography (8 cm 14 cm), eluting with n-hexane-EtOAc of increasing polarity (10:0 1 L; 8:2 1 L, 6:4 750 mL, 4:6 500 mL, 0:10 500 mL), which were collected into 150 mL fractions and subsequently combined based on their TLC into 7 fractions A–G. Fraction B (3.0 g) was subjected to column chromatography on silica gel (25–40 mm,
H. Fouotsa et al. / Phytochemistry Letters 5 (2012) 236–239
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compounds. After 9 days of incubation, each sample was examined for the development of specific fluorescence (excitation, 330 nm; emission, 440 nm), against sample blank on a microtitre plate spectrophotometer (Spectra Max, Molecular Devices). Rutin (Harris et al., 2011) was used as a positive control (IC50 = 294.5 1.5 mM). The percent inhibition of AGE formation in the test sample versus control was calculated for each inhibitor compound by using the following formula:
4.0 cm 60 cm) eluting with n-hexane/CH2Cl2 and CH2Cl2/MeOH by increasing polarity, collected 150 mL of each fraction and subsequently combined on the basis of similar TLC into 4 fractions B1–B4. 8-hydroxycudraxanthone G (6, 7 mg), morusignin I (7, 11 mg) were obtained from fraction B2 by using preparative thin layer chromatography (PTLC) following by further purification on silica gel chromatography (25–40 mm; 3.0 cm 15 cm) eluting with n-hexane/CH2Cl2 solvent system by increasing the polarity. Fraction B3 (2.5 g) was then further purified using column chromatography and silica gel (25–40 mm; 4.0 cm 60 cm) eluting with n-hexane/CH2Cl2 to afford euxanthone (5, 9 mg). Fraction C (700 mg) was subjected to column chromatography on silica gel (25–40 mm; 3 cm 15 cm) eluting with n-hexane/CH2Cl2 (150 mL) and subsequently combined on the basis of TLC to yield 10 fractions C1–C10. C2 (70 mg) was further purified by silica gel in the same column chromatography using n-hexane/CH2Cl2/MeOH by increasing polarity then with silica gel Normal Phase HPLC eluting with 25% ethyl acetate-hexane to afford 4-prenyl-2-(3,7dimethyl-2,6-octadienyl)-1,3,5,8-tetrahydroxyxanthone (2, 7 mg). Fraction C3 and C4 (200 mg) were combined due to similar TLC and then subjected to silica gel column chromatography (25–40 mm; 3.0 cm 15 cm) using n-hexane/CH2Cl2/MeOH by increasing polarity and then silica gel Normal Phase HPLC with n-hexane/ ethyl acetate (7.5–3.5) to afford the new xanthone (1, 5 mg) and gartanin (4, 10 mg). The high chlorophyll containing fraction D (3.0 g) was not investigated. Fractions E–G (4.0 g) were combined on the basis of TLC and subjected to Sephadex LH-20 chromatography eluting with methanol. The resultant fraction was then subjected to PTLC and further purification was done using silica gel column chromatography (25–40 mm; 4.0 cm 60 cm) to afford smeathxanthone A (3, 15 mg).
The inhibitory activity of a-chymotrypsin was performed in 50 mM Tris–HCl buffer pH 7.6 with 10 mM CaCl2 as reported by Cannell et al. (1988) with slight modification. The enzyme; achymotrypsin (12 Units/mL prepared in buffer mentioned above) with the test compound (0.5 mM) prepared in DMSO (final concentration 7%) was incubated at 30 8C for 25 min. The reaction was started by the addition of the substrate N-succinyl-Lphenylalanine-p-nitroanilide (SPpNA; 0.4 mM prepared in the buffer as above). The change in absorbance by released pnitroanilide was continuously monitored at 410 nm. Positive controls without test compound and negative controls without enzyme or with standard inhibitors were run in parallel. The percent inhibition was calculated as: % inhibition = 100 (OD of test sample/OD of the control) 100. Chymostatin was used as a positive control (IC50 = 5.7 0.13 mM).
3.4. In vitro a-glucosidase inhibition assay
3.7. Statistical analysis
a-Glucosidase activity was assayed in 0.1 M sodium phosphate buffer (pH 6.8) with p-nitrophenyl-a-D-glucopyranoside as a substrate. The concentration of a-glucosidase was 0.2 U/mL in each experiment. The enzyme (20 mL) along with 100 mL of
Results are presented as means SEM from three experiments as indicated in each figure legend. IC50 values were determined by using EZ-FIT, Enzyme kinetics software by Perrella Scientific, Inc. USA. All reagents, chemicals, and solvents used were of analytical grade. Bovine serum albumin (BSA) was purchased from Merck Marker Pvt. Ltd. Rutin, methyl glyoxal (MGO) (40% aqueous solution), a-glucosidase (from Saccharomyces cerevisiae), p-nitrophenyl-a-D-glucopyranoside, 1-deoxynojirimycin, sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), dimethyl sulfoxide (DMSO), a-chymotrypsin (from bovine pancreas), N-succinyl-L-phenylalanine-p-nitroanilide (SppNA) all were purchased from Sigma Aldrich.
phosphate buffered saline was incubated with various concentrations of tested compounds at 37 8C. The pre-incubation time was 15 min, then 20 mL of substrate (0.7 mM) was added and reaction was carried out at 37 8C for 30 min. Enzymatic activity was quantified by measuring the absorbance of p-nitrophenol at 400 nm on a microtitre plate spectrophotometer (Spectra Max, Molecular Devices, USA). One unit of a-glucosidase was defined as the amount of enzyme liberating 1.0 mmol of p-nitrophenol per minute under the conditions specified (Choudhary et al., 2010). 1Deoxynojirimycin was used as the positive control (IC50 = 441 0.01 mM). The percent inhibition of p-nitrophenol formation in the test sample versus control was calculated for each compound by using the following formula:
% inhibition ¼ 100
OD of test sample 100 OD of the control
% inhibition ¼
1 fluorescence of test sample 100 fluorescence of the control
3.6. In vitro a-chymotrypsin inhibition assay
3.8. Caroxanthone (1) Yellow powder, UV(MeOH): lmax 352, 264, 241 and 201 nm IR (KBr): ymax 3730, 3370, 2922, 1739 and 1622 cm1. EI-MS: m/ z = 312 [M]+ (98), 297 (98), 269 (96), 257 (100), 244 (14); HR-EIMS: m/z = 312.0978 (calc 312.0998) for C18H16O5. 1H (CDCl3, 500 MHz) and 13C (CDCl3, 125 MHz). See Table 3.
OH
1
9
3.5. In vitro antiglycation assay The BSA-MGO assay was performed by using the method described by Rahbar and Figarola (2003) with slight modifications. Triplicate samples of BSA at 10 mg/mL, 14 mM MGO, in 0.1 M phosphate buffer (pH 7.4) containing sodium azide (30 mM) were incubated under aseptic conditions, with each well containing 50 mL BSA, 50 mL MGO, and 20 mL test sample, at 37 8C for 9 days in the presence or absence of various concentrations of the
OH
O
8
2
8a
5
10a
OH
O 1
4a
4 1'
Fig. 1. Structure compound 1.
H. Fouotsa et al. / Phytochemistry Letters 5 (2012) 236–239 Table 3 1 H-(600 MHz), HMBC and
13
C NMR (125 MHz) data for 1 in CDCl3.
Position
13
1
1 2 3 4 4a 10a 5 6 7 8 8a 9 9a 10 20 30 40 50 1-OH 5-OH 8-OH
158.8 106.1 137.6 124.0 153.8 142.9 135.7 123.7 109.8 154.0 107.6 186.2 107.3 27.0 121.3 133.8 17.8 27.8 – – –
– 6.88 7.49 – – – – 7.28 6.69 – – – – 3.37 5.30 – 1.72 1.75 12.14 5.21 11.16
C
H [m, J (Hz)]
HMBC correlations
(d, 8.5) (d, 8.5)
158.8; 107.3; 124.0 158.8; 153.8; 27.0
(d, 8.3) (d, 8.3)
135.7; 142.9; 154.0 135.7; 154.0; 107.6
(d, 7.5) (t)
121.3; 137.6; 133.8; 124.0; 153.8 17.8; 25.8; 124.0
(s) (s) (s) (s) (s)
121.3; 121.3; 158.8; 135.7; 154.0;
133.8; 133.8; 107.3; 142.9 107.6;
27.8 17.8 106.1 109.8
Acknowledgments FOUOTSA Hugues is thankful to the TWAS for financial support for ICCBS-TWAS fellowship at the H.E.J. R.I., ICCBS, University of Karachi, Pakistan. Dr. Alain Meli Lannang would like to thank TWAS Research Grand N: 09-092 RG/CHE/AF/AC_I; UNESCO: 3240230323 of 30 April 2010, and the Alexander von Humboldt Stiftung to provided postdoctoral fellowship. We would like to acknowledge Dr. John A. Beutler, National Cancer Institute, Frederick, MD 21702, USA for English editing of the manuscript. This paper is also dedicated to the memory of Professor David Lontsi who passed away on December 22, 2008. References Ampofo, A.S., Waterman, G.P., 1986. Xanthones from three Garcinia species. Phytochemistry 25, 2351–2355.
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